University of Veterinary Medicine Hannover
Department of Small Animal Medicine and Surgery
Central venous catheter induced thrombosis in dogs ndash occurrence
under an antithrombotic treatment regimen and possible indicators
THESIS
Submitted in partial fulfilment of the requirements for the degree
DOCTOR OF PHILOSOPHY
(PhD)
awarded by the University of Veterinary Medicine Hannover
by
Joseacute Mauricio Pereira Mora
(Cartago Costa Rica)
Hannover Germany 2020
Supervisor Prof Dr Reinhard Mischke
Supervision Group Prof Dr Reinhard Mischke
Prof Dr Manfred Kietzmann
PD Dr Mario von Depka Prondzinski
1st Evaluation Prof Dr Reinhard Mischke
Department of Small Animal Medicine and Surgery
University of Veterinary Medicine Hannover Germany
Prof Dr Manfred Kietzmann
Institute for Pharmacology Toxicology and Pharmacy
University of Veterinary Medicine Hannover Germany
PD Dr Mario von Depka Prondzinski
Werlhof Institute Medical Care Center
Hannover Germany
2nd Evaluation
Prof Dr Andreas Moritz
Clinical Pathophysiology and Clinical Laboratory Diagnostics
Justus-Liebig-University Giessen Germany
Date of final exam November 9th 2020
Parts of the thesis have been published as full paper previously in journals
Pereira JM Rohn K Mischke R 2020 Reference intervals for rotational
thromboelastometry measurements using the ROTEMreg delta device in dogs Res Vet
Sci 130 26ndash32
Parts of the thesis have been presented in abstracts related to the poster and oral
presentations at congresses
Pereira JM Mischke R 2017 Reference intervals for thromboelastometric
measurements using the ROTEMreg delta device in dogs In 10th Graduate School
DaysHannover Graduate School for Veterinary Pathobiology Neuroinfectology and
Translational Medicine 01ndash02122017 Bad Salzdetfurth Germany Stiftung
Tieraumlrztliche Hochschule Hannover P 48 P 08
Pereira JM Mischke R 2018 Reference intervals for thromboelastometric
measurements using the ROTEMreg delta device in dogs In 26 Jahrestagung der DVG-
Fachgruppe Innere Medizin und klinische Labordiagnostik 02ndash030218 Hannover
Germany Tierarztl Prax Ausg K Kleintiere Heimtiere 46(02) P18-A16
(DOI 101055s-0038-1647663)
Pereira JM Hewicker-Trautwein M von Depka Prondzinski M Mischke R 2018
Central venous catheter associated thrombosis in dogs In 11th Graduate School
DaysHannover Graduate School for Veterinary Pathobiology Neuroinfectology and
Translational Medicine 3011ndash021217 Hannover Germany Stiftung Tieraumlrztliche
Hochschule Hannover V07
Pereira JM Hewicker-Trautwein M von Depka Prondzinski M Mischke R 2019
Central venous catheter associated thrombosis in dogs In 27 Jahrestagung der DVG-
Fachgruppe Innere Medizin und klinische Labordiagnostik 01ndash020219 Munich
Germany Tierarztl Prax Ausg K Kleintiere Heimtiere 47(02) P14-139 (DOI
101055s-0039-1679119)
Pereira JM Rohn K Mischke R 2019 Reference intervals for rotational
thromboelastometric measurements using the ROTEMreg delta device in dogs In 35th
World Veterinary Association Congress 27 ndash300319 San Joseacute Costa Rica
Pereira JM Rohn K Mischke R 2019 Reference intervals for rotational
thromboelastometric measurements using the ROTEMreg delta device in dogs Abstracts
of the ESVCP-ECVCP Annual Congress 25ndash280919 Arnhem The Netherlands Vet
Clin Pathol 48 813 P 57
Pereira JM Hewicker-Trautwein M von Depka Prondzinski M Mischke R 2020
Central venous catheter associated thrombosis in dogs In 20 Annual ECVIM-CA
Congress 03ndash050920 Barcelona Spain J Vet Int Med (in press)
Sponsorship Joseacute Mauricio Pereira received a scholarship from the Universidad Nacional
de Costa Rica (reference Code JB-C-1107-2016)
Table of contents
Table of contents
Chapter 1 Introduction 1
Epidemiology of CVC related thrombosis 1
Pathogenesis of CVC related thrombosis 2
Diagnosis of CVC related thrombosis 3
Heparin thromboprophylaxis for CVC related thrombosis 5
Prediction of CVC related thrombosis 7
Aims of the study 8
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs 9
ABSTRACT 11
1 Introduction 12
2 Materials and methods 14
3 Results 20
4 Discussion 26
5 Conclusions 31
6 Acknowledgments 31
7 References 32
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes 37
ABSTRACT 40
1 Introduction 42
2 Material and methods 44
3 Results 54
Table of contents
4 Discussion 62
5 Conclusions 67
6 Acknowledgments 67
7 References 68
Chapter 4 General discussion 72
Incidence of CVC related thrombosis 72
Heparin thromboprophylaxis for CVC related thrombosis 76
Prediction of CVC related thrombosis 77
Conclusion 78
Chapter 5 Summary 79
Chapter 6 Zusammenfassung 82
Chapter 7 References 86
Acknowledgements 91
List of abbreviations
i
List of abbreviations
CV coefficient of variation
CVC central venous catheter
EJV external jugular vein
ETP endogenous thrombin potential
HDL high-density lipoprotein
IV intravenous
LDL low-density lipoprotein
LMWH low molecular weight heparin
ROTEM rotational elastometry
SC subcutaneous
TGA thrombin generation assay
UFH unfractionated heparin
Chapters 2 and 3 have their own abbreviations included
List of figures and tables
ii
List of figures and tables
Chapter 2
Table 1 Coefficients of variation () for rotational thromboelastometry using the
ROTEMreg delta analyser in normal blood samples 20
Table 2 Coefficients of variation () for rotational thromboelastometry using the
ROTEMreg delta analyser in canine blood samples with haemostatic alterations 20
Table 3 Reference values (median reference interval based on 25 - and 975 -
quantiles) for thromboelastometric measurements with the ROTEMreg delta analyser in
dogs (n=125 95 confidence intervals in brackets) 23
Table 4 Median values (minimum and maximum in brackets) for different ROTEMreg
variables using ex-tem reagent stratified by sex and neuter status 24
Table 5 Median values (minimum and maximum in brackets) for different ROTEMreg
variables using the in-tem reagent stratified by sex and neutering status 25
Chapter 3
Fig 1 Definition of different grades of central venous catheter induced thrombosis of the
jugular vein as detected using colour Doppler ultrasound 48
Fig 2 Definition and illustration of different grades of central venous catheter induced
(extraluminal) thrombosis as detected using electron microscopy 53
Fig 1S (suppl material) Definition and illustration of different grades of intraluminal
thrombosis in central venous catheters as detected using electron microscopy 53
Table 1S (Supplementary material) Heparin activities (IUml median values
minimumndashmaximum) measured in 27 dogs receiving 75 or 150 IUkg unfractionated
(standard) heparin TID subcutaneously 2 hours after the first injection on the day
Comparison of samples collected via puncture of the peripheral veins and those collected
from the central venous catheter (CVC) using the three-syringe-technique 56
List of figures and tables
iii
Fig 3ab Heparin activities (box- and whisker plot arithmetic means) in dogs receiving
unfractionated heparin at a dosage of 75 IUkg BW tid (a) or 150 IUkg BW tid (b) two
hours after the first daily heparin administration 57
Table 1 Heparin activities (IUml median values minimumndashmaximum) in 28 dogs
receiving 75 or 150 IUkg unfractionated (standard) heparin TID subcutaneously with
(grades 1ndash3) or without (grade 0) sonographically detectable thrombus Blood collections
were performed 2 hours after the first daily injection 57
Table 2 Heparin activities (IUml median values minimumndashmaximum) in 27 dogs
receiving 75 or 150 IUkg unfractionated (standard) heparin TID subcutaneously with
(score 1ndash3) or without (score 0) detectable thrombus via electron microscopy 58
Table 3 Comparison of initial results of haemostasis parameters (median values
minimumndashmaximum in dogs with (grades 1ndash3) and without (grade 0) sonographically
detectable central venous catheter-induced thromboses 60
Table 4 Comparison of results of haemostasis parameters (median values minimumndash
maximum) on the last day in dogs with (grades 1ndash3) and without (grade 0) central venous
catheter-associated thrombus formation as detected by electron microscopy 61
Chapter 1 Introduction
1
Chapter 1 Introduction
Central venous catheters (CVC) are mainly used at critical care units in human and small
animal patients requiring infusion of large amounts of fluids or blood parenteral nutrition
support routine drug therapy haemodialysis chemotherapy plasmapheresis or
periodical blood sampling (Geerts 2014 Vose et al 2019) CVC related thrombosis is
one of the most severe complications (apart from infections and pneumothorax) of CVCs
in humans (Comerlato et al 2017 Evans and Ratchford 2018 Geerts 2014) leading to
patientrsquos distress CVC dysfunction venous stenosis and in some cases to the
development of thromboembolism (Geerts 2014) In the human literature there are
various studies dealing with epidemiologic and prophylactic aspects which are described
below in detail In contrast to the best of the authoracutes knowledge studies about extra
luminal CVC related thrombosis in dogs are rare in the available veterinary literature
Studies are mainly dealing with development of intraluminal CVC thrombi occurring
frequently in dogs and cats (Langston et al 2014) and leading to CVC malfunction (Vose
et al 2019)
Epidemiology of CVC related thrombosis
CVC related thrombosis represents 70ndash80 of all upper extremity thrombosis cases and
approximately 10 of thromboembolism cases in humans (Kucher 2011) The incidence
of CVC related thrombosis reported in humans based in 25 studies ranged widely from
2 to 67 (median = 30 ) (van Rooden et al 2005) The incidence of CVC related
thrombosis detected by colour Doppler ultrasound of the internal jugular vein reported in
3 studies performed in humans was 2 (Harter et al 2002) 30 (Lordick et al 2003)
and 56 (Wu et al 1999)
Chapter 1 Introduction
2
The incidence of CVC related thrombosis in humans can vary between studies according
to the CVC type used entry site of the CVC (jugular femoral subclavian axillary
cephalic vein) and time period with the CVC in place the diagnostic thrombus detection
technique (venography or ultrasonography) and criteria for diagnosis duration of follow
up diseases and presence of predisposing factors (eg previous venous thrombosis
history presence of cancer) and finally prophylactic anticoagulation treatment (Geerts
2014 Liu et al 2015 van Rooden et al 2005)
To the best of the authoracutes knowledge data about the incidence of CVC related
thrombosis detected by colour Doppler ultrasound into the lumen of the jugular vein in
dogs are rare in the available veterinary literature In one study performed in 81 dogs and
12 cats (Adamantos et al 2010) the incidence of vein thrombosis detected by palpation
or ultrasound was 2 and the incidence of intraluminal CVC thrombosis (presumed by
the inability to draw blood back) was 15 Additionally regarding to intraluminal CVC
thrombosis an incidence of 8 was reported in one case control study performed in 24
dogs receiving CVC flushing with heparinised solution (Vose et al 2019) Data about
incidence in other animal species was also found In another case-control study performed
on 37 pigs thrombosis in the caval vein related to CVC placed in the external jugular
vein (EJV) was evaluated by histological analysis however no ultrasound detection was
performed (Kohler and Kirkman 1998) In this study in pigs 94 of patients with CVC
developed thrombi in the vena cava based on sections at the site of the end of the catheter
tip and 05 and 1 cm proximal of the first section (Kohler and Kirkman 1998)
Pathogenesis of CVC related thrombosis
Two types of CVC related thrombosis are distinguished in the human medicine literature
(Geerts 2014 van Rooden et al 2005) First symptomatic thrombosis is defined as
Chapter 1 Introduction
3
thrombosis detected by diagnostic images (ultrasonography venography) when
symptoms and signs (eg localised swelling pain redness) are present (Lee and
Kamphuisen 2012) Second subclinical thrombosis is defined as thrombosis
demonstrated by the previously mentioned diagnostic images without overt symptoms or
signs (Boersma et al 2008) Additionally the CVC related thrombi can be classified as
occlusive thrombus into the CVC lumen peri-catheter related fibrin sheaths and mural
thrombi (Geerts 2014)
During the CVC insertion the puncture of the skin on the CVC insertion site and the
rupture of the blood vessels integrity activate the coagulation cascade and the CVC acts
as a matrix for adherence and accumulation of fibrin and cellular components (Smith et
al 2012) The attachment of proteins and blood cells to the internal andor external wall
of the CVC can lead to (1) (partial or complete) occlusion of the catheter lumen and (2)
sometimes if a thrombus formation starts into the lumen of the vein (attached to the vein
wall or to the CVC) a partial or complete obstruction of the blood flow andor embolism
can develop (Langston and Eatroff 2018) Additionally a study performed in humans
(Lordick et al 2003) demonstrated a relationship between CVC related thrombosis and
microbial colonisation of the catheter leading to CVC related systemic infections This
relationship was explained among others by the ease of microorganisms like
Staphylococcus aureus and Staphylococcus epidermidis to adhere to CVC related
thrombin sheaths (Boersma et al 2008)
Diagnosis of CVC related thrombosis
Venous thrombosis is commonly diagnosed in human and small animal medicine
(especially in dogs) with vascular ultrasonography with or without use of the colour
Doppler technique which is normally used to evaluate the blood flow through the blood
Chapter 1 Introduction
4
vessels (Evans and Ratchford 2018 Laurenson et al 2010) According to the literature
in humans cited below the CVC related thrombosis detected by colour Doppler
ultrasound is present if any of the following aspects are observed (1) well defined
thrombus visualised in the lumen of the vein (2) impossibility to perform compression
of the vein by slight pressure with the ultrasound probe andor (3) no detectable blood
flow signal by colour Doppler ultrasound (Bonnet et al 1989 Koumlksoy et al 1995)
Similarly in dogs the CVC related thrombosis is usually diagnosed by colour Doppler
ultrasonography through the detection of the thrombus within the lumen of the vein
andor the obstruction (partial or complete) of the normal blood flow (Bliss et al 2002)
Especially in humans the contrast venography technique is used as gold standard to
confirm thrombosis For this purpose a contrast solution is injected through a peripheral
vein to assess its passage through the bloodstream by x-ray However this diagnostic
technique is normally used only in the case of a strong clinical suspicion of venous
thrombosis that cannot be detected by ultrasonography (Laurenson et al 2010 Langston
and Eatroff 2018) In a study performed in 44 human patients requiring a CVC (Koumlksoy
et al 1995) a sensitivity of 94 and a specificity of 96 were determined for the colour
Doppler ultrasound diagnosis when it was compared with the diagnosis performed with
the contrast venography technique That results make the colour Doppler ultrasound an
accurate diagnosis method to detect CVC related thrombosis in humans (Koumlksoy et al
1995 van Rooden et al 2003)
Advanced diagnostic image techniques like computed tomography or magnetic resonance
tomography are also accurate to perform the diagnosis of CVC related thrombosis in
humans (Whitesell and Steenburg 2014) In dogs multidetector computed tomography
venography was used in one study for the diagnosis of CVC related thrombosis allowing
the identification of catheter related sheaths around the CVC external wall (Wang at al
Chapter 1 Introduction
5
2015) However the high costs of these advanced diagnostic imaging procedures and the
requirement to perform at least sedation or general anesthesia are disadvantages when
compared with ultrasonography
Finally electron microscopy (ELMI) is a diagnostic technique that has been used in
humans (Lucas et al 2017) and experimental animals (Xiang et al 1998 Xiang et al
2001) mainly to perform a microstructural evaluation of thrombi developed into the
lumen of the CVC or in the vein containing the CVC ELMI allows the ex vivo diagnosis
of very small thrombi or sleeve related thrombosis that perhaps cannot be detected by
ultrasonography or venography during the evaluation of patients with CVCs (Xiang et al
2001) However one point to consider is that it is possible that compact thrombi
developed on the CVC can be detached during the CVC removal from the vein and
thereby before its analysis by ELMI (Xiang et al 2001)
Heparin thromboprophylaxis for CVC related thrombosis
Application of CVCs in human medicine is commonly accompanied by prophylactic
anticoagulation in order to decrease the risk of development of partial or complete vein
occlusion andor thromboembolism and thereby to improve the patientrsquos comfort
(Baumann-Kreuziger et al 2015) Apart from vitamin K antagonists (Mismetti et al
2003) unfractionated heparin (UFH) (Abdelfeki et al 2004) and low molecular weight
heparin (LMWH) (Karthaus et al 2006) are anticoagulants that have been used on
studies in humans administrated by intravenous (IV) infusion or by subcutaneous (SC)
injection to prevent thrombi development into the lumen of the veins containing the CVC
(review article Baumann-Kreuziger et al 2015) Different heparin dosages have been
used as thromboprophylactic anticoagulation in humans with CVC (review article Lee
and Kamphuisen 2012) For example in five different case-control studies performed in
Chapter 1 Introduction
6
human haemato-oncological patients UFH at a daily dosage of 100 IUkg given by IV
infusion (Abdelfeki et al 2004) and LMWH at SC daily dosages of 40 mg enoxaparin
(Verso et al 2005) 5000 IU AXa dalteparin (Karthaus et al 2006 De Cicco et al 2009)
and 2850 IU AXa nadroparin (Niers et al 2007) were used Only in part of these studies
it is stated that systemic heparin was combined with flushing of the CVC using
heparinised saline solution to reduce the risk of catheter occlusion (Karthaus et al 2006)
In 3 of the 5 previously mentioned studies no statistically significant difference was found
in the incidence of CVC related thrombosis between patients receiving the
anticoagulation prophylaxis compared with the control groups without systemic
anticoagulation (Verso et al 2005 p = 035 Karthaus et al 2006 p gt 005 Niers et al
2007 p = 049) In contrast in the residual two studies a lower incidence of CVC related
thrombosis was found in the group receiving an anticoagulation prophylaxis compared
with the control groups (Abdelfeki et al 2004 150 vs 126 p = 003 De Cicco et
al 2009 400 vs 526 p = 005)
Obviously there is no consensus in the available human literature regarding the beneficial
use of anticoagulation prophylaxis to prevent CVC related thrombosis and this may
reflect great variations for example according to the study design period trial drug
administration route (Lee and Kamphuisen 2012) and the moment of the initial
anticoagulant dosage administration (Decicco et al 2009)
To the best of the authoracutes knowledge studies about thromboprophylaxis for extraluminal
CVC related thrombosis in dogs cannot be found in the available veterinary literature
However similarly to humans prophylactic anticoagulation with heparinised saline
(administrated through CVC flushing) to prevent CVC occlusion by intraluminal thrombi
was used in one case-control study performed in 24 dogs (Vose et al 2019) In the cited
study 12 dogs received dosages of 3 ml of heparinised saline solution (10 IU UFHml) to
Chapter 1 Introduction
7
flush the catheter every 6 hours during 72 hours whereas 12 control dogs received
flushing with the same volume of non-heparinised saline solution administered at the
same frequency and no statistically significant difference was found
Prediction of CVC related thrombosis
Studies regarding the relationship between laboratory biomarkers and CVC related
thrombosis are available in the human literature (Boersma et al 2016 Cheng et al 2013
Liu et al 2015) however to the best of the authoracutes knowledge literature about laboratory
tests used as predictors for CVC related thrombosis in small animals cannot be found at
all Availability of approved blood tests which can predict the risk of thrombosis
development associated to CVC already at the moment of the CVC placement are
important in order to identify an increased risk of CVC related thrombosis (Boersma et
al 2016) In the study performed by Boersma et al (2016) in 168 human patients without
indication of anticoagulation prophylaxis elevated leukocyte counts high levels of
plasminogen activator inhibitor and of coagulation factor VIII (all measured on blood
samples taken after CVC placement) were associated with an increased incidence of
symptomatic CVC-related thrombosis diagnosed by ultrasonography In another study
(Liu et al 2015) high blood levels of lactate dehydrogenase and low concentrations of
HDL and albumin were associated with the incidence for CVC related thrombosis
diagnosed by ultrasound in 324 senile patients which was in total 617 Additionally
dyslipidemia (high triglycerides low HDL and high LDLHDL ratio) was associated
with CVC related thrombosis (diagnosed by ultrasound and computer tomography
angiography) in human patients undergoing haemodialysis (Cheng et al 2013) Studies
about predictive tests for CVC related thrombosis in dogs were not found in the available
literature to the best of the authoracutes knowledge
Chapter 1 Introduction
8
Aims of the study
The aims of the preliminary methodological study of the present scientific project were
(1) to define reference intervals for viscoelastic analyses of canine haemostasis using the
ROTEMreg delta analyser and (2) to determine the precision (repeatability) of this
method And the aims of the main study of the present scientific project were (1) to
determine the incidence of catheter-induced thrombosis in canine intensive care patients
(2) to evaluate the efficacy of a prophylactic heparin regimen routinely used in the Small
Animal Clinic of the University of Veterinary Medicine Hannover and (3) to assess
whether selected haemostasis test results are predictive for an increased risk for
thrombosis formation
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
9
Chapter 2 Reference intervals for rotational thromboelastometry measurements
using the ROTEMreg delta device in dogs
Pereira JM Rohn K Mischke R
Contributions of the authors
Pereira JM performed (1) the patients data and blood samples collection (2)
haematology and ROTEMreg analysis (3) generated the reference intervals and
coefficients of variation included in the study and (4) wrote the manuscript
Rohn K performed the statistical analysis
Mischke R performed the clinical chemistry and coagulation tests and was involved in
designing and supervision of the study analysis and discussion of the data and its
statistical analysis and revision edition and correction of the manuscript
Original source Pereira J M Rohn K amp Mischke R 2020 Reference intervals
for rotational thromboelastometry measurements using the ROTEMreg delta device
in dogs Res Vet Sci 130 26ndash32 httpsdoiorg101016jrvsc202001019
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
10
Reference intervals for rotational thromboelastometry measurements using the
ROTEMreg delta device in dogs
Pereira JMa Rohn Kb Mischke Ra
aSmall Animal Clinic University of Veterinary Medicine Hannover Buumlnteweg 9 D-
30559 Hannover Germany
bInstitute of Biometry Epidemiology and Information Processing University of
Veterinary Medicine Hannover Buumlnteweg 2 30559 Hannover Germany
Corresponding author
E-mail ReinhardMischketiho-hannoverde
Original source Pereira J M Rohn K amp Mischke R 2020 Reference intervals
for rotational thromboelastometry measurements using the ROTEMreg delta device
in dogs Res Vet Sci 130 26ndash32 httpsdoiorg101016jrvsc202001019
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
11
ABSTRACT
Aims of the present study were to define reference intervals for viscoelastic analyses of
canine haemostasis using the ROTEMreg delta analyser and as a secondary aspect to
determine the precision (repeatability) of this method Blood samples from 125 clinically
healthy dogs were included Measurements were performed with commercially available
activating reagents (ex-tem in-tem and kaolin solution) as well as without activation
Additional fourfold measurements were done in 3 of the normal blood samples and in 3
samples with haemostatic alterations to evaluate the precision of the method
Coefficients of Variation (CVs) for most of the ROTEM variables were lt 10 Clot
formation time and maximum clot elasticity showed a wide inter-individual variation in
comparison with alpha angle and maximum clot firmness A multivariate analysis on
various ROTEM parameters revealed particularly a significant influence of neuter status
and a significant interrelationship between the factors sex and neuter status for
measurements with different activating reagents These results reflected the fact that
significant differences occurred only between intact and neutered females but not in
males No or only occasionally significant differences were found between groups of sex
age and size
In conclusion CVs demonstrated that the method delivers repeatable results in canine
citrated whole blood Established reference intervals should deliver valuable orientation
for the evaluation of viscoelastic properties of clotting whole blood in dogs using the
ROTEM delta analyser Neuter status in females appeared to be the most relevant
influencing factor and should be considered for the interpretation of ROTEM delta test
results
Keywords Viscoelastic measurements reference values canine age sex neuter status
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
12
1 Introduction
Heamostasis is a physiologic and dynamic process in which plasma soluble coagulation
factors blood cells and blood vessels are involved Clot formation and possible
subsequent clot lysis as well as vascular tissue repair aim to prevent haemorrhages on the
one hand and thromboses on the other hand (Kol and Borjesson 2010 Versteeg et al
2013) Conventional assays of coagulation such as prothrombin time (PT) activated
partial thromboplastin time (aPTT) and thrombin time TT have limitations especially
cellular elements are not incorporated into the test and thereby these tests reflect only
limitedly in vivo functionality of haemostasis (McMichael and Smith 2011 Crochemore
et al 2018)
The rotational thromboelastometry (ROTEMreg) currently termed as thromboelastometry
(TEM) and thromboelastography (TEG) are methods for the rapid evaluation of the
overall haemostasis capacity through the measurement of viscoelastic properties of
clotting whole blood (Kol and Borjesson 2010 Theusinger et al 2010 Sankarankutty
et al 2012 Versteeg et al 2013) In TEG the cup with the blood sample is rotating
whereas the torsion wire is fixed In ROTEMTEM the cup is fixed whereas the pin is
rotating Changes in torque ie resistance to rotation by the fibrin bridge created between
pin and cup in the clotting blood are detected electromechanically in TEG and optically
in ROTEM (Bollinger et al 2012) and displayed as a graph that indicates the time for the
formation of blood clot and its strength (Bagge et al 2016) The resulting graph reflects
the initiation and propagation of the coagulation system fibrin-platelet interactions and
clot lysis
TEG was first described in 1948 by Hellmut Harten (Hartert 1948) as an in vitro method
to evaluate the clot formation process while resembling the in vivo blood flow The
development of the actually used instrument types TEG and ROTEMregTEM were based
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
13
on that original principle Viscoelastic measurements have been widely used since the
1980s in human medicine to evaluate haemostasis and hypercoagulable states in surgical
patients (eg cardiac and hepatic surgeries) and in general in critical care units (Kol and
Borjesson 2010 Nogami 2016 Wikkelso et al 2016 Crochemore et al 2017) In
veterinary medicine viscoelastic measurements using TEG and ROTEMTEM have been
validated for use in dogs cats and horses and used to study haemostasis in different
diseases (eg infectious diseases liver diseases) and medical conditions (eg
orthopaedic surgery hyperfibrinolysis) and also to investigate the effect of
anticoagulants and other drugs (eg corticosteroids) on haemostasis (Kol and Borjesson
2010 Bruno et al 2015a 2015b Woodman et al 2015 Conversy et al 2017 Sigrist et
al 2018 Turner et al 2019) The methodology seems to be especially suitable to detect
hypercoagulability (Wiinberg et al 2008 Kol and Borjesson 2010 Marschner et al
2018)
In the available veterinary literature information about reference values for TEG were
found using kaolin as activation reagent (Bauer et al 2009) Falco et al (2012)
determined reference values for the ROTEM analyser in dogs using ex-tem in-tem and
fib-tem reagents for selected ROTEM parameters in a study on forty five healthy dogs
However data related to reference intervals obtained in an adequate number of samples
(at least 120) according to the American Society for Veterinary Clinical Pathology
(ASVCP) (Friedrichs et al 2012) and the National Committee for Clinical Laboratory
Standards (NCCLS) (Horn and Pesce 2003) could not be found in the available literature
The aim of this study is to determine reference values for the ROTEMreg delta device in
dogs using citrated whole blood with different clot activator reagents (ex-tem in-tem
kaolin) and without activator
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
14
2 Materials and methods
21 Study design
Reference intervals Single thromboelastometric measurements were performed in 125
clinically healthy dogs using the ROTEMreg analyser and different activating reagents (ex-
tem in-tem and kaolin) as well as without activation Reference values were calculated
and influences of age groups (young adult and senior) sex neuter status and size
(groups small medium and large) were assessed
Precision analysis Additional fourfold measurements using ex-tem or in-tem activating
reagents respectively were performed on 3 of the normal and also in 3 abnormal blood
samples each in order to assess within-run imprecision for these measurements
22 Animals
The clinically healthy dogs with a body weight of at least 5 kg were recruited by launching
a call in the University of Veterinary Medicine Hannover network for students and staff
members Interested dog owners received a free health check of their animals (clinical
examination and blood tests specified below) in combination with the blood collection
In addition individual privately owned patient dogs of the Small Animal Clinic with
undisturbed general health were also included if blood was collected due to another
indication (eg general health check pre-anaesthetic examination test for hereditary
diseases etc) Before taking the blood samples all dogs were fasted for at least 12 h with
free access to water 125 animals for which the examination of the health status (clinical
examination routine haematology and clinical chemistry basis coagulation profile) did
not reveal any deviations from the reference were included in the study whereas 3 of the
128 initially recruited animals had to be excluded due to laboratory abnormalities
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
15
Haematological and clinical chemistry profiles performed in order to confirm the health
status included white and red cell count haematocrit platelet counts and reticulocytes
plasma activities of alanine aminotransferase glutamate dehydrogenase alkaline
phosphatase and pseudocholinesterase as well as concentrations of total bilirubin urea
creatinine glucose total protein albumin cholesterol and ionised calcium A basis
coagulation profile including PT PTT and TT was also performed to exclude significant
abnormalities within the blood coagulation system
The 125 healthy dogs enclosed in the establishment of reference values included 48 mixed
breed dogs and the following pedigree dogs 13 Labrador Retrievers 6 Australian
Shepherds 4 Havanese dogs 3 from each of the following breeds American
Staffordshire Terriers and Cocker Spaniels 2 from each of the following breeds Beagles
Border Collies Dalmatians Elo dogs (breed not recognised by Feacutedeacuteration cynologique
international [FCI]) German Longhaired Pointers German Shorthaired Pointers Giant
Schnauzers and Golden Retrievers 1 from each of the following breeds Belgian
Malinois Bolonka Zwetna Briard Brussels Griffon American English Coonhound
Italian Mastiff Flat Coated Retriever French Bulldog German Shepherd Great Dane
Greater Swiss Mountain Dog Giant Spitz Goldendoodle Hovawart Hungarian Vizsla
Italian Bloodhound Lycanis Wolfdog Miniature American Shepherd Miniature
Schnauzer Old German Shepherd Podenco Poodle Pyrenean Shepherd Rhodesian
Ridgeback Rottweiler Schafpudel (breed not recognised by FCI) Small
Munsterlander Spanish Mastiff Tibet Terrier Weimaraner West Highland White
Terrier and Wirehaired Pointer
These dogs were aged between 6 and 154 months (median = 410 months) and had body
weights ranging from 50 to 707 kg (median = 226 kg) 30 were castrated males 33
intact males 41 spayed females and 21 intact females According to the height at withers
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
16
the dogs were divided into 3 different size groups small (le40 cm n = 22) medium (41ndash
60 cm n = 77) and large (gt60 cm n = 26) Dogs were also divided in three different age
groups young (le 24 months n = 34) adult (25ndash84 months n = 59) and senior dogs (gt84
months n = 32)
The three blood samples with haemostatic abnormalities were taken from two dogs with
complex haemostatic disorders (Disseminated Intravascular Coagulation [DIC])
including reduced platelet count and abnormal PT and APTT and one dog with
thrombocytopenia
Animals were treated in accordance with the German Animal Welfare Law The
experimental design was approved by the Animal Welfare officer of the University of
Veterinary Medicine Hannover Foundation and by the ethics committee of the
responsible authority (Lower Saxony State Office for Consumer Protection and Food
Safety reference number 17A101)
23 Blood sampling
The venipuncture area was shaved and then disinfected with alcohol The saphenous or
cephalic vein was visualised with slight pressure and punctured with disposable cannulas
(diameter 09 or 10 mm) The continuously running blood was directly collected from
the needle into the tubes containing the respective anticoagulants From each dog
approximately 10 ml of blood were collected including approximately 8 ml of citrated
blood for the measurement of haemostasis tests (ROTEM PT aPTT TT) in tubes
containing sodium citrate 313 (Eifelfangoreg GmbH amp Co KG Bad Neuenahr-
Ahrweiler Germany) in a ratio of 1 volume9 volumes of blood In addition
approximately 1 ml of EDTA blood for haematological analysis and approximately 1 ml
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
17
of lithium heparin blood for clinical chemical measurements were collected into
commercial 13 ml tubes (Sarstedt AG amp Co Nuumlmbrecht Germany)
Citrated blood was initially kept at room temperature (20-25 C) in order to avoid errors
within the preanalytical phase (Toulon et al 2017) Two ml of citrated blood were used
for the ROTEMreg analysis the residual citrate blood was centrifuged for 10 min at 3800
rpm and 5 degC then the plasma was separated by careful pipetting and it was centrifuged
a second time under the same conditions to get platelet poor plasma which was stored in
aliquots of 500 microl and 250 microl at -80 degC for coagulation tests
24 Rotational elastometry
Viscoelastic measurements using the ROTEMreg delta for the establishment of reference
intervals were performed approximately 15 min after the blood collection using activating
reagents (liquid reagents) provided by the manufacturer of the instrument (r-ex-temreg in-
temreg) and a kaolin solution (5 g1 as part of the PTT reagent CK Prest [Diagnostica
Stago SAS Asniegraveres sur Seine Cedex France]) In addition non-activated analyses
were performed using isotonic NaCl solution instead of the activating reagent in the fourth
channel of the device Fourfold measurements for precision analysis were performed
simultaneously in the four channels of the device in separate runs
According to the standard test protocol provided by the manufacturer of the ROTEMreg
delta (Werfen GmbHInstrumentation Laboratory Munich Germany) 20 μl of the
reagent for recalcification (star-temreg20) and 20 μl of the activating reagent were pipetted
into a cuvette using the automated pipette provided with the device Immediately
thereafter 300 μl of citrated whole blood (prewarmed to 37 degC for five minutes in the
heating device of the instrument and gently rocked to resuspend possibly sedimented
cells) were added to the test preparation in the cuvette using the automated pipette and
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
18
then the cuvette was laced carefully in the ROTEMreg device whereby the rotating pin
entered into the test approach
Different parameters are provided automatically by the ROTEMreg delta device and the
following variables (described according to the Pentapharm GmbH ROTEMreg delta
Manual [2009]) were used in the study
Clotting time (CT) the time (in s) from the beginning of the test (by adding the clot
activator) until the formation of fibrin starts (defined as the moment when the curve
achieves an amplitude of 2 mm) This Parameter facilitates the decision to replace clotting
factors when it is required
Clot formation time (CFT) describes the next phase of clotting by the formation of a
stable clot through the activation of thrombocytes and fibrin formation It is defined as
the time (in s) between the 2 mm amplitude and 20 mm amplitude of the curve
Alpha angle (α angle) is the angle (in degrees) between the middle axis and the tangent
to the clotting curve through the 2 mm amplitude point
Amplitude after ldquoXXrdquo minutes (eg A30 after 30 min) is the amplitude (in mm) at a
certain time after the CT has finished
Maximum clot firmness (MCF) corresponds to the maximum amplitude (in mm) reached
before the clot is dissolved by fibrinolysis This parameter measures the firmness of the
clot and its quality Like CFT and α angle it facilitates the decision for substitution therapy
with platelet concentrate andor fibrinogen especially in humans
Maximum clot elasticity (MCE) is a parameter calculated from MCF and may be superior
at high amplitudes as compared to the MCF
Lysis index at 30 45 and 60 min (LI30 LI45 LI60) represents the fibrinolysis (in
percentage) 30 45 and 60 min after the CT It may facilitate the decision for or against a
therapy with anti-fibrinolytic drugs
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
19
Maximum lysis (ML) describes the degree of fibrinolysis relative to the MCF achieved
during the measurement (percent loss of clot firmness)
25 Statistical methods
Original data obtained from the ROTEMreg delta device software were transferred into the
Microsoftreg Excelreg formal (Microsoft Office Professional Plus 2013) The Statistical
analyses were performed using the SAS Enterprise Guide Version 71 (SAS Institute Inc
Cary NC USA)
Coefficients of Variation (CVs) for selected relevant parameters of the ROTEM delta
analysis using ex-tem and in-tem regents were calculated for the fourfold measurements
on 3 normal and 3 abnormal blood samples in order to assess within-run imprecision CVs
for the results of each sample were calculated by dividing the standard deviation of the
data set by its arithmetic mean Finally the median of the CVs for each group of 3 samples
was calculated The reference values were calculated according to recommendations of
the American College of Veterinary Clinical Pathology (ACVCP) based on non-
parametric methods (25 and 975 quantiles) supplemented by 95 confidence
intervals (Friedrichs et al 2012)
Data were analysed for Standard normal distribution by Kolmogorov Smirnov test and
assessment of residuals of the analysis of variance (ANOVA) Because these tests
indicated almost normally distributed values for the studied ROTEM variables data were
analysed by a fourway ANOVA stratified by the activating reagent (ex-tem in-tem
kaolin or without activation) to test the influence of size age sex and neuter status on
several parameters and taking all interactions into account Post hoc t-tests were
performed when indicated The significance level was set to P lt 05
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
20
3 Results
31 Precision of the method
For all the parameters evaluated in the study the CVs for the fourfold measurement in
healthy dogs were less than 10 (Table 1) In the case of dogs with haemostatic
abnormalities CVs of the vast majority of ROTEM variables were also less than 10
except the variables CFT and ML irrespective whether ex-tem or in-tem reagents were
used (Table 2)
Table 1 Coefficients of variation () for rotational thromboelastometry using the
ROTEMreg delta analyser in normal blood samples
Activator CT (s) CFT (s) α (deg) A30 (mm) MCF (mm) MCE(mm) LI30 () LI45 () LI60 () ML ()
Ex-temreg 875 629 262 213 179 634 000 020 037 534
In-temreg 245 596 166 117 135 299 000 000 046 216
Abbreviations CT ndash clotting time CFT ndash clot formation time α ndash alpha angle A30 ndash
amplitude after 30 min MCF ndash maximum clot firmness MCE ndash maximum clot elasticity
LI 30 LI 45 LI 60 ndash Lysis index at 30 45 and 60 min ML ndash maximum lysis
Median values of the measurements of 3 samples
Table 2 Coefficients of variation () for rotational thromboelastometry using the
ROTEMreg delta analyser in canine blood samples with haemostatic alterations
Activator CT (s) CFT (s) α (deg) A30 (mm) MCF (mm) MCE(mm) LI30 () LI45 () LI60 () ML ()
Ex-temreg 668 1478 393 218 268 582 0 018 000 848
In-temreg 228 799 461 219 251 371 0 0 017 283
Median values of the measurements of 3 samples from patients with DIC (n = 2)
thrombocytopenia (n = 1) abbreviations please see legend to Table 1
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
21
32 Reference intervals
Activation by ex-tem reagent (tissue factor) resulted in faster CTs when compared to
activation via the intrinsic pathway using ellagic acid (in-tem) or kaolin reagents whereas
the CFT was much less affected by the activating reagent (Table 3) Especially CFT and
MCE showed a wide inter-individual variation in comparison for example with α angle
and MCF regardless of the used activating reagent (Table 3)
33 Influence of age sex neuter status and size
ANOVA did not reveal statistically significant differences in ROTEM delta parameters
between dog groups of variable size or sex (P gt 05) Age had influence only on CT and
only when the in-tem reagent was used (P = 0169 ANOVA) but not when using the ex-
tem reagent (P = 7105) Post hoc comparisons using t-test of the in-tem results between
age groups revealed shorter CTs in the group of senior dogs when compared with young
dogs (P = 0194) but not between adult dogs and these two groups ANOVA showed
particularly influences of the neuter status and a significant interrelationship between the
factors sex and neuter status on different ROTEM delta parameters using ex-tem and in-
tem respectively as activator (Tables 4 and 5) ANOVA revealed also a significant
influence of neuter status on different ROTEM parameters when kaolin was used as
activating reagent (CFT α angle MCF MCE) whereas non-activated measurements
were not subject to any significant influence of the assessed factors (results not shown)
The interrelationship between sex and neuter status mentioned above reflected the fact
that significant differences between neutered and not neutered dogs were only found in
females The statistical analysis using the t-test to compare intact vs neutered dogs
stratified by sex showed statistically significantly (P lt 05) longer CTs and CFTs and
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
22
lower α angle A30 MCF and MCE values in spayed when compared to intact females
(Tables 4 and 5)
Chapter 2 Reference intervals for rotational thromboelastometry measurements using the ROTEMreg delta device in dogs
23
Table 3 Reference values (median reference interval based on 25 - and 975 - quantiles) for thromboelastometric measurements with the ROTEMreg delta analyser in
dogs (n=125 95 confidence intervals in brackets) Abbreviations please see legend to Table 1
Activator
Statistical
para-meter
CT
(s)
CFT
(s)
Α
(deg)
A30
(mm)
MCF
(mm)
MCE
(mm)
LI30
()
LI45
()
LI60
()
ML
()
Ex-temreg
Median 450
[430ndash470]
110
[105ndash115]
680
[670ndash690]
600
[580ndash610]
610
[600ndash620]
154
[147ndash164]
100
[960ndash100]
100
[100ndash100]
970
[960ndash980]
220
[210ndash240]
25 340
[270ndash350]
650
[490ndash710]
540
[420ndash560]
460
[410ndash480]
460
[420-490]
870
[720ndash950]
100
[100-100]
870
[800ndash960]
700
[640ndash820]
800
[100ndash110]
975 830
[790ndash990]
220
[201ndash315]
790
[770ndash800]
770
[690ndash770]
730
[700ndash770]
264
[230-334]
100
[100ndash100]
100
[100ndash100]
100
[990ndash100]
590
[530ndash680]
In-tem
Median 190
[187ndash201]
116
[111ndash125]
680
[670ndash690]
570
[560ndash580]
590
[580ndash600]
143
[135ndash149]
100
[100ndash100]
100
[100ndash100]
980
[980ndash980]
180
[170ndash190]
25 135
[112ndash142]
730
[590ndash800]
510
[460ndash570]
450
[410ndash470]
480
[410ndash500]
920
[710ndash101]
100
[950ndash100]
990
[820ndash990]
930
[740ndash930]
500
[100ndash700]
975 283
[268ndash291]
251
[198ndash227]
750
[750ndash780]
660
[660ndash690]
680
[660ndash690]
210
[195ndash227]
100
[100ndash100]
100
[100ndash100]
100
[100ndash100]
270
[260ndash380]
Activator
Statistical
para-meter
CT
(s)
CFT
(s)
Α
(deg)
A30
(mm)
MCF
(mm)
MCE
(mm)
LI30
()
LI45
()
LI60
()
ML
()
Kaolin
Median 162
[154ndash173]
870
[820ndash910]
740
[720ndash750]
500
[470ndash530]
550
[530ndash560]
121
[115ndash127]
960
[940ndash980]
840
[790ndash880]
710
[640ndash770]
500
[450ndash540]
25
109
[990ndash115]
470
[440ndash580]
560
[490ndash600]
110
[700ndash230]
390
[380ndash400]
640
[600ndash680]
270
[140ndash480]
110
[800ndash260]
800
[400ndash170]
200
[160ndash240]
975 246
[230ndash337]
214
[185ndash290]
810
[790ndash810]
640
[630ndash690]
650
[650ndash690]
187
[182ndash223]
100
[100ndash100]
990
[980ndash100]
940
[930ndash990]
970
[930ndash100]
Without activation
Median 607
[495ndash662]
221
[194ndash265]
510
[460ndash550]
520
[500ndash530]
540
[520ndash550]
115
[109ndash123]
100
[100ndash100]
100
[100ndash100]
980
[980ndash990]
170
[160ndash190]
25 203
[123ndash215]
860
[550ndash930]
190
[120ndash230]
350
[230ndash370]
370
[260ndash400]
600
[350ndash670]
100
[950ndash100]
980
[890ndash990]
930
[840ndash950]
400
[000ndash500]
975 1356
[1305ndash1721]
811
[688ndash1333]
730
[710ndash790]
660
[640ndash720]
660
[640ndash730]
197
[179ndash265]
100
[100ndash100]
100
[100ndash100]
100
[100ndash100]
290
[280ndash320]
Chapter 2 Reference intervals for rotational thromboelastometry measurements using the ROTEMreg delta device in dogs
24
Table 4 Median values (minimum and maximum in brackets) for different ROTEMreg variables using ex-tem reagent stratified by sex and neuter status
Group
CT
(s)
CFT
(s)
Α
(deg)
A30
(mm)
MCF
(mm)
MCE
(mm)
LI30
()
LI45
()
LI60
()
ML
()
Female intact 430
(320ndash530)
960
(590ndash198)
710
(570ndash800)
620
(480ndash730)
640
(510ndash740)
179
(103ndash282)
100
(100ndash100)
100
(980ndash100)
960
(800ndash100)
230
(100ndash640)
Female spayed 460
(270ndash830)
121
(650ndash224)
660
(520ndash770)
580
(430ndash690)
590
(440ndash700)
146
(790ndash230)
100
(960ndash100)
100
(800ndash100)
970
(690ndash100)
240
(100ndash590)
p-value (t-test)
Female
intact vs spayed
00025 00063 00049 00023 00016 00015 na na na na
Male intact 450
(320ndash990)
105
(650ndash315)
690
(420ndash790)
590
(420ndash700)
610
(440ndash710)
154
(770ndash244)
100
(100ndash100)
100
(870ndash100)
970
(650ndash100)
220
(700ndash680)
Male castrated 460
(340ndash930)
111
(490ndash255)
685
(480ndash800)
590
(410ndash770)
600
(420ndash770)
152
(720ndash334)
100
(100ndash100)
100
(830ndash100)
980
(640ndash100)
205
(500ndash590)
p-value (t-test)
Male
Intact vs castrated
08326 05106 04756 01799 01568 02163 na na na na
p-values (ANOVA)
Factor Sex
05428 04847 06612 02206 01887 01630 08489 07893 07689 02758
Factor neuter status 01872 00167 00173 00039 00037 00028 07141 04737 09356 05948
Interrelationship
sexneuter status 00383 00968 00782 00488 00472 00319 04943 08611 05422 00160
na ndash not analysed all significant P-values (lt 005) are highlighted in bold print
Chapter 2 Reference intervals for rotational thromboelastometry measurements using the ROTEMreg delta device in dogs
25
Table 5 Median values (minimum and maximum in brackets) for different ROTEMreg variables using the in-tem reagent stratified by sex and neutering status
Group
CT
(s)
CFT
(s)
Α
(deg)
A30
(mm)
MCF
(mm)
MCE
(mm)
LI30
()
LI45
()
LI60
()
ML
()
Female intact 186
(126ndash238)
940
(590ndash164)
720
(620ndash780)
610
(490ndash690)
620
(530ndash690)
166
(113ndash227)
100
(100ndash100)
100
(990ndash100)
980
(930ndash100)
170
(100ndash300)
Female spayed 201
(149ndash291)
1220
(820ndash262)
670
(500ndash740)
550
(450ndash660)
570
(480ndash680)
133
(910ndash214)
100
(950ndash100)
100
(820ndash100)
980
(740ndash100)
190
(500ndash380)
p-value (t-test)
Female
intact vs spayed
na na lt0001 00009 00005 00005 na na na na
Male intact 199
(117ndash283)
122
(730ndash277)
680
(460ndash750)
570
(430ndash660)
590
(450ndash660)
146
(810ndash192)
100
(100ndash100)
100
(990ndash100)
980
(930ndash100)
180
(600ndash270)
Male castrated 181
(112ndash288)
123
(820ndash222)
670
(540ndash740)
565
(410ndash630)
585
(410ndash640)
138
(710ndash180)
100
(100ndash100)
100
(970ndash100)
980
(890ndash100)
180
(800ndash260)
p-value (t-test)
Male
intact vs castrated
na na 08232 01401 01162 00849 na na na na
p-values (ANOVA)
Factor Sex
02105 03317 03370 00877 01258 00510 09564 07832 08117 03638
Factor neuter status 04289 00660 00619 00107 00088 00054 07152 09795 07056 08299
Interrelationship
sexneuter status
04310 00550 00408 00707 01733 00996 05240 05982 08047 04578
na ndash not analysed all significant P-values (lt 005) are highlighted in bold print
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
26
4 Discussion
41 Coefficients of variation
In accordance with the literature CVs below 10 indicate adequate repeatability in intra-
assays evaluations (Steiner et al 2003 Hua et al 2018) According to our results
obtained from the fourfold measurements in citrated whole blood ROTEM delta
measurements on healthy dogs and dogs with haemostatic alterations fulfil these
requirements with only a few exceptions concerning CFT and ML values in abnormal
samples
Our results regarding repeatability are similar to those obtained for several TEM
parameters in previous studies performed in animals In one study the imprecision of the
ROTEM analyser was evaluated by duplicate measurements in 10 blood samples of
healthy dogs and as result of that evaluation a good precision (CVs below 10 ) was
calculated for CT CFT MCF and α angle but in contrary to our results a low
repeatability was found for ML (CVs of 233 [ex-tem] and 285 [in-tem] own values
534 and 216 ) (Falco et al 2012)
In a previous feline study using ex-tem and in-tem as activating reagents for the ROTEM
delta repeatability was assessed similarly to the present study based on fourfold
measurements of blood samples of two healthy individuals (Doumlderlein and Mischke
2015) The results also revealed CVs below 10 for most of the parameters evaluated
The partially low repeatability of ML values may reflect the vulnerability of this variable
which is not only influenced by the complex clot formation process but also by its
subsequent lysis Reproducibility of ex-tem or in-tem activated measurements of the
ROTEM delta analyser which was not investigated in our study was studied in blood
from healthy and unhealthy horses by two different operators using three different
ROTEM devices Results indicated acceptable reproducibility (Junge et al 2016)
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
27
42 Reference intervals
The number of 125 healthy dogs used in the present study to create reference intervals is
regarded adequate according to the guidelines suggested by the American Society of
Veterinary Clinical Pathology (ASVCP) and the National Committee for Clinical
Laboratory Standards (NCCLS) who recommend to include at least 120 individuals
(Friedrichs et al 2012) To the best of our knowledge reference intervals for the most
relevant ROTEMreg delta parameters obtained in an adequate number of samples of whole
citrated canine blood have not been published so far A previous study was performed on
a smaller number of 45 dogs (Falco et al 2012) The median values for reference
intervals obtained in the present study are partially similar to those calculated in the
mentioned study based on 45 healthy dogs (eg ex- tem CT 610 s450 s [Falco et al
2012own results] CFT 150 s 110 s α angle 620deg680deg MCF 530 m 610 mm ML
180 220 in-tem CT 235 s190 s CFT 136 s116 s α angle 660deg680deg MCF
560 mm590 mm ML 30 180 ) However in comparison to our results the
reference intervals in the mentioned study are slightly wider (eg ex-tem CT 29ndash92
s34ndash83 s [Falco et al 2012own results] CFT 540ndash275 s650ndash220 s α angle 470ndash
790deg540ndash790deg MCF 360ndash730 mm460ndash730 mm ML 000ndash560 800ndash590
in-tem CT 126ndash363 s135ndash283 s CFT 470ndash224 s730ndash251 s aα angle 550ndash
810deg510ndash750deg MCF 500-ndash750 mm480ndash680 mm ML 000ndash400 500ndash270 )
although the previous study used the 5 and 95 quantiles to define limits of the
reference range whereas we calculated the commonly used 25 and 975 quantiles
to capture the central 95 of reference values (Friedrichs et al 2012)
Although it is desirable that each laboratory should specifically determine its own
reference intervals the results obtained in this study may provide an orientation for other
laboratories to interpret ROTEM delta parameters analysed in citrated canine blood In
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
28
order to test transference of the reference ranges to another laboratory a validation
procedure with measurement of 20 samples should be performed (Friedrichs et al 2012)
Although intra-individual control values (subject-based reference values) are regarded
superior to population based reference values (Wiinber et al 2007) the latter have to be
routinely used in clinical patients due to lacking individual controls especially for specific
tests such as ROTEM analyses In addition the calculated reference values cannot be
used for the fully automated device ROTEM sigma because - according to an actual
human study - values of part of the parameters do not correlate well between both
instruments (Gillissen et al 2019)
43 Influence of size age sex and neuter status
In previous studies in humans (Attard et al 2013) and animals (Mischke 1994 Borrelli
et al 2017) age has been described as an influencing factor for different haemostasis
parameters We also found an influence of age on the CT with shorter times in senior
dogs (gt 84 months) compared with young dogs (up to 24 months) which was limited to
measurements activated by the in-tem reagent
This age dependency confirms in principle a previous study on 20 healthy dogs
(Barthelemy et al 2015) which found shorter CTs in elderly dogs and results of a human
study where individuals with an age gt 45 years showed shorter CTs compared with
individuals lt 45 years old (Sucher et al 2011) However in the cited studies this effect
was only seen when ex-tem but not when the in-tem reagent was used
Based on the age distribution of our dogs which were primarily recruited to establish
reference intervals we cannot deliver information regarding very young dogs A human
study focusing on ROTEM reference ranges in children demonstrated significant
differences for nearly all ROTEM parameters with the most striking differences in
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
29
subjects aged 0-3 months (Oswald et al 2010) The limited number of dogs with an age
lt 1 year which did not allow to consider these separately in the present study may also
be one reason for the different results in a feline study (Doumlderlein and Mischke 2015) In
the cited study on healthy cats age showed an influence on CT but contrary to our study
the CT was shorter in young individuals (6-12 months) compared with older ones In
addition this was only the case in measurements using kaolin and in non-activated
measurements When compared to humans and cats uni- or multivariate analyses
considering factors age and breed or size based on do populations including different
breeds have the limitation that they can be biased by the variate life expectancy between
small medium and large dog breeds (Willems et al 2017) Apart from age we found a
significant influence only for the neuter status in bitches reflected by a significant
interaction effect between factors neuter status and sex in the ANOVA analysis
Especially for CFT values (eg activated by ex-tem median values of 121 s [spayed] vs
96 s [intact]) this difference seems to be also clinically relevant with respect to the
interpretation of ROTEM results In contrast although also significantly different
absolute and relative differences of CT values were small (eg activated by ex-tem 46 s
[spayed] vs 43 s [intact]) These differences may not only be considered for the result
interpretation of clinical patients but indicate also the necessity for sex-matched patient
groups in scientific studies
The obviously more active haemostasis system in intact females is probably related to an
influence of sex hormones (specifically estrogens) on the coagulation system
Accordingly ex vivo and in vivo studies demonstrated the activating properties of
endogenous estrogens on the haemostasis system In one ex vivo study addition of
endogenous estrogens (estrone [E1] 17szlig-estradiol [E2] and estriol [E3]) to blood samples
from men resulted in shorter mean values of the reaction time in TEG analyses
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
30
corresponding to CT in ROTEM measurements (452-537 s vs 698 s [control]) and of
the TEG variable kinetics (corresponding to the CFT in ROTEM analyses) (Swanepoel
et al 2017) In a further study subcutaneous injection of E2 into male rats induced a
significant increase in activities of coagulation factors X (by 8 ) and XI (by 10-24 )
however did not show any significant effect on PT aPTT and factor VII activity (Franco-
Murillo and Jaimez 2017)
In the cited studies also the effects of the synthetic estrogen ethinylestradiol [EE] were
studied which interestingly seems to have an inhibiting effect as indicated by prolonged
reaction time and kinetics (TEG) compared with the control in the human ex vivo study
and significantly longer PTs and aPTTs and lower factor VII and X activities in the study
on rats As expected we did not find a significant influence of size on ROTEM parameters
in dogs To the best of the authoracutes knowledge size has not been examined as an
influencing factor on viscoelastic measurements or any other haemostatic variable in
dogs Apart from Greyhounds there is limited literature on breed comparisons of
haemostatic variables (Clemons and Meyers 1984 Mischke 1994 Nielsen et al 2007
Nielsen et al 2010 Boyd et al 2018) indicating very limited breed differences for
example for factor VII (Mischke 1994) However these also do not indicate systematic
influence of size and in addition possible breed-specific effects have to be considered
As already stated one limitation of the study is the limited number of young animals
which does not allow to verify reference values for animals in this stage of life In
addition with respect to the main aim of the study possible influencing factors such as
platelet count and haematocrit were not considered in the statistical analysis Finally the
relatively small number of dogs used for the precision analysis which express the fact
that this was only a secondary aspect of the study may be regarded as a limitation At the
same time these considerations provide space for further investigations
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
31
5 Conclusions
Results of the precision analysis revealed acceptable results and thereby confirm that the
ROTEMreg delta analyser delivers repeatable results to measure overall haemostasis in
canine citrated blood samples The established reference values should offer a reliable
basis for the interpretation of thromboelastometric measurements in whole blood using
the device ROTEMreg delta in dogs with suspected haemostasis disorders Neuter status
in female dogs appeared to be a relevant influencing factor on most important ROTEMreg
parameters and should be considered for result interpretation and with respect to
experimental designs
6 Acknowledgments
Jose Mauricio Pereira received a scholarship from the Universidad Nacional de Costa
Rica (reference code JB-C-1107-2016)
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
32
7 References
1 Attard C van der Straaten T Karlafitis V Monagle P Ignjativic V 2013 Developmental
hemostasis age-specific differences in the levels of hemostatic proteins J Thromb and
Haemost 11 1850ndash1854
2 Bagge A Schoumltt U Kander T 2016 Effects of naturopathic medicines on Multiplate and
ROTEMreg A prospective experimental pilot study in healthy volunteers BMC Complemen
Altern Med 17 (16) 64
3 Bartheacutelemy A Rannou B Forterre M Verwaerde P Bonnet-Garin JM Pouzot-Nevoret
C Goy-Thollot I 2015 Differences between coagulation and cytokine profiles in dogs of
different ages Vet J 205 410ndash412
4 Bauer N Eralp O Moritz A 2009 Establishment of reference intervals for kaolin-activated
thromboelastography in dogs including an assessment of the effects of sex and anticoagulat use
J Vet Diagn Investi 21 641ndash648
5 Bollinger D Seeberger MD Tanaka KA 2012 Principles and practice of
thrromboelastography in clinical coagulation management and transfusio practice Transfus Med
Rev 26 1ndash13
6 Borrelli A Botto A Maurella C Falco S Pagani E Miniscaldo B Tarducci A Bruno
B 2017 Thromboelastometric assesment of hemostasis in newborn Piemontese calves J Vet
Diagn Invest 29 293ndash297
7 Boyd CJ Claus MA Raisis AL Hosqood G Sharp CR SmartL 2018
Hypocoagulability and platelet dysfunction are exacerbated by synthetic colloids in a canine
hemorrhagic shock model Front Vet Sci 5 279
8 Bruno B Maurella C Falco S Tarducci A Zanatta R DrsquoAngelo A Borrelli A 2015a
Thromboelastometric evaluation of hemostasis in dogs infected with Leishmania infantum J Vet
Emerg Crit Care (San Antonio) 25 502ndash511
9 Bruno B Maurella C Falco S Tarducci A Zanatta R Gianella P DrsquoAngelo A Piras L
Di Bella A Borrelli A 2015b Assessment of coagulation utilizing thromboelastometry in dogs
undergoing orthopedic surgery J Vet Emerg Crit Care (San Antonio) 25 358ndash363
10 Clemons RM Meyers KM 1984 Acquisition and aggregation of canine blood platelets Basic
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
33
mechanisms of function and differences because of breed origin Am J Vet Res 45 137ndash144
11 Conversy B Blais MC Dunn M Gara-Boivin C del Castillo JRE 2017 Anticoagulant
activity of oral rivaroxaban in healthy dogs Vet J 223 5ndash11
12 Crochemore T Piza FMT Rodriacuteguez RDR Guerra JCC Ferraz LJR Correcirca TD
2017 A new era of thromboelastometry Einstein (Sao Paulo) 15 380ndash385
13 Crochemore T Correcirca TD Lance MD Solomon C Neto AS Guerra JCC Lellis PS
Bernz LM Nunes N Mancio CM Yokoyama APH Silva E 2018 Thromboelastometry
profile in critically ill patients A single-center retrospective observational study PLoS One 13
e0192965
14 Doumlderlein E Mischke R 2015 Reference intervals for thromboelastometry with the ROTEMreg
delta in cats Res Vet Sci 100 271ndash276
15 Falco S Bruno B Maurella C Bellino C DacuteAngello A Gianella P Tarducci A Zannata
R Borrelli A 2012 In vitro evaluation of canine hemostasis following dilution with
hydroxyethyl starch (13004) via thromboelastometry J Vet Emerg Crit Care (San Antonio)
22 640ndash645
16 Franco-Murillo Y Jaimez R 2017 Inhibitory effect of ethinylestradiol on coagulation factors
in rats Exp Anim 66 107ndash113
17 Friedrichs KR Harr KE Freeman KP Szladovits B Walton RM Barnhart KF Blanco-
Chavez J 2012 ASVCP reference interval guidelines Determination of de novo reference
intervals in veterinary species and other related topics Vet Clin Pathol 41 441ndash453
18 Gillissen A va den Akker T Caram-Deelder C Henriquez DDCA Bloemenkamp
RWM Eikenboom J van der Bom JG de Maat MPM 2019 Comparison of
thromboelastometry by ROTEMreg Delta and ROTEMreg Sigma in women with postpartum
haemorrhage Scand J Clin Lab Invest 79 32ndash38
19 Harttert H 1948 Blutgerinnungstudien mit der Thrombelastographie einem neuen
Untersuchungsverfahren Klin Wochenschr 26 577ndash583
20 Horn P Pesce A 2003 Reference intervals and update Clin Chim Acta 334 5ndash23
21 Hua Yanhun Qiu W Xiao Q Wu Q 2018 Precision (repeatability and reproducibility) of
ocular parameters obtained by the Tomey OA-2000 biometer compared to the IOLMaster in
healthy eyes PLoS One 13 e0193023
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
34
22 Junge H Ringer S Mayer N Schwarzwald C 2016 Assesment of method reliability and
determination of reference intervals for rotational thromboelastometry in horses J Vet Emerg
Crit Care (San Antonio) 26 691ndash703
23 Kol A Borjesson DL 2010 Application of thrombelastographythromboelastometry to
veterinary medicine Vet Clin Pathol 39 405ndash416
24 Marschner CB Wiinberg B Tarnow I Markussen B Kuumlhnel L Bochsen L Kristensen
AT 2018 The influence of inflammation and hematocrit on clot strength in canine
thromboelastographic hypercoagulability J Vet Emerg Crit Care (San Antonio) 28 20ndash30
25 McMichael MA Smith SA 2011 Viscoelastic coagulation testing technology applications
and limitations Vet Clin Pathol 40 140ndash153
26 Mischke R 1994 Activity of coagulation factors II V VII and X in healthy dogs- dependence
on age sex and breed Berl Muumlnch Tieraumlrztl Wochenschr 107 289ndash294
27 Nielsen A Zois N Pedersen H Olse L Tarnow I 2007 Platelet function in dogs bread
differences and effect of acetylsalicylic acid administration Vet Clin Pathol 36 267ndash273
28 Nielsen L Kjelgaard‐Hansen M Jensen A Kristensen A 2010 Breed‐specific variation of
hematologic and biochemical analytes in healthy adult Bernese Mountain dogs Vet Clin Pathol
39 20ndash28
29 Nogami K 2016 The utility of thromboelastography in inherited and acquired bleeding
disorders Br J Haematol 174 503ndash514
30 Oswald E Stalzer B Heitz E Weiss M Schmugge M Strasak A Innerhofer P Haas T
2010 Thromboelastometry (ROTEMreg) in children Age-related reference ranges and correlations
with standard coagulation tests Br J Anaesth 105 827ndash835
31 Sankarankutty A Nascimento B Teodoro da Luz L Rizoli S 2012 TEGreg and ROTEMreg
in trauma similar test but different results World J Emerg Surg 7 (Suppl 1) S3
32 Sigrist NE Schefer RJJ Kutter APN 2018 Characteristics of hyperfibrinolysis in dogs and
cats demonstrated by rotational Thromboelastometry (ROTEM) Vet J 242 67-73
33 Steiner J Teague S Williams D 2003 Development and analytic validation of an enzime-
linked immunosorbent assay for the measurement of canine pancreatic lipase immunoreactivity in
serum Can J Vet Res 68 161ndash168
34 Sucker C Tharra K Lithmate J Scharf RE Zotz RB 2011 Rotation thromboelastography
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
35
(ROTEM) parameters are influenced by age gender and oral contraception Perfusion 26 334ndash
340
35 Swanepoel A Emmerson O Pretorius E 2017 The effect of endogenous and synthetic
estrogens on whole blood clot formation and erythrocyte structure Microsc Microanal 23 599ndash
606
36 Theusinger OM Nuumlrnberg J Asmis LM Seifert B Spahn DR 2010 Rotation
thromboelastometry (ROTEM) stability and reproducibility over time Eur J Cardiothorac Surg
37 677ndash683
37 Toulon P Metge S Hangard M Zwahlen S Piaulenne S Besson V 2017 Impact of
different storage times at room temperature of unspun citrated blood samples on routine
coagulation tests results Results of a bicenter study and review of the literature Int J Lab
Hematol 3 458ndash468
38 Turner JS Kutter APN Sigrist NE 2019 Correlation of rotational thrromboelastometry
(ROTEM) parameters with platelet count and their ability to predict thrombocytopenia in dogs
Res Vet Sci 126 45ndash50
39 Versteeg HH Heemskerk JWM Levi M Reitsma PH 2013 New Fundamentals in
Hemostasis Physiol Rev 93 327ndash358
40 Wiinberg B Jensen AL Kjelgaard-Hansen M Rojkjaer R Johansson PI Gade LP
Gram DX Kristensen AT 2007 Study on biological variation of haemostatic parameters in
clinically healthy dogs Vet J 174 62ndash68
41 Wiinberg B Jensen AL Johansson PL Rozanski E Tranholm M Kristensen AT 2008
Thromboelastographic evaluation of hemostatic function in dogs with disseminated intravascular
coagulation J Vet Intern Med 22 357ndash365
42 Wikkelsoslash A Wetterslev J Moslashller AM Afshari A 2016 Thromboelastography (TEG) or
thromboelastometry (ROTEMreg) to monitor haemostatic treatment versus usual care in adults or
children with bleeding Cochrane Database Syst Rev Art
httpsdoiorg10100214651858CD007871pub3 No CD007871
43 Willems A Paepe D Marynissen S Smets P Van de Maele I Picavet P Duchateau L
Daminet S 2017 Results of screening of apparently healthy senior and geriatric dogs J Vet
Intern Med 31 81ndash92
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
36
44 Woodman J Wagg CR Boysen SR Leguillete R Mizen K Roy MF 2015 Evaluation
of coagulation via thromboelastography in healthy horses administered dexamethasone Can Vet
J 56 1271ndash1274
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
37
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of
standard heparin prophylaxis and correlation to haemostatic changes
Pereira JM Hewicker-Trautwein M Rohn K von Depka Prondzinski M
Mischke R
Contributions of the authors
Pereira JM performed (1) the ultrasound evaluations and blood sampling of the
patients under study (2) haematology analysis (3) injection of the anticoagulation
prophylaxis (partially) to the patients under study (4) analysis of samples (collaboration)
at Werlhof Institute Medical Care Center (5) analysis of some haemostasis test
(collaboration) (6) statistical analysis (partially) (7) data documentation (8) and wrote
the manuscript
Hewicker-Trautwein M performed the electron microscopy analysis
Rohn K performed the statistical analysis
von Depka Prondzinski M performed some haemostasis test (thrombin generation
assay and endogenous thrombin potential)
Mischke R performed the coagulation and haemostasis test was involved in designing
and supervision of the study analysis and discussion of the data and its statistical analysis
and revision edition and correction of the manuscript
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
38
Central venous catheter induced thrombosis in dogs ndash efficacy of standard heparin
prophylaxis and correlation to haemostatic changes
Joseacute Mauricio Pereira1 Marion Hewicker-Trautwein2 Karl Rohn3 Mario von Depka
Prondzinski4 Reinhard Mischke1
1Small Animal Clinic University of Veterinary Medicine Hannover Foundation
Buumlnteweg 9 D-30559 Hannover Germany
2Institute for Pathology University of Veterinary Medicine Hannover Foundation
Buumlnteweg 17 D-30559 Hannover Germany
3Institute of Biometry Epidemiology and Information Processing University of
Veterinary Medicine Hannover Foundation Buumlnteweg 2 30559 Hannover Germany
4Werlhof Institute Schillerstrasse 23 30159 Hannover Germany
Abbreviations anti-Xa anti-factor Xa BW body weight CVC central venous catheter
EJV external jugular vein ELMI electron microscopy IV intravenous JV jugular vein
LMWH low molecular weight heparin ML maximum lysis (ROTEM) PIVC
peripheral intravenous catheter ROTEM rotational elastometry SC subcutaneous
TGA thrombin generation assay TID three times a day TT thrombin time UFH
unfractionated heparin
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
39
Corresponding author
E-mail addresses matiscrgmailcom (JM Pereira) MarionHewicker-
Trautweintiho-hannoverde (M Hewicker-Trautwein) KarlRohntiho-hannoverde
(K Rohn) mailwerlhof-institutde (M von Depka Prondzinski)
ReinhardMischketiho-hannoverde (R Mischke)
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
40
ABSTRACT
Central venous catheters (CVC) are of importance for intensive care management of
canine patients Thrombosis of the jugular vein is a severe complication related to the
CVC use However only little is known about the incidence of CVC-associated
thromboses in dogs and the necessity and efficacy of prophylactic antithrombotic
regimens The objectives of the present study were (1) to determine the incidence of
catheter-induced thrombosis in canine intensive care patients and thereby (2) to evaluate
the efficacy of a routine prophylactic heparin treatment and finally (3) to assess whether
initial changes of selected haemostasis parameters are predictive for an increased risk for
thrombosis formation 29 dogs hospitalised in the Small Animal Clinic University of
Veterinary Medicine Hannover Foundation and receiving a central venous catheter
(CVC) in the external jugular vein (EJV) for medical reasons were included in the final
assessment 17 dogs received the standard antithrombotic dosage regimen of
unfractionated heparin (UFH 150 IUKG BW TID sc) and 12 dogs (usually surgical
patients) the reduced dosage (75 IUKG BW TID sc) A colour Doppler ultrasound of
the respective EJV in order to detect thrombus formation and blood collection for
haemostasis tests were performed immediately before the installation procedure (day 0)
and on days 1 3 5 7 and so on after the CVC insertion Haemostasis tests included
prothrombin time activated partial thromboplastin time thrombin time antithrombin
activity D-dimer concentration thrombin generation and rotational elastometry as well
as the heparin activity using a chromogenic anti-factor Xa test Finally electron
microscopy (ELMI) of sections of the removed CVC was performed In 8 dogs
sonographic examination revealed thrombus formation in the lumen of EJV and in 1927
CVCs assessed using ELMI thrombi on the external CVC surface were detected without
significant correlation between both methods Plasmatic heparin activities showed great
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
41
variability but no significant differences were found between dogs with or without CVC-
associated thrombus formation From the initially performed haemostasis tests high
fibrinogen concentrations and low maximum lysis values as measured by the ROTEM
delta device (ex-tem reagent) were associated with sonographically detectable thrombi
The results of the present study indicate that CVC-associated thromboses occur (eg 28
[829] of the patients as detected by ultrasound) although severe thromboses (high
grades) are relatively rare in intensive care canine patients receiving a routine
anticoagulant treatment A high fibrinogen concentration and low maximum lysis
(ROTEM ex-tem) may be useful indicators for an increased thrombotic risk in individual
patients which may require special anticoagulatory treatment
Keywords CVC complication venal occlusion antithrombotic treatment ultrasound
electron microscopy rotational elastometry
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
42
1 Introduction
Central venous catheters (CVCs) are very important tools for care of patients in critical
care units allowing parenteral nutrition support therapy administration of high volumes
of intravenous fluids safe drugs administration that cannot be given through peripheral
veins plasmapheresis haemodialysis blood transfusions as well as periodical blood
sampling (Geerts 2014 Vose et al 2019) CVC related thrombosis is one of the most
severe complications (apart from infections and pneumothorax) of CVCs in humans
(Geerts 2014 Comerlato et al 2017 Evans and Ratchford 2018) leading to patientrsquos
distress CVC dysfunction venous stenosis and in some cases to the development of
thromboembolism (Geerts 2014)
In the human literature there are various studies dealing with epidemiology with reference
to different anticoagulatory regimens including placebo-controlled studies (Lee and
Kamphuisen 2012) In humans CVC related thromboses represent 70ndash80 of all upper
extremity thrombosis cases and approximately 10 of thromboembolism cases in
humans (Kucher 2011) The incidence of CVC related thrombosis reported in humans
based on 25 studies ranged widely from 2 to 67 (median = 30 ) (van Rooden et al
2005 review article) This wide range of calculated incidences of CVC related
thromboses in humans reflects the use of different CVC types different locations of entry
site of the CVC (jugular femoral subclavian axillary cephalic vein) different thrombus
detection technique (venography or ultrasonography) unequal durations of follow up
varying diseases of patients (eg haematological oncological) and presence of
predisposing factors (eg previous venous thrombosis history presence of cancer) (van
Rooden et al 2005 Geerts 2014 Liu et al 2015)
Part of the authors empirically observed CVC-associated thrombosis of the external
jugular vein (EJV) as a relevant complication in their canine intensive care patients which
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
43
awakened their interest in that topic To the best of the authoracutes knowledge studies about
extra luminal CVC related thromboses in dogs are rare in the available veterinary
literature One study evaluating the complications associated with 100 CVCs placed in
the jugular vein of dogs and cats reported only 2 cases of thrombosis (confirmed by
palpation or ultrasound) but obviously did not assess the appearance of subclinical CVC
related thromboses (Adamantos et al 2010) Thereby the incidence of CVC-induced
thromboses in dogs is unknown and systematic studies regarding an effective
anticoagulatory management are lacking
In this context it is of interest whether a possible development of thrombosis or an
increased risk (despite the prevention by a standard application of unfractionated heparin
[UFH]) can be predicted by means of changes of haemostasis related parameters during
the initial examination In one study on 168 human patients without indication of
anticoagulation prophylaxis for example elevated leukocyte counts high plasminogen
activator inhibitor and high coagulation factor VIII levels (all measured on blood samples
taken after CVC placement) were associated with an increased incidence of symptomatic
CVC-related thrombosis diagnosed by ultrasonography (Boersma et al 2016)
Therefore aims of the present study were (1) to determine the incidence of catheter-
induced thrombosis in canine intensive care patients (as detected by ultrasonography and
electron microscopy ELMI) and thereby (2) to evaluate the efficacy of a standard
antithrombotic regimen with unfractionated heparin (UFH) routinely used in the Small
Animal Clinic of the University of Veterinary Medicine Hannover and (3) to assess
whether selected haemostasis test results are predictive for an increased risk for
thrombosis formation
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
44
2 Material and methods
21 Study design
Canine in-patients of the Small Animal Clinic University of Veterinary Medicine
Hannover receiving a CVC in the EJV for medical reasons from March 2017 to
December 2019 were included Anticoagulatory treatment was performed according to
the standard protocol of the clinic Colour Doppler ultrasonographic controls of the EJV
and blood samplings (for heparin activity basic haematology and haemostasis tests) were
done immediately before the catheter was placed (day 0) and on days 1 3 5 etc (2 hours
after the first daily UFH injection) until the CVC was removed Only patients were
included which could be examined at least until day 3 Haemostasis tests included
prothrombin time activated partial thromboplastin time thrombin time fibrinogen
concentration antithrombin activity fibrin monomers D-dimers thrombin generation
assay (TGA) and rotational elastometry as well as the heparin activity using a
chromogenic anti-factor Xa test Finally ELMI of sections of the CVC after its removal
was performed
22 Animals
Originally 32 patients hospitalised in the Small Animal Clinic and receiving a CVC in the
EJV for medical reasons entered the study Three of them were subsequently excluded
because they did not reach the defined minimum study period of 3 days Two were
euthanised and one referred back to the referring vet before day 3 22 dogs weighing more
than 15 kg were included so that the withdrawal of about 10 ml of blood on several days
was not a significant burden for the patient (lt 1 of the total blood volume) Five dogs
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
45
with a body weight of less than 15 kg but at least 8 kg were included with special
consideration of the amount of blood taken (no more than 6 ml on individual days)
All animals received standard anticoagulatory treatment with UFH 17 dogs with internal
diseases received 150 IUkg BW TID subcutaneously and 12 dogs (post surgery) received
a reduced dosage of 75 IUkg BW TID subcutaneously
The 29 dogs enclosed in the study included 4 mixed breed dogs and the following
pedigree dogs 5 Labrador Retrievers 3 German Shepherds 2 Australian Shepherds and
one from each of the following breeds Akita Inu Asian Shepherd Bavarian Mountain
Hound Beagle Belgian Shepherd Briard Canaan dog Dalmatian Golden Retriever
Havanese dog Hovawart Rhodesian Ridgeback Schnauzer Shar Pei and Wolfspitz
These dogs were aged between 2 and 164 months (median 77 months) and had body
weights ranging from 81 and 63 kg (median 27 kg) 9 were castrated males 8 intact
males 7 spayed females and 5 intact females
The diseases of the 29 patients requiring a CVC included 3 from each of the following
diseases hepatopathy and esophagus dilatation 2 from each of the following diseases
bacterial cystitis diabetes mellitus and 1 from each of the following diseases abdominal
wall perforation aspiration pneumonia cystitis intestinal round cells sarcoma carcinoma
of the nasal cavity chronic renal insufficiency cystic duct rupture heartworm disease
ivermectine intoxication lymphoma multifocal alveolar histiocytosis pancreatitis post-
surgical bleeding stomach dilatation stomach foreign body tetanus thoracic
pyogranulomatous inflammation thrombocytopenia and trigeminal nerve paresis
Animals were treated in accordance with the German Animal Welfare Law The
experimental design was approved by the Animal Welfare officer of the University of
Veterinary Medicine and reported to the responsible authority (Lower Saxony State
Office for Consumer Protection and Food Safety reference number 17A101)
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
46
23 Positioning and handling of the central venous catheter
Two types of single lumen radiopaque polyurethane CVC (different on length and
diameter) were used during the study The CVC type I (n = 2529) was Cavafixreg Certoreg
Splittocanreg CVC 32 cm length 11 x 17 mm diameter16 G (BBraun Melsungen AG)
the CVC type II used (n = 429) was Certofixreg Mono S 215 15 cm length 08 x 14 mm
diameter18G (BBraun Melsungen AG) Each CVC was inserted into the EJV using a
modified Seldinger technique as follows the skin of the neck was shaved and disinfected
with antiseptic soap alcohol and iodine Local anesthesia (Lidocaine HCl 2) was
administrated subcutaneously on the catheter insertion site (a middle point in the lateral
neck over the jugular sulcus) A small skin incision was performed in the insertion site
and one peripheral plastic cannula with a metallic needle inside (14 G for CVC type I
and 20 G for CVC type II) was inserted in the EJV in cranio-caudal orientation (Hundley
et al 2018) Once the EJV was reached the needle was removed and a flexible wire
guide was inserted through the peripheral cannula following an external landmark
(4thintercostal space) (Reminga et al 2018) The peripheral cannula was removed and
the respective CVC was inserted slowly and with gentle pressure into the EJV guided
with the flexible wire and following the same external landmark to reach the right
position After the insertion of the CVC the flexible wire guide was removed The correct
positioning of the CVC was checked with a latero-lateral thorax x-ray ensuring that the
CVC tip was placed at the junction of the cranial cava vein with the right atrium Each
CVC was fixed to the neck skin (adjacent to the insertion point) with a monofilament
non-absorbable suture (gauge 3-0) Finally a bandage elaborated with Povidone-Iodine
gauze (Betaisodonareg) padded synthetic band (Artiflexreg Natur) and elastic band
(Elastomullreg) was used to protect the CVC output (Claude et al 2010) and changed
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
47
daily Several times day the catheter was flushed with isotonic sodium chloride
containing 1 IUml UFH
24 Heparin preparation and administration
Heparin-sodium-25000-ratiopharm (injection solution containing 5000 IUml sodium
heparin [mucosa]) was used SC injection was performed over the lateral thorax wall
using 1 mL syringes with fine graduation marks
25 Ultrasonography
Colour Doppler ultrasonographic examinations were performed in lateral position using
a LO-GIQ E9 ultrasound device (GE Healthcare Chicago Illinois USA) with a 15
Megahertz linear ultrasound probe The development of a thrombus or fibrin debris in the
EJV lumen around the CVC or inside it was assessed at each control Once the
ultrasound was finished a new protective bandage was placed around the neck of the dog
According to the percentage of lumen occlusion of the EJV (evaluated in cross sectional
views during the ultrasound scanning) the thrombi were classified in four different grades
(Fig 1)
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
48
of the residual vein lumen C ndash catheter T ndash thrombus
Fig 1 Definition of different grades of central venous catheter induced thrombosis of the
jugular vein as detected using colour Doppler ultrasound
26 Blood collection
At each time point approx 6ndash10 ml of blood (according to the body weight) was collected
This included approx 1 ml of EDTA blood for hematological analysis and approx 5ndash9
ml of citrated blood (tubes containing one part of 011 molL [38 ] sodium citrate to
nine parts of blood) for the measurement of hemostasis tests in citrated blood (ROTEM)
or citrated plasma Blood samples were collected by puncture of peripheral veins with
disposable cannulas (09 x 40 mm [20G] or 10 x 40mm [21G]) directly into sample tubes
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
49
or from the CVC using the three-syringe-technique (Villalta-Garciacutea et al 2015) in
situations when the peripheral venipuncture was not indicated due to phlebitis or irritation
andor inflammation of the surrounding skin Briefly 2ndash3 ml of blood were taken in a
sterile disposable syringe and immediately afterwards the blood sample was taken in a
new disposable syringe and transferred into the test tubes containing the respective
anticoagulants finally the first 2-3 ml taken were re-infused to the dog through the CVC
followed by flushing with a solution of sodium chlorideUFH (02) Immediately after
blood collection each sample tube was swayed gently until blood and anticoagulant had
been mixed thoroughly To prepare citrated platelet poor plasma citrated blood was
centrifuged at 16000 g for 10 min at room temperature using a microcentrifuge The
plasma was transferred into plastic tubes and the centrifugation procedure was repeated
This platelet poor plasma was then frozen in aliquots at -70 degC until used for analysis
Directly before analysis the plastic tubes were thawed at 37 degC in a water bath
27 Laboratory tests
Haematological analyses were made automatically with Adviareg 2120i (Siemens
Healthcare GmbH Germany)
Prothrombin time activated partial thromboplastin time and thrombin time were
measured with the autoanalyser Amax Destiny Plus (Tcoag Deutschland GmbH
Germany) and commercial reagents (Thromborel S Siemens Healthcare Diagnostics
Products GmbH Marburg Germany CK Prestreg and Test Thrombin Reagent
Diagnostica Stago SAS Asniegraveres sur Seine Cedex France) Whereas the last two tests
were measured according to the manufacturer`s instructions a modified test optimised
for canine plasma was used to measure prothrombin time (25 microl citrated plasma 120 with
imidazole buffer + 25 microl of a human fibrinogen solution 2 gl prewarmed for 120 s at 37
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
50
oC addition of 25 microl of activating reagent Thromborel S) and the results converted into a
percentage activity based on a calibration curve prepared with canine pooled plasma The
fibrinogen concentration was measured with the method according to Clauss using a test
kit from Stago Diagnostica SAS D-dimer concentration was measured with a
quantitative commercial human latex turbidimetric method in the autoanalyser Cobas
c311 using a completely assembled kit and human standards (Roche Diagnostics
Mannheim Germany) Antithrombin activity was also measured with a completely
assembled chromogenic test kit in the autoanalyser Cobas c311 and calibrated with a
canine pool plasma (n = 100)
The thrombin generation assay (Technothrombinreg TGA (Technoclone GbmH Vienna
Austria) determines thrombin generation based on monitoring of the fluorescence
generated by cleavage of a fluorogenic substrate by thrombin over time after activation
of the coagulation cascade by different concentrations of tissue factor and negatively
charged phospholipid in platelet poor plasma From the fluorescence development over
time the concentration of thrombin in the sample can be calculated using a thrombin
calibration curve The increase in thrombin concentration with time then allows to
calculate generation of thrombin in the sample and to plot thrombin values over time for
the whole coagulation process
The rotational thromboelastometry analysis with ROTEM delta device (Werfen Munich
Germany) was started approx 15 minutes after the blood collection To perform the
analysis and according to the automated ROTEM software 20 microl of recalcification reagent
(200 mmoll calcium chloride solution star-tem) and 20 microl of the respective activating
reagent (ex-tem in-tem) were pipetted into and carefully mixed in a pre-warmed cuvette
using the automated pipette provided in the device immediately afterwards 300 microl of
citrated whole blood (previously gently revolved) were added and mixed with the
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
51
reagents previously deposited Finally the cuvette was carefully placed and fixed to an
oscillating pin (oscillation movements on left and right through an angle of 475deg) in the
ROTEM delta device The interaction between blood cells and subsequently the bond
between fibrin and platelets promoted by the biomechanical changes in pH electrolytes
and temperature start the clot formation with its subsequent retraction and lysis The
magnitude of the resistance produced by the clot over the oscillating pin is quantitatively
represented by different parameters measured during the analysis and also qualitatively
in a graphic illustration (Bagge et al 2016 Crochemore et al 2017)
A chromogenic substrate test (Coatest Heparin Chromogenix-Instrumentation
Laboratory Haemochrom Diagnostica Essen Germany) was used for measurements of
UFH in the auto-analyser Cobas c311 The test application was created based on
manufacturers` instructions Different dilutions of the same batch of the commercial
sodium UFH preparation used in the experiment with normal canine pool plasma
(prepared from identical aliquots of 10 healthy adult dogs) served as standards for the
calibration of the measurement
28 Electron microscopy
Selected areas on the last third of the CVC (5ndash10 cm according with the CVC) with
macroscopically detectable thrombi were analysed and some of the CVCs were cut in
two different places The cut samples were preserved in a fixative medium composed of
1 glutaraldehyde and 4 formalin Once the sample was fixed it was prepared
according to the osmium thiocarbohydrazide procedure (OTOTO-procedure) (Malick et
al 1976) The fixed CVCs were dehydrated in seven series as follow first three series of
120 minutes each one on 30 50 and 70 ethanol respectively followed by two series
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
52
of 120 min each one on 90 ethanol and then two series of 24 hours each one on 100
ethanol After the dehydration process the samples were placed two times during 24
hours each one on isoamyl acetate and then placed on in a critical point drier Finally the
samples were coated with gold in a splutter-coater with 25 kV acceleration voltage in an
argon atmosphere with a current of 20 mA for one minute The samples were examined
in an EVO 15 scanning electron microscope (ZEISS International Carl Zeiss Jena
Germany) and photographed with magnifications of 600x and 6000x avoiding mechanical
artifacts (Toskala et al 1995) Thrombi on the external wall of the CVC were analysed
and measured using the software ImageJ (National Institutes of Health and the Laboratory
for Optical and Computational Instrumentation University of Wisconsin USA)
According to their extension (surface size) and thickness (related to the CVC wall
thickness in a cross sectional view) thrombi on the external wall and additionally within
the lumen of the CVC were each classified in four grades (Fig 2 Fig 1S [suppl
material]) In case of two cuts the highest degree of thrombosis detected at one of the two
sites was used for the final assessment
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
53
surface sizepercent of CVC wall thickness
Fig 2 Definition and illustration of different grades of central venous catheter induced
(extraluminal) thrombosis as detected using electron microscopy
Fig 1S (suppl material) Definition and illustration of different grades of intraluminal
thrombosis in central venous catheters as detected using electron microscopy
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
54
29 Statistical methods
Comparison between thrombus graduation either based on sonographic and ELMI
findings respectively was performed using the McNemar test with calculation of the
concordance index (κ) Standard normal distribution was assessed using the
Kolomogorov-Smirnov test
Due to non-normal distribution of part of data sets heparin activities and results of
haemostasis assays are reported using non-parametric variables (median minimumndash
maximum box and whisker plots) Heparin activities and results of haemostasis tests
between dogs with or without thrombosis were compared using Mann-Whitney-U test
3 Results
31 Sonographic detection of thrombus formation
Thrombus formation was sonographically detected in 829 patients during the study
period Based on examination on the final day in half of these eight patients (n = 4) the
thrombus formation was scored grade 1 in 3 dogs grade 2 and only in one grade 3 In all
of these 8 dogs this was the maximum grade during the study period One dog with fibrin
precipitates was excluded from further analysis because it could not be clearly assigned
to one of the groups In 78 cases thrombi were attached mainly to the venous wall
whereas only in one case was attached to the CVC external wall Seven of these 8 dogs
which developed thrombi received the standard heparin dosage (150 IUkg TID 41
[717] of dogs receiving this dosage) and only one the reduced heparin dosage (75 IUkg
TID 8 [112] of dogs receiving this dosage)
The time point of first detection of thrombus formation varied between days 3 and 11
(median day 8) In addition detritus (presumably composed mainly of fibrin) was
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
55
detectable in the lumen of the EJV in one further dog and within the lumen of the CVC
in further 5 dogs
32 Thrombus formation by electron microscopy
27 from 29 CVC catheter tips were analysed by electron microscopy 827 samples were
without detectable thrombus (grade 0) and 1927 with detectable thrombi 72 showed
thrombus formation of grade 1 427 of grade 2 and 827 of grade 3 There was no
concordance between thrombus graduation based on sonographic and the ELMI
graduation respectively The concordance index (McNemar test) was negative (κ = -
00718) indicating that the degree of agreement is less than by chance
Of the in total 19 dogs with detectable thrombus formation by electron microscopy 11
dogs received 150 IUkg TID (in total 16 dogs of these 27 dogs received that heparin
dosage) and 8 received 75 IUkg TID (in total 11 of these 27 dogs received that heparin
dosage 73 811) and Of the 12 patients with grade 2 and 3 thrombus formation 5 dogs
received 75 IUkg TID and 7 dogs 150 IUkg
The supplementary analysis of intraluminal thrombus formation revealed no detectable
thrombi in 7 cases and thrombus formation of grade 1 (lt 25 occlusion) in 11 cases of
grade 2 (25ndash50 ) in 3 cases and of grade 3 (gt 50 ) in 6 cases
33 Heparin activities and relationship to thrombus formation
Heparin activities varied remarkably in both dosage groups (Fig 3 ab) There was no
significant difference of heparin activities between blood samples collected via
venipuncture or from the CVC (Table 1S supplementary material) In addition there was
no significant difference between heparin activities in dogs with or without
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
56
sonographically or electron microscopically detectable thrombosis (Tables 1 and 2)
Table 1 refers to sonographically detectable thromboses and is based on median heparin
activities calculated from the measurements on day 1 and following days per patient
firstly for the total number of patients and secondly for the patients receiving 150 IUkg
TID In addition in the eight dogs heparin activities on the day of first thrombus detection
(023 IUml 019ndash036 IUml) were not significantly different from the median values
(Table 1 P = 05214 Wilcoxon test) Table 2 compares heparin activities (median values
of all days and results on the final day) between dogs with and without electron
microscopically detectable CVC-associated thrombi
Table 1S (Supplementary material) Heparin activities (IUml median values
minimumndashmaximum) measured in 27 dogs receiving 75 or 150 IUkg unfractionated
(standard) heparin TID subcutaneously 2 hours after the first injection on the day
Comparison of samples collected via puncture of the peripheral veins and those collected
from the central venous catheter (CVC) using the three-syringe-technique
Peripheral vein
puncture CVC
Mann-Whitney-U
test
Total number
(75 and 150
IUkg TID)
019
(00ndash108)
(n = 65)
023
(000ndash105)
(n = 65)
P = 03033
75 IUkg TID
010
(000ndash108)
(n = 25)
012
(000ndash023)
(n = 21)
P = 06907
150 IUkg
TID
030
(000ndash096)
(n = 40)
036
(000ndash105)
(n = 44)
P = 01223
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
57
Fig 3ab Heparin activities (box- and whisker plot arithmetic means) in dogs receiving
unfractionated heparin at a dosage of 75 IUkg BW tid (a) or 150 IUkg BW tid (b) two
hours after the first daily heparin administration
day 1 represents the first day after initiation of heparin treatment
only days were considered with at least results from 5 patients
Table 1 Heparin activities (IUml median values minimumndashmaximum) in 28 dogs
receiving 75 or 150 IUkg unfractionated (standard) heparin TID subcutaneously with
(grades 1ndash3) or without (grade 0) sonographically detectable thrombus Blood collections
were performed 2 hours after the first daily injection
Grade 0
(sonography)
Grades 1ndash3
(sonography)
Mann-Whitney U
test
Total number
(75 and 150 IUkg
TID)
020 (000ndash072)
(n = 20)
027 (018ndash059)
(n = 8)
P = 03212
150 IUkg TID 042 (000ndash072)
(n = 10)
031 (018ndash059)
(n = 7)
P = 04593
one dog with fibrin precipitates in the lumen of the jugular vein but without defined thrombus formation
was not considered because it could not be clearly assigned to one of the two groups
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
58
Table 2 Heparin activities (IUml median values minimumndashmaximum) in 27 dogs
receiving 75 or 150 IUkg unfractionated (standard) heparin TID subcutaneously with
(score 1ndash3) or without (score 0) detectable thrombus via electron microscopy
Grade 0
(electron
microscop)
(n = 8)
Grades 1ndash3
(electron
microscop)
(n = 19)
Mann-Whitney U
test
Median value of
different days 028 (005ndash060)
022 (000ndash072)
P = 05770
Final day 020 (001ndash045)
015 (000ndash105)
P = 06706
34 Relationship between haemostasis tests and thrombus formation
From the initially performed haemostasis tests high fibrinogen concentrations and low
maximum lysis values as measured by the ROTEM delta device (using the ex-tem
reagent) were associated with sonographically detectable thrombi (Tab 3) Additional
analyses based on the median values of different days for individual patients revealed that
low maximum lysis values as measured by the ROTEM delta device (using the in-tem
reagent) were associated with sonographically detectable thrombi (maximum lysis score
0 850 [100ndash175 ] score 1ndash3 375 [100ndash900 ] median [minimumndash
maximum] P = 00166) but no other test Comparison of the results of haemostasis tests
on the final day between dogs with and without sonographically detectable thromboses
did not show any significant difference (P gt 005)
Comparison of results of haemostasis tests on the final day in dogs with detectable
thrombosis via ELMI and dogs without significant thrombus formation showed several
significant differences including lower fibrinogen concentrations and longer clotting
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
59
times (ex-tem reagent) and clot formation times (ex-tem and in-tem reagents) using the
ROTEM delta analyser in general expressing a less coagulable status (Tab 4) If values
of dogs graded 0 and 1 (n =15) are compared to those of grades 2 and 3 (n = 12) the last-
mentioned had longer clotting times (ROTEM delta in-tem reagent) as well as longer clot
formation times and lower alpha angles (ROTEM delta in-tem reagent) (P lt 005 results
not shown)
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
60
Table 3 Comparison of initial results of haemostasis parameters (median values
minimumndashmaximum in dogs with (grades 1ndash3) and without (grade 0) sonographically
detectable central venous catheter-induced thromboses
Parameter Unit
Sonographic
thrombus grade
P-value
(Mann-Whitney
U test)
0
(n = 20)
123
(n = 8)
Haematocrit 355 (228ndash641)
382 (245ndash499)
06111
Platelet count x103microl 292 (420ndash483)
218 (860ndash458)
02320
Prothrombin time 944 (359ndash169)
896 (471ndash136)
05758
Activated partial thromboplastin
time s 128
(955ndash249) 140
(117ndash164) 02322
Thrombin time s 129 (121ndash178)
141 (650ndash160)
02028
Fibrinogen gl 448 (153ndash770)
747 (153ndash116)
00235
Antithrombin activity 639 (289ndash107)
670 (464ndash746)
05249
D-dimers microgml 006 (000ndash148)
015 (003ndash372)
03997
Thrombin generation assay nmol 130 (000ndash365)
912 (190ndash277)
08787
Clotting time (ROTEM ex-tem) s 420 (290ndash930)
430 (170ndash159)
07989
Clot formation time (ROTEM ex-
tem) s 665
(310ndash143) 470
(240ndash800) 00633
Alpha angle (ROTEM ex-tem) degrees 795 (620ndash840)
815 (750ndash860)
01667
Max clot firmness (ROTEM ex-
tem) mm 750
(570ndash860) 775
(700ndash830) 01753
Max clot elasticity (ROTEM ex-
tem) - 299
(131ndash619) 347
(235ndash484) 01544
Maximum lysis (ROTEM ex-tem) 135 (300ndash290)
400 (000ndash800)
00037
Clotting time (ROTEM in-tem) s 208 (840ndash810)
249 (720ndash371)
05085
Clot formation time (ROTEM in-
tem) s 835
(370ndash634) 745
(390ndash235) 08988
Alpha angle (ROTEM in-tem) degrees 745 (250ndash820)
750 (500ndash820)
09797
Max clot firmness (ROTEM in-
tem) mm 700
(540ndash810) 740
(610ndash770) 03587
Max clot elasticity (ROTEM in-
tem) - 235
(118ndash424) 287
(156ndash332) 04764
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
61
Maximum lysis (ROTEM in-tem) 850 (000ndash210)
350 (000ndash220)
01327
Table 4 Comparison of results of haemostasis parameters (median values minimumndash
maximum) on the last day in dogs with (grades 1ndash3) and without (grade 0) central venous
catheter-associated thrombus formation as detected by electron microscopy
Parameter Unit
Thrombus grade
(ELMI)
P-value
(Mann Whitney
U test)
0
(n = 8)
123
(n = 19)
Haematocrit 322 (241ndash454)
330 (216ndash510)
05953
Platelet count 103microl 446 (321ndash592)
268 (101ndash675)
00558
Prothrombin time 145 (104ndash264)
120 (561ndash148)
00462
Activated partial thromboplastin
time s 123
(109ndash140) 136
(109ndash188) 00665
Thrombin time s 129 (121ndash178)
141 (650ndash160)
02028
Fibrinogen gl 465 (275ndash866)
347 (189ndash992)
00491
Antithrombin activity 793 (448ndash981)
692 (398ndash889)
04572
D-dimers microgml 018 (000ndash173)
007 (000ndash118)
01920
Thrombin generation assay nmol 501 (000ndash160)
93 (000ndash287)
09562
Clotting time (ROTEM ex-tem) s 360
(270ndash410) 400
(310ndash620) 00238
Clot formation time (ROTEM ex-
tem) s 395
(120ndash740) 650
(240ndash174) 00275
Alpha angle (ROTEM ex-tem) degrees 835 (760ndash880)
790 (580ndash850)
00484
Max clot firmness (ROTEM ex-
tem) mm 780
(700ndash850) 720
(550ndash860) 00586
Max clot elasticity (ROTEM ex-
tem) - 346
(239ndash555) 254
(121ndash637) 00495
Maximum lysis (ROTEM ex-tem) 105 (200ndash190)
110 (000ndash380)
03796
Clotting time (ROTEM in-tem) s 178 (100ndash222)
201 (139ndash554)
00525
Clot formation time (ROTEM in-
tem) s 435
(330ndash111) 790
(350ndash882) 00209
Alpha angle (ROTEM in-tem) degrees 815 (710ndash830)
740 (240ndash830)
00191
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
62
Max clot firmness (ROTEM in-
tem) mm 785
(660ndash810) 690
(270ndash840) 00586
Max clot elasticity (ROTEM in-
tem) - 362
(192ndash434) 218
(370ndash507) 00559
Maximum lysis (ROTEM in-tem) 950 (200ndash190)
800 (100ndash180)
06890
4 Discussion
The main result of our study is that under the study conditions part of the dogs developed
CVC-associated thromboses However only a very low part of these thromboses were
obviously of a clinically significant degree regarding the amount of occlusion of the EJV
and none of the dogs had a symptomatic thrombosis whose definition includes presence
of clinical signs such as localised swelling pain and redness (Boersma et al 2008 Lee
and Kamphuisen 2012) In the present study we particularly refer to the extraluminal
thromboses with regard to the assessment of the antithrombotic management
One major problem of the statistical analysis and result presentation was that the two
methods used to define the occurrence and degree of thrombus formation (ultrasound and
ELMI) revealed discrepant results Therefore we performed a statistical analysis
independently regarding thrombus formation with either of the two methods It is well
known that ELMI which has already been used to detect CVC induced thromboses in
humans (Lucas et al 2017) and experimental animals (Xiang et al 1998 Xiang et al
2001) is a very sensitive diagnostic technique which allows the ex vivo diagnosis of very
small or fibrin rich thrombi that perhaps cannot be detected by ultrasonography or
venography during the in vivo evaluation of patients (Xiang et al 2001) A major reason
for the discrepant results between the two methods used to detect CVC associated
thromboses is that different CVC segments are evaluated Using ultrasonography it was
only possible to evaluate the EJV in the neck area of the patient and thereby only its
proximal third In contrast ELMI assessed mainly the CVC tip and a human study
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
63
revealed that even different positions of the catheter tip are associated with various risks
for development of CVC associated thrombosis (Ballard et al 2016) One additional point
to consider which also may contribute to the discrepancy is that it is possible that thrombi
developed on the CVC surface can be detached during the CVC removal from the vein
and thereby before its analysis by ELMI (Xiang et al 2001)
The contrast venography technique is used as gold standard to confirm thrombosis in
human medicine However this diagnostic technique is normally used only in the case of
a strong clinical suspicion of venous thrombosis that cannot be detected by
ultrasonography (Laurenson et al 2010 Langston and Eatroff 2018) Our study protocol
with privately owned clinic patients did not allow to use this method In a study performed
in 44 human patients requiring a CVC (Koumlksoy et al 1995) a sensitivity of 94 and a
specificity of 96 were determined for the colour Doppler ultrasound diagnosis when it
was compared with the diagnosis performed with the contrast venography technique and
that makes the colour Doppler ultrasound an accurate diagnosis method to detect CVC
related thrombosis even in humans (Koumlksoy et al 1995 van Rooden et al 2003)
The incidences of CVC related thrombosis detected by colour Doppler ultrasound of the
internal jugular vein reported in 3 human studies were 2 30 and 56 (Harter et al
2002 Lordick et al 2003 Wu et al 1999) and although with a wide range the median
value of these rates (30 ) is very well suited to the incidence of 28 (829) which we
calculated for our canine study based on sonographic findings The wide variation of
incidences of CVC related thrombosis in different human studies can in general be
explained by differences regarding the type and entry site of the CVC (jugular femoral
subclavian axillary cephalic vein) the time period with the CVC in place the thrombus
detection technique (venography or ultrasonography comment in the cited 3 studies
consistently the last-mentioned method was used) and criteria for diagnosis of
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
64
thrombosis the duration of follow up diseases and presence of predisposing factors in
these patients (eg previous venous thrombosis history presence of cancer) and finally
prophylactic treatment with anticoagulants (van Rooden et al 2005 Geerts 2014 Liu et
al 2015)
Different pathomechanisms contribute to the development of CVC induced thromboses
During the CVC insertion the puncture of the skin on the CVC insertion site and the
rupture of the blood vessels integrity activate the coagulation cascade and the CVC acts
as a matrix for adherence and accumulation of fibrin and cellular components (Smith et
al 2012) The attachment of proteins and blood cells to the internal andor external wall
of the CVC can lead to a partial or complete occlusion of the catheter lumen and
sometimes to a partial or complete obstruction of the blood flow andor
thromboembolism (Langston and Eatroff 2018) There is a strong interrelationship
between thrombus and biofilm formation (Smith et al 2012) Prevention of thrombus
formation has thereby also relevance regarding the increased risk for microbial
colonisation of the catheter (Boersma et al 2008) leading at least in humans to an
increased number of CVC related systemic infections (Lordick et al 2003)
In this context it has to be considered that CVC produced from polyurethane which are
routinely used in our clinic and thereby in the study are usually associated with a
relatively high thrombosis potential and thereby definitely provide an effective test for
an antithrombotic regimen Silicone catheters have a significantly reduced risk for
thrombotic complications but this advantage has to be purchased by a higher rate of
mechanical failure (eg rupture of the catheter) (Wildgruber et al 2016) Promising
experimental approaches work on surface modifications of the standard catheters For
example one group was able to demonstrate that a coating of the surface of the catheter
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
65
with a zwitterionic polymeric sulfobetaine showed a significant reduction of thrombus
formation in vitro with human blood and in an in vivo canine model (Smith et al 2012)
Due to ethical reasons we did not performed a placebo-controlled study However
interestingly not all human studies report systemic anticoagulation and part of the human
studies was even placebo-controlled (review Lee and Kamphuisen 2012) Obviously
there is no consensus in the available human literature regarding the beneficial use of
antithrombotic prophylaxis to prevent CVC related thrombosis Several studies did not
find a statistically significant difference in the incidence of CVC related thrombosis
between patients receiving the antithrombotic prophylaxis compared with the control
groups without systemic anticoagulation (Verso et al 2005 P = 035 Karthaus et al
2006 P gt 005 Niers et al 2007 P = 049) whereas in other studies a lower incidence
of CVC related thrombosis was found in the group receiving anticoagulants as
prophylaxis compared with the control groups (Abdelfeki et al 2004 150 vs 126
P = 003 De Cicco et al 2009 400 vs 526 P = 005)
Although it is well known that subcutaneously administered UFH has a limited and
variable bioavailability and thereby leads to unpredictable blood activities (Hirsh and
Raschke 2004) the extremely wide range of blood activities after a defined dosage in our
study was relatively unexpected Blood was collected 2 hours after the first UFH
injection and thereby at the time of suspected maximum heparin activity considering the
relatively low UGH dosage (Mischke and Jacobs 2001) The cited study was performed
on healthy experimental dogs Possible additional influencing factors in critical ill
patients include circulatory insufficiency which may reduce and delay absorption from
subcutanenous tissue decreased clearance function of the liver and kidneys as well as
altered blood plasma concentrations of heparin binding plasma proteins such as
fibrinogen and platelet factor 4 and alterations of the heparin binding capacity of
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
66
endothelial cells which altogether are reasons for a great inter- but also intrapatient
variability (Hirsh and Raschke 2004) In individual patients this can lead to heparin
resistance It has also to be considered that antithrombin activities which are necessary
for an adequate heparin effect (Bjoumlrk and Lindahl 1982) were reduced in our patients
Availability of approved blood tests which can predict the risk of development of CVC
associated thrombosis already at the moment of the CVC placement are important
(Boersma et al 2016) One of the most interesting results was that dogs which developed
sonographically detectable thrombi had nearly twofold higher fibrinogen levels Well in
agreement with our results one study performed in anticoagulated human patients
demonstrated that high fibrinogen levels were associated with an increased risk of
perioperative venous thromboembolism (P = 0004) (Kato et al 2016) The cited study
does not exclusively refer to CVC associated thrombosis but 76 (1621) of the venous
thromboses were suggested to be associated with CVCs Fibrinogen measurement a test
which is widely available is therefore a useful and practicable indicator of an increased
risk for CVC induced thromboses and may advice the clinician to treat these patients
especially carefully (eg with low molecular weight heparin) In the study performed by
Boersma et al (2016) in 168 human patients without indication of anticoagulation
prophylaxis elevated leukocyte counts high levels of plasminogen activator inhibitor
and of coagulation factor VIII were associated with an increased incidence of
symptomatic CVC related thrombosis diagnosed by ultrasonography A limitation of that
study is that all measurements were performed on blood samples taken after CVC
placement Interestingly in our study ROTEM results were partly associated with
thrombus formation which reflects the well-known fact that results of viscoelastic
measurements are a good indicator of hypercoagulability and thromboembolic events
(Harahsheh and Ho 2018) It is of further interest and possibly unexpeted that on the final
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
67
day of examination dogs with ELMI signs of thromboses had partly lower coagulation
activities than dogs without thromboses Due to the low number of dogs without
detectable thrombi using ELMI this implausible result has to be interpreted with caution
One possible explanation could be that temporary hypercoagulability in these dogs may
have finally resulted in consumption of haemostasis substrates
Limitations of the present study were as a consequence of the study design based on
clinical patients the relatively low total number of patients inclusion of different UFH
treatment regimens and the lack of a control group
5 Conclusions
The results of the present study indicate that the used UFH regimen was not completely
effective to prevent CVC-associated thromboses which were detectable in 28 [829]
of the patients by ultrasound However severe thromboses (high grades) are relatively
rare in intensive care canine patients receiving a routine anticoagulant treatment Because
heparin plasma activities were not different in dogs with or without CVC-associated
thrombus formation other local and systemic factors must play a significant role as well
A high fibrinogen concentration and low maximum lysis (ROTEM ex-tem) may be
useful indicators for an increased thrombotic risk in individual patients which may
require special anticoagulatory treatment
6 Acknowledgments
Jose Mauricio Pereira received a scholarship from the Universidad Nacional de Costa
Rica (reference code JB-C-1107-2016)
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
68
7 References
1 Abdelkefi A Ben Othman T Kammoun L Chelli M Ben Romdhane N Kriaa A Ladeb S
Torjman L Lakhal A Achour W Ben Hassen A Hsaiumlri M Ladeb F Ben Abdeladhim A
2004 Prevention of central venous line-related thrombosis by continuous infusion of low-dose
unfractionated heparin in patients with haemato-oncological disease A randomized controlled trial
Thromb Haemost 92 654ndash661
2 Adamantos S Brodbelt D Moores A L 2010 Prospective evaluation of complications associated
with jugular venous catheter use in a veterinary hospital J Small Anim Pract 51 254ndash257
3 Bagge A Schoumltt U Kander T 2016 Effects of naturopathic medicines on Multiplate and ROTEM
a prospective experimental pilot study in healthy volunteers BMC Complement Altern Med 16 64
4 Ballard DH Samra NS Gifford KM Roller R Wolfe BM Owings JT 2016 Distance of the
internal central venous catheter tip from the right atrium is positively correlated with central venous
thrombosis Emerg Radiol 23 269ndash273
5 Boersma RS Jie KS Verbon A van Pampus EC Schouten HC 2008 Thrombotic and
infectious complications of central venous catheters in patients with hematological malignancies Ann
Oncol 19 433ndash442
6 Boersma R S Hamulyak K van Oerle R Tuinenburg A Ten Cate-Hoek AJ Schouten HC
2016 Biomarkers for prediction of central venous catheter related-thrombosis in patients with
hematological malignancies Clin Appl Thromb Hemost 22 779ndash784
7 Bjoumlrk I Lindahl U 1982 Mechanism of the anticoagulant action of heparin Mol Cell Biochem
48 161ndash182
8 Claude A Riedesel D Riedesel E 2010 Electrocardiography-guided and retrospective analysis of
central venous catheter placement in the dog Vet Anaesth Analg 37 97ndash105
9 Comerlato PH Rebelatto TF Santiago de Almeida FA Klein LB Boniatti M M Schaan
DB Rados VD 2017 Complications of central venous catheter insertion in a teaching hospital
Rev Assoc Med Bras 63 613ndash620
10 Crochemore T Piza FMT Rodriacutegues RDR Guerra JCC Ferraz LJR Correcirca TD 2017
A new era of thromboelastometry Einstein (Sao Paulo) 15 380ndash385
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
69
11 De Cicco M Matovic M Balestreri L Steffan A Pacenzia R Malafronte M Fantin D
Bertuzzi CA Fabiani F Morassut S Bidoli E Veronesi A 2009 Early and short‐term
acenocumarine or dalteparin for the prevention of central vein catheter‐related thrombosis in cancer
patients a randomized controlled study based on serial venographies Ann Oncol 20 1936ndash1942
12 Evans NS Ratchford EV 2018 Catheter related-venous thrombosis Vasc Med 23 411ndash413
13 Geerts W 2014 Central venous catheterndashrelated thrombosis Hematology Am Soc Hematol Educ
Program 2014 306ndash311
14 Harahsheh Y Ho KM 2018 Use of viscoelastic tests to predict clinical thromboembolic events A
systematic review and meta‐analysis Eur J Haematol 100 113ndash123
15 Harter C Salwender HJ Bach A Egerer G Goldschmidt H Ho AD 2002 Catheter-related
infection and thrombosis of the internal jugular vein in hematologic-oncologic patients undergoing
chemotherapy a prospective comparison of silver-coated and uncoated catheters Cancer 94 245ndash251
16 Hirsh J Raschke R 2004 Heparin and low-molecular-weight heparin The Seventh ACCP
Conference on Antithrombotic and Thrombolytic Therapy Chest 126 (Suppl) 188S-203S
17 Hundley D Brooks A Thomovsky E Jhonson P Freeman L Schafbuch R Heng HG Moore
G 2018 Comparison of ultrasound-guided and landmark-based techniques for central venous
catheterization via the external jugular vein in healthy anesthetized dogs Am J Vet Res 79 628ndash
636
18 Kato F Takeuchi H Matsuda S Kawakubo H Omori T Kitagawa Y 2016 Incidence of and
risk factors for venous thromboembolism during surgical treatment for esophageal cancer a single-
institution study Surg Today 46 445ndash452
19 Karthaus M Kretzschmar A Kroning H Biakhov M Irwin D Marschner N Slabber C
Fountzilas G Garin A Abecasis NG Baronius W Steger GG Sudhoff T Giorgetti C
Reichardt P 2006 Dalteparin for prevention of catheter‐related complications in cancer patients with
central venous catheters final results of a double‐blind placebo‐controlled phase III trial Ann Oncol
17 289ndash296
20 Koumlksoy C Kuzu A Kutlay J Erden I Ozcan H Ergicircn K 1995 The diagnostic value of colour
Doppler ultrasound in central venous catheter related thrombosis Clin Radiol 50 687-689
21 Kucher N 2011 Clinical practice Deep-vein thrombosis of the upper extremities N Engl J Med
364 861ndash869
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
70
22 Langston CE Eatroff AE 2018 Hemodialysis catheter-associated fibrin sheath in a dog J Vet
Emerg Crit Care 28 366ndash371
23 Laurenson MP Hopper K Herrera MA Johnson EG 2010 Concurrent diseases and conditions
in dogs with splenic vein thrombosis J Vet Intern Med 24 1298ndash1304
24 Lee AYY Kamphuisen PW 2012 Epidemiology and prevention of catheter‐related thrombosis in
patients with cancer J Thromb Haemost 10 1491ndash1499
25 Liu G Fu ZQ Zhu P Li SJ 2015 Central venous catheter-related thrombosis in senile male
patients New risk factors and predictors J Huazhong Univ Sci Technolog Med Sci 35 445ndash449
26 Lordick F Hentrich M Decker T Hennig M Pohlmann H Hartenstein R Peschel C 2003
Ultrasound screening for internal jugular vein thrombosis aids the detection of central venous catheter-
related infections in patients with haemato-oncological diseases a prospective observational study Br
J Haematol 120 1073ndash1078
27 Lucas TC Silva EED Souza DOD Santos ARD Lara MO 2017 Microstructural
evaluation by confocal and electron microscopy in thrombi developed in central venous catheters Rev
Esc Enferm USP 51 1ndash9
28 Malick L Wilson R Stetson D 1976 Modified thiocarbo- hydrazide procedure for scanning
electron microscopy routine use for normal pathological and experimental tissue Stain Technol 50
265ndash269
29 Mischke R Jacobs C 2001 The monitoring of heparin administration by screening tests in
experimental dogs Res Vet Sci 70 101ndash108
30 Niers TM Di Nisio M Klerk CP Baarslag HJ Buller HR Biemond BJ 2007 Prevention
of catheter‐related venous thrombosis with nadroparin in patients receiving chemotherapy for
hematologic malignancies a randomized placebo‐controlled study J Thromb Haemost 5 1878ndash
1882
31 Reminga C Silverstein D Drobatz K Clarke D 2018 Evaluation of the placement and
maintenance of central venous jugular catheters in critically ill dogs and cats J Vet Emerg Crit Care
28 232ndash243
32 Smith RS Zhang Z Bouchard M Li J Lapp HS Brotske GR Lucchino DL Weaver D
Roth LA Coury A Biggerstaff J Sukavaneshvar S Langer R Loose C 2012 Vascular
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
71
catheters with a nonleaching poly-sulfobetaine surface modification reduce thrombus formation and
microbial attachment Sci Transl Med 4 153ra132 doi101126scitranslmed3004120
33 Toskala E Nuutinen J Rautiainen M 1995 Scanning electron microscopy findings on human
respiratory cilia in chronic sinusitis and in recurrent respiratory infections J Laryngol Otol 109 509ndash
514
34 Van Rooden CJ Tesselaar ME Osanto S Rosendaal FR Huisman MV 2005 Deep vein
thrombosis associated with central venous catheters - a review J Thromb Haemost 3 2409ndash2419
35 Verso M Agnelli G Bertoglio S Di Somma FC Paoletti F Ageno W Bazzan M Parise P
Quintavalle R Naglieri E Santoro A Imberti D Soraru M Mosca S 2005 Enoxaparin for the
prevention of venous thromboembolism associated with central vein catheter a double‐blind placebo‐
controlled randomized study in cancer patients J Clin Oncol 23 4057ndash4062
36 Villalta-Garciacutea P Loacutepez-Herranz M Mazo-Pascual S Honrubia-Fernaacutendez T Jaacutentildeez-Escalada
L Fernaacutendez-Peacuterez C 2015 Reliability of blood test results in samples obtained using a 2-mL discard
volume from the proximal lumen of a triple-lumen central venous catheter in the critically ill patient
Nurs Crit Care 22 298ndash304
37 Vose J Odunayo A Price JM Daves M Schildt JC Tolbert MK 2019 Comparison of
heparinized saline and 09 sodium chloride for maintaining central venous catheter patency in healthy
dogs PeerJ 7 e7072 httpsdoiorg107717peerj7072
38 Wildgruber M Lueg C Borgmeyer S Karimov I Braun U Kiechle M Meier R Koehler M
Ettl J Berger H 2016 Polyurethane versus silicone catheters for central venous port devices
implanted at the forearm Eur J Cancer 59 113ndash124 doi101016jejca201602011
39 Wu X Studer W Skarvan K Seeberger MD 1999 High incidence of intravenous thrombi after
short-term central venous catheterization of the internal jugular vein J Clin Anesth 11 482ndash485
40 Xiang DZ Verbeken EK Van Lommel AT Stas M De Wever I 1998 Composition and
formation of the sleeve enveloping a central venous catheter J Vasc Surg 28 260ndash271
41 Xiang DZ Verbeken EK Van Lommel AT Stas M De Wever I 2001 Sleeve-related
thrombosis a new form of catheter-related thrombosis Thromb Res 104 7ndash14
Chapter 4 General discussion
72
Chapter 4 General discussion
Incidence of CVC related thrombosis
In our study the incidence of CVC related thrombosis detected by ultrasound in critical
care canine patients was 28 To the best of the authoracutes knowledge similar studies
performed in dogs are rare in the available veterinary literature In one study performed
in 81 dogs and 12 cats (Adamantos et al 2010) an incidence of 2 was found for vein
thrombosis related to CVC This low incidence can be due to a lacking of systematic
ultrasound evaluations of the jugular vein containing the CVC In the mentioned study in
dogs and cats the vein thrombosis was confirmed by palpation or ultrasound presumably
after the presentation of clinical symptoms however in many cases as in our study
thrombus into the jugular vein can be developed without manifestation of clinical
symptoms what makes a routinely ultrasound evaluation of the vein containing the CVC
very important Our results are well aligned with the findings in humans in which a
median incidence of 30 was found for thrombosis related to CVC based on 25 studies
performed in patients requiring a CVC (review article van Rooden et al 2005) In the
mentioned 25 human studies the incidence of CVC related thrombosis varied within a
wide range (2ndash67 ) according to factors related to (1) the CVC (eg material type
entry site [jugular femoral subclavian axillary cephalic vein] time period in place (2)
diagnostic thrombus detection technique (ultrasonography or venography) and criteria
(3) patient collective (diseases and presence of predisposing factors [eg previous venous
thrombosis history presence of cancer] and (4) prophylactic antithrombotic treatment
(Geerts 2014 Liu et al 2015 van Rooden et al 2005)
The incidence of CVC related thrombosis detected by colour Doppler ultrasound in our
study fits also with the median incidence (30 ) found in three studies performed in
humans in which colour Doppler ultrasound evaluations of the jugular vein containing a
Chapter 4 General discussion
73
CVC were performed (Harter et al 2002 Lordick et al 2003 Wu et al 1999) In these
three studies the individual incidence of CVC related thrombosis was 2 (Harter et al
2002) 30 (Lordick et al 2003) and 56 (Wu et al 1999) The high thrombus
incidence reported in the study performed by Wu et al (1999) could be due to the criteria
for diagnosis of thrombosis because in that study both fibrin sleeves and compact
thrombi were considered as cases of thrombosis related to CVC The incidence for
compact thrombi was 25 which is more similar to our results In the study performed
by Harter et al (2002) the CVC related thrombosis incidence (2 ) was very low
compared with the incidence found in our study In that study the jugular veins were
examined just before or less than 24 hours after CVC removal (median period of CVC
use = 1025 days) In our study the ultrasound evaluations were performed before the
CVC placement (day 0) and then on days 1 3 5 etc until the CVC removal (median
period of CVC use = 7 days median interval until thrombus detection = 7 days) In our
study we detected thrombus formation as early as on the 3rd day so it could be possible
that in the cited study possibly only temporarily existing thrombi could have been missed
by ultrasonography in the final stage and thereby for the incidence calculation However
this scenario is not very likely because results of the present study show that
sonographically detectable thrombi during the observation period had in all cases their
maximum grade on the final day
In the study performed by Lordick et al (2003) the CVC related thrombosis incidence
determined for 43 human patients was very similar to our study and in both studies only
compact thrombi detectable by ultrasound were considered for assessment of the
incidence In the mentioned study all the human patients were screened for the presence
of thrombosis every 4 days after CVC insertion with real time B-mode ultrasound
(Lordick et al 2003)
Chapter 4 General discussion
74
In our study 70 of CVCs analysed by ELMI showed thrombi of grade 1ndash3 on the
external CVC wall To the best of the authoracutes knowledge studies about incidence of CVC
related thrombosis on the outside surface of the CVC detected by ELMI cannot be found
in the human and small animal literature In one study performed in humans (Lucas et al
2017) thrombus formation was analysed by ELMI in 78 central venous catheters That
study focussed on microstructural assessment of thrombus formation inside the distal
openings of the CVC which were found in 85 of the evaluated CVCs The percentage
of occurrence of thrombosis in the mentioned study in humans was nearly similar to the
incidence of intraluminal thrombus formation as detected in the presented study by ELMI
(74 ) In general intraluminal thrombi were not specially considered in the present
study because they mainly express the efficacy of the flushing technique rather than the
effectivity of the antithrombotic treatment In one study performed in rats the
microstructure of thrombi developed in the jugular vein containing the CVC was also
evaluated by ELMI (Xiang et al 1998) In the mentioned study pericatheter thrombosis
was detected on the proximal section of the CVC in 100 of rats (n = 12) after 1 and 3
days of catheterisation The analyses of the CVCs placed in rats were performed with the
catheter inside the vein in contrast to our study where the CVC was removed from the
jugular vein for the respective analysis Xiang et al (2001) hypothesised in one study
performed in rabbits and rats in which CVCs were placed in the jugular vein that fibrin
rich thrombi associated to the CVC can be detached during the catheter removal and this
could explain the difference between the thrombosis incidence assessed in our study and
that of the study performed by Xiang et al (1998)
In our study the incidence of CVC related thrombosis detected by ultrasonography in the
lumen of the EJV did not correlate with the incidence of CVC related thrombosis detected
by ELMI on the external wall of the CVC This was mainly due to the fact that the CVC
Chapter 4 General discussion
75
segment evaluated with both methods was different Using ultrasonography it was only
possible to evaluate the EJV in the neck area of the patient and thereby only the proximal
third of the CVC In contrast with the ELMI analysis only the distal section of the CVC
(last third) was evaluated because macroscopically detected thrombi mainly developed
near the CVC tip which was routinely positioned at the junction of the cranial cava vein
with the right atrium In a retrospective review of medical records of 169 human patients
requiring a CVC (Ballard et al 2016) the position of the CVC tip was determined by
radiologic evaluation and it was determined that the catheter tip position can be a
predisposing factor for central venous thrombosis development Thereby it could be of
interest to evaluate in further studies in dogs if a high incidence of thrombosis detected
by ELMI as was shown in our study could be related to the CVC tip position
In our study polyurethane catheters were tested which are widely used and are the
standard CVCs used in the Small Animal Clinic University of Veterinary Medicine
Hannover In vivo and in vitro studies have assessed the thrombogenicity of different
catheter materials (Borow and Crowley 1985 Smith et al 2012) In general terms
polyurethane catheters (central or peripheral) had shown a higher thrombogenicity
compared with coated (eg with hydromer) and silicone catheters Due to this property
of polyurethane catheters they can be supposed as a good test system for antithrombotic
regimens as evaluated in our study On the other hand despite silicone catheters have been
associated with a lower thrombogenicity their mechanical flexibility could be a
disadvantage in small clinical practice due to the increased risk of rupture detected even
in humans (Wildgruber et al 2016)
Chapter 4 General discussion
76
Heparin thromboprophylaxis for CVC related thrombosis
The occurence of CVC related thrombosis detected by colour Doppler ultrasound and
ELMI in our study demonstrates that the UFH in the given dosage was not completely
effective to prevent CVC related thrombosis in canine intensive care patients In human
medicine LMWH is commonly used in prophylactic anticoagulation regimens however
studies performed in human patients requiring a CVC and receiving a prophylactic
anticoagulation (with LMWH or UFH) did not achieve consensus regarding the efficacy
of prophylactic heparin to prevent CVC related thrombosis (Abdelfeki et al 2004
Lordick et al 2003 Wu et al 1999) To the best of the authoracutes knowledge studies
performed in dogs evaluating systemic prophylactic anticoagulation to prevent CVC
related thrombosis cannot be found However according to the evidence found in the
human literature and the results of our study further investigations using comparative
groups with different drugs dosages andor administration routes are required to establish
an effective antithrombotic treatment to prevent CVC related thrombosis
The plasma heparin activities measured in canine patients in our study varied significantly
after a defined subcutaneous dosage The limited and variable bioavailability of SC
administered UFH is well documented in humans (Hirsh and Raschke 2004)
Considerable variations of the circulatory status of the liver- and kidney function and
concentration of heparin-binding proteins in intensive care patients obviously amplify
this phenomenon leading to an extremely high inter-individual and intra-individual
variation of the heparin blood levels in our patients A different application route (IV)
may have reduced the influence of part of these factors and therefore could have led to
more predictable UFH blood levels However continuous infusion requires more
technical equipment and is also associated with a wide range of heparin activities in
canine patients (Scott et al 2009)
Chapter 4 General discussion
77
Prediction of CVC related thrombosis
Of 7 haemostasis tests (including ROTEM with 12 different parameters [6 for each
reagent ex-tem and in-tem]) evaluated as possible predictors of CVC related thrombosis
in our study initial values of high fibrinogen and low maximum lysis (ROTEM ex-tem)
were associated with sonographically detectable thrombus formation Some studies in
humans have established the association of laboratory biomarkers as predictors of risk
thrombosis (Boersma et al 2016 Cheng et al 2013 Kato et al 2016 Liu et al 2015)
One study performed in anticoagulated human patients showed ndash well in agreement with
our results ndash that high fibrinogen levels were associated with an increased risk of
perioperative venous thromboembolism (P = 0004) (Kato et al 2016) The cited study
does not exclusively refer to CVC associated thrombosis but 762 (1621) of the
venous thromboses were suggested to be associated with CVCs Fibrinogen concentration
measurements are normally available in the human and veterinary practice and the
association of this parameter with a high risk of development of CVC related thrombosis
can be useful to choose special antithrombotic treatment in individual critical care patients
to prevent such severe complications
Thrombin generation assay (TGA) abnormalities such as higher velocity and endogenous
thrombin potential (ETP) values measured initially ie before any anticoagulation
therapy have been associated with a higher thromboembolism risk in general (ie without
specific association with CVCs) in one study performed on 105 human patients (Espitia
et al 2017) In contrast in our study the initial TGA assay results did not show an
association with formation of CVC related thrombosis
The ROTEM parameters evaluated in the present study were partially associated with
formation of CVC thrombosis detected by colour Doppler ultrasound This finding
confirms the results obtained in humans in which ROTEM measurements are regarded as
Chapter 4 General discussion
78
a valuable indicator of hypercoagulability (Harahsheh and Ho 2018 Tuumlrk et al 2018)
However in our study individual ROTEM parameters showed partly lower coagulation
activities in dogs with CVC related thrombosis detected by ELMI at the final day of
evaluation Because the number of patients without signs of thrombosis detected by ELMI
was low compared with those who showed signs these implausible results should be
interpreted carefully The usefulness of ROTEM analysis which has also many other
indications demonstrates that is was worth to perform a detailed methodological approval
of the device including the establishment of reference values in the preliminary study
Conclusion
In conclusion the prethrombotic or thrombotic changes detected in our study did not
correlate between ultrasonography and ELMI however both methods show accurately
signs of CVC related thrombosis The UFH in the given dosage was not completely
effective to prevent CVC induced thrombus formation in canine intensive care patients
Plasma heparin activities varied significantly after a defined subcutaneous dosage
However plasma heparin activities were not different between dogs with and without
detectable CVC associated thromboses Initial high fibrinogen values and low maximum
lysis (ROTEM ex-tem) values were associated with sonographically detectable thrombi
and may be useful as indicators for an increased thrombotic risk
Finally our study in canine intensive care patients is to the best of the authoracutes
knowledge the first study in the available veterinary literature performed to evaluate the
efficacy of an antithrombotic treatment with UFH to prevent CVC related thrombosis
Chapter 5 Summary
79
Chapter 5 Summary
Central venous catheter induced thrombosis in dogs ndash occurrence under an
antithrombotic treatment regimen and possible indicators
Joseacute Mauricio Pereira Mora
Central venous catheters (CVC) are of importance for intensive care management of
canine patients Thrombosis of the jugular vein is a severe complication related to the
CVC use However only little is known about the incidence of CVC-associated
thromboses in dogs and the necessity and efficacy of prophylactic antithrombotic
regimens
Therefore the aims of the present study were (1) to determine the incidence of catheter-
induced thrombosis in canine intensive care patients (2) to evaluate the efficacy of a
prophylactic heparin regimen routinely used in the Small Animal Clinic of the University
of Veterinary Medicine Hannover and (3) to assess whether selected haemostasis test
results are predictive for an increased risk for thrombosis formation
The thesis also includes a preliminary methodological study on the use of rotational
elastometry in dogs using the ROTEM delta device In this preliminary study reference
intervals were determined based on 125 healthy dogs for different clot activator reagents
and for measurements without activator Coefficients of variation (CVs) were calculated
based on results of fourfold measurements of 3 normal and 3 abnormal blood samples in
order to assess precision of the method Influence of age groups sex neuter status and
size were also assessed The median CVs for most of the parameters evaluated in the
precision analysis were less than 10 and this confirmed that the ROTEM delta analyser
delivers repeatable results Regarding the influence of age sex neuter status and animal
Chapter 5 Summary
80
size only neuter status in female dogs appeared to be a relevant influencing factor on
most of important ROTEM parameters
The main study of the thesis comprises 29 patients hospitalised at the Small Animal Clinic
requiring a CVC for medical reasons These patients received a prophylactic
anticoagulation treatment with unfractionated heparin (UFH 150 IU [surgical dogs
received 75 IU]kg BW TID subcutaneously) A colour Doppler ultrasound of the
respective external jugular vein (EJV) in order to detect thrombus formation in the lumen
of the EJV and blood collection for haemostasis tests were performed directly before the
installation procedure (day 0) and on days 1 3 5 7 and so on after the CVC insertion
Haemostasis tests included prothrombin time activated partial thromboplastin time
thrombin time antithrombin activity D-dimer concentration thrombin generation and
rotational elastometry as well as the heparin activity using a chromogenic anti-factor Xa
test Finally when the CVC was removed electron microscopy (ELMI) of sections of the
CVC with macroscopically visible thrombi in the last third (5ndash10 cm) of the CVC was
performed 3 different grades were defined for CVC-associated (extraluminal) thrombi
detected by either ultrasound or ELMI respectively
829 patients evaluated by colour Doppler ultrasound during the study developed thrombi
into the lumen of EJV (4 x grade 1 3 x grade 2 and 1 x grade 3) 1927 CVCs assessed
using ELMI showed detectable thrombi on the external CVC surface (7 x grade 1 4 x
grade 2 and 8 x grade 3) There was no significant correlation between thrombus
formation detected by ultrasound or ELMI Thus statistical group comparisons of
patients with or without CVC-associated thrombus formation was performed
independently for both diagnostic procedures The plasmatic heparin activities showed
great variability but no significant differences were found between dogs with or without
CVC-associated thrombus formation regardless whether this was based on sonographic
Chapter 5 Summary
81
or ELMI findings From the initially performed haemostasis tests high fibrinogen
concentrations and low maximum lysis values as measured by the ROTEM delta device
(using the ex-tem reagent) were associated with sonographically detectable thrombi
In conclusion the preliminary methodological study on the ROTEM delta in dogs shows
that the method delivers repeatable results in canine citrated whole blood and the
reference values obtained should offer a reliable basis for the interpretation of test results
of this device in dogs with suspected haemostasis disorders The results of the main study
indicate that CVC-associated thromboses occur (eg 28 [829] of the patients as
detected by ultrasound) although severe thromboses (high grades) are relatively rare in
intensive care canine patients receiving a routine anticoagulant treatment Thereby
prophylactic antithrombotic protocol (unfractionated heparin [UFH]) in the given dosage
was not completely effective to prevent CVC induced thrombus formation in canine
intensive care patients A high fibrinogen concentration and low maximum lysis
(ROTEM ex-tem) may be useful indicators for an increased thrombotic risk in individual
patients which may require special anticoagulatory treatment Further studies are
required to evaluate whether different thromboprophylactic treatment regimens eg with
low molecular weight heparin are more effective in preventing CVC-associated
thromboses in dogs
Chapter 6 Zusammenfassung
82
Chapter 6 Zusammenfassung
Zentralvenenkatheter-induzierte Thrombose bei Hunden - Auftreten unter einem
antithrombotischen Behandlungsschema und moumlgliche Indikatoren
Joseacute Mauricio Pereira Mora
Zentralvenenkatheter (ZVK) sind fuumlr das Intensivmanagement von Hunden von
Bedeutung Die Entstehung einer Thrombose der Vena jugularis ist eine schwerwiegende
Komplikation im Zusammenhang mit der Verwendung eines ZVKs Uumlber die Inzidenz
von ZVK-assoziierten Thrombosen bei Hunden und die Notwendigkeit und Wirksamkeit
prophylaktischer antithrombotischer Protokolle liegen jedoch nur wenige fundierte
Angaben in der Literatur vor
Daher waren die Ziele der vorliegenden Studie (1) die Inzidenz der Katheter-induzierten
Thrombose bei caninen Intensivpatienten zu bestimmen (2) die Wirksamkeit eines
prophylaktischen Heparin-Regimes zu bewerten das routinemaumlszligig in der Klinik fuumlr
Kleintiere der Stiftung Tieraumlrztliche Hochschule Hannover angewendet wird und (3) um
zu beurteilen ob ausgewaumlhlte Haumlmostasetestergebnisse ein erhoumlhtes Risiko fuumlr die
Thrombosebildung vorhersagen koumlnnen
Die PhD-Arbeit enthaumllt zudem eine vorbereitende methodische Studie zur Verwendung
der Rotationselastometrie bei Hunden mit Hilfe des ROTEM delta-Geraumltes In dieser
Vorstudie wurden Referenzintervalle basierend auf 125 gesunden Hunden fuumlr
verschiedene Gerinnungsaktivatorreagenzien und fuumlr Messungen ohne Aktivator
bestimmt Um die Genauigkeit der Methode einzustufen wurden Variationskoeffizienten
(VKs) basierend auf den Ergebnissen von Vierfachmessungen von drei normalen und drei
abnormalen Blutproben berechnet Der Einfluss von Alter Geschlecht Kastration und
Groumlszlige wurde ebenfalls getestet Die Medianwerte der VKs fuumlr die meisten in der
Chapter 6 Zusammenfassung
83
Praumlzisionsanalyse bewerteten Parameter betrugen weniger als 10 und dies bestaumltigte
dass das ROTEM delta-Analysegeraumlt wiederholbare Ergebnisse liefert In Bezug auf den
Einfluss von Alter Geschlecht Kastration und Tiergroumlszlige erwies sich nur eine Kastration
bei Huumlndinnen als relevanter Einflussfaktor fuumlr die meisten wichtigen ROTEM-
Parameter
Die Hauptstudie der Arbeit umfasst 29 Patienten welche zum Zeitpunkt der
Untersuchung stationaumlr in der Kleintierklinik aufgenommen worden waren und aus
medizinischer Indikation einen ZVK benoumltigten Diese Patienten erhielten eine
prophylaktische Antikoagulation mit unfraktioniertem Heparin (UFH 150 IE
[chirurgische Hunde erhielten 75 IE]kg KG 3 x taumlglich subkutan) Bildgebende
Untersuchungen mittels Farbdoppler-Ultraschall der jeweiligen Vena jugularis externa
(VJE) zum Nachweis der Thrombusbildung im Lumen der VJE und Blutentnahmen fuumlr
Haumlmostasetests wurden jeweils direkt vor dem Installationsvorgang (Tag 0) sowie an den
Tagen 1 3 5 und 7 nach Legen des ZVKs durchgefuumlhrt Haumlmostasetests umfassten die
Prothrombinzeit die aktivierte partielle Thromboplastinzeit die Thrombinzeit die
Antithrombinaktivitaumlt die D-Dimer-Konzentration die Thrombingenerierung und die
Rotationselastometrie sowie die Heparinaktivitaumlt unter Verwendung eines chromogenen
Anti-Faktor-Xa-Tests Schlieszliglich wurde nach Entfernung des ZVKs eine
Elektronenmikroskopie (ELMI) von Abschnitten aus dem letzten Drittel (5ndash10 cm) des
ZVKs mit makroskopisch sichtbaren Thromben durchgefuumlhrt Fuumlr ZVK-assoziierte
(extraluminale) Thromben die entweder durch Ultraschall oder ELMI nachgewiesen
wurden wurden drei verschiedene Grade definiert
829 Patienten die waumlhrend der Studie mittels Farbdoppler-Ultraschall untersucht
wurden entwickelten Thromben im Lumen der VJE (4 x Grad 1 3 x Grad 2 und 1 x Grad
3) 1927 ZVKs die unter Verwendung von ELMI bewertet wurden zeigten
Chapter 6 Zusammenfassung
84
nachweisbare Thromben auf der aumluszligeren ZVK-Oberflaumlche (7 x Grad 1 4 x Grad 2 und 8
x Grad 3) Es gab keine signifikante Korrelation zwischen der durch Ultraschall oder
ELMI festgestellten Thrombusbildung Daher wurden statistische Gruppenvergleiche
von Patienten mit oder ohne ZVK-assoziierter Thrombusbildung fuumlr beide diagnostische
Verfahren unabhaumlngig voneinander durchgefuumlhrt Die plasmatischen Heparinaktivitaumlten
zeigten eine groszlige Variabilitaumlt es wurden jedoch keine signifikanten Unterschiede
zwischen Hunden mit oder ohne ZVK-assoziierter Thrombusbildung gefunden
unabhaumlngig davon ob diese auf sonographischen oder ELMI-Befunden beruhte Von den
initial durchgefuumlhrten Haumlmostasetests waren hohe Fibrinogenkonzentrationen und
niedrige maximale Lyse-Werte der Messungen mit ROTEM delta (ex-tem Reagenz) mit
sonographisch nachweisbaren Thromben assoziiert
Zusammenfassend zeigt die vorbereitende methodische Studie zum ROTEM delta bei
Hunden dass die Methode wiederholbare Ergebnisse in mit Zitrat
antikoaguliertemVollblut liefert Die erarbeiteten Referenzwerte sollten eine zuverlaumlssige
Grundlage fuumlr die Interpretation der Testergebnisse dieses Geraumlts bei Hunden mit
Verdacht auf Haumlmostasestoumlrungen bieten Die Ergebnisse der Hauptstudie zeigen dass
ZVK-assoziierte Thrombosen auftreten (z B 28 [829] der Patienten bezogen auf den
Ultraschall-gestuumltzten Nachweis) obwohl schwere Thrombosen (hohe Grade) bei
intensivmedizinisch versorgten Hundepatienten die eine Routineantikoagulation
erhalten relativ selten sind Somit war das prophylaktische antithrombotische Protokoll
(unfraktioniertes Heparin [UFH]) in der angewendeten Dosierung nicht vollstaumlndig
wirksam um die ZVK-induzierte Thrombusbildung bei caninen Intensivpatienten zu
verhindern Eine hohe Fibrinogenkonzentration und eine niedrige maximale Lyse
(ROTEM ex-tem) koumlnnen als Indikatoren eines erhoumlhten Thromboserisikos bei einzelnen
Patienten nuumltzlich sein fuumlr die moumlglicherweise eine spezielle gerinnungshemmende
Chapter 6 Zusammenfassung
85
Behandlung notwendig ist Weitere Studien sind erforderlich um zu untersuchen ob
verschiedene thromboprophylaktische Behandlungsschemata z B mit
niedermolekularem Heparin bezuumlglich der Verhinderung von ZVK-assoziierten
Thrombosen bei Hunden wirksamer sind
Chapter 7 References
86
Chapter 7 References
1 Abdelkefi A Ben Othman T Kammoun L Chelli M Ben Romdhane N Kriaa A Ladeb
S Torjman L Lakhal A Achour W Ben Hassen A Hsaiumlri M Ladeb F Ben Abdeladhim
A 2004 Prevention of central venous line-related thrombosis by continuous infusion of low-dose
unfractionated heparin in patients with haemato-oncological disease A randomized controlled
trial Thromb Haemost 92 654ndash661
2 Adamantos S Brodbelt D Moores A L 2010 Prospective evaluation of complications
associated with jugular venous catheter use in a veterinary hospital J Small Anim Pract 51 254ndash
257
3 Ballard D H Samra N S Gifford K M Roller R Wolfe B M Owings J T 2016
Distance of the internal central venous catheter tip from the right atrium is positively correlated
with central venous thrombosis Emerg Radiol 23 269ndash273
4 Baumann-Kreuziger L Onwuemene O Kolesar E Crowther M Lim W 2015 Systematic
review of anticoagulant treatment of catheter-related thrombosis Thromb Res 136 1103ndash1109
5 Bliss SP Bliss SK Harvey HJ 2002 Use of recombinant tissue‐plasminogen activator in a
dog with chylothorax secondary to catheter‐associated thrombosis of the cranial vena cava J Am
Anim Hosp Assoc 38 431ndash435
6 Boersma RS Hamulyak K van Oerle R Tuinenburg A Ten Cate-Hoek AJ Schouten
HC 2016 Biomarkers for prediction of central venous catheter related-thrombosis in patients
with hematological malignancies Clin Appl Thromb Hemost 22 779ndash784
7 Boersma RS Jie KS Verbon A van Pampus EC Schouten HC 2008 Thrombotic and
infectious complications of central venous catheters in patients with hematological
malignancies Ann Oncol 19 433ndash442
8 Bonnet F Loriferne JF Texier JP Texier M Salvat A Vasile N 1989 Evaluation of
Doppler examination for diagnosis of catheter-related deep vein thrombosis Intensive Care Med
15 238ndash240
9 Borow M Crowley J G 1985 Evaluation of central venous catheter thrombogenicity Acta
Anaesthesiol Scand Suppl 81 59ndash64
10 Cheng Y Cui T Fu P Liu F Zhou L 2013 Dyslipidemia is associated with tunneled-cuffed
catheter-related central venous thrombosis in hemodialysis patients a retrospective multicenter
Chapter 7 References
87
study Artif Organs 37 155ndash161
11 Comerlato PH Rebelatto TF Santiago de Almeida FA Klein LB Boniatti MM
Schaan BD Rados DV 2017 Complications of central venous catheter insertion in a teaching
hospital Rev Assoc Med Bras 63 613ndash620
12 De Cicco M Matovic M Balestreri L Steffan A Pacenzia R Malafronte M Fantin
D Bertuzzi CA Fabiani F Morassut S Bidoli E Veronesi A 2009 Early and short‐term
acenocumarine or dalteparin for the prevention of central vein catheter‐related thrombosis in
cancer patients a randomized controlled study based on serial venographies Ann
Oncol 20 1936ndash1942
13 Espitia O Fouassier M Hardouin JB Pistorius MA Agard C Planchon B Trossaert M
Pottier P 2017 Thrombin generation assay in hospitalized nonsurgical patients A new tool to
assess venous thromboembolism risk Clin Appl Thromb Hemost 23 45ndash51
14 Evans NS Ratchford EV 2018 Catheter related-venous thrombosis Vasc Med 23 411ndash413
15 Geerts W 2014 Central Venous Catheter-related thrombosis Hematology Am Soc Hematol
Educ Program 2014 306ndash311
16 Harahsheh Y Ho KM 2018 Use of viscoelastic tests to predict clinical thromboembolic
events A systematic review and meta‐analysis Eur J Haematol 100 113ndash123
17 Harter C Salwender HJ Bach A Egerer G Goldschmidt H Ho AD 2002 Catheter-
related infection and thrombosis of the internal jugular vein in hematologic-oncologic patients
undergoing chemotherapy a prospective comparison of silver-coated and uncoated
catheters Cancer 94 245ndash251
18 Hirsh J Raschke R 2004 Heparin and low-molecular-weight heparin The Seventh ACCP
Conference on Antithrombotic and Thrombolytic Therapy Chest 126 (Suppl) 188Sndash203S
19 Karthaus M Kretzschmar A Kroning H Biakhov M Irwin D Marschner N Slabber
C Fountzilas G Garin A Abecasis NG Baronius W Steger GG Sudhoff T Giorgetti
C Reichardt P 2006 Dalteparin for prevention of catheter‐related complications in cancer
patients with central venous catheters final results of a double‐blind placebo‐controlled phase III
trial Ann Oncol 17 289ndash296
20 Kato F Takeuchi H Matsuda S Kawakubo H Omori T Kitagawa Y 2016 Incidence of
and risk factors for venous thromboembolism during surgical treatment for esophageal cancer a
Chapter 7 References
88
single-institution study Surg Today 46 445ndash452
21 Kohler TR Kirkman TR 1998 Central venous catheter failure is induced by injury and can
be prevented by stabilizing the catheter tip J Vasc Surg 28 59ndash66
22 Koumlksoy C Kuzu A Kutlay J Erden I Ozcan H Ergicircn K 1995 The diagnostic value of
colour Doppler ultrasound in central venous catheter related thrombosis Clin Radiol 50 687ndash
689
23 Kucher N 2011 Clinical practice Deep-vein thrombosis of the upper extremities N Engl J
Med 364 861ndash869
24 Langston CE Eatroff AE 2018 Hemodialysis catheter-associated fibrin sheath in a dog J
Vet Emerg Crit Care 28 366ndash371
25 Langston CE Eatroff AE Poeppel K 2014 Use of tissue plasminogen activator in catheters
used for extracorporeal renal replacement therapy J Vet Intern Med 28 270ndash276
26 Laurenson MP Hopper K Herrera MA Johnson EG 2010 Concurrent diseases and
conditions in dogs with splenic vein thrombosis J Vet Intern Med 24 1298ndash1304
27 Lee AYY Kamphuisen PW 2012 Epidemiology and prevention of catheter‐related
thrombosis in patients with cancer J Thromb Haemost 10 1491ndash1499
28 Liu G Fu ZQ Zhu P Li SJ 2015 Central venous catheter-related thrombosis in senile male
patients New risk factors and predictors J Huazhong Univ Sci Technolog Med Sci 35 445ndash
449
29 Lordick F Hentrich M Decker T Hennig M Pohlmann H Hartenstein R Peschel C
2003 Ultrasound screening for internal jugular vein thrombosis aids the detection of central
venous catheter-related infections in patients with haemato-oncological diseases a prospective
observational study Br J Haematol 120 1073ndash1078
30 Lucas TC Silva EED Souza DOD Santos ARD Lara MO 2017 Microstructural
evaluation by confocal and electron microscopy in thrombi developed in central venous catheters
Rev Esc Enferm USP 51 1ndash9
31 Mismetti P Mille D Laporte S Charlet V Buchmuller‐Cordier A Jacquin JP Fournel
P Dutrey‐Dupagne C Decousus H 2003 Low‐molecular‐weight heparin (nadroparin) and
very low doses of warfarin in the prevention of upper extremity thrombosis in cancer patients with
indwelling long‐term central venous catheters a pilot randomized trial Haematologica 88 67ndash
Chapter 7 References
89
73
32 Niers TM Di Nisio M Klerk CP Baarslag HJ Buller HR Biemond BJ 2007
Prevention of catheter‐related venous thrombosis with nadroparin in patients receiving
chemotherapy for hematologic malignancies a randomized placebo‐controlled study J Thromb
Haemost 5 1878ndash1882
33 Scott K C Hansen B D DeFrancesco T C (2009) Coagulation effects of low molecular
weight heparin compared with heparin in dogs considered to be at risk for clinically significant
venous thrombosis J Vet Emerg Crit Care 19 74ndash80
34 Smith RS Zhang Z Bouchard M Li J Lapp HS Brotske GR Lucchino DL Weaver
D Roth LA Coury A Biggerstaff J Sukavaneshvar S Langer R Loose C 2012
Vascular catheters with a nonleaching poly-sulfobetaine surface modification reduce thrombus
formation and microbial attachment Sci Transl Med 4 153
35 Tuumlrk S M Cansu D Uuml Teke H Uuml Kaşifoğlu T Meltem Akay O Bilgin M amp Korkmaz
C 2018 Can we predict thrombotic tendency in rheumatoid arthritis A thromboelastographic
analysis (with ROTEM) Clin Rheumatol 37 2341ndash2349
36 van Rooden CJ Rosendaal FR Barge RM van Oostayen JA van der Meer FJM
Meinders AE Huisman MV 2003 Central venous catheter related thrombosis in haematology
patients and prediction of risk by screening with Doppler-ultrasound Br J Haematol 123 507ndash
512
37 van Rooden CJ Tesselaar ME Osanto S Rosendaal FR Huisman MV 2005 Deep vein
thrombosis associated with central venous catheters - a review J Thromb Haemost 3 2409ndash
2419
38 Verso M Agnelli G Bertoglio S Di Somma FC Paoletti F Ageno W Bazzan M Parise
P Quintavalle R Naglieri E Santoro A Imberti D Soraru M Mosca S 2005 Enoxaparin
for the prevention of venous thromboembolism associated with central vein catheter a double‐
blind placebo‐controlled randomized study in cancer patients J Clin Oncol 23 4057ndash4062
39 Vose J Odunayo A Price JM Daves M Schildt JC Tolbert MK 2019 Comparison of
heparinized saline and 09 sodium chloride for maintaining central venous catheter patency in
healthy dogs PeerJ 7e7072 httpdoi107717peerj7072
40 Wang LH Wei F Jia L Zhi Lu Wang B Dong HY Yu HY Sun GJ YangJ Li B
Chapter 7 References
90
Meng J Zhang RN Bi XQ Chen HY Jiang AL 2015 Fibrin sheath formation and
intimal thickening after catheter placement in dog model role of hemodynamic wall shear stress J
Vasc Access 16 275ndash284
41 Wildgruber M Lueg C Borgmeyer S Karimov I Braun U Kiechle M Meier R Koehler
M Ettl J Berger H 2016 Polyurethane versus silicone catheters for central venous port devices
implanted at the forearm Eur J Cancer 59 113ndash124
42 Whitesell RT Steenburg SD 2014 Imaging findings of acute intravascular thrombus on non-
enhanced computed tomography Emerg Radiol 21 271ndash277
43 Wu X Studer W Skarvan K Seeberger MD 1999 High incidence of intravenous thrombi
after short-term central venous catheterization of the internal jugular vein J Clin Anesth 11
482ndash485
44 Xiang DZ Verbeken EK Van Lommel AT Stas M De Wever I 1998 Composition and
formation of the sleeve enveloping a central venous catheter J Vasc Surg 28 260ndash271
45 Xiang DZ Verbeken EK Van Lommel AT Stas M De Wever I 2001 Sleeve-related
thrombosis a new form of catheter-related thrombosis Thromb Res 104 7ndash14
91
Acknowledgements
First I thank God for every day in my life for His guide and because He was is
and will be always with me and that certainty gives me strength to take a step
forward
My sincere thanks to Prof Dr Reinhard Mischke for the opportunity to perform
my PhD under his supervision for his support and guidance for all that I learned
with him and for his friendship
I also thank to Prof Dr Manfred Kietzmann and PD Dr Mario von Depka
Prondzinski for being part of my supervision group for their time and beneficial
advices and discussion during my PhD
My gratitude to Prof Hewicker-Trautwein and Mrs Rohn for their valuable work
and availability to help me
I thank the Werlhof Institute Medical Care Center Hannover for the valuable
cooperation especially to Mrs Ober-Cassebaum Anne and Franziska for all their
help
I thank all the staff of the Small Animal Clinic of the University of Veterinary
Medicine Hannover (TiHo) My special gratitude to the internal medicine and
critical care surgery neurology ultrasound and anesthesia departments and to
the TiHo students all their cooperation made possible my research
I thank the haematology clinical chemistry and haemostasis laboratory
department of the Small Animal Clinic TiHo especially to Dirk because he was
always available for me thanks for his guide and friendship all his explanations
in the laboratory were of great value but also the conversations about different
topics of the life were of big value for me
92
I thank the Hannover Graduate School for Veterinary Pathobiology
Neuroinfectology and translational Medicine (HGNI) for the well-organized
PhD program and especially thanks to Prof Dr Beatrice Grummer Dr Tina
Selle and Mrs Tania Czeslik for his guide and help during my studies
My sincere thanks to Mrs Maritta Ledwoch for her guidance at the International
office of the TiHo ldquoI remember you since my externship in Germanyrdquo
I also thank the Student Union Hannover for all the help with the accommodation
for me and my family
I want to thank the Universidad Nacional de Costa Rica and therefore to Costa
Rica (my loved country) for the financial support to perform my PhD studies
but also for my professional formation as a veterinarian Special thanks to the
Veterinary Medicine School from Universidad Nacional de Costa Rica To all the
academic and administrative staff for their support and for believing in me
Sincerely thanks to Dr Mauricio Jimenez Soto my mentor my adviser but most
important my friend Thanks for believing in me and for always being by my side
ldquoAgradezco profundamente a mis papaacutes ldquodon Tito (Papillo) y dontildea Eida (Mami)rdquo
Todo lo bueno que hay en mi proviene de Dios y de ellos Gracias a mis hermanos
(as) cuntildeados (as) sobrinos y a toda la familia relacionada a ellos Siempre han
sido un gran apoyo y los amordquo
ldquoGracias a dontildea Alba Fran y David Gracias por toda la ayuda y por estar siempre
pendientesrdquo
I would like to thank all my friends that during a short part or during my whole
life have been with me For all the good time shared really thanks
To Willy thanks ldquogordordquo
93
I sincerely thank to Adriano and Johanna for their help and hospitality Thanks
Adriano for your guidance and support during my beginning at the TiHo for make
me feel in Costa Rica with your friendship for the tourist walks and for the bike
training I missed the coffee times
I want to thank Ina the first person to give me a smile at the start of my PhD
Thanks for your hugs and friendship
I also thank Franz for his help and for being a very nice partner Thanks for help
me always when I requested you
ldquoFinalmente quiero expresar mi gratitud y amor a Karo Faacutetima y Abigaiacutel A Karo
por tu sacrificio y soporte por las palabras de aliento por estar a mi lado en las
buenas pero en especial en las malas A Faacute por tu sacrificio por tus sonrisas por
tus abrazos y besos por bailar con migo siempre por ser mi modelo e inspiracioacuten
A Abi por ser una nueva razoacuten in mi vida para seguir adelante por todo lo que
deseo compartir con tigo por mostrarme junto con Faacutetima la felicidad en una
sonrisardquo
Supervisor Prof Dr Reinhard Mischke
Supervision Group Prof Dr Reinhard Mischke
Prof Dr Manfred Kietzmann
PD Dr Mario von Depka Prondzinski
1st Evaluation Prof Dr Reinhard Mischke
Department of Small Animal Medicine and Surgery
University of Veterinary Medicine Hannover Germany
Prof Dr Manfred Kietzmann
Institute for Pharmacology Toxicology and Pharmacy
University of Veterinary Medicine Hannover Germany
PD Dr Mario von Depka Prondzinski
Werlhof Institute Medical Care Center
Hannover Germany
2nd Evaluation
Prof Dr Andreas Moritz
Clinical Pathophysiology and Clinical Laboratory Diagnostics
Justus-Liebig-University Giessen Germany
Date of final exam November 9th 2020
Parts of the thesis have been published as full paper previously in journals
Pereira JM Rohn K Mischke R 2020 Reference intervals for rotational
thromboelastometry measurements using the ROTEMreg delta device in dogs Res Vet
Sci 130 26ndash32
Parts of the thesis have been presented in abstracts related to the poster and oral
presentations at congresses
Pereira JM Mischke R 2017 Reference intervals for thromboelastometric
measurements using the ROTEMreg delta device in dogs In 10th Graduate School
DaysHannover Graduate School for Veterinary Pathobiology Neuroinfectology and
Translational Medicine 01ndash02122017 Bad Salzdetfurth Germany Stiftung
Tieraumlrztliche Hochschule Hannover P 48 P 08
Pereira JM Mischke R 2018 Reference intervals for thromboelastometric
measurements using the ROTEMreg delta device in dogs In 26 Jahrestagung der DVG-
Fachgruppe Innere Medizin und klinische Labordiagnostik 02ndash030218 Hannover
Germany Tierarztl Prax Ausg K Kleintiere Heimtiere 46(02) P18-A16
(DOI 101055s-0038-1647663)
Pereira JM Hewicker-Trautwein M von Depka Prondzinski M Mischke R 2018
Central venous catheter associated thrombosis in dogs In 11th Graduate School
DaysHannover Graduate School for Veterinary Pathobiology Neuroinfectology and
Translational Medicine 3011ndash021217 Hannover Germany Stiftung Tieraumlrztliche
Hochschule Hannover V07
Pereira JM Hewicker-Trautwein M von Depka Prondzinski M Mischke R 2019
Central venous catheter associated thrombosis in dogs In 27 Jahrestagung der DVG-
Fachgruppe Innere Medizin und klinische Labordiagnostik 01ndash020219 Munich
Germany Tierarztl Prax Ausg K Kleintiere Heimtiere 47(02) P14-139 (DOI
101055s-0039-1679119)
Pereira JM Rohn K Mischke R 2019 Reference intervals for rotational
thromboelastometric measurements using the ROTEMreg delta device in dogs In 35th
World Veterinary Association Congress 27 ndash300319 San Joseacute Costa Rica
Pereira JM Rohn K Mischke R 2019 Reference intervals for rotational
thromboelastometric measurements using the ROTEMreg delta device in dogs Abstracts
of the ESVCP-ECVCP Annual Congress 25ndash280919 Arnhem The Netherlands Vet
Clin Pathol 48 813 P 57
Pereira JM Hewicker-Trautwein M von Depka Prondzinski M Mischke R 2020
Central venous catheter associated thrombosis in dogs In 20 Annual ECVIM-CA
Congress 03ndash050920 Barcelona Spain J Vet Int Med (in press)
Sponsorship Joseacute Mauricio Pereira received a scholarship from the Universidad Nacional
de Costa Rica (reference Code JB-C-1107-2016)
Table of contents
Table of contents
Chapter 1 Introduction 1
Epidemiology of CVC related thrombosis 1
Pathogenesis of CVC related thrombosis 2
Diagnosis of CVC related thrombosis 3
Heparin thromboprophylaxis for CVC related thrombosis 5
Prediction of CVC related thrombosis 7
Aims of the study 8
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs 9
ABSTRACT 11
1 Introduction 12
2 Materials and methods 14
3 Results 20
4 Discussion 26
5 Conclusions 31
6 Acknowledgments 31
7 References 32
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes 37
ABSTRACT 40
1 Introduction 42
2 Material and methods 44
3 Results 54
Table of contents
4 Discussion 62
5 Conclusions 67
6 Acknowledgments 67
7 References 68
Chapter 4 General discussion 72
Incidence of CVC related thrombosis 72
Heparin thromboprophylaxis for CVC related thrombosis 76
Prediction of CVC related thrombosis 77
Conclusion 78
Chapter 5 Summary 79
Chapter 6 Zusammenfassung 82
Chapter 7 References 86
Acknowledgements 91
List of abbreviations
i
List of abbreviations
CV coefficient of variation
CVC central venous catheter
EJV external jugular vein
ETP endogenous thrombin potential
HDL high-density lipoprotein
IV intravenous
LDL low-density lipoprotein
LMWH low molecular weight heparin
ROTEM rotational elastometry
SC subcutaneous
TGA thrombin generation assay
UFH unfractionated heparin
Chapters 2 and 3 have their own abbreviations included
List of figures and tables
ii
List of figures and tables
Chapter 2
Table 1 Coefficients of variation () for rotational thromboelastometry using the
ROTEMreg delta analyser in normal blood samples 20
Table 2 Coefficients of variation () for rotational thromboelastometry using the
ROTEMreg delta analyser in canine blood samples with haemostatic alterations 20
Table 3 Reference values (median reference interval based on 25 - and 975 -
quantiles) for thromboelastometric measurements with the ROTEMreg delta analyser in
dogs (n=125 95 confidence intervals in brackets) 23
Table 4 Median values (minimum and maximum in brackets) for different ROTEMreg
variables using ex-tem reagent stratified by sex and neuter status 24
Table 5 Median values (minimum and maximum in brackets) for different ROTEMreg
variables using the in-tem reagent stratified by sex and neutering status 25
Chapter 3
Fig 1 Definition of different grades of central venous catheter induced thrombosis of the
jugular vein as detected using colour Doppler ultrasound 48
Fig 2 Definition and illustration of different grades of central venous catheter induced
(extraluminal) thrombosis as detected using electron microscopy 53
Fig 1S (suppl material) Definition and illustration of different grades of intraluminal
thrombosis in central venous catheters as detected using electron microscopy 53
Table 1S (Supplementary material) Heparin activities (IUml median values
minimumndashmaximum) measured in 27 dogs receiving 75 or 150 IUkg unfractionated
(standard) heparin TID subcutaneously 2 hours after the first injection on the day
Comparison of samples collected via puncture of the peripheral veins and those collected
from the central venous catheter (CVC) using the three-syringe-technique 56
List of figures and tables
iii
Fig 3ab Heparin activities (box- and whisker plot arithmetic means) in dogs receiving
unfractionated heparin at a dosage of 75 IUkg BW tid (a) or 150 IUkg BW tid (b) two
hours after the first daily heparin administration 57
Table 1 Heparin activities (IUml median values minimumndashmaximum) in 28 dogs
receiving 75 or 150 IUkg unfractionated (standard) heparin TID subcutaneously with
(grades 1ndash3) or without (grade 0) sonographically detectable thrombus Blood collections
were performed 2 hours after the first daily injection 57
Table 2 Heparin activities (IUml median values minimumndashmaximum) in 27 dogs
receiving 75 or 150 IUkg unfractionated (standard) heparin TID subcutaneously with
(score 1ndash3) or without (score 0) detectable thrombus via electron microscopy 58
Table 3 Comparison of initial results of haemostasis parameters (median values
minimumndashmaximum in dogs with (grades 1ndash3) and without (grade 0) sonographically
detectable central venous catheter-induced thromboses 60
Table 4 Comparison of results of haemostasis parameters (median values minimumndash
maximum) on the last day in dogs with (grades 1ndash3) and without (grade 0) central venous
catheter-associated thrombus formation as detected by electron microscopy 61
Chapter 1 Introduction
1
Chapter 1 Introduction
Central venous catheters (CVC) are mainly used at critical care units in human and small
animal patients requiring infusion of large amounts of fluids or blood parenteral nutrition
support routine drug therapy haemodialysis chemotherapy plasmapheresis or
periodical blood sampling (Geerts 2014 Vose et al 2019) CVC related thrombosis is
one of the most severe complications (apart from infections and pneumothorax) of CVCs
in humans (Comerlato et al 2017 Evans and Ratchford 2018 Geerts 2014) leading to
patientrsquos distress CVC dysfunction venous stenosis and in some cases to the
development of thromboembolism (Geerts 2014) In the human literature there are
various studies dealing with epidemiologic and prophylactic aspects which are described
below in detail In contrast to the best of the authoracutes knowledge studies about extra
luminal CVC related thrombosis in dogs are rare in the available veterinary literature
Studies are mainly dealing with development of intraluminal CVC thrombi occurring
frequently in dogs and cats (Langston et al 2014) and leading to CVC malfunction (Vose
et al 2019)
Epidemiology of CVC related thrombosis
CVC related thrombosis represents 70ndash80 of all upper extremity thrombosis cases and
approximately 10 of thromboembolism cases in humans (Kucher 2011) The incidence
of CVC related thrombosis reported in humans based in 25 studies ranged widely from
2 to 67 (median = 30 ) (van Rooden et al 2005) The incidence of CVC related
thrombosis detected by colour Doppler ultrasound of the internal jugular vein reported in
3 studies performed in humans was 2 (Harter et al 2002) 30 (Lordick et al 2003)
and 56 (Wu et al 1999)
Chapter 1 Introduction
2
The incidence of CVC related thrombosis in humans can vary between studies according
to the CVC type used entry site of the CVC (jugular femoral subclavian axillary
cephalic vein) and time period with the CVC in place the diagnostic thrombus detection
technique (venography or ultrasonography) and criteria for diagnosis duration of follow
up diseases and presence of predisposing factors (eg previous venous thrombosis
history presence of cancer) and finally prophylactic anticoagulation treatment (Geerts
2014 Liu et al 2015 van Rooden et al 2005)
To the best of the authoracutes knowledge data about the incidence of CVC related
thrombosis detected by colour Doppler ultrasound into the lumen of the jugular vein in
dogs are rare in the available veterinary literature In one study performed in 81 dogs and
12 cats (Adamantos et al 2010) the incidence of vein thrombosis detected by palpation
or ultrasound was 2 and the incidence of intraluminal CVC thrombosis (presumed by
the inability to draw blood back) was 15 Additionally regarding to intraluminal CVC
thrombosis an incidence of 8 was reported in one case control study performed in 24
dogs receiving CVC flushing with heparinised solution (Vose et al 2019) Data about
incidence in other animal species was also found In another case-control study performed
on 37 pigs thrombosis in the caval vein related to CVC placed in the external jugular
vein (EJV) was evaluated by histological analysis however no ultrasound detection was
performed (Kohler and Kirkman 1998) In this study in pigs 94 of patients with CVC
developed thrombi in the vena cava based on sections at the site of the end of the catheter
tip and 05 and 1 cm proximal of the first section (Kohler and Kirkman 1998)
Pathogenesis of CVC related thrombosis
Two types of CVC related thrombosis are distinguished in the human medicine literature
(Geerts 2014 van Rooden et al 2005) First symptomatic thrombosis is defined as
Chapter 1 Introduction
3
thrombosis detected by diagnostic images (ultrasonography venography) when
symptoms and signs (eg localised swelling pain redness) are present (Lee and
Kamphuisen 2012) Second subclinical thrombosis is defined as thrombosis
demonstrated by the previously mentioned diagnostic images without overt symptoms or
signs (Boersma et al 2008) Additionally the CVC related thrombi can be classified as
occlusive thrombus into the CVC lumen peri-catheter related fibrin sheaths and mural
thrombi (Geerts 2014)
During the CVC insertion the puncture of the skin on the CVC insertion site and the
rupture of the blood vessels integrity activate the coagulation cascade and the CVC acts
as a matrix for adherence and accumulation of fibrin and cellular components (Smith et
al 2012) The attachment of proteins and blood cells to the internal andor external wall
of the CVC can lead to (1) (partial or complete) occlusion of the catheter lumen and (2)
sometimes if a thrombus formation starts into the lumen of the vein (attached to the vein
wall or to the CVC) a partial or complete obstruction of the blood flow andor embolism
can develop (Langston and Eatroff 2018) Additionally a study performed in humans
(Lordick et al 2003) demonstrated a relationship between CVC related thrombosis and
microbial colonisation of the catheter leading to CVC related systemic infections This
relationship was explained among others by the ease of microorganisms like
Staphylococcus aureus and Staphylococcus epidermidis to adhere to CVC related
thrombin sheaths (Boersma et al 2008)
Diagnosis of CVC related thrombosis
Venous thrombosis is commonly diagnosed in human and small animal medicine
(especially in dogs) with vascular ultrasonography with or without use of the colour
Doppler technique which is normally used to evaluate the blood flow through the blood
Chapter 1 Introduction
4
vessels (Evans and Ratchford 2018 Laurenson et al 2010) According to the literature
in humans cited below the CVC related thrombosis detected by colour Doppler
ultrasound is present if any of the following aspects are observed (1) well defined
thrombus visualised in the lumen of the vein (2) impossibility to perform compression
of the vein by slight pressure with the ultrasound probe andor (3) no detectable blood
flow signal by colour Doppler ultrasound (Bonnet et al 1989 Koumlksoy et al 1995)
Similarly in dogs the CVC related thrombosis is usually diagnosed by colour Doppler
ultrasonography through the detection of the thrombus within the lumen of the vein
andor the obstruction (partial or complete) of the normal blood flow (Bliss et al 2002)
Especially in humans the contrast venography technique is used as gold standard to
confirm thrombosis For this purpose a contrast solution is injected through a peripheral
vein to assess its passage through the bloodstream by x-ray However this diagnostic
technique is normally used only in the case of a strong clinical suspicion of venous
thrombosis that cannot be detected by ultrasonography (Laurenson et al 2010 Langston
and Eatroff 2018) In a study performed in 44 human patients requiring a CVC (Koumlksoy
et al 1995) a sensitivity of 94 and a specificity of 96 were determined for the colour
Doppler ultrasound diagnosis when it was compared with the diagnosis performed with
the contrast venography technique That results make the colour Doppler ultrasound an
accurate diagnosis method to detect CVC related thrombosis in humans (Koumlksoy et al
1995 van Rooden et al 2003)
Advanced diagnostic image techniques like computed tomography or magnetic resonance
tomography are also accurate to perform the diagnosis of CVC related thrombosis in
humans (Whitesell and Steenburg 2014) In dogs multidetector computed tomography
venography was used in one study for the diagnosis of CVC related thrombosis allowing
the identification of catheter related sheaths around the CVC external wall (Wang at al
Chapter 1 Introduction
5
2015) However the high costs of these advanced diagnostic imaging procedures and the
requirement to perform at least sedation or general anesthesia are disadvantages when
compared with ultrasonography
Finally electron microscopy (ELMI) is a diagnostic technique that has been used in
humans (Lucas et al 2017) and experimental animals (Xiang et al 1998 Xiang et al
2001) mainly to perform a microstructural evaluation of thrombi developed into the
lumen of the CVC or in the vein containing the CVC ELMI allows the ex vivo diagnosis
of very small thrombi or sleeve related thrombosis that perhaps cannot be detected by
ultrasonography or venography during the evaluation of patients with CVCs (Xiang et al
2001) However one point to consider is that it is possible that compact thrombi
developed on the CVC can be detached during the CVC removal from the vein and
thereby before its analysis by ELMI (Xiang et al 2001)
Heparin thromboprophylaxis for CVC related thrombosis
Application of CVCs in human medicine is commonly accompanied by prophylactic
anticoagulation in order to decrease the risk of development of partial or complete vein
occlusion andor thromboembolism and thereby to improve the patientrsquos comfort
(Baumann-Kreuziger et al 2015) Apart from vitamin K antagonists (Mismetti et al
2003) unfractionated heparin (UFH) (Abdelfeki et al 2004) and low molecular weight
heparin (LMWH) (Karthaus et al 2006) are anticoagulants that have been used on
studies in humans administrated by intravenous (IV) infusion or by subcutaneous (SC)
injection to prevent thrombi development into the lumen of the veins containing the CVC
(review article Baumann-Kreuziger et al 2015) Different heparin dosages have been
used as thromboprophylactic anticoagulation in humans with CVC (review article Lee
and Kamphuisen 2012) For example in five different case-control studies performed in
Chapter 1 Introduction
6
human haemato-oncological patients UFH at a daily dosage of 100 IUkg given by IV
infusion (Abdelfeki et al 2004) and LMWH at SC daily dosages of 40 mg enoxaparin
(Verso et al 2005) 5000 IU AXa dalteparin (Karthaus et al 2006 De Cicco et al 2009)
and 2850 IU AXa nadroparin (Niers et al 2007) were used Only in part of these studies
it is stated that systemic heparin was combined with flushing of the CVC using
heparinised saline solution to reduce the risk of catheter occlusion (Karthaus et al 2006)
In 3 of the 5 previously mentioned studies no statistically significant difference was found
in the incidence of CVC related thrombosis between patients receiving the
anticoagulation prophylaxis compared with the control groups without systemic
anticoagulation (Verso et al 2005 p = 035 Karthaus et al 2006 p gt 005 Niers et al
2007 p = 049) In contrast in the residual two studies a lower incidence of CVC related
thrombosis was found in the group receiving an anticoagulation prophylaxis compared
with the control groups (Abdelfeki et al 2004 150 vs 126 p = 003 De Cicco et
al 2009 400 vs 526 p = 005)
Obviously there is no consensus in the available human literature regarding the beneficial
use of anticoagulation prophylaxis to prevent CVC related thrombosis and this may
reflect great variations for example according to the study design period trial drug
administration route (Lee and Kamphuisen 2012) and the moment of the initial
anticoagulant dosage administration (Decicco et al 2009)
To the best of the authoracutes knowledge studies about thromboprophylaxis for extraluminal
CVC related thrombosis in dogs cannot be found in the available veterinary literature
However similarly to humans prophylactic anticoagulation with heparinised saline
(administrated through CVC flushing) to prevent CVC occlusion by intraluminal thrombi
was used in one case-control study performed in 24 dogs (Vose et al 2019) In the cited
study 12 dogs received dosages of 3 ml of heparinised saline solution (10 IU UFHml) to
Chapter 1 Introduction
7
flush the catheter every 6 hours during 72 hours whereas 12 control dogs received
flushing with the same volume of non-heparinised saline solution administered at the
same frequency and no statistically significant difference was found
Prediction of CVC related thrombosis
Studies regarding the relationship between laboratory biomarkers and CVC related
thrombosis are available in the human literature (Boersma et al 2016 Cheng et al 2013
Liu et al 2015) however to the best of the authoracutes knowledge literature about laboratory
tests used as predictors for CVC related thrombosis in small animals cannot be found at
all Availability of approved blood tests which can predict the risk of thrombosis
development associated to CVC already at the moment of the CVC placement are
important in order to identify an increased risk of CVC related thrombosis (Boersma et
al 2016) In the study performed by Boersma et al (2016) in 168 human patients without
indication of anticoagulation prophylaxis elevated leukocyte counts high levels of
plasminogen activator inhibitor and of coagulation factor VIII (all measured on blood
samples taken after CVC placement) were associated with an increased incidence of
symptomatic CVC-related thrombosis diagnosed by ultrasonography In another study
(Liu et al 2015) high blood levels of lactate dehydrogenase and low concentrations of
HDL and albumin were associated with the incidence for CVC related thrombosis
diagnosed by ultrasound in 324 senile patients which was in total 617 Additionally
dyslipidemia (high triglycerides low HDL and high LDLHDL ratio) was associated
with CVC related thrombosis (diagnosed by ultrasound and computer tomography
angiography) in human patients undergoing haemodialysis (Cheng et al 2013) Studies
about predictive tests for CVC related thrombosis in dogs were not found in the available
literature to the best of the authoracutes knowledge
Chapter 1 Introduction
8
Aims of the study
The aims of the preliminary methodological study of the present scientific project were
(1) to define reference intervals for viscoelastic analyses of canine haemostasis using the
ROTEMreg delta analyser and (2) to determine the precision (repeatability) of this
method And the aims of the main study of the present scientific project were (1) to
determine the incidence of catheter-induced thrombosis in canine intensive care patients
(2) to evaluate the efficacy of a prophylactic heparin regimen routinely used in the Small
Animal Clinic of the University of Veterinary Medicine Hannover and (3) to assess
whether selected haemostasis test results are predictive for an increased risk for
thrombosis formation
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
9
Chapter 2 Reference intervals for rotational thromboelastometry measurements
using the ROTEMreg delta device in dogs
Pereira JM Rohn K Mischke R
Contributions of the authors
Pereira JM performed (1) the patients data and blood samples collection (2)
haematology and ROTEMreg analysis (3) generated the reference intervals and
coefficients of variation included in the study and (4) wrote the manuscript
Rohn K performed the statistical analysis
Mischke R performed the clinical chemistry and coagulation tests and was involved in
designing and supervision of the study analysis and discussion of the data and its
statistical analysis and revision edition and correction of the manuscript
Original source Pereira J M Rohn K amp Mischke R 2020 Reference intervals
for rotational thromboelastometry measurements using the ROTEMreg delta device
in dogs Res Vet Sci 130 26ndash32 httpsdoiorg101016jrvsc202001019
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
10
Reference intervals for rotational thromboelastometry measurements using the
ROTEMreg delta device in dogs
Pereira JMa Rohn Kb Mischke Ra
aSmall Animal Clinic University of Veterinary Medicine Hannover Buumlnteweg 9 D-
30559 Hannover Germany
bInstitute of Biometry Epidemiology and Information Processing University of
Veterinary Medicine Hannover Buumlnteweg 2 30559 Hannover Germany
Corresponding author
E-mail ReinhardMischketiho-hannoverde
Original source Pereira J M Rohn K amp Mischke R 2020 Reference intervals
for rotational thromboelastometry measurements using the ROTEMreg delta device
in dogs Res Vet Sci 130 26ndash32 httpsdoiorg101016jrvsc202001019
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
11
ABSTRACT
Aims of the present study were to define reference intervals for viscoelastic analyses of
canine haemostasis using the ROTEMreg delta analyser and as a secondary aspect to
determine the precision (repeatability) of this method Blood samples from 125 clinically
healthy dogs were included Measurements were performed with commercially available
activating reagents (ex-tem in-tem and kaolin solution) as well as without activation
Additional fourfold measurements were done in 3 of the normal blood samples and in 3
samples with haemostatic alterations to evaluate the precision of the method
Coefficients of Variation (CVs) for most of the ROTEM variables were lt 10 Clot
formation time and maximum clot elasticity showed a wide inter-individual variation in
comparison with alpha angle and maximum clot firmness A multivariate analysis on
various ROTEM parameters revealed particularly a significant influence of neuter status
and a significant interrelationship between the factors sex and neuter status for
measurements with different activating reagents These results reflected the fact that
significant differences occurred only between intact and neutered females but not in
males No or only occasionally significant differences were found between groups of sex
age and size
In conclusion CVs demonstrated that the method delivers repeatable results in canine
citrated whole blood Established reference intervals should deliver valuable orientation
for the evaluation of viscoelastic properties of clotting whole blood in dogs using the
ROTEM delta analyser Neuter status in females appeared to be the most relevant
influencing factor and should be considered for the interpretation of ROTEM delta test
results
Keywords Viscoelastic measurements reference values canine age sex neuter status
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
12
1 Introduction
Heamostasis is a physiologic and dynamic process in which plasma soluble coagulation
factors blood cells and blood vessels are involved Clot formation and possible
subsequent clot lysis as well as vascular tissue repair aim to prevent haemorrhages on the
one hand and thromboses on the other hand (Kol and Borjesson 2010 Versteeg et al
2013) Conventional assays of coagulation such as prothrombin time (PT) activated
partial thromboplastin time (aPTT) and thrombin time TT have limitations especially
cellular elements are not incorporated into the test and thereby these tests reflect only
limitedly in vivo functionality of haemostasis (McMichael and Smith 2011 Crochemore
et al 2018)
The rotational thromboelastometry (ROTEMreg) currently termed as thromboelastometry
(TEM) and thromboelastography (TEG) are methods for the rapid evaluation of the
overall haemostasis capacity through the measurement of viscoelastic properties of
clotting whole blood (Kol and Borjesson 2010 Theusinger et al 2010 Sankarankutty
et al 2012 Versteeg et al 2013) In TEG the cup with the blood sample is rotating
whereas the torsion wire is fixed In ROTEMTEM the cup is fixed whereas the pin is
rotating Changes in torque ie resistance to rotation by the fibrin bridge created between
pin and cup in the clotting blood are detected electromechanically in TEG and optically
in ROTEM (Bollinger et al 2012) and displayed as a graph that indicates the time for the
formation of blood clot and its strength (Bagge et al 2016) The resulting graph reflects
the initiation and propagation of the coagulation system fibrin-platelet interactions and
clot lysis
TEG was first described in 1948 by Hellmut Harten (Hartert 1948) as an in vitro method
to evaluate the clot formation process while resembling the in vivo blood flow The
development of the actually used instrument types TEG and ROTEMregTEM were based
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
13
on that original principle Viscoelastic measurements have been widely used since the
1980s in human medicine to evaluate haemostasis and hypercoagulable states in surgical
patients (eg cardiac and hepatic surgeries) and in general in critical care units (Kol and
Borjesson 2010 Nogami 2016 Wikkelso et al 2016 Crochemore et al 2017) In
veterinary medicine viscoelastic measurements using TEG and ROTEMTEM have been
validated for use in dogs cats and horses and used to study haemostasis in different
diseases (eg infectious diseases liver diseases) and medical conditions (eg
orthopaedic surgery hyperfibrinolysis) and also to investigate the effect of
anticoagulants and other drugs (eg corticosteroids) on haemostasis (Kol and Borjesson
2010 Bruno et al 2015a 2015b Woodman et al 2015 Conversy et al 2017 Sigrist et
al 2018 Turner et al 2019) The methodology seems to be especially suitable to detect
hypercoagulability (Wiinberg et al 2008 Kol and Borjesson 2010 Marschner et al
2018)
In the available veterinary literature information about reference values for TEG were
found using kaolin as activation reagent (Bauer et al 2009) Falco et al (2012)
determined reference values for the ROTEM analyser in dogs using ex-tem in-tem and
fib-tem reagents for selected ROTEM parameters in a study on forty five healthy dogs
However data related to reference intervals obtained in an adequate number of samples
(at least 120) according to the American Society for Veterinary Clinical Pathology
(ASVCP) (Friedrichs et al 2012) and the National Committee for Clinical Laboratory
Standards (NCCLS) (Horn and Pesce 2003) could not be found in the available literature
The aim of this study is to determine reference values for the ROTEMreg delta device in
dogs using citrated whole blood with different clot activator reagents (ex-tem in-tem
kaolin) and without activator
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
14
2 Materials and methods
21 Study design
Reference intervals Single thromboelastometric measurements were performed in 125
clinically healthy dogs using the ROTEMreg analyser and different activating reagents (ex-
tem in-tem and kaolin) as well as without activation Reference values were calculated
and influences of age groups (young adult and senior) sex neuter status and size
(groups small medium and large) were assessed
Precision analysis Additional fourfold measurements using ex-tem or in-tem activating
reagents respectively were performed on 3 of the normal and also in 3 abnormal blood
samples each in order to assess within-run imprecision for these measurements
22 Animals
The clinically healthy dogs with a body weight of at least 5 kg were recruited by launching
a call in the University of Veterinary Medicine Hannover network for students and staff
members Interested dog owners received a free health check of their animals (clinical
examination and blood tests specified below) in combination with the blood collection
In addition individual privately owned patient dogs of the Small Animal Clinic with
undisturbed general health were also included if blood was collected due to another
indication (eg general health check pre-anaesthetic examination test for hereditary
diseases etc) Before taking the blood samples all dogs were fasted for at least 12 h with
free access to water 125 animals for which the examination of the health status (clinical
examination routine haematology and clinical chemistry basis coagulation profile) did
not reveal any deviations from the reference were included in the study whereas 3 of the
128 initially recruited animals had to be excluded due to laboratory abnormalities
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
15
Haematological and clinical chemistry profiles performed in order to confirm the health
status included white and red cell count haematocrit platelet counts and reticulocytes
plasma activities of alanine aminotransferase glutamate dehydrogenase alkaline
phosphatase and pseudocholinesterase as well as concentrations of total bilirubin urea
creatinine glucose total protein albumin cholesterol and ionised calcium A basis
coagulation profile including PT PTT and TT was also performed to exclude significant
abnormalities within the blood coagulation system
The 125 healthy dogs enclosed in the establishment of reference values included 48 mixed
breed dogs and the following pedigree dogs 13 Labrador Retrievers 6 Australian
Shepherds 4 Havanese dogs 3 from each of the following breeds American
Staffordshire Terriers and Cocker Spaniels 2 from each of the following breeds Beagles
Border Collies Dalmatians Elo dogs (breed not recognised by Feacutedeacuteration cynologique
international [FCI]) German Longhaired Pointers German Shorthaired Pointers Giant
Schnauzers and Golden Retrievers 1 from each of the following breeds Belgian
Malinois Bolonka Zwetna Briard Brussels Griffon American English Coonhound
Italian Mastiff Flat Coated Retriever French Bulldog German Shepherd Great Dane
Greater Swiss Mountain Dog Giant Spitz Goldendoodle Hovawart Hungarian Vizsla
Italian Bloodhound Lycanis Wolfdog Miniature American Shepherd Miniature
Schnauzer Old German Shepherd Podenco Poodle Pyrenean Shepherd Rhodesian
Ridgeback Rottweiler Schafpudel (breed not recognised by FCI) Small
Munsterlander Spanish Mastiff Tibet Terrier Weimaraner West Highland White
Terrier and Wirehaired Pointer
These dogs were aged between 6 and 154 months (median = 410 months) and had body
weights ranging from 50 to 707 kg (median = 226 kg) 30 were castrated males 33
intact males 41 spayed females and 21 intact females According to the height at withers
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
16
the dogs were divided into 3 different size groups small (le40 cm n = 22) medium (41ndash
60 cm n = 77) and large (gt60 cm n = 26) Dogs were also divided in three different age
groups young (le 24 months n = 34) adult (25ndash84 months n = 59) and senior dogs (gt84
months n = 32)
The three blood samples with haemostatic abnormalities were taken from two dogs with
complex haemostatic disorders (Disseminated Intravascular Coagulation [DIC])
including reduced platelet count and abnormal PT and APTT and one dog with
thrombocytopenia
Animals were treated in accordance with the German Animal Welfare Law The
experimental design was approved by the Animal Welfare officer of the University of
Veterinary Medicine Hannover Foundation and by the ethics committee of the
responsible authority (Lower Saxony State Office for Consumer Protection and Food
Safety reference number 17A101)
23 Blood sampling
The venipuncture area was shaved and then disinfected with alcohol The saphenous or
cephalic vein was visualised with slight pressure and punctured with disposable cannulas
(diameter 09 or 10 mm) The continuously running blood was directly collected from
the needle into the tubes containing the respective anticoagulants From each dog
approximately 10 ml of blood were collected including approximately 8 ml of citrated
blood for the measurement of haemostasis tests (ROTEM PT aPTT TT) in tubes
containing sodium citrate 313 (Eifelfangoreg GmbH amp Co KG Bad Neuenahr-
Ahrweiler Germany) in a ratio of 1 volume9 volumes of blood In addition
approximately 1 ml of EDTA blood for haematological analysis and approximately 1 ml
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
17
of lithium heparin blood for clinical chemical measurements were collected into
commercial 13 ml tubes (Sarstedt AG amp Co Nuumlmbrecht Germany)
Citrated blood was initially kept at room temperature (20-25 C) in order to avoid errors
within the preanalytical phase (Toulon et al 2017) Two ml of citrated blood were used
for the ROTEMreg analysis the residual citrate blood was centrifuged for 10 min at 3800
rpm and 5 degC then the plasma was separated by careful pipetting and it was centrifuged
a second time under the same conditions to get platelet poor plasma which was stored in
aliquots of 500 microl and 250 microl at -80 degC for coagulation tests
24 Rotational elastometry
Viscoelastic measurements using the ROTEMreg delta for the establishment of reference
intervals were performed approximately 15 min after the blood collection using activating
reagents (liquid reagents) provided by the manufacturer of the instrument (r-ex-temreg in-
temreg) and a kaolin solution (5 g1 as part of the PTT reagent CK Prest [Diagnostica
Stago SAS Asniegraveres sur Seine Cedex France]) In addition non-activated analyses
were performed using isotonic NaCl solution instead of the activating reagent in the fourth
channel of the device Fourfold measurements for precision analysis were performed
simultaneously in the four channels of the device in separate runs
According to the standard test protocol provided by the manufacturer of the ROTEMreg
delta (Werfen GmbHInstrumentation Laboratory Munich Germany) 20 μl of the
reagent for recalcification (star-temreg20) and 20 μl of the activating reagent were pipetted
into a cuvette using the automated pipette provided with the device Immediately
thereafter 300 μl of citrated whole blood (prewarmed to 37 degC for five minutes in the
heating device of the instrument and gently rocked to resuspend possibly sedimented
cells) were added to the test preparation in the cuvette using the automated pipette and
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
18
then the cuvette was laced carefully in the ROTEMreg device whereby the rotating pin
entered into the test approach
Different parameters are provided automatically by the ROTEMreg delta device and the
following variables (described according to the Pentapharm GmbH ROTEMreg delta
Manual [2009]) were used in the study
Clotting time (CT) the time (in s) from the beginning of the test (by adding the clot
activator) until the formation of fibrin starts (defined as the moment when the curve
achieves an amplitude of 2 mm) This Parameter facilitates the decision to replace clotting
factors when it is required
Clot formation time (CFT) describes the next phase of clotting by the formation of a
stable clot through the activation of thrombocytes and fibrin formation It is defined as
the time (in s) between the 2 mm amplitude and 20 mm amplitude of the curve
Alpha angle (α angle) is the angle (in degrees) between the middle axis and the tangent
to the clotting curve through the 2 mm amplitude point
Amplitude after ldquoXXrdquo minutes (eg A30 after 30 min) is the amplitude (in mm) at a
certain time after the CT has finished
Maximum clot firmness (MCF) corresponds to the maximum amplitude (in mm) reached
before the clot is dissolved by fibrinolysis This parameter measures the firmness of the
clot and its quality Like CFT and α angle it facilitates the decision for substitution therapy
with platelet concentrate andor fibrinogen especially in humans
Maximum clot elasticity (MCE) is a parameter calculated from MCF and may be superior
at high amplitudes as compared to the MCF
Lysis index at 30 45 and 60 min (LI30 LI45 LI60) represents the fibrinolysis (in
percentage) 30 45 and 60 min after the CT It may facilitate the decision for or against a
therapy with anti-fibrinolytic drugs
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
19
Maximum lysis (ML) describes the degree of fibrinolysis relative to the MCF achieved
during the measurement (percent loss of clot firmness)
25 Statistical methods
Original data obtained from the ROTEMreg delta device software were transferred into the
Microsoftreg Excelreg formal (Microsoft Office Professional Plus 2013) The Statistical
analyses were performed using the SAS Enterprise Guide Version 71 (SAS Institute Inc
Cary NC USA)
Coefficients of Variation (CVs) for selected relevant parameters of the ROTEM delta
analysis using ex-tem and in-tem regents were calculated for the fourfold measurements
on 3 normal and 3 abnormal blood samples in order to assess within-run imprecision CVs
for the results of each sample were calculated by dividing the standard deviation of the
data set by its arithmetic mean Finally the median of the CVs for each group of 3 samples
was calculated The reference values were calculated according to recommendations of
the American College of Veterinary Clinical Pathology (ACVCP) based on non-
parametric methods (25 and 975 quantiles) supplemented by 95 confidence
intervals (Friedrichs et al 2012)
Data were analysed for Standard normal distribution by Kolmogorov Smirnov test and
assessment of residuals of the analysis of variance (ANOVA) Because these tests
indicated almost normally distributed values for the studied ROTEM variables data were
analysed by a fourway ANOVA stratified by the activating reagent (ex-tem in-tem
kaolin or without activation) to test the influence of size age sex and neuter status on
several parameters and taking all interactions into account Post hoc t-tests were
performed when indicated The significance level was set to P lt 05
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
20
3 Results
31 Precision of the method
For all the parameters evaluated in the study the CVs for the fourfold measurement in
healthy dogs were less than 10 (Table 1) In the case of dogs with haemostatic
abnormalities CVs of the vast majority of ROTEM variables were also less than 10
except the variables CFT and ML irrespective whether ex-tem or in-tem reagents were
used (Table 2)
Table 1 Coefficients of variation () for rotational thromboelastometry using the
ROTEMreg delta analyser in normal blood samples
Activator CT (s) CFT (s) α (deg) A30 (mm) MCF (mm) MCE(mm) LI30 () LI45 () LI60 () ML ()
Ex-temreg 875 629 262 213 179 634 000 020 037 534
In-temreg 245 596 166 117 135 299 000 000 046 216
Abbreviations CT ndash clotting time CFT ndash clot formation time α ndash alpha angle A30 ndash
amplitude after 30 min MCF ndash maximum clot firmness MCE ndash maximum clot elasticity
LI 30 LI 45 LI 60 ndash Lysis index at 30 45 and 60 min ML ndash maximum lysis
Median values of the measurements of 3 samples
Table 2 Coefficients of variation () for rotational thromboelastometry using the
ROTEMreg delta analyser in canine blood samples with haemostatic alterations
Activator CT (s) CFT (s) α (deg) A30 (mm) MCF (mm) MCE(mm) LI30 () LI45 () LI60 () ML ()
Ex-temreg 668 1478 393 218 268 582 0 018 000 848
In-temreg 228 799 461 219 251 371 0 0 017 283
Median values of the measurements of 3 samples from patients with DIC (n = 2)
thrombocytopenia (n = 1) abbreviations please see legend to Table 1
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
21
32 Reference intervals
Activation by ex-tem reagent (tissue factor) resulted in faster CTs when compared to
activation via the intrinsic pathway using ellagic acid (in-tem) or kaolin reagents whereas
the CFT was much less affected by the activating reagent (Table 3) Especially CFT and
MCE showed a wide inter-individual variation in comparison for example with α angle
and MCF regardless of the used activating reagent (Table 3)
33 Influence of age sex neuter status and size
ANOVA did not reveal statistically significant differences in ROTEM delta parameters
between dog groups of variable size or sex (P gt 05) Age had influence only on CT and
only when the in-tem reagent was used (P = 0169 ANOVA) but not when using the ex-
tem reagent (P = 7105) Post hoc comparisons using t-test of the in-tem results between
age groups revealed shorter CTs in the group of senior dogs when compared with young
dogs (P = 0194) but not between adult dogs and these two groups ANOVA showed
particularly influences of the neuter status and a significant interrelationship between the
factors sex and neuter status on different ROTEM delta parameters using ex-tem and in-
tem respectively as activator (Tables 4 and 5) ANOVA revealed also a significant
influence of neuter status on different ROTEM parameters when kaolin was used as
activating reagent (CFT α angle MCF MCE) whereas non-activated measurements
were not subject to any significant influence of the assessed factors (results not shown)
The interrelationship between sex and neuter status mentioned above reflected the fact
that significant differences between neutered and not neutered dogs were only found in
females The statistical analysis using the t-test to compare intact vs neutered dogs
stratified by sex showed statistically significantly (P lt 05) longer CTs and CFTs and
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
22
lower α angle A30 MCF and MCE values in spayed when compared to intact females
(Tables 4 and 5)
Chapter 2 Reference intervals for rotational thromboelastometry measurements using the ROTEMreg delta device in dogs
23
Table 3 Reference values (median reference interval based on 25 - and 975 - quantiles) for thromboelastometric measurements with the ROTEMreg delta analyser in
dogs (n=125 95 confidence intervals in brackets) Abbreviations please see legend to Table 1
Activator
Statistical
para-meter
CT
(s)
CFT
(s)
Α
(deg)
A30
(mm)
MCF
(mm)
MCE
(mm)
LI30
()
LI45
()
LI60
()
ML
()
Ex-temreg
Median 450
[430ndash470]
110
[105ndash115]
680
[670ndash690]
600
[580ndash610]
610
[600ndash620]
154
[147ndash164]
100
[960ndash100]
100
[100ndash100]
970
[960ndash980]
220
[210ndash240]
25 340
[270ndash350]
650
[490ndash710]
540
[420ndash560]
460
[410ndash480]
460
[420-490]
870
[720ndash950]
100
[100-100]
870
[800ndash960]
700
[640ndash820]
800
[100ndash110]
975 830
[790ndash990]
220
[201ndash315]
790
[770ndash800]
770
[690ndash770]
730
[700ndash770]
264
[230-334]
100
[100ndash100]
100
[100ndash100]
100
[990ndash100]
590
[530ndash680]
In-tem
Median 190
[187ndash201]
116
[111ndash125]
680
[670ndash690]
570
[560ndash580]
590
[580ndash600]
143
[135ndash149]
100
[100ndash100]
100
[100ndash100]
980
[980ndash980]
180
[170ndash190]
25 135
[112ndash142]
730
[590ndash800]
510
[460ndash570]
450
[410ndash470]
480
[410ndash500]
920
[710ndash101]
100
[950ndash100]
990
[820ndash990]
930
[740ndash930]
500
[100ndash700]
975 283
[268ndash291]
251
[198ndash227]
750
[750ndash780]
660
[660ndash690]
680
[660ndash690]
210
[195ndash227]
100
[100ndash100]
100
[100ndash100]
100
[100ndash100]
270
[260ndash380]
Activator
Statistical
para-meter
CT
(s)
CFT
(s)
Α
(deg)
A30
(mm)
MCF
(mm)
MCE
(mm)
LI30
()
LI45
()
LI60
()
ML
()
Kaolin
Median 162
[154ndash173]
870
[820ndash910]
740
[720ndash750]
500
[470ndash530]
550
[530ndash560]
121
[115ndash127]
960
[940ndash980]
840
[790ndash880]
710
[640ndash770]
500
[450ndash540]
25
109
[990ndash115]
470
[440ndash580]
560
[490ndash600]
110
[700ndash230]
390
[380ndash400]
640
[600ndash680]
270
[140ndash480]
110
[800ndash260]
800
[400ndash170]
200
[160ndash240]
975 246
[230ndash337]
214
[185ndash290]
810
[790ndash810]
640
[630ndash690]
650
[650ndash690]
187
[182ndash223]
100
[100ndash100]
990
[980ndash100]
940
[930ndash990]
970
[930ndash100]
Without activation
Median 607
[495ndash662]
221
[194ndash265]
510
[460ndash550]
520
[500ndash530]
540
[520ndash550]
115
[109ndash123]
100
[100ndash100]
100
[100ndash100]
980
[980ndash990]
170
[160ndash190]
25 203
[123ndash215]
860
[550ndash930]
190
[120ndash230]
350
[230ndash370]
370
[260ndash400]
600
[350ndash670]
100
[950ndash100]
980
[890ndash990]
930
[840ndash950]
400
[000ndash500]
975 1356
[1305ndash1721]
811
[688ndash1333]
730
[710ndash790]
660
[640ndash720]
660
[640ndash730]
197
[179ndash265]
100
[100ndash100]
100
[100ndash100]
100
[100ndash100]
290
[280ndash320]
Chapter 2 Reference intervals for rotational thromboelastometry measurements using the ROTEMreg delta device in dogs
24
Table 4 Median values (minimum and maximum in brackets) for different ROTEMreg variables using ex-tem reagent stratified by sex and neuter status
Group
CT
(s)
CFT
(s)
Α
(deg)
A30
(mm)
MCF
(mm)
MCE
(mm)
LI30
()
LI45
()
LI60
()
ML
()
Female intact 430
(320ndash530)
960
(590ndash198)
710
(570ndash800)
620
(480ndash730)
640
(510ndash740)
179
(103ndash282)
100
(100ndash100)
100
(980ndash100)
960
(800ndash100)
230
(100ndash640)
Female spayed 460
(270ndash830)
121
(650ndash224)
660
(520ndash770)
580
(430ndash690)
590
(440ndash700)
146
(790ndash230)
100
(960ndash100)
100
(800ndash100)
970
(690ndash100)
240
(100ndash590)
p-value (t-test)
Female
intact vs spayed
00025 00063 00049 00023 00016 00015 na na na na
Male intact 450
(320ndash990)
105
(650ndash315)
690
(420ndash790)
590
(420ndash700)
610
(440ndash710)
154
(770ndash244)
100
(100ndash100)
100
(870ndash100)
970
(650ndash100)
220
(700ndash680)
Male castrated 460
(340ndash930)
111
(490ndash255)
685
(480ndash800)
590
(410ndash770)
600
(420ndash770)
152
(720ndash334)
100
(100ndash100)
100
(830ndash100)
980
(640ndash100)
205
(500ndash590)
p-value (t-test)
Male
Intact vs castrated
08326 05106 04756 01799 01568 02163 na na na na
p-values (ANOVA)
Factor Sex
05428 04847 06612 02206 01887 01630 08489 07893 07689 02758
Factor neuter status 01872 00167 00173 00039 00037 00028 07141 04737 09356 05948
Interrelationship
sexneuter status 00383 00968 00782 00488 00472 00319 04943 08611 05422 00160
na ndash not analysed all significant P-values (lt 005) are highlighted in bold print
Chapter 2 Reference intervals for rotational thromboelastometry measurements using the ROTEMreg delta device in dogs
25
Table 5 Median values (minimum and maximum in brackets) for different ROTEMreg variables using the in-tem reagent stratified by sex and neutering status
Group
CT
(s)
CFT
(s)
Α
(deg)
A30
(mm)
MCF
(mm)
MCE
(mm)
LI30
()
LI45
()
LI60
()
ML
()
Female intact 186
(126ndash238)
940
(590ndash164)
720
(620ndash780)
610
(490ndash690)
620
(530ndash690)
166
(113ndash227)
100
(100ndash100)
100
(990ndash100)
980
(930ndash100)
170
(100ndash300)
Female spayed 201
(149ndash291)
1220
(820ndash262)
670
(500ndash740)
550
(450ndash660)
570
(480ndash680)
133
(910ndash214)
100
(950ndash100)
100
(820ndash100)
980
(740ndash100)
190
(500ndash380)
p-value (t-test)
Female
intact vs spayed
na na lt0001 00009 00005 00005 na na na na
Male intact 199
(117ndash283)
122
(730ndash277)
680
(460ndash750)
570
(430ndash660)
590
(450ndash660)
146
(810ndash192)
100
(100ndash100)
100
(990ndash100)
980
(930ndash100)
180
(600ndash270)
Male castrated 181
(112ndash288)
123
(820ndash222)
670
(540ndash740)
565
(410ndash630)
585
(410ndash640)
138
(710ndash180)
100
(100ndash100)
100
(970ndash100)
980
(890ndash100)
180
(800ndash260)
p-value (t-test)
Male
intact vs castrated
na na 08232 01401 01162 00849 na na na na
p-values (ANOVA)
Factor Sex
02105 03317 03370 00877 01258 00510 09564 07832 08117 03638
Factor neuter status 04289 00660 00619 00107 00088 00054 07152 09795 07056 08299
Interrelationship
sexneuter status
04310 00550 00408 00707 01733 00996 05240 05982 08047 04578
na ndash not analysed all significant P-values (lt 005) are highlighted in bold print
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
26
4 Discussion
41 Coefficients of variation
In accordance with the literature CVs below 10 indicate adequate repeatability in intra-
assays evaluations (Steiner et al 2003 Hua et al 2018) According to our results
obtained from the fourfold measurements in citrated whole blood ROTEM delta
measurements on healthy dogs and dogs with haemostatic alterations fulfil these
requirements with only a few exceptions concerning CFT and ML values in abnormal
samples
Our results regarding repeatability are similar to those obtained for several TEM
parameters in previous studies performed in animals In one study the imprecision of the
ROTEM analyser was evaluated by duplicate measurements in 10 blood samples of
healthy dogs and as result of that evaluation a good precision (CVs below 10 ) was
calculated for CT CFT MCF and α angle but in contrary to our results a low
repeatability was found for ML (CVs of 233 [ex-tem] and 285 [in-tem] own values
534 and 216 ) (Falco et al 2012)
In a previous feline study using ex-tem and in-tem as activating reagents for the ROTEM
delta repeatability was assessed similarly to the present study based on fourfold
measurements of blood samples of two healthy individuals (Doumlderlein and Mischke
2015) The results also revealed CVs below 10 for most of the parameters evaluated
The partially low repeatability of ML values may reflect the vulnerability of this variable
which is not only influenced by the complex clot formation process but also by its
subsequent lysis Reproducibility of ex-tem or in-tem activated measurements of the
ROTEM delta analyser which was not investigated in our study was studied in blood
from healthy and unhealthy horses by two different operators using three different
ROTEM devices Results indicated acceptable reproducibility (Junge et al 2016)
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
27
42 Reference intervals
The number of 125 healthy dogs used in the present study to create reference intervals is
regarded adequate according to the guidelines suggested by the American Society of
Veterinary Clinical Pathology (ASVCP) and the National Committee for Clinical
Laboratory Standards (NCCLS) who recommend to include at least 120 individuals
(Friedrichs et al 2012) To the best of our knowledge reference intervals for the most
relevant ROTEMreg delta parameters obtained in an adequate number of samples of whole
citrated canine blood have not been published so far A previous study was performed on
a smaller number of 45 dogs (Falco et al 2012) The median values for reference
intervals obtained in the present study are partially similar to those calculated in the
mentioned study based on 45 healthy dogs (eg ex- tem CT 610 s450 s [Falco et al
2012own results] CFT 150 s 110 s α angle 620deg680deg MCF 530 m 610 mm ML
180 220 in-tem CT 235 s190 s CFT 136 s116 s α angle 660deg680deg MCF
560 mm590 mm ML 30 180 ) However in comparison to our results the
reference intervals in the mentioned study are slightly wider (eg ex-tem CT 29ndash92
s34ndash83 s [Falco et al 2012own results] CFT 540ndash275 s650ndash220 s α angle 470ndash
790deg540ndash790deg MCF 360ndash730 mm460ndash730 mm ML 000ndash560 800ndash590
in-tem CT 126ndash363 s135ndash283 s CFT 470ndash224 s730ndash251 s aα angle 550ndash
810deg510ndash750deg MCF 500-ndash750 mm480ndash680 mm ML 000ndash400 500ndash270 )
although the previous study used the 5 and 95 quantiles to define limits of the
reference range whereas we calculated the commonly used 25 and 975 quantiles
to capture the central 95 of reference values (Friedrichs et al 2012)
Although it is desirable that each laboratory should specifically determine its own
reference intervals the results obtained in this study may provide an orientation for other
laboratories to interpret ROTEM delta parameters analysed in citrated canine blood In
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
28
order to test transference of the reference ranges to another laboratory a validation
procedure with measurement of 20 samples should be performed (Friedrichs et al 2012)
Although intra-individual control values (subject-based reference values) are regarded
superior to population based reference values (Wiinber et al 2007) the latter have to be
routinely used in clinical patients due to lacking individual controls especially for specific
tests such as ROTEM analyses In addition the calculated reference values cannot be
used for the fully automated device ROTEM sigma because - according to an actual
human study - values of part of the parameters do not correlate well between both
instruments (Gillissen et al 2019)
43 Influence of size age sex and neuter status
In previous studies in humans (Attard et al 2013) and animals (Mischke 1994 Borrelli
et al 2017) age has been described as an influencing factor for different haemostasis
parameters We also found an influence of age on the CT with shorter times in senior
dogs (gt 84 months) compared with young dogs (up to 24 months) which was limited to
measurements activated by the in-tem reagent
This age dependency confirms in principle a previous study on 20 healthy dogs
(Barthelemy et al 2015) which found shorter CTs in elderly dogs and results of a human
study where individuals with an age gt 45 years showed shorter CTs compared with
individuals lt 45 years old (Sucher et al 2011) However in the cited studies this effect
was only seen when ex-tem but not when the in-tem reagent was used
Based on the age distribution of our dogs which were primarily recruited to establish
reference intervals we cannot deliver information regarding very young dogs A human
study focusing on ROTEM reference ranges in children demonstrated significant
differences for nearly all ROTEM parameters with the most striking differences in
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
29
subjects aged 0-3 months (Oswald et al 2010) The limited number of dogs with an age
lt 1 year which did not allow to consider these separately in the present study may also
be one reason for the different results in a feline study (Doumlderlein and Mischke 2015) In
the cited study on healthy cats age showed an influence on CT but contrary to our study
the CT was shorter in young individuals (6-12 months) compared with older ones In
addition this was only the case in measurements using kaolin and in non-activated
measurements When compared to humans and cats uni- or multivariate analyses
considering factors age and breed or size based on do populations including different
breeds have the limitation that they can be biased by the variate life expectancy between
small medium and large dog breeds (Willems et al 2017) Apart from age we found a
significant influence only for the neuter status in bitches reflected by a significant
interaction effect between factors neuter status and sex in the ANOVA analysis
Especially for CFT values (eg activated by ex-tem median values of 121 s [spayed] vs
96 s [intact]) this difference seems to be also clinically relevant with respect to the
interpretation of ROTEM results In contrast although also significantly different
absolute and relative differences of CT values were small (eg activated by ex-tem 46 s
[spayed] vs 43 s [intact]) These differences may not only be considered for the result
interpretation of clinical patients but indicate also the necessity for sex-matched patient
groups in scientific studies
The obviously more active haemostasis system in intact females is probably related to an
influence of sex hormones (specifically estrogens) on the coagulation system
Accordingly ex vivo and in vivo studies demonstrated the activating properties of
endogenous estrogens on the haemostasis system In one ex vivo study addition of
endogenous estrogens (estrone [E1] 17szlig-estradiol [E2] and estriol [E3]) to blood samples
from men resulted in shorter mean values of the reaction time in TEG analyses
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
30
corresponding to CT in ROTEM measurements (452-537 s vs 698 s [control]) and of
the TEG variable kinetics (corresponding to the CFT in ROTEM analyses) (Swanepoel
et al 2017) In a further study subcutaneous injection of E2 into male rats induced a
significant increase in activities of coagulation factors X (by 8 ) and XI (by 10-24 )
however did not show any significant effect on PT aPTT and factor VII activity (Franco-
Murillo and Jaimez 2017)
In the cited studies also the effects of the synthetic estrogen ethinylestradiol [EE] were
studied which interestingly seems to have an inhibiting effect as indicated by prolonged
reaction time and kinetics (TEG) compared with the control in the human ex vivo study
and significantly longer PTs and aPTTs and lower factor VII and X activities in the study
on rats As expected we did not find a significant influence of size on ROTEM parameters
in dogs To the best of the authoracutes knowledge size has not been examined as an
influencing factor on viscoelastic measurements or any other haemostatic variable in
dogs Apart from Greyhounds there is limited literature on breed comparisons of
haemostatic variables (Clemons and Meyers 1984 Mischke 1994 Nielsen et al 2007
Nielsen et al 2010 Boyd et al 2018) indicating very limited breed differences for
example for factor VII (Mischke 1994) However these also do not indicate systematic
influence of size and in addition possible breed-specific effects have to be considered
As already stated one limitation of the study is the limited number of young animals
which does not allow to verify reference values for animals in this stage of life In
addition with respect to the main aim of the study possible influencing factors such as
platelet count and haematocrit were not considered in the statistical analysis Finally the
relatively small number of dogs used for the precision analysis which express the fact
that this was only a secondary aspect of the study may be regarded as a limitation At the
same time these considerations provide space for further investigations
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
31
5 Conclusions
Results of the precision analysis revealed acceptable results and thereby confirm that the
ROTEMreg delta analyser delivers repeatable results to measure overall haemostasis in
canine citrated blood samples The established reference values should offer a reliable
basis for the interpretation of thromboelastometric measurements in whole blood using
the device ROTEMreg delta in dogs with suspected haemostasis disorders Neuter status
in female dogs appeared to be a relevant influencing factor on most important ROTEMreg
parameters and should be considered for result interpretation and with respect to
experimental designs
6 Acknowledgments
Jose Mauricio Pereira received a scholarship from the Universidad Nacional de Costa
Rica (reference code JB-C-1107-2016)
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
32
7 References
1 Attard C van der Straaten T Karlafitis V Monagle P Ignjativic V 2013 Developmental
hemostasis age-specific differences in the levels of hemostatic proteins J Thromb and
Haemost 11 1850ndash1854
2 Bagge A Schoumltt U Kander T 2016 Effects of naturopathic medicines on Multiplate and
ROTEMreg A prospective experimental pilot study in healthy volunteers BMC Complemen
Altern Med 17 (16) 64
3 Bartheacutelemy A Rannou B Forterre M Verwaerde P Bonnet-Garin JM Pouzot-Nevoret
C Goy-Thollot I 2015 Differences between coagulation and cytokine profiles in dogs of
different ages Vet J 205 410ndash412
4 Bauer N Eralp O Moritz A 2009 Establishment of reference intervals for kaolin-activated
thromboelastography in dogs including an assessment of the effects of sex and anticoagulat use
J Vet Diagn Investi 21 641ndash648
5 Bollinger D Seeberger MD Tanaka KA 2012 Principles and practice of
thrromboelastography in clinical coagulation management and transfusio practice Transfus Med
Rev 26 1ndash13
6 Borrelli A Botto A Maurella C Falco S Pagani E Miniscaldo B Tarducci A Bruno
B 2017 Thromboelastometric assesment of hemostasis in newborn Piemontese calves J Vet
Diagn Invest 29 293ndash297
7 Boyd CJ Claus MA Raisis AL Hosqood G Sharp CR SmartL 2018
Hypocoagulability and platelet dysfunction are exacerbated by synthetic colloids in a canine
hemorrhagic shock model Front Vet Sci 5 279
8 Bruno B Maurella C Falco S Tarducci A Zanatta R DrsquoAngelo A Borrelli A 2015a
Thromboelastometric evaluation of hemostasis in dogs infected with Leishmania infantum J Vet
Emerg Crit Care (San Antonio) 25 502ndash511
9 Bruno B Maurella C Falco S Tarducci A Zanatta R Gianella P DrsquoAngelo A Piras L
Di Bella A Borrelli A 2015b Assessment of coagulation utilizing thromboelastometry in dogs
undergoing orthopedic surgery J Vet Emerg Crit Care (San Antonio) 25 358ndash363
10 Clemons RM Meyers KM 1984 Acquisition and aggregation of canine blood platelets Basic
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
33
mechanisms of function and differences because of breed origin Am J Vet Res 45 137ndash144
11 Conversy B Blais MC Dunn M Gara-Boivin C del Castillo JRE 2017 Anticoagulant
activity of oral rivaroxaban in healthy dogs Vet J 223 5ndash11
12 Crochemore T Piza FMT Rodriacuteguez RDR Guerra JCC Ferraz LJR Correcirca TD
2017 A new era of thromboelastometry Einstein (Sao Paulo) 15 380ndash385
13 Crochemore T Correcirca TD Lance MD Solomon C Neto AS Guerra JCC Lellis PS
Bernz LM Nunes N Mancio CM Yokoyama APH Silva E 2018 Thromboelastometry
profile in critically ill patients A single-center retrospective observational study PLoS One 13
e0192965
14 Doumlderlein E Mischke R 2015 Reference intervals for thromboelastometry with the ROTEMreg
delta in cats Res Vet Sci 100 271ndash276
15 Falco S Bruno B Maurella C Bellino C DacuteAngello A Gianella P Tarducci A Zannata
R Borrelli A 2012 In vitro evaluation of canine hemostasis following dilution with
hydroxyethyl starch (13004) via thromboelastometry J Vet Emerg Crit Care (San Antonio)
22 640ndash645
16 Franco-Murillo Y Jaimez R 2017 Inhibitory effect of ethinylestradiol on coagulation factors
in rats Exp Anim 66 107ndash113
17 Friedrichs KR Harr KE Freeman KP Szladovits B Walton RM Barnhart KF Blanco-
Chavez J 2012 ASVCP reference interval guidelines Determination of de novo reference
intervals in veterinary species and other related topics Vet Clin Pathol 41 441ndash453
18 Gillissen A va den Akker T Caram-Deelder C Henriquez DDCA Bloemenkamp
RWM Eikenboom J van der Bom JG de Maat MPM 2019 Comparison of
thromboelastometry by ROTEMreg Delta and ROTEMreg Sigma in women with postpartum
haemorrhage Scand J Clin Lab Invest 79 32ndash38
19 Harttert H 1948 Blutgerinnungstudien mit der Thrombelastographie einem neuen
Untersuchungsverfahren Klin Wochenschr 26 577ndash583
20 Horn P Pesce A 2003 Reference intervals and update Clin Chim Acta 334 5ndash23
21 Hua Yanhun Qiu W Xiao Q Wu Q 2018 Precision (repeatability and reproducibility) of
ocular parameters obtained by the Tomey OA-2000 biometer compared to the IOLMaster in
healthy eyes PLoS One 13 e0193023
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
34
22 Junge H Ringer S Mayer N Schwarzwald C 2016 Assesment of method reliability and
determination of reference intervals for rotational thromboelastometry in horses J Vet Emerg
Crit Care (San Antonio) 26 691ndash703
23 Kol A Borjesson DL 2010 Application of thrombelastographythromboelastometry to
veterinary medicine Vet Clin Pathol 39 405ndash416
24 Marschner CB Wiinberg B Tarnow I Markussen B Kuumlhnel L Bochsen L Kristensen
AT 2018 The influence of inflammation and hematocrit on clot strength in canine
thromboelastographic hypercoagulability J Vet Emerg Crit Care (San Antonio) 28 20ndash30
25 McMichael MA Smith SA 2011 Viscoelastic coagulation testing technology applications
and limitations Vet Clin Pathol 40 140ndash153
26 Mischke R 1994 Activity of coagulation factors II V VII and X in healthy dogs- dependence
on age sex and breed Berl Muumlnch Tieraumlrztl Wochenschr 107 289ndash294
27 Nielsen A Zois N Pedersen H Olse L Tarnow I 2007 Platelet function in dogs bread
differences and effect of acetylsalicylic acid administration Vet Clin Pathol 36 267ndash273
28 Nielsen L Kjelgaard‐Hansen M Jensen A Kristensen A 2010 Breed‐specific variation of
hematologic and biochemical analytes in healthy adult Bernese Mountain dogs Vet Clin Pathol
39 20ndash28
29 Nogami K 2016 The utility of thromboelastography in inherited and acquired bleeding
disorders Br J Haematol 174 503ndash514
30 Oswald E Stalzer B Heitz E Weiss M Schmugge M Strasak A Innerhofer P Haas T
2010 Thromboelastometry (ROTEMreg) in children Age-related reference ranges and correlations
with standard coagulation tests Br J Anaesth 105 827ndash835
31 Sankarankutty A Nascimento B Teodoro da Luz L Rizoli S 2012 TEGreg and ROTEMreg
in trauma similar test but different results World J Emerg Surg 7 (Suppl 1) S3
32 Sigrist NE Schefer RJJ Kutter APN 2018 Characteristics of hyperfibrinolysis in dogs and
cats demonstrated by rotational Thromboelastometry (ROTEM) Vet J 242 67-73
33 Steiner J Teague S Williams D 2003 Development and analytic validation of an enzime-
linked immunosorbent assay for the measurement of canine pancreatic lipase immunoreactivity in
serum Can J Vet Res 68 161ndash168
34 Sucker C Tharra K Lithmate J Scharf RE Zotz RB 2011 Rotation thromboelastography
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
35
(ROTEM) parameters are influenced by age gender and oral contraception Perfusion 26 334ndash
340
35 Swanepoel A Emmerson O Pretorius E 2017 The effect of endogenous and synthetic
estrogens on whole blood clot formation and erythrocyte structure Microsc Microanal 23 599ndash
606
36 Theusinger OM Nuumlrnberg J Asmis LM Seifert B Spahn DR 2010 Rotation
thromboelastometry (ROTEM) stability and reproducibility over time Eur J Cardiothorac Surg
37 677ndash683
37 Toulon P Metge S Hangard M Zwahlen S Piaulenne S Besson V 2017 Impact of
different storage times at room temperature of unspun citrated blood samples on routine
coagulation tests results Results of a bicenter study and review of the literature Int J Lab
Hematol 3 458ndash468
38 Turner JS Kutter APN Sigrist NE 2019 Correlation of rotational thrromboelastometry
(ROTEM) parameters with platelet count and their ability to predict thrombocytopenia in dogs
Res Vet Sci 126 45ndash50
39 Versteeg HH Heemskerk JWM Levi M Reitsma PH 2013 New Fundamentals in
Hemostasis Physiol Rev 93 327ndash358
40 Wiinberg B Jensen AL Kjelgaard-Hansen M Rojkjaer R Johansson PI Gade LP
Gram DX Kristensen AT 2007 Study on biological variation of haemostatic parameters in
clinically healthy dogs Vet J 174 62ndash68
41 Wiinberg B Jensen AL Johansson PL Rozanski E Tranholm M Kristensen AT 2008
Thromboelastographic evaluation of hemostatic function in dogs with disseminated intravascular
coagulation J Vet Intern Med 22 357ndash365
42 Wikkelsoslash A Wetterslev J Moslashller AM Afshari A 2016 Thromboelastography (TEG) or
thromboelastometry (ROTEMreg) to monitor haemostatic treatment versus usual care in adults or
children with bleeding Cochrane Database Syst Rev Art
httpsdoiorg10100214651858CD007871pub3 No CD007871
43 Willems A Paepe D Marynissen S Smets P Van de Maele I Picavet P Duchateau L
Daminet S 2017 Results of screening of apparently healthy senior and geriatric dogs J Vet
Intern Med 31 81ndash92
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
36
44 Woodman J Wagg CR Boysen SR Leguillete R Mizen K Roy MF 2015 Evaluation
of coagulation via thromboelastography in healthy horses administered dexamethasone Can Vet
J 56 1271ndash1274
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
37
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of
standard heparin prophylaxis and correlation to haemostatic changes
Pereira JM Hewicker-Trautwein M Rohn K von Depka Prondzinski M
Mischke R
Contributions of the authors
Pereira JM performed (1) the ultrasound evaluations and blood sampling of the
patients under study (2) haematology analysis (3) injection of the anticoagulation
prophylaxis (partially) to the patients under study (4) analysis of samples (collaboration)
at Werlhof Institute Medical Care Center (5) analysis of some haemostasis test
(collaboration) (6) statistical analysis (partially) (7) data documentation (8) and wrote
the manuscript
Hewicker-Trautwein M performed the electron microscopy analysis
Rohn K performed the statistical analysis
von Depka Prondzinski M performed some haemostasis test (thrombin generation
assay and endogenous thrombin potential)
Mischke R performed the coagulation and haemostasis test was involved in designing
and supervision of the study analysis and discussion of the data and its statistical analysis
and revision edition and correction of the manuscript
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
38
Central venous catheter induced thrombosis in dogs ndash efficacy of standard heparin
prophylaxis and correlation to haemostatic changes
Joseacute Mauricio Pereira1 Marion Hewicker-Trautwein2 Karl Rohn3 Mario von Depka
Prondzinski4 Reinhard Mischke1
1Small Animal Clinic University of Veterinary Medicine Hannover Foundation
Buumlnteweg 9 D-30559 Hannover Germany
2Institute for Pathology University of Veterinary Medicine Hannover Foundation
Buumlnteweg 17 D-30559 Hannover Germany
3Institute of Biometry Epidemiology and Information Processing University of
Veterinary Medicine Hannover Foundation Buumlnteweg 2 30559 Hannover Germany
4Werlhof Institute Schillerstrasse 23 30159 Hannover Germany
Abbreviations anti-Xa anti-factor Xa BW body weight CVC central venous catheter
EJV external jugular vein ELMI electron microscopy IV intravenous JV jugular vein
LMWH low molecular weight heparin ML maximum lysis (ROTEM) PIVC
peripheral intravenous catheter ROTEM rotational elastometry SC subcutaneous
TGA thrombin generation assay TID three times a day TT thrombin time UFH
unfractionated heparin
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
39
Corresponding author
E-mail addresses matiscrgmailcom (JM Pereira) MarionHewicker-
Trautweintiho-hannoverde (M Hewicker-Trautwein) KarlRohntiho-hannoverde
(K Rohn) mailwerlhof-institutde (M von Depka Prondzinski)
ReinhardMischketiho-hannoverde (R Mischke)
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
40
ABSTRACT
Central venous catheters (CVC) are of importance for intensive care management of
canine patients Thrombosis of the jugular vein is a severe complication related to the
CVC use However only little is known about the incidence of CVC-associated
thromboses in dogs and the necessity and efficacy of prophylactic antithrombotic
regimens The objectives of the present study were (1) to determine the incidence of
catheter-induced thrombosis in canine intensive care patients and thereby (2) to evaluate
the efficacy of a routine prophylactic heparin treatment and finally (3) to assess whether
initial changes of selected haemostasis parameters are predictive for an increased risk for
thrombosis formation 29 dogs hospitalised in the Small Animal Clinic University of
Veterinary Medicine Hannover Foundation and receiving a central venous catheter
(CVC) in the external jugular vein (EJV) for medical reasons were included in the final
assessment 17 dogs received the standard antithrombotic dosage regimen of
unfractionated heparin (UFH 150 IUKG BW TID sc) and 12 dogs (usually surgical
patients) the reduced dosage (75 IUKG BW TID sc) A colour Doppler ultrasound of
the respective EJV in order to detect thrombus formation and blood collection for
haemostasis tests were performed immediately before the installation procedure (day 0)
and on days 1 3 5 7 and so on after the CVC insertion Haemostasis tests included
prothrombin time activated partial thromboplastin time thrombin time antithrombin
activity D-dimer concentration thrombin generation and rotational elastometry as well
as the heparin activity using a chromogenic anti-factor Xa test Finally electron
microscopy (ELMI) of sections of the removed CVC was performed In 8 dogs
sonographic examination revealed thrombus formation in the lumen of EJV and in 1927
CVCs assessed using ELMI thrombi on the external CVC surface were detected without
significant correlation between both methods Plasmatic heparin activities showed great
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
41
variability but no significant differences were found between dogs with or without CVC-
associated thrombus formation From the initially performed haemostasis tests high
fibrinogen concentrations and low maximum lysis values as measured by the ROTEM
delta device (ex-tem reagent) were associated with sonographically detectable thrombi
The results of the present study indicate that CVC-associated thromboses occur (eg 28
[829] of the patients as detected by ultrasound) although severe thromboses (high
grades) are relatively rare in intensive care canine patients receiving a routine
anticoagulant treatment A high fibrinogen concentration and low maximum lysis
(ROTEM ex-tem) may be useful indicators for an increased thrombotic risk in individual
patients which may require special anticoagulatory treatment
Keywords CVC complication venal occlusion antithrombotic treatment ultrasound
electron microscopy rotational elastometry
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
42
1 Introduction
Central venous catheters (CVCs) are very important tools for care of patients in critical
care units allowing parenteral nutrition support therapy administration of high volumes
of intravenous fluids safe drugs administration that cannot be given through peripheral
veins plasmapheresis haemodialysis blood transfusions as well as periodical blood
sampling (Geerts 2014 Vose et al 2019) CVC related thrombosis is one of the most
severe complications (apart from infections and pneumothorax) of CVCs in humans
(Geerts 2014 Comerlato et al 2017 Evans and Ratchford 2018) leading to patientrsquos
distress CVC dysfunction venous stenosis and in some cases to the development of
thromboembolism (Geerts 2014)
In the human literature there are various studies dealing with epidemiology with reference
to different anticoagulatory regimens including placebo-controlled studies (Lee and
Kamphuisen 2012) In humans CVC related thromboses represent 70ndash80 of all upper
extremity thrombosis cases and approximately 10 of thromboembolism cases in
humans (Kucher 2011) The incidence of CVC related thrombosis reported in humans
based on 25 studies ranged widely from 2 to 67 (median = 30 ) (van Rooden et al
2005 review article) This wide range of calculated incidences of CVC related
thromboses in humans reflects the use of different CVC types different locations of entry
site of the CVC (jugular femoral subclavian axillary cephalic vein) different thrombus
detection technique (venography or ultrasonography) unequal durations of follow up
varying diseases of patients (eg haematological oncological) and presence of
predisposing factors (eg previous venous thrombosis history presence of cancer) (van
Rooden et al 2005 Geerts 2014 Liu et al 2015)
Part of the authors empirically observed CVC-associated thrombosis of the external
jugular vein (EJV) as a relevant complication in their canine intensive care patients which
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
43
awakened their interest in that topic To the best of the authoracutes knowledge studies about
extra luminal CVC related thromboses in dogs are rare in the available veterinary
literature One study evaluating the complications associated with 100 CVCs placed in
the jugular vein of dogs and cats reported only 2 cases of thrombosis (confirmed by
palpation or ultrasound) but obviously did not assess the appearance of subclinical CVC
related thromboses (Adamantos et al 2010) Thereby the incidence of CVC-induced
thromboses in dogs is unknown and systematic studies regarding an effective
anticoagulatory management are lacking
In this context it is of interest whether a possible development of thrombosis or an
increased risk (despite the prevention by a standard application of unfractionated heparin
[UFH]) can be predicted by means of changes of haemostasis related parameters during
the initial examination In one study on 168 human patients without indication of
anticoagulation prophylaxis for example elevated leukocyte counts high plasminogen
activator inhibitor and high coagulation factor VIII levels (all measured on blood samples
taken after CVC placement) were associated with an increased incidence of symptomatic
CVC-related thrombosis diagnosed by ultrasonography (Boersma et al 2016)
Therefore aims of the present study were (1) to determine the incidence of catheter-
induced thrombosis in canine intensive care patients (as detected by ultrasonography and
electron microscopy ELMI) and thereby (2) to evaluate the efficacy of a standard
antithrombotic regimen with unfractionated heparin (UFH) routinely used in the Small
Animal Clinic of the University of Veterinary Medicine Hannover and (3) to assess
whether selected haemostasis test results are predictive for an increased risk for
thrombosis formation
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
44
2 Material and methods
21 Study design
Canine in-patients of the Small Animal Clinic University of Veterinary Medicine
Hannover receiving a CVC in the EJV for medical reasons from March 2017 to
December 2019 were included Anticoagulatory treatment was performed according to
the standard protocol of the clinic Colour Doppler ultrasonographic controls of the EJV
and blood samplings (for heparin activity basic haematology and haemostasis tests) were
done immediately before the catheter was placed (day 0) and on days 1 3 5 etc (2 hours
after the first daily UFH injection) until the CVC was removed Only patients were
included which could be examined at least until day 3 Haemostasis tests included
prothrombin time activated partial thromboplastin time thrombin time fibrinogen
concentration antithrombin activity fibrin monomers D-dimers thrombin generation
assay (TGA) and rotational elastometry as well as the heparin activity using a
chromogenic anti-factor Xa test Finally ELMI of sections of the CVC after its removal
was performed
22 Animals
Originally 32 patients hospitalised in the Small Animal Clinic and receiving a CVC in the
EJV for medical reasons entered the study Three of them were subsequently excluded
because they did not reach the defined minimum study period of 3 days Two were
euthanised and one referred back to the referring vet before day 3 22 dogs weighing more
than 15 kg were included so that the withdrawal of about 10 ml of blood on several days
was not a significant burden for the patient (lt 1 of the total blood volume) Five dogs
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
45
with a body weight of less than 15 kg but at least 8 kg were included with special
consideration of the amount of blood taken (no more than 6 ml on individual days)
All animals received standard anticoagulatory treatment with UFH 17 dogs with internal
diseases received 150 IUkg BW TID subcutaneously and 12 dogs (post surgery) received
a reduced dosage of 75 IUkg BW TID subcutaneously
The 29 dogs enclosed in the study included 4 mixed breed dogs and the following
pedigree dogs 5 Labrador Retrievers 3 German Shepherds 2 Australian Shepherds and
one from each of the following breeds Akita Inu Asian Shepherd Bavarian Mountain
Hound Beagle Belgian Shepherd Briard Canaan dog Dalmatian Golden Retriever
Havanese dog Hovawart Rhodesian Ridgeback Schnauzer Shar Pei and Wolfspitz
These dogs were aged between 2 and 164 months (median 77 months) and had body
weights ranging from 81 and 63 kg (median 27 kg) 9 were castrated males 8 intact
males 7 spayed females and 5 intact females
The diseases of the 29 patients requiring a CVC included 3 from each of the following
diseases hepatopathy and esophagus dilatation 2 from each of the following diseases
bacterial cystitis diabetes mellitus and 1 from each of the following diseases abdominal
wall perforation aspiration pneumonia cystitis intestinal round cells sarcoma carcinoma
of the nasal cavity chronic renal insufficiency cystic duct rupture heartworm disease
ivermectine intoxication lymphoma multifocal alveolar histiocytosis pancreatitis post-
surgical bleeding stomach dilatation stomach foreign body tetanus thoracic
pyogranulomatous inflammation thrombocytopenia and trigeminal nerve paresis
Animals were treated in accordance with the German Animal Welfare Law The
experimental design was approved by the Animal Welfare officer of the University of
Veterinary Medicine and reported to the responsible authority (Lower Saxony State
Office for Consumer Protection and Food Safety reference number 17A101)
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
46
23 Positioning and handling of the central venous catheter
Two types of single lumen radiopaque polyurethane CVC (different on length and
diameter) were used during the study The CVC type I (n = 2529) was Cavafixreg Certoreg
Splittocanreg CVC 32 cm length 11 x 17 mm diameter16 G (BBraun Melsungen AG)
the CVC type II used (n = 429) was Certofixreg Mono S 215 15 cm length 08 x 14 mm
diameter18G (BBraun Melsungen AG) Each CVC was inserted into the EJV using a
modified Seldinger technique as follows the skin of the neck was shaved and disinfected
with antiseptic soap alcohol and iodine Local anesthesia (Lidocaine HCl 2) was
administrated subcutaneously on the catheter insertion site (a middle point in the lateral
neck over the jugular sulcus) A small skin incision was performed in the insertion site
and one peripheral plastic cannula with a metallic needle inside (14 G for CVC type I
and 20 G for CVC type II) was inserted in the EJV in cranio-caudal orientation (Hundley
et al 2018) Once the EJV was reached the needle was removed and a flexible wire
guide was inserted through the peripheral cannula following an external landmark
(4thintercostal space) (Reminga et al 2018) The peripheral cannula was removed and
the respective CVC was inserted slowly and with gentle pressure into the EJV guided
with the flexible wire and following the same external landmark to reach the right
position After the insertion of the CVC the flexible wire guide was removed The correct
positioning of the CVC was checked with a latero-lateral thorax x-ray ensuring that the
CVC tip was placed at the junction of the cranial cava vein with the right atrium Each
CVC was fixed to the neck skin (adjacent to the insertion point) with a monofilament
non-absorbable suture (gauge 3-0) Finally a bandage elaborated with Povidone-Iodine
gauze (Betaisodonareg) padded synthetic band (Artiflexreg Natur) and elastic band
(Elastomullreg) was used to protect the CVC output (Claude et al 2010) and changed
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
47
daily Several times day the catheter was flushed with isotonic sodium chloride
containing 1 IUml UFH
24 Heparin preparation and administration
Heparin-sodium-25000-ratiopharm (injection solution containing 5000 IUml sodium
heparin [mucosa]) was used SC injection was performed over the lateral thorax wall
using 1 mL syringes with fine graduation marks
25 Ultrasonography
Colour Doppler ultrasonographic examinations were performed in lateral position using
a LO-GIQ E9 ultrasound device (GE Healthcare Chicago Illinois USA) with a 15
Megahertz linear ultrasound probe The development of a thrombus or fibrin debris in the
EJV lumen around the CVC or inside it was assessed at each control Once the
ultrasound was finished a new protective bandage was placed around the neck of the dog
According to the percentage of lumen occlusion of the EJV (evaluated in cross sectional
views during the ultrasound scanning) the thrombi were classified in four different grades
(Fig 1)
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
48
of the residual vein lumen C ndash catheter T ndash thrombus
Fig 1 Definition of different grades of central venous catheter induced thrombosis of the
jugular vein as detected using colour Doppler ultrasound
26 Blood collection
At each time point approx 6ndash10 ml of blood (according to the body weight) was collected
This included approx 1 ml of EDTA blood for hematological analysis and approx 5ndash9
ml of citrated blood (tubes containing one part of 011 molL [38 ] sodium citrate to
nine parts of blood) for the measurement of hemostasis tests in citrated blood (ROTEM)
or citrated plasma Blood samples were collected by puncture of peripheral veins with
disposable cannulas (09 x 40 mm [20G] or 10 x 40mm [21G]) directly into sample tubes
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
49
or from the CVC using the three-syringe-technique (Villalta-Garciacutea et al 2015) in
situations when the peripheral venipuncture was not indicated due to phlebitis or irritation
andor inflammation of the surrounding skin Briefly 2ndash3 ml of blood were taken in a
sterile disposable syringe and immediately afterwards the blood sample was taken in a
new disposable syringe and transferred into the test tubes containing the respective
anticoagulants finally the first 2-3 ml taken were re-infused to the dog through the CVC
followed by flushing with a solution of sodium chlorideUFH (02) Immediately after
blood collection each sample tube was swayed gently until blood and anticoagulant had
been mixed thoroughly To prepare citrated platelet poor plasma citrated blood was
centrifuged at 16000 g for 10 min at room temperature using a microcentrifuge The
plasma was transferred into plastic tubes and the centrifugation procedure was repeated
This platelet poor plasma was then frozen in aliquots at -70 degC until used for analysis
Directly before analysis the plastic tubes were thawed at 37 degC in a water bath
27 Laboratory tests
Haematological analyses were made automatically with Adviareg 2120i (Siemens
Healthcare GmbH Germany)
Prothrombin time activated partial thromboplastin time and thrombin time were
measured with the autoanalyser Amax Destiny Plus (Tcoag Deutschland GmbH
Germany) and commercial reagents (Thromborel S Siemens Healthcare Diagnostics
Products GmbH Marburg Germany CK Prestreg and Test Thrombin Reagent
Diagnostica Stago SAS Asniegraveres sur Seine Cedex France) Whereas the last two tests
were measured according to the manufacturer`s instructions a modified test optimised
for canine plasma was used to measure prothrombin time (25 microl citrated plasma 120 with
imidazole buffer + 25 microl of a human fibrinogen solution 2 gl prewarmed for 120 s at 37
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
50
oC addition of 25 microl of activating reagent Thromborel S) and the results converted into a
percentage activity based on a calibration curve prepared with canine pooled plasma The
fibrinogen concentration was measured with the method according to Clauss using a test
kit from Stago Diagnostica SAS D-dimer concentration was measured with a
quantitative commercial human latex turbidimetric method in the autoanalyser Cobas
c311 using a completely assembled kit and human standards (Roche Diagnostics
Mannheim Germany) Antithrombin activity was also measured with a completely
assembled chromogenic test kit in the autoanalyser Cobas c311 and calibrated with a
canine pool plasma (n = 100)
The thrombin generation assay (Technothrombinreg TGA (Technoclone GbmH Vienna
Austria) determines thrombin generation based on monitoring of the fluorescence
generated by cleavage of a fluorogenic substrate by thrombin over time after activation
of the coagulation cascade by different concentrations of tissue factor and negatively
charged phospholipid in platelet poor plasma From the fluorescence development over
time the concentration of thrombin in the sample can be calculated using a thrombin
calibration curve The increase in thrombin concentration with time then allows to
calculate generation of thrombin in the sample and to plot thrombin values over time for
the whole coagulation process
The rotational thromboelastometry analysis with ROTEM delta device (Werfen Munich
Germany) was started approx 15 minutes after the blood collection To perform the
analysis and according to the automated ROTEM software 20 microl of recalcification reagent
(200 mmoll calcium chloride solution star-tem) and 20 microl of the respective activating
reagent (ex-tem in-tem) were pipetted into and carefully mixed in a pre-warmed cuvette
using the automated pipette provided in the device immediately afterwards 300 microl of
citrated whole blood (previously gently revolved) were added and mixed with the
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
51
reagents previously deposited Finally the cuvette was carefully placed and fixed to an
oscillating pin (oscillation movements on left and right through an angle of 475deg) in the
ROTEM delta device The interaction between blood cells and subsequently the bond
between fibrin and platelets promoted by the biomechanical changes in pH electrolytes
and temperature start the clot formation with its subsequent retraction and lysis The
magnitude of the resistance produced by the clot over the oscillating pin is quantitatively
represented by different parameters measured during the analysis and also qualitatively
in a graphic illustration (Bagge et al 2016 Crochemore et al 2017)
A chromogenic substrate test (Coatest Heparin Chromogenix-Instrumentation
Laboratory Haemochrom Diagnostica Essen Germany) was used for measurements of
UFH in the auto-analyser Cobas c311 The test application was created based on
manufacturers` instructions Different dilutions of the same batch of the commercial
sodium UFH preparation used in the experiment with normal canine pool plasma
(prepared from identical aliquots of 10 healthy adult dogs) served as standards for the
calibration of the measurement
28 Electron microscopy
Selected areas on the last third of the CVC (5ndash10 cm according with the CVC) with
macroscopically detectable thrombi were analysed and some of the CVCs were cut in
two different places The cut samples were preserved in a fixative medium composed of
1 glutaraldehyde and 4 formalin Once the sample was fixed it was prepared
according to the osmium thiocarbohydrazide procedure (OTOTO-procedure) (Malick et
al 1976) The fixed CVCs were dehydrated in seven series as follow first three series of
120 minutes each one on 30 50 and 70 ethanol respectively followed by two series
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
52
of 120 min each one on 90 ethanol and then two series of 24 hours each one on 100
ethanol After the dehydration process the samples were placed two times during 24
hours each one on isoamyl acetate and then placed on in a critical point drier Finally the
samples were coated with gold in a splutter-coater with 25 kV acceleration voltage in an
argon atmosphere with a current of 20 mA for one minute The samples were examined
in an EVO 15 scanning electron microscope (ZEISS International Carl Zeiss Jena
Germany) and photographed with magnifications of 600x and 6000x avoiding mechanical
artifacts (Toskala et al 1995) Thrombi on the external wall of the CVC were analysed
and measured using the software ImageJ (National Institutes of Health and the Laboratory
for Optical and Computational Instrumentation University of Wisconsin USA)
According to their extension (surface size) and thickness (related to the CVC wall
thickness in a cross sectional view) thrombi on the external wall and additionally within
the lumen of the CVC were each classified in four grades (Fig 2 Fig 1S [suppl
material]) In case of two cuts the highest degree of thrombosis detected at one of the two
sites was used for the final assessment
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
53
surface sizepercent of CVC wall thickness
Fig 2 Definition and illustration of different grades of central venous catheter induced
(extraluminal) thrombosis as detected using electron microscopy
Fig 1S (suppl material) Definition and illustration of different grades of intraluminal
thrombosis in central venous catheters as detected using electron microscopy
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
54
29 Statistical methods
Comparison between thrombus graduation either based on sonographic and ELMI
findings respectively was performed using the McNemar test with calculation of the
concordance index (κ) Standard normal distribution was assessed using the
Kolomogorov-Smirnov test
Due to non-normal distribution of part of data sets heparin activities and results of
haemostasis assays are reported using non-parametric variables (median minimumndash
maximum box and whisker plots) Heparin activities and results of haemostasis tests
between dogs with or without thrombosis were compared using Mann-Whitney-U test
3 Results
31 Sonographic detection of thrombus formation
Thrombus formation was sonographically detected in 829 patients during the study
period Based on examination on the final day in half of these eight patients (n = 4) the
thrombus formation was scored grade 1 in 3 dogs grade 2 and only in one grade 3 In all
of these 8 dogs this was the maximum grade during the study period One dog with fibrin
precipitates was excluded from further analysis because it could not be clearly assigned
to one of the groups In 78 cases thrombi were attached mainly to the venous wall
whereas only in one case was attached to the CVC external wall Seven of these 8 dogs
which developed thrombi received the standard heparin dosage (150 IUkg TID 41
[717] of dogs receiving this dosage) and only one the reduced heparin dosage (75 IUkg
TID 8 [112] of dogs receiving this dosage)
The time point of first detection of thrombus formation varied between days 3 and 11
(median day 8) In addition detritus (presumably composed mainly of fibrin) was
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
55
detectable in the lumen of the EJV in one further dog and within the lumen of the CVC
in further 5 dogs
32 Thrombus formation by electron microscopy
27 from 29 CVC catheter tips were analysed by electron microscopy 827 samples were
without detectable thrombus (grade 0) and 1927 with detectable thrombi 72 showed
thrombus formation of grade 1 427 of grade 2 and 827 of grade 3 There was no
concordance between thrombus graduation based on sonographic and the ELMI
graduation respectively The concordance index (McNemar test) was negative (κ = -
00718) indicating that the degree of agreement is less than by chance
Of the in total 19 dogs with detectable thrombus formation by electron microscopy 11
dogs received 150 IUkg TID (in total 16 dogs of these 27 dogs received that heparin
dosage) and 8 received 75 IUkg TID (in total 11 of these 27 dogs received that heparin
dosage 73 811) and Of the 12 patients with grade 2 and 3 thrombus formation 5 dogs
received 75 IUkg TID and 7 dogs 150 IUkg
The supplementary analysis of intraluminal thrombus formation revealed no detectable
thrombi in 7 cases and thrombus formation of grade 1 (lt 25 occlusion) in 11 cases of
grade 2 (25ndash50 ) in 3 cases and of grade 3 (gt 50 ) in 6 cases
33 Heparin activities and relationship to thrombus formation
Heparin activities varied remarkably in both dosage groups (Fig 3 ab) There was no
significant difference of heparin activities between blood samples collected via
venipuncture or from the CVC (Table 1S supplementary material) In addition there was
no significant difference between heparin activities in dogs with or without
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
56
sonographically or electron microscopically detectable thrombosis (Tables 1 and 2)
Table 1 refers to sonographically detectable thromboses and is based on median heparin
activities calculated from the measurements on day 1 and following days per patient
firstly for the total number of patients and secondly for the patients receiving 150 IUkg
TID In addition in the eight dogs heparin activities on the day of first thrombus detection
(023 IUml 019ndash036 IUml) were not significantly different from the median values
(Table 1 P = 05214 Wilcoxon test) Table 2 compares heparin activities (median values
of all days and results on the final day) between dogs with and without electron
microscopically detectable CVC-associated thrombi
Table 1S (Supplementary material) Heparin activities (IUml median values
minimumndashmaximum) measured in 27 dogs receiving 75 or 150 IUkg unfractionated
(standard) heparin TID subcutaneously 2 hours after the first injection on the day
Comparison of samples collected via puncture of the peripheral veins and those collected
from the central venous catheter (CVC) using the three-syringe-technique
Peripheral vein
puncture CVC
Mann-Whitney-U
test
Total number
(75 and 150
IUkg TID)
019
(00ndash108)
(n = 65)
023
(000ndash105)
(n = 65)
P = 03033
75 IUkg TID
010
(000ndash108)
(n = 25)
012
(000ndash023)
(n = 21)
P = 06907
150 IUkg
TID
030
(000ndash096)
(n = 40)
036
(000ndash105)
(n = 44)
P = 01223
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
57
Fig 3ab Heparin activities (box- and whisker plot arithmetic means) in dogs receiving
unfractionated heparin at a dosage of 75 IUkg BW tid (a) or 150 IUkg BW tid (b) two
hours after the first daily heparin administration
day 1 represents the first day after initiation of heparin treatment
only days were considered with at least results from 5 patients
Table 1 Heparin activities (IUml median values minimumndashmaximum) in 28 dogs
receiving 75 or 150 IUkg unfractionated (standard) heparin TID subcutaneously with
(grades 1ndash3) or without (grade 0) sonographically detectable thrombus Blood collections
were performed 2 hours after the first daily injection
Grade 0
(sonography)
Grades 1ndash3
(sonography)
Mann-Whitney U
test
Total number
(75 and 150 IUkg
TID)
020 (000ndash072)
(n = 20)
027 (018ndash059)
(n = 8)
P = 03212
150 IUkg TID 042 (000ndash072)
(n = 10)
031 (018ndash059)
(n = 7)
P = 04593
one dog with fibrin precipitates in the lumen of the jugular vein but without defined thrombus formation
was not considered because it could not be clearly assigned to one of the two groups
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
58
Table 2 Heparin activities (IUml median values minimumndashmaximum) in 27 dogs
receiving 75 or 150 IUkg unfractionated (standard) heparin TID subcutaneously with
(score 1ndash3) or without (score 0) detectable thrombus via electron microscopy
Grade 0
(electron
microscop)
(n = 8)
Grades 1ndash3
(electron
microscop)
(n = 19)
Mann-Whitney U
test
Median value of
different days 028 (005ndash060)
022 (000ndash072)
P = 05770
Final day 020 (001ndash045)
015 (000ndash105)
P = 06706
34 Relationship between haemostasis tests and thrombus formation
From the initially performed haemostasis tests high fibrinogen concentrations and low
maximum lysis values as measured by the ROTEM delta device (using the ex-tem
reagent) were associated with sonographically detectable thrombi (Tab 3) Additional
analyses based on the median values of different days for individual patients revealed that
low maximum lysis values as measured by the ROTEM delta device (using the in-tem
reagent) were associated with sonographically detectable thrombi (maximum lysis score
0 850 [100ndash175 ] score 1ndash3 375 [100ndash900 ] median [minimumndash
maximum] P = 00166) but no other test Comparison of the results of haemostasis tests
on the final day between dogs with and without sonographically detectable thromboses
did not show any significant difference (P gt 005)
Comparison of results of haemostasis tests on the final day in dogs with detectable
thrombosis via ELMI and dogs without significant thrombus formation showed several
significant differences including lower fibrinogen concentrations and longer clotting
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
59
times (ex-tem reagent) and clot formation times (ex-tem and in-tem reagents) using the
ROTEM delta analyser in general expressing a less coagulable status (Tab 4) If values
of dogs graded 0 and 1 (n =15) are compared to those of grades 2 and 3 (n = 12) the last-
mentioned had longer clotting times (ROTEM delta in-tem reagent) as well as longer clot
formation times and lower alpha angles (ROTEM delta in-tem reagent) (P lt 005 results
not shown)
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
60
Table 3 Comparison of initial results of haemostasis parameters (median values
minimumndashmaximum in dogs with (grades 1ndash3) and without (grade 0) sonographically
detectable central venous catheter-induced thromboses
Parameter Unit
Sonographic
thrombus grade
P-value
(Mann-Whitney
U test)
0
(n = 20)
123
(n = 8)
Haematocrit 355 (228ndash641)
382 (245ndash499)
06111
Platelet count x103microl 292 (420ndash483)
218 (860ndash458)
02320
Prothrombin time 944 (359ndash169)
896 (471ndash136)
05758
Activated partial thromboplastin
time s 128
(955ndash249) 140
(117ndash164) 02322
Thrombin time s 129 (121ndash178)
141 (650ndash160)
02028
Fibrinogen gl 448 (153ndash770)
747 (153ndash116)
00235
Antithrombin activity 639 (289ndash107)
670 (464ndash746)
05249
D-dimers microgml 006 (000ndash148)
015 (003ndash372)
03997
Thrombin generation assay nmol 130 (000ndash365)
912 (190ndash277)
08787
Clotting time (ROTEM ex-tem) s 420 (290ndash930)
430 (170ndash159)
07989
Clot formation time (ROTEM ex-
tem) s 665
(310ndash143) 470
(240ndash800) 00633
Alpha angle (ROTEM ex-tem) degrees 795 (620ndash840)
815 (750ndash860)
01667
Max clot firmness (ROTEM ex-
tem) mm 750
(570ndash860) 775
(700ndash830) 01753
Max clot elasticity (ROTEM ex-
tem) - 299
(131ndash619) 347
(235ndash484) 01544
Maximum lysis (ROTEM ex-tem) 135 (300ndash290)
400 (000ndash800)
00037
Clotting time (ROTEM in-tem) s 208 (840ndash810)
249 (720ndash371)
05085
Clot formation time (ROTEM in-
tem) s 835
(370ndash634) 745
(390ndash235) 08988
Alpha angle (ROTEM in-tem) degrees 745 (250ndash820)
750 (500ndash820)
09797
Max clot firmness (ROTEM in-
tem) mm 700
(540ndash810) 740
(610ndash770) 03587
Max clot elasticity (ROTEM in-
tem) - 235
(118ndash424) 287
(156ndash332) 04764
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
61
Maximum lysis (ROTEM in-tem) 850 (000ndash210)
350 (000ndash220)
01327
Table 4 Comparison of results of haemostasis parameters (median values minimumndash
maximum) on the last day in dogs with (grades 1ndash3) and without (grade 0) central venous
catheter-associated thrombus formation as detected by electron microscopy
Parameter Unit
Thrombus grade
(ELMI)
P-value
(Mann Whitney
U test)
0
(n = 8)
123
(n = 19)
Haematocrit 322 (241ndash454)
330 (216ndash510)
05953
Platelet count 103microl 446 (321ndash592)
268 (101ndash675)
00558
Prothrombin time 145 (104ndash264)
120 (561ndash148)
00462
Activated partial thromboplastin
time s 123
(109ndash140) 136
(109ndash188) 00665
Thrombin time s 129 (121ndash178)
141 (650ndash160)
02028
Fibrinogen gl 465 (275ndash866)
347 (189ndash992)
00491
Antithrombin activity 793 (448ndash981)
692 (398ndash889)
04572
D-dimers microgml 018 (000ndash173)
007 (000ndash118)
01920
Thrombin generation assay nmol 501 (000ndash160)
93 (000ndash287)
09562
Clotting time (ROTEM ex-tem) s 360
(270ndash410) 400
(310ndash620) 00238
Clot formation time (ROTEM ex-
tem) s 395
(120ndash740) 650
(240ndash174) 00275
Alpha angle (ROTEM ex-tem) degrees 835 (760ndash880)
790 (580ndash850)
00484
Max clot firmness (ROTEM ex-
tem) mm 780
(700ndash850) 720
(550ndash860) 00586
Max clot elasticity (ROTEM ex-
tem) - 346
(239ndash555) 254
(121ndash637) 00495
Maximum lysis (ROTEM ex-tem) 105 (200ndash190)
110 (000ndash380)
03796
Clotting time (ROTEM in-tem) s 178 (100ndash222)
201 (139ndash554)
00525
Clot formation time (ROTEM in-
tem) s 435
(330ndash111) 790
(350ndash882) 00209
Alpha angle (ROTEM in-tem) degrees 815 (710ndash830)
740 (240ndash830)
00191
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
62
Max clot firmness (ROTEM in-
tem) mm 785
(660ndash810) 690
(270ndash840) 00586
Max clot elasticity (ROTEM in-
tem) - 362
(192ndash434) 218
(370ndash507) 00559
Maximum lysis (ROTEM in-tem) 950 (200ndash190)
800 (100ndash180)
06890
4 Discussion
The main result of our study is that under the study conditions part of the dogs developed
CVC-associated thromboses However only a very low part of these thromboses were
obviously of a clinically significant degree regarding the amount of occlusion of the EJV
and none of the dogs had a symptomatic thrombosis whose definition includes presence
of clinical signs such as localised swelling pain and redness (Boersma et al 2008 Lee
and Kamphuisen 2012) In the present study we particularly refer to the extraluminal
thromboses with regard to the assessment of the antithrombotic management
One major problem of the statistical analysis and result presentation was that the two
methods used to define the occurrence and degree of thrombus formation (ultrasound and
ELMI) revealed discrepant results Therefore we performed a statistical analysis
independently regarding thrombus formation with either of the two methods It is well
known that ELMI which has already been used to detect CVC induced thromboses in
humans (Lucas et al 2017) and experimental animals (Xiang et al 1998 Xiang et al
2001) is a very sensitive diagnostic technique which allows the ex vivo diagnosis of very
small or fibrin rich thrombi that perhaps cannot be detected by ultrasonography or
venography during the in vivo evaluation of patients (Xiang et al 2001) A major reason
for the discrepant results between the two methods used to detect CVC associated
thromboses is that different CVC segments are evaluated Using ultrasonography it was
only possible to evaluate the EJV in the neck area of the patient and thereby only its
proximal third In contrast ELMI assessed mainly the CVC tip and a human study
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
63
revealed that even different positions of the catheter tip are associated with various risks
for development of CVC associated thrombosis (Ballard et al 2016) One additional point
to consider which also may contribute to the discrepancy is that it is possible that thrombi
developed on the CVC surface can be detached during the CVC removal from the vein
and thereby before its analysis by ELMI (Xiang et al 2001)
The contrast venography technique is used as gold standard to confirm thrombosis in
human medicine However this diagnostic technique is normally used only in the case of
a strong clinical suspicion of venous thrombosis that cannot be detected by
ultrasonography (Laurenson et al 2010 Langston and Eatroff 2018) Our study protocol
with privately owned clinic patients did not allow to use this method In a study performed
in 44 human patients requiring a CVC (Koumlksoy et al 1995) a sensitivity of 94 and a
specificity of 96 were determined for the colour Doppler ultrasound diagnosis when it
was compared with the diagnosis performed with the contrast venography technique and
that makes the colour Doppler ultrasound an accurate diagnosis method to detect CVC
related thrombosis even in humans (Koumlksoy et al 1995 van Rooden et al 2003)
The incidences of CVC related thrombosis detected by colour Doppler ultrasound of the
internal jugular vein reported in 3 human studies were 2 30 and 56 (Harter et al
2002 Lordick et al 2003 Wu et al 1999) and although with a wide range the median
value of these rates (30 ) is very well suited to the incidence of 28 (829) which we
calculated for our canine study based on sonographic findings The wide variation of
incidences of CVC related thrombosis in different human studies can in general be
explained by differences regarding the type and entry site of the CVC (jugular femoral
subclavian axillary cephalic vein) the time period with the CVC in place the thrombus
detection technique (venography or ultrasonography comment in the cited 3 studies
consistently the last-mentioned method was used) and criteria for diagnosis of
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
64
thrombosis the duration of follow up diseases and presence of predisposing factors in
these patients (eg previous venous thrombosis history presence of cancer) and finally
prophylactic treatment with anticoagulants (van Rooden et al 2005 Geerts 2014 Liu et
al 2015)
Different pathomechanisms contribute to the development of CVC induced thromboses
During the CVC insertion the puncture of the skin on the CVC insertion site and the
rupture of the blood vessels integrity activate the coagulation cascade and the CVC acts
as a matrix for adherence and accumulation of fibrin and cellular components (Smith et
al 2012) The attachment of proteins and blood cells to the internal andor external wall
of the CVC can lead to a partial or complete occlusion of the catheter lumen and
sometimes to a partial or complete obstruction of the blood flow andor
thromboembolism (Langston and Eatroff 2018) There is a strong interrelationship
between thrombus and biofilm formation (Smith et al 2012) Prevention of thrombus
formation has thereby also relevance regarding the increased risk for microbial
colonisation of the catheter (Boersma et al 2008) leading at least in humans to an
increased number of CVC related systemic infections (Lordick et al 2003)
In this context it has to be considered that CVC produced from polyurethane which are
routinely used in our clinic and thereby in the study are usually associated with a
relatively high thrombosis potential and thereby definitely provide an effective test for
an antithrombotic regimen Silicone catheters have a significantly reduced risk for
thrombotic complications but this advantage has to be purchased by a higher rate of
mechanical failure (eg rupture of the catheter) (Wildgruber et al 2016) Promising
experimental approaches work on surface modifications of the standard catheters For
example one group was able to demonstrate that a coating of the surface of the catheter
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
65
with a zwitterionic polymeric sulfobetaine showed a significant reduction of thrombus
formation in vitro with human blood and in an in vivo canine model (Smith et al 2012)
Due to ethical reasons we did not performed a placebo-controlled study However
interestingly not all human studies report systemic anticoagulation and part of the human
studies was even placebo-controlled (review Lee and Kamphuisen 2012) Obviously
there is no consensus in the available human literature regarding the beneficial use of
antithrombotic prophylaxis to prevent CVC related thrombosis Several studies did not
find a statistically significant difference in the incidence of CVC related thrombosis
between patients receiving the antithrombotic prophylaxis compared with the control
groups without systemic anticoagulation (Verso et al 2005 P = 035 Karthaus et al
2006 P gt 005 Niers et al 2007 P = 049) whereas in other studies a lower incidence
of CVC related thrombosis was found in the group receiving anticoagulants as
prophylaxis compared with the control groups (Abdelfeki et al 2004 150 vs 126
P = 003 De Cicco et al 2009 400 vs 526 P = 005)
Although it is well known that subcutaneously administered UFH has a limited and
variable bioavailability and thereby leads to unpredictable blood activities (Hirsh and
Raschke 2004) the extremely wide range of blood activities after a defined dosage in our
study was relatively unexpected Blood was collected 2 hours after the first UFH
injection and thereby at the time of suspected maximum heparin activity considering the
relatively low UGH dosage (Mischke and Jacobs 2001) The cited study was performed
on healthy experimental dogs Possible additional influencing factors in critical ill
patients include circulatory insufficiency which may reduce and delay absorption from
subcutanenous tissue decreased clearance function of the liver and kidneys as well as
altered blood plasma concentrations of heparin binding plasma proteins such as
fibrinogen and platelet factor 4 and alterations of the heparin binding capacity of
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
66
endothelial cells which altogether are reasons for a great inter- but also intrapatient
variability (Hirsh and Raschke 2004) In individual patients this can lead to heparin
resistance It has also to be considered that antithrombin activities which are necessary
for an adequate heparin effect (Bjoumlrk and Lindahl 1982) were reduced in our patients
Availability of approved blood tests which can predict the risk of development of CVC
associated thrombosis already at the moment of the CVC placement are important
(Boersma et al 2016) One of the most interesting results was that dogs which developed
sonographically detectable thrombi had nearly twofold higher fibrinogen levels Well in
agreement with our results one study performed in anticoagulated human patients
demonstrated that high fibrinogen levels were associated with an increased risk of
perioperative venous thromboembolism (P = 0004) (Kato et al 2016) The cited study
does not exclusively refer to CVC associated thrombosis but 76 (1621) of the venous
thromboses were suggested to be associated with CVCs Fibrinogen measurement a test
which is widely available is therefore a useful and practicable indicator of an increased
risk for CVC induced thromboses and may advice the clinician to treat these patients
especially carefully (eg with low molecular weight heparin) In the study performed by
Boersma et al (2016) in 168 human patients without indication of anticoagulation
prophylaxis elevated leukocyte counts high levels of plasminogen activator inhibitor
and of coagulation factor VIII were associated with an increased incidence of
symptomatic CVC related thrombosis diagnosed by ultrasonography A limitation of that
study is that all measurements were performed on blood samples taken after CVC
placement Interestingly in our study ROTEM results were partly associated with
thrombus formation which reflects the well-known fact that results of viscoelastic
measurements are a good indicator of hypercoagulability and thromboembolic events
(Harahsheh and Ho 2018) It is of further interest and possibly unexpeted that on the final
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
67
day of examination dogs with ELMI signs of thromboses had partly lower coagulation
activities than dogs without thromboses Due to the low number of dogs without
detectable thrombi using ELMI this implausible result has to be interpreted with caution
One possible explanation could be that temporary hypercoagulability in these dogs may
have finally resulted in consumption of haemostasis substrates
Limitations of the present study were as a consequence of the study design based on
clinical patients the relatively low total number of patients inclusion of different UFH
treatment regimens and the lack of a control group
5 Conclusions
The results of the present study indicate that the used UFH regimen was not completely
effective to prevent CVC-associated thromboses which were detectable in 28 [829]
of the patients by ultrasound However severe thromboses (high grades) are relatively
rare in intensive care canine patients receiving a routine anticoagulant treatment Because
heparin plasma activities were not different in dogs with or without CVC-associated
thrombus formation other local and systemic factors must play a significant role as well
A high fibrinogen concentration and low maximum lysis (ROTEM ex-tem) may be
useful indicators for an increased thrombotic risk in individual patients which may
require special anticoagulatory treatment
6 Acknowledgments
Jose Mauricio Pereira received a scholarship from the Universidad Nacional de Costa
Rica (reference code JB-C-1107-2016)
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
68
7 References
1 Abdelkefi A Ben Othman T Kammoun L Chelli M Ben Romdhane N Kriaa A Ladeb S
Torjman L Lakhal A Achour W Ben Hassen A Hsaiumlri M Ladeb F Ben Abdeladhim A
2004 Prevention of central venous line-related thrombosis by continuous infusion of low-dose
unfractionated heparin in patients with haemato-oncological disease A randomized controlled trial
Thromb Haemost 92 654ndash661
2 Adamantos S Brodbelt D Moores A L 2010 Prospective evaluation of complications associated
with jugular venous catheter use in a veterinary hospital J Small Anim Pract 51 254ndash257
3 Bagge A Schoumltt U Kander T 2016 Effects of naturopathic medicines on Multiplate and ROTEM
a prospective experimental pilot study in healthy volunteers BMC Complement Altern Med 16 64
4 Ballard DH Samra NS Gifford KM Roller R Wolfe BM Owings JT 2016 Distance of the
internal central venous catheter tip from the right atrium is positively correlated with central venous
thrombosis Emerg Radiol 23 269ndash273
5 Boersma RS Jie KS Verbon A van Pampus EC Schouten HC 2008 Thrombotic and
infectious complications of central venous catheters in patients with hematological malignancies Ann
Oncol 19 433ndash442
6 Boersma R S Hamulyak K van Oerle R Tuinenburg A Ten Cate-Hoek AJ Schouten HC
2016 Biomarkers for prediction of central venous catheter related-thrombosis in patients with
hematological malignancies Clin Appl Thromb Hemost 22 779ndash784
7 Bjoumlrk I Lindahl U 1982 Mechanism of the anticoagulant action of heparin Mol Cell Biochem
48 161ndash182
8 Claude A Riedesel D Riedesel E 2010 Electrocardiography-guided and retrospective analysis of
central venous catheter placement in the dog Vet Anaesth Analg 37 97ndash105
9 Comerlato PH Rebelatto TF Santiago de Almeida FA Klein LB Boniatti M M Schaan
DB Rados VD 2017 Complications of central venous catheter insertion in a teaching hospital
Rev Assoc Med Bras 63 613ndash620
10 Crochemore T Piza FMT Rodriacutegues RDR Guerra JCC Ferraz LJR Correcirca TD 2017
A new era of thromboelastometry Einstein (Sao Paulo) 15 380ndash385
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
69
11 De Cicco M Matovic M Balestreri L Steffan A Pacenzia R Malafronte M Fantin D
Bertuzzi CA Fabiani F Morassut S Bidoli E Veronesi A 2009 Early and short‐term
acenocumarine or dalteparin for the prevention of central vein catheter‐related thrombosis in cancer
patients a randomized controlled study based on serial venographies Ann Oncol 20 1936ndash1942
12 Evans NS Ratchford EV 2018 Catheter related-venous thrombosis Vasc Med 23 411ndash413
13 Geerts W 2014 Central venous catheterndashrelated thrombosis Hematology Am Soc Hematol Educ
Program 2014 306ndash311
14 Harahsheh Y Ho KM 2018 Use of viscoelastic tests to predict clinical thromboembolic events A
systematic review and meta‐analysis Eur J Haematol 100 113ndash123
15 Harter C Salwender HJ Bach A Egerer G Goldschmidt H Ho AD 2002 Catheter-related
infection and thrombosis of the internal jugular vein in hematologic-oncologic patients undergoing
chemotherapy a prospective comparison of silver-coated and uncoated catheters Cancer 94 245ndash251
16 Hirsh J Raschke R 2004 Heparin and low-molecular-weight heparin The Seventh ACCP
Conference on Antithrombotic and Thrombolytic Therapy Chest 126 (Suppl) 188S-203S
17 Hundley D Brooks A Thomovsky E Jhonson P Freeman L Schafbuch R Heng HG Moore
G 2018 Comparison of ultrasound-guided and landmark-based techniques for central venous
catheterization via the external jugular vein in healthy anesthetized dogs Am J Vet Res 79 628ndash
636
18 Kato F Takeuchi H Matsuda S Kawakubo H Omori T Kitagawa Y 2016 Incidence of and
risk factors for venous thromboembolism during surgical treatment for esophageal cancer a single-
institution study Surg Today 46 445ndash452
19 Karthaus M Kretzschmar A Kroning H Biakhov M Irwin D Marschner N Slabber C
Fountzilas G Garin A Abecasis NG Baronius W Steger GG Sudhoff T Giorgetti C
Reichardt P 2006 Dalteparin for prevention of catheter‐related complications in cancer patients with
central venous catheters final results of a double‐blind placebo‐controlled phase III trial Ann Oncol
17 289ndash296
20 Koumlksoy C Kuzu A Kutlay J Erden I Ozcan H Ergicircn K 1995 The diagnostic value of colour
Doppler ultrasound in central venous catheter related thrombosis Clin Radiol 50 687-689
21 Kucher N 2011 Clinical practice Deep-vein thrombosis of the upper extremities N Engl J Med
364 861ndash869
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
70
22 Langston CE Eatroff AE 2018 Hemodialysis catheter-associated fibrin sheath in a dog J Vet
Emerg Crit Care 28 366ndash371
23 Laurenson MP Hopper K Herrera MA Johnson EG 2010 Concurrent diseases and conditions
in dogs with splenic vein thrombosis J Vet Intern Med 24 1298ndash1304
24 Lee AYY Kamphuisen PW 2012 Epidemiology and prevention of catheter‐related thrombosis in
patients with cancer J Thromb Haemost 10 1491ndash1499
25 Liu G Fu ZQ Zhu P Li SJ 2015 Central venous catheter-related thrombosis in senile male
patients New risk factors and predictors J Huazhong Univ Sci Technolog Med Sci 35 445ndash449
26 Lordick F Hentrich M Decker T Hennig M Pohlmann H Hartenstein R Peschel C 2003
Ultrasound screening for internal jugular vein thrombosis aids the detection of central venous catheter-
related infections in patients with haemato-oncological diseases a prospective observational study Br
J Haematol 120 1073ndash1078
27 Lucas TC Silva EED Souza DOD Santos ARD Lara MO 2017 Microstructural
evaluation by confocal and electron microscopy in thrombi developed in central venous catheters Rev
Esc Enferm USP 51 1ndash9
28 Malick L Wilson R Stetson D 1976 Modified thiocarbo- hydrazide procedure for scanning
electron microscopy routine use for normal pathological and experimental tissue Stain Technol 50
265ndash269
29 Mischke R Jacobs C 2001 The monitoring of heparin administration by screening tests in
experimental dogs Res Vet Sci 70 101ndash108
30 Niers TM Di Nisio M Klerk CP Baarslag HJ Buller HR Biemond BJ 2007 Prevention
of catheter‐related venous thrombosis with nadroparin in patients receiving chemotherapy for
hematologic malignancies a randomized placebo‐controlled study J Thromb Haemost 5 1878ndash
1882
31 Reminga C Silverstein D Drobatz K Clarke D 2018 Evaluation of the placement and
maintenance of central venous jugular catheters in critically ill dogs and cats J Vet Emerg Crit Care
28 232ndash243
32 Smith RS Zhang Z Bouchard M Li J Lapp HS Brotske GR Lucchino DL Weaver D
Roth LA Coury A Biggerstaff J Sukavaneshvar S Langer R Loose C 2012 Vascular
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
71
catheters with a nonleaching poly-sulfobetaine surface modification reduce thrombus formation and
microbial attachment Sci Transl Med 4 153ra132 doi101126scitranslmed3004120
33 Toskala E Nuutinen J Rautiainen M 1995 Scanning electron microscopy findings on human
respiratory cilia in chronic sinusitis and in recurrent respiratory infections J Laryngol Otol 109 509ndash
514
34 Van Rooden CJ Tesselaar ME Osanto S Rosendaal FR Huisman MV 2005 Deep vein
thrombosis associated with central venous catheters - a review J Thromb Haemost 3 2409ndash2419
35 Verso M Agnelli G Bertoglio S Di Somma FC Paoletti F Ageno W Bazzan M Parise P
Quintavalle R Naglieri E Santoro A Imberti D Soraru M Mosca S 2005 Enoxaparin for the
prevention of venous thromboembolism associated with central vein catheter a double‐blind placebo‐
controlled randomized study in cancer patients J Clin Oncol 23 4057ndash4062
36 Villalta-Garciacutea P Loacutepez-Herranz M Mazo-Pascual S Honrubia-Fernaacutendez T Jaacutentildeez-Escalada
L Fernaacutendez-Peacuterez C 2015 Reliability of blood test results in samples obtained using a 2-mL discard
volume from the proximal lumen of a triple-lumen central venous catheter in the critically ill patient
Nurs Crit Care 22 298ndash304
37 Vose J Odunayo A Price JM Daves M Schildt JC Tolbert MK 2019 Comparison of
heparinized saline and 09 sodium chloride for maintaining central venous catheter patency in healthy
dogs PeerJ 7 e7072 httpsdoiorg107717peerj7072
38 Wildgruber M Lueg C Borgmeyer S Karimov I Braun U Kiechle M Meier R Koehler M
Ettl J Berger H 2016 Polyurethane versus silicone catheters for central venous port devices
implanted at the forearm Eur J Cancer 59 113ndash124 doi101016jejca201602011
39 Wu X Studer W Skarvan K Seeberger MD 1999 High incidence of intravenous thrombi after
short-term central venous catheterization of the internal jugular vein J Clin Anesth 11 482ndash485
40 Xiang DZ Verbeken EK Van Lommel AT Stas M De Wever I 1998 Composition and
formation of the sleeve enveloping a central venous catheter J Vasc Surg 28 260ndash271
41 Xiang DZ Verbeken EK Van Lommel AT Stas M De Wever I 2001 Sleeve-related
thrombosis a new form of catheter-related thrombosis Thromb Res 104 7ndash14
Chapter 4 General discussion
72
Chapter 4 General discussion
Incidence of CVC related thrombosis
In our study the incidence of CVC related thrombosis detected by ultrasound in critical
care canine patients was 28 To the best of the authoracutes knowledge similar studies
performed in dogs are rare in the available veterinary literature In one study performed
in 81 dogs and 12 cats (Adamantos et al 2010) an incidence of 2 was found for vein
thrombosis related to CVC This low incidence can be due to a lacking of systematic
ultrasound evaluations of the jugular vein containing the CVC In the mentioned study in
dogs and cats the vein thrombosis was confirmed by palpation or ultrasound presumably
after the presentation of clinical symptoms however in many cases as in our study
thrombus into the jugular vein can be developed without manifestation of clinical
symptoms what makes a routinely ultrasound evaluation of the vein containing the CVC
very important Our results are well aligned with the findings in humans in which a
median incidence of 30 was found for thrombosis related to CVC based on 25 studies
performed in patients requiring a CVC (review article van Rooden et al 2005) In the
mentioned 25 human studies the incidence of CVC related thrombosis varied within a
wide range (2ndash67 ) according to factors related to (1) the CVC (eg material type
entry site [jugular femoral subclavian axillary cephalic vein] time period in place (2)
diagnostic thrombus detection technique (ultrasonography or venography) and criteria
(3) patient collective (diseases and presence of predisposing factors [eg previous venous
thrombosis history presence of cancer] and (4) prophylactic antithrombotic treatment
(Geerts 2014 Liu et al 2015 van Rooden et al 2005)
The incidence of CVC related thrombosis detected by colour Doppler ultrasound in our
study fits also with the median incidence (30 ) found in three studies performed in
humans in which colour Doppler ultrasound evaluations of the jugular vein containing a
Chapter 4 General discussion
73
CVC were performed (Harter et al 2002 Lordick et al 2003 Wu et al 1999) In these
three studies the individual incidence of CVC related thrombosis was 2 (Harter et al
2002) 30 (Lordick et al 2003) and 56 (Wu et al 1999) The high thrombus
incidence reported in the study performed by Wu et al (1999) could be due to the criteria
for diagnosis of thrombosis because in that study both fibrin sleeves and compact
thrombi were considered as cases of thrombosis related to CVC The incidence for
compact thrombi was 25 which is more similar to our results In the study performed
by Harter et al (2002) the CVC related thrombosis incidence (2 ) was very low
compared with the incidence found in our study In that study the jugular veins were
examined just before or less than 24 hours after CVC removal (median period of CVC
use = 1025 days) In our study the ultrasound evaluations were performed before the
CVC placement (day 0) and then on days 1 3 5 etc until the CVC removal (median
period of CVC use = 7 days median interval until thrombus detection = 7 days) In our
study we detected thrombus formation as early as on the 3rd day so it could be possible
that in the cited study possibly only temporarily existing thrombi could have been missed
by ultrasonography in the final stage and thereby for the incidence calculation However
this scenario is not very likely because results of the present study show that
sonographically detectable thrombi during the observation period had in all cases their
maximum grade on the final day
In the study performed by Lordick et al (2003) the CVC related thrombosis incidence
determined for 43 human patients was very similar to our study and in both studies only
compact thrombi detectable by ultrasound were considered for assessment of the
incidence In the mentioned study all the human patients were screened for the presence
of thrombosis every 4 days after CVC insertion with real time B-mode ultrasound
(Lordick et al 2003)
Chapter 4 General discussion
74
In our study 70 of CVCs analysed by ELMI showed thrombi of grade 1ndash3 on the
external CVC wall To the best of the authoracutes knowledge studies about incidence of CVC
related thrombosis on the outside surface of the CVC detected by ELMI cannot be found
in the human and small animal literature In one study performed in humans (Lucas et al
2017) thrombus formation was analysed by ELMI in 78 central venous catheters That
study focussed on microstructural assessment of thrombus formation inside the distal
openings of the CVC which were found in 85 of the evaluated CVCs The percentage
of occurrence of thrombosis in the mentioned study in humans was nearly similar to the
incidence of intraluminal thrombus formation as detected in the presented study by ELMI
(74 ) In general intraluminal thrombi were not specially considered in the present
study because they mainly express the efficacy of the flushing technique rather than the
effectivity of the antithrombotic treatment In one study performed in rats the
microstructure of thrombi developed in the jugular vein containing the CVC was also
evaluated by ELMI (Xiang et al 1998) In the mentioned study pericatheter thrombosis
was detected on the proximal section of the CVC in 100 of rats (n = 12) after 1 and 3
days of catheterisation The analyses of the CVCs placed in rats were performed with the
catheter inside the vein in contrast to our study where the CVC was removed from the
jugular vein for the respective analysis Xiang et al (2001) hypothesised in one study
performed in rabbits and rats in which CVCs were placed in the jugular vein that fibrin
rich thrombi associated to the CVC can be detached during the catheter removal and this
could explain the difference between the thrombosis incidence assessed in our study and
that of the study performed by Xiang et al (1998)
In our study the incidence of CVC related thrombosis detected by ultrasonography in the
lumen of the EJV did not correlate with the incidence of CVC related thrombosis detected
by ELMI on the external wall of the CVC This was mainly due to the fact that the CVC
Chapter 4 General discussion
75
segment evaluated with both methods was different Using ultrasonography it was only
possible to evaluate the EJV in the neck area of the patient and thereby only the proximal
third of the CVC In contrast with the ELMI analysis only the distal section of the CVC
(last third) was evaluated because macroscopically detected thrombi mainly developed
near the CVC tip which was routinely positioned at the junction of the cranial cava vein
with the right atrium In a retrospective review of medical records of 169 human patients
requiring a CVC (Ballard et al 2016) the position of the CVC tip was determined by
radiologic evaluation and it was determined that the catheter tip position can be a
predisposing factor for central venous thrombosis development Thereby it could be of
interest to evaluate in further studies in dogs if a high incidence of thrombosis detected
by ELMI as was shown in our study could be related to the CVC tip position
In our study polyurethane catheters were tested which are widely used and are the
standard CVCs used in the Small Animal Clinic University of Veterinary Medicine
Hannover In vivo and in vitro studies have assessed the thrombogenicity of different
catheter materials (Borow and Crowley 1985 Smith et al 2012) In general terms
polyurethane catheters (central or peripheral) had shown a higher thrombogenicity
compared with coated (eg with hydromer) and silicone catheters Due to this property
of polyurethane catheters they can be supposed as a good test system for antithrombotic
regimens as evaluated in our study On the other hand despite silicone catheters have been
associated with a lower thrombogenicity their mechanical flexibility could be a
disadvantage in small clinical practice due to the increased risk of rupture detected even
in humans (Wildgruber et al 2016)
Chapter 4 General discussion
76
Heparin thromboprophylaxis for CVC related thrombosis
The occurence of CVC related thrombosis detected by colour Doppler ultrasound and
ELMI in our study demonstrates that the UFH in the given dosage was not completely
effective to prevent CVC related thrombosis in canine intensive care patients In human
medicine LMWH is commonly used in prophylactic anticoagulation regimens however
studies performed in human patients requiring a CVC and receiving a prophylactic
anticoagulation (with LMWH or UFH) did not achieve consensus regarding the efficacy
of prophylactic heparin to prevent CVC related thrombosis (Abdelfeki et al 2004
Lordick et al 2003 Wu et al 1999) To the best of the authoracutes knowledge studies
performed in dogs evaluating systemic prophylactic anticoagulation to prevent CVC
related thrombosis cannot be found However according to the evidence found in the
human literature and the results of our study further investigations using comparative
groups with different drugs dosages andor administration routes are required to establish
an effective antithrombotic treatment to prevent CVC related thrombosis
The plasma heparin activities measured in canine patients in our study varied significantly
after a defined subcutaneous dosage The limited and variable bioavailability of SC
administered UFH is well documented in humans (Hirsh and Raschke 2004)
Considerable variations of the circulatory status of the liver- and kidney function and
concentration of heparin-binding proteins in intensive care patients obviously amplify
this phenomenon leading to an extremely high inter-individual and intra-individual
variation of the heparin blood levels in our patients A different application route (IV)
may have reduced the influence of part of these factors and therefore could have led to
more predictable UFH blood levels However continuous infusion requires more
technical equipment and is also associated with a wide range of heparin activities in
canine patients (Scott et al 2009)
Chapter 4 General discussion
77
Prediction of CVC related thrombosis
Of 7 haemostasis tests (including ROTEM with 12 different parameters [6 for each
reagent ex-tem and in-tem]) evaluated as possible predictors of CVC related thrombosis
in our study initial values of high fibrinogen and low maximum lysis (ROTEM ex-tem)
were associated with sonographically detectable thrombus formation Some studies in
humans have established the association of laboratory biomarkers as predictors of risk
thrombosis (Boersma et al 2016 Cheng et al 2013 Kato et al 2016 Liu et al 2015)
One study performed in anticoagulated human patients showed ndash well in agreement with
our results ndash that high fibrinogen levels were associated with an increased risk of
perioperative venous thromboembolism (P = 0004) (Kato et al 2016) The cited study
does not exclusively refer to CVC associated thrombosis but 762 (1621) of the
venous thromboses were suggested to be associated with CVCs Fibrinogen concentration
measurements are normally available in the human and veterinary practice and the
association of this parameter with a high risk of development of CVC related thrombosis
can be useful to choose special antithrombotic treatment in individual critical care patients
to prevent such severe complications
Thrombin generation assay (TGA) abnormalities such as higher velocity and endogenous
thrombin potential (ETP) values measured initially ie before any anticoagulation
therapy have been associated with a higher thromboembolism risk in general (ie without
specific association with CVCs) in one study performed on 105 human patients (Espitia
et al 2017) In contrast in our study the initial TGA assay results did not show an
association with formation of CVC related thrombosis
The ROTEM parameters evaluated in the present study were partially associated with
formation of CVC thrombosis detected by colour Doppler ultrasound This finding
confirms the results obtained in humans in which ROTEM measurements are regarded as
Chapter 4 General discussion
78
a valuable indicator of hypercoagulability (Harahsheh and Ho 2018 Tuumlrk et al 2018)
However in our study individual ROTEM parameters showed partly lower coagulation
activities in dogs with CVC related thrombosis detected by ELMI at the final day of
evaluation Because the number of patients without signs of thrombosis detected by ELMI
was low compared with those who showed signs these implausible results should be
interpreted carefully The usefulness of ROTEM analysis which has also many other
indications demonstrates that is was worth to perform a detailed methodological approval
of the device including the establishment of reference values in the preliminary study
Conclusion
In conclusion the prethrombotic or thrombotic changes detected in our study did not
correlate between ultrasonography and ELMI however both methods show accurately
signs of CVC related thrombosis The UFH in the given dosage was not completely
effective to prevent CVC induced thrombus formation in canine intensive care patients
Plasma heparin activities varied significantly after a defined subcutaneous dosage
However plasma heparin activities were not different between dogs with and without
detectable CVC associated thromboses Initial high fibrinogen values and low maximum
lysis (ROTEM ex-tem) values were associated with sonographically detectable thrombi
and may be useful as indicators for an increased thrombotic risk
Finally our study in canine intensive care patients is to the best of the authoracutes
knowledge the first study in the available veterinary literature performed to evaluate the
efficacy of an antithrombotic treatment with UFH to prevent CVC related thrombosis
Chapter 5 Summary
79
Chapter 5 Summary
Central venous catheter induced thrombosis in dogs ndash occurrence under an
antithrombotic treatment regimen and possible indicators
Joseacute Mauricio Pereira Mora
Central venous catheters (CVC) are of importance for intensive care management of
canine patients Thrombosis of the jugular vein is a severe complication related to the
CVC use However only little is known about the incidence of CVC-associated
thromboses in dogs and the necessity and efficacy of prophylactic antithrombotic
regimens
Therefore the aims of the present study were (1) to determine the incidence of catheter-
induced thrombosis in canine intensive care patients (2) to evaluate the efficacy of a
prophylactic heparin regimen routinely used in the Small Animal Clinic of the University
of Veterinary Medicine Hannover and (3) to assess whether selected haemostasis test
results are predictive for an increased risk for thrombosis formation
The thesis also includes a preliminary methodological study on the use of rotational
elastometry in dogs using the ROTEM delta device In this preliminary study reference
intervals were determined based on 125 healthy dogs for different clot activator reagents
and for measurements without activator Coefficients of variation (CVs) were calculated
based on results of fourfold measurements of 3 normal and 3 abnormal blood samples in
order to assess precision of the method Influence of age groups sex neuter status and
size were also assessed The median CVs for most of the parameters evaluated in the
precision analysis were less than 10 and this confirmed that the ROTEM delta analyser
delivers repeatable results Regarding the influence of age sex neuter status and animal
Chapter 5 Summary
80
size only neuter status in female dogs appeared to be a relevant influencing factor on
most of important ROTEM parameters
The main study of the thesis comprises 29 patients hospitalised at the Small Animal Clinic
requiring a CVC for medical reasons These patients received a prophylactic
anticoagulation treatment with unfractionated heparin (UFH 150 IU [surgical dogs
received 75 IU]kg BW TID subcutaneously) A colour Doppler ultrasound of the
respective external jugular vein (EJV) in order to detect thrombus formation in the lumen
of the EJV and blood collection for haemostasis tests were performed directly before the
installation procedure (day 0) and on days 1 3 5 7 and so on after the CVC insertion
Haemostasis tests included prothrombin time activated partial thromboplastin time
thrombin time antithrombin activity D-dimer concentration thrombin generation and
rotational elastometry as well as the heparin activity using a chromogenic anti-factor Xa
test Finally when the CVC was removed electron microscopy (ELMI) of sections of the
CVC with macroscopically visible thrombi in the last third (5ndash10 cm) of the CVC was
performed 3 different grades were defined for CVC-associated (extraluminal) thrombi
detected by either ultrasound or ELMI respectively
829 patients evaluated by colour Doppler ultrasound during the study developed thrombi
into the lumen of EJV (4 x grade 1 3 x grade 2 and 1 x grade 3) 1927 CVCs assessed
using ELMI showed detectable thrombi on the external CVC surface (7 x grade 1 4 x
grade 2 and 8 x grade 3) There was no significant correlation between thrombus
formation detected by ultrasound or ELMI Thus statistical group comparisons of
patients with or without CVC-associated thrombus formation was performed
independently for both diagnostic procedures The plasmatic heparin activities showed
great variability but no significant differences were found between dogs with or without
CVC-associated thrombus formation regardless whether this was based on sonographic
Chapter 5 Summary
81
or ELMI findings From the initially performed haemostasis tests high fibrinogen
concentrations and low maximum lysis values as measured by the ROTEM delta device
(using the ex-tem reagent) were associated with sonographically detectable thrombi
In conclusion the preliminary methodological study on the ROTEM delta in dogs shows
that the method delivers repeatable results in canine citrated whole blood and the
reference values obtained should offer a reliable basis for the interpretation of test results
of this device in dogs with suspected haemostasis disorders The results of the main study
indicate that CVC-associated thromboses occur (eg 28 [829] of the patients as
detected by ultrasound) although severe thromboses (high grades) are relatively rare in
intensive care canine patients receiving a routine anticoagulant treatment Thereby
prophylactic antithrombotic protocol (unfractionated heparin [UFH]) in the given dosage
was not completely effective to prevent CVC induced thrombus formation in canine
intensive care patients A high fibrinogen concentration and low maximum lysis
(ROTEM ex-tem) may be useful indicators for an increased thrombotic risk in individual
patients which may require special anticoagulatory treatment Further studies are
required to evaluate whether different thromboprophylactic treatment regimens eg with
low molecular weight heparin are more effective in preventing CVC-associated
thromboses in dogs
Chapter 6 Zusammenfassung
82
Chapter 6 Zusammenfassung
Zentralvenenkatheter-induzierte Thrombose bei Hunden - Auftreten unter einem
antithrombotischen Behandlungsschema und moumlgliche Indikatoren
Joseacute Mauricio Pereira Mora
Zentralvenenkatheter (ZVK) sind fuumlr das Intensivmanagement von Hunden von
Bedeutung Die Entstehung einer Thrombose der Vena jugularis ist eine schwerwiegende
Komplikation im Zusammenhang mit der Verwendung eines ZVKs Uumlber die Inzidenz
von ZVK-assoziierten Thrombosen bei Hunden und die Notwendigkeit und Wirksamkeit
prophylaktischer antithrombotischer Protokolle liegen jedoch nur wenige fundierte
Angaben in der Literatur vor
Daher waren die Ziele der vorliegenden Studie (1) die Inzidenz der Katheter-induzierten
Thrombose bei caninen Intensivpatienten zu bestimmen (2) die Wirksamkeit eines
prophylaktischen Heparin-Regimes zu bewerten das routinemaumlszligig in der Klinik fuumlr
Kleintiere der Stiftung Tieraumlrztliche Hochschule Hannover angewendet wird und (3) um
zu beurteilen ob ausgewaumlhlte Haumlmostasetestergebnisse ein erhoumlhtes Risiko fuumlr die
Thrombosebildung vorhersagen koumlnnen
Die PhD-Arbeit enthaumllt zudem eine vorbereitende methodische Studie zur Verwendung
der Rotationselastometrie bei Hunden mit Hilfe des ROTEM delta-Geraumltes In dieser
Vorstudie wurden Referenzintervalle basierend auf 125 gesunden Hunden fuumlr
verschiedene Gerinnungsaktivatorreagenzien und fuumlr Messungen ohne Aktivator
bestimmt Um die Genauigkeit der Methode einzustufen wurden Variationskoeffizienten
(VKs) basierend auf den Ergebnissen von Vierfachmessungen von drei normalen und drei
abnormalen Blutproben berechnet Der Einfluss von Alter Geschlecht Kastration und
Groumlszlige wurde ebenfalls getestet Die Medianwerte der VKs fuumlr die meisten in der
Chapter 6 Zusammenfassung
83
Praumlzisionsanalyse bewerteten Parameter betrugen weniger als 10 und dies bestaumltigte
dass das ROTEM delta-Analysegeraumlt wiederholbare Ergebnisse liefert In Bezug auf den
Einfluss von Alter Geschlecht Kastration und Tiergroumlszlige erwies sich nur eine Kastration
bei Huumlndinnen als relevanter Einflussfaktor fuumlr die meisten wichtigen ROTEM-
Parameter
Die Hauptstudie der Arbeit umfasst 29 Patienten welche zum Zeitpunkt der
Untersuchung stationaumlr in der Kleintierklinik aufgenommen worden waren und aus
medizinischer Indikation einen ZVK benoumltigten Diese Patienten erhielten eine
prophylaktische Antikoagulation mit unfraktioniertem Heparin (UFH 150 IE
[chirurgische Hunde erhielten 75 IE]kg KG 3 x taumlglich subkutan) Bildgebende
Untersuchungen mittels Farbdoppler-Ultraschall der jeweiligen Vena jugularis externa
(VJE) zum Nachweis der Thrombusbildung im Lumen der VJE und Blutentnahmen fuumlr
Haumlmostasetests wurden jeweils direkt vor dem Installationsvorgang (Tag 0) sowie an den
Tagen 1 3 5 und 7 nach Legen des ZVKs durchgefuumlhrt Haumlmostasetests umfassten die
Prothrombinzeit die aktivierte partielle Thromboplastinzeit die Thrombinzeit die
Antithrombinaktivitaumlt die D-Dimer-Konzentration die Thrombingenerierung und die
Rotationselastometrie sowie die Heparinaktivitaumlt unter Verwendung eines chromogenen
Anti-Faktor-Xa-Tests Schlieszliglich wurde nach Entfernung des ZVKs eine
Elektronenmikroskopie (ELMI) von Abschnitten aus dem letzten Drittel (5ndash10 cm) des
ZVKs mit makroskopisch sichtbaren Thromben durchgefuumlhrt Fuumlr ZVK-assoziierte
(extraluminale) Thromben die entweder durch Ultraschall oder ELMI nachgewiesen
wurden wurden drei verschiedene Grade definiert
829 Patienten die waumlhrend der Studie mittels Farbdoppler-Ultraschall untersucht
wurden entwickelten Thromben im Lumen der VJE (4 x Grad 1 3 x Grad 2 und 1 x Grad
3) 1927 ZVKs die unter Verwendung von ELMI bewertet wurden zeigten
Chapter 6 Zusammenfassung
84
nachweisbare Thromben auf der aumluszligeren ZVK-Oberflaumlche (7 x Grad 1 4 x Grad 2 und 8
x Grad 3) Es gab keine signifikante Korrelation zwischen der durch Ultraschall oder
ELMI festgestellten Thrombusbildung Daher wurden statistische Gruppenvergleiche
von Patienten mit oder ohne ZVK-assoziierter Thrombusbildung fuumlr beide diagnostische
Verfahren unabhaumlngig voneinander durchgefuumlhrt Die plasmatischen Heparinaktivitaumlten
zeigten eine groszlige Variabilitaumlt es wurden jedoch keine signifikanten Unterschiede
zwischen Hunden mit oder ohne ZVK-assoziierter Thrombusbildung gefunden
unabhaumlngig davon ob diese auf sonographischen oder ELMI-Befunden beruhte Von den
initial durchgefuumlhrten Haumlmostasetests waren hohe Fibrinogenkonzentrationen und
niedrige maximale Lyse-Werte der Messungen mit ROTEM delta (ex-tem Reagenz) mit
sonographisch nachweisbaren Thromben assoziiert
Zusammenfassend zeigt die vorbereitende methodische Studie zum ROTEM delta bei
Hunden dass die Methode wiederholbare Ergebnisse in mit Zitrat
antikoaguliertemVollblut liefert Die erarbeiteten Referenzwerte sollten eine zuverlaumlssige
Grundlage fuumlr die Interpretation der Testergebnisse dieses Geraumlts bei Hunden mit
Verdacht auf Haumlmostasestoumlrungen bieten Die Ergebnisse der Hauptstudie zeigen dass
ZVK-assoziierte Thrombosen auftreten (z B 28 [829] der Patienten bezogen auf den
Ultraschall-gestuumltzten Nachweis) obwohl schwere Thrombosen (hohe Grade) bei
intensivmedizinisch versorgten Hundepatienten die eine Routineantikoagulation
erhalten relativ selten sind Somit war das prophylaktische antithrombotische Protokoll
(unfraktioniertes Heparin [UFH]) in der angewendeten Dosierung nicht vollstaumlndig
wirksam um die ZVK-induzierte Thrombusbildung bei caninen Intensivpatienten zu
verhindern Eine hohe Fibrinogenkonzentration und eine niedrige maximale Lyse
(ROTEM ex-tem) koumlnnen als Indikatoren eines erhoumlhten Thromboserisikos bei einzelnen
Patienten nuumltzlich sein fuumlr die moumlglicherweise eine spezielle gerinnungshemmende
Chapter 6 Zusammenfassung
85
Behandlung notwendig ist Weitere Studien sind erforderlich um zu untersuchen ob
verschiedene thromboprophylaktische Behandlungsschemata z B mit
niedermolekularem Heparin bezuumlglich der Verhinderung von ZVK-assoziierten
Thrombosen bei Hunden wirksamer sind
Chapter 7 References
86
Chapter 7 References
1 Abdelkefi A Ben Othman T Kammoun L Chelli M Ben Romdhane N Kriaa A Ladeb
S Torjman L Lakhal A Achour W Ben Hassen A Hsaiumlri M Ladeb F Ben Abdeladhim
A 2004 Prevention of central venous line-related thrombosis by continuous infusion of low-dose
unfractionated heparin in patients with haemato-oncological disease A randomized controlled
trial Thromb Haemost 92 654ndash661
2 Adamantos S Brodbelt D Moores A L 2010 Prospective evaluation of complications
associated with jugular venous catheter use in a veterinary hospital J Small Anim Pract 51 254ndash
257
3 Ballard D H Samra N S Gifford K M Roller R Wolfe B M Owings J T 2016
Distance of the internal central venous catheter tip from the right atrium is positively correlated
with central venous thrombosis Emerg Radiol 23 269ndash273
4 Baumann-Kreuziger L Onwuemene O Kolesar E Crowther M Lim W 2015 Systematic
review of anticoagulant treatment of catheter-related thrombosis Thromb Res 136 1103ndash1109
5 Bliss SP Bliss SK Harvey HJ 2002 Use of recombinant tissue‐plasminogen activator in a
dog with chylothorax secondary to catheter‐associated thrombosis of the cranial vena cava J Am
Anim Hosp Assoc 38 431ndash435
6 Boersma RS Hamulyak K van Oerle R Tuinenburg A Ten Cate-Hoek AJ Schouten
HC 2016 Biomarkers for prediction of central venous catheter related-thrombosis in patients
with hematological malignancies Clin Appl Thromb Hemost 22 779ndash784
7 Boersma RS Jie KS Verbon A van Pampus EC Schouten HC 2008 Thrombotic and
infectious complications of central venous catheters in patients with hematological
malignancies Ann Oncol 19 433ndash442
8 Bonnet F Loriferne JF Texier JP Texier M Salvat A Vasile N 1989 Evaluation of
Doppler examination for diagnosis of catheter-related deep vein thrombosis Intensive Care Med
15 238ndash240
9 Borow M Crowley J G 1985 Evaluation of central venous catheter thrombogenicity Acta
Anaesthesiol Scand Suppl 81 59ndash64
10 Cheng Y Cui T Fu P Liu F Zhou L 2013 Dyslipidemia is associated with tunneled-cuffed
catheter-related central venous thrombosis in hemodialysis patients a retrospective multicenter
Chapter 7 References
87
study Artif Organs 37 155ndash161
11 Comerlato PH Rebelatto TF Santiago de Almeida FA Klein LB Boniatti MM
Schaan BD Rados DV 2017 Complications of central venous catheter insertion in a teaching
hospital Rev Assoc Med Bras 63 613ndash620
12 De Cicco M Matovic M Balestreri L Steffan A Pacenzia R Malafronte M Fantin
D Bertuzzi CA Fabiani F Morassut S Bidoli E Veronesi A 2009 Early and short‐term
acenocumarine or dalteparin for the prevention of central vein catheter‐related thrombosis in
cancer patients a randomized controlled study based on serial venographies Ann
Oncol 20 1936ndash1942
13 Espitia O Fouassier M Hardouin JB Pistorius MA Agard C Planchon B Trossaert M
Pottier P 2017 Thrombin generation assay in hospitalized nonsurgical patients A new tool to
assess venous thromboembolism risk Clin Appl Thromb Hemost 23 45ndash51
14 Evans NS Ratchford EV 2018 Catheter related-venous thrombosis Vasc Med 23 411ndash413
15 Geerts W 2014 Central Venous Catheter-related thrombosis Hematology Am Soc Hematol
Educ Program 2014 306ndash311
16 Harahsheh Y Ho KM 2018 Use of viscoelastic tests to predict clinical thromboembolic
events A systematic review and meta‐analysis Eur J Haematol 100 113ndash123
17 Harter C Salwender HJ Bach A Egerer G Goldschmidt H Ho AD 2002 Catheter-
related infection and thrombosis of the internal jugular vein in hematologic-oncologic patients
undergoing chemotherapy a prospective comparison of silver-coated and uncoated
catheters Cancer 94 245ndash251
18 Hirsh J Raschke R 2004 Heparin and low-molecular-weight heparin The Seventh ACCP
Conference on Antithrombotic and Thrombolytic Therapy Chest 126 (Suppl) 188Sndash203S
19 Karthaus M Kretzschmar A Kroning H Biakhov M Irwin D Marschner N Slabber
C Fountzilas G Garin A Abecasis NG Baronius W Steger GG Sudhoff T Giorgetti
C Reichardt P 2006 Dalteparin for prevention of catheter‐related complications in cancer
patients with central venous catheters final results of a double‐blind placebo‐controlled phase III
trial Ann Oncol 17 289ndash296
20 Kato F Takeuchi H Matsuda S Kawakubo H Omori T Kitagawa Y 2016 Incidence of
and risk factors for venous thromboembolism during surgical treatment for esophageal cancer a
Chapter 7 References
88
single-institution study Surg Today 46 445ndash452
21 Kohler TR Kirkman TR 1998 Central venous catheter failure is induced by injury and can
be prevented by stabilizing the catheter tip J Vasc Surg 28 59ndash66
22 Koumlksoy C Kuzu A Kutlay J Erden I Ozcan H Ergicircn K 1995 The diagnostic value of
colour Doppler ultrasound in central venous catheter related thrombosis Clin Radiol 50 687ndash
689
23 Kucher N 2011 Clinical practice Deep-vein thrombosis of the upper extremities N Engl J
Med 364 861ndash869
24 Langston CE Eatroff AE 2018 Hemodialysis catheter-associated fibrin sheath in a dog J
Vet Emerg Crit Care 28 366ndash371
25 Langston CE Eatroff AE Poeppel K 2014 Use of tissue plasminogen activator in catheters
used for extracorporeal renal replacement therapy J Vet Intern Med 28 270ndash276
26 Laurenson MP Hopper K Herrera MA Johnson EG 2010 Concurrent diseases and
conditions in dogs with splenic vein thrombosis J Vet Intern Med 24 1298ndash1304
27 Lee AYY Kamphuisen PW 2012 Epidemiology and prevention of catheter‐related
thrombosis in patients with cancer J Thromb Haemost 10 1491ndash1499
28 Liu G Fu ZQ Zhu P Li SJ 2015 Central venous catheter-related thrombosis in senile male
patients New risk factors and predictors J Huazhong Univ Sci Technolog Med Sci 35 445ndash
449
29 Lordick F Hentrich M Decker T Hennig M Pohlmann H Hartenstein R Peschel C
2003 Ultrasound screening for internal jugular vein thrombosis aids the detection of central
venous catheter-related infections in patients with haemato-oncological diseases a prospective
observational study Br J Haematol 120 1073ndash1078
30 Lucas TC Silva EED Souza DOD Santos ARD Lara MO 2017 Microstructural
evaluation by confocal and electron microscopy in thrombi developed in central venous catheters
Rev Esc Enferm USP 51 1ndash9
31 Mismetti P Mille D Laporte S Charlet V Buchmuller‐Cordier A Jacquin JP Fournel
P Dutrey‐Dupagne C Decousus H 2003 Low‐molecular‐weight heparin (nadroparin) and
very low doses of warfarin in the prevention of upper extremity thrombosis in cancer patients with
indwelling long‐term central venous catheters a pilot randomized trial Haematologica 88 67ndash
Chapter 7 References
89
73
32 Niers TM Di Nisio M Klerk CP Baarslag HJ Buller HR Biemond BJ 2007
Prevention of catheter‐related venous thrombosis with nadroparin in patients receiving
chemotherapy for hematologic malignancies a randomized placebo‐controlled study J Thromb
Haemost 5 1878ndash1882
33 Scott K C Hansen B D DeFrancesco T C (2009) Coagulation effects of low molecular
weight heparin compared with heparin in dogs considered to be at risk for clinically significant
venous thrombosis J Vet Emerg Crit Care 19 74ndash80
34 Smith RS Zhang Z Bouchard M Li J Lapp HS Brotske GR Lucchino DL Weaver
D Roth LA Coury A Biggerstaff J Sukavaneshvar S Langer R Loose C 2012
Vascular catheters with a nonleaching poly-sulfobetaine surface modification reduce thrombus
formation and microbial attachment Sci Transl Med 4 153
35 Tuumlrk S M Cansu D Uuml Teke H Uuml Kaşifoğlu T Meltem Akay O Bilgin M amp Korkmaz
C 2018 Can we predict thrombotic tendency in rheumatoid arthritis A thromboelastographic
analysis (with ROTEM) Clin Rheumatol 37 2341ndash2349
36 van Rooden CJ Rosendaal FR Barge RM van Oostayen JA van der Meer FJM
Meinders AE Huisman MV 2003 Central venous catheter related thrombosis in haematology
patients and prediction of risk by screening with Doppler-ultrasound Br J Haematol 123 507ndash
512
37 van Rooden CJ Tesselaar ME Osanto S Rosendaal FR Huisman MV 2005 Deep vein
thrombosis associated with central venous catheters - a review J Thromb Haemost 3 2409ndash
2419
38 Verso M Agnelli G Bertoglio S Di Somma FC Paoletti F Ageno W Bazzan M Parise
P Quintavalle R Naglieri E Santoro A Imberti D Soraru M Mosca S 2005 Enoxaparin
for the prevention of venous thromboembolism associated with central vein catheter a double‐
blind placebo‐controlled randomized study in cancer patients J Clin Oncol 23 4057ndash4062
39 Vose J Odunayo A Price JM Daves M Schildt JC Tolbert MK 2019 Comparison of
heparinized saline and 09 sodium chloride for maintaining central venous catheter patency in
healthy dogs PeerJ 7e7072 httpdoi107717peerj7072
40 Wang LH Wei F Jia L Zhi Lu Wang B Dong HY Yu HY Sun GJ YangJ Li B
Chapter 7 References
90
Meng J Zhang RN Bi XQ Chen HY Jiang AL 2015 Fibrin sheath formation and
intimal thickening after catheter placement in dog model role of hemodynamic wall shear stress J
Vasc Access 16 275ndash284
41 Wildgruber M Lueg C Borgmeyer S Karimov I Braun U Kiechle M Meier R Koehler
M Ettl J Berger H 2016 Polyurethane versus silicone catheters for central venous port devices
implanted at the forearm Eur J Cancer 59 113ndash124
42 Whitesell RT Steenburg SD 2014 Imaging findings of acute intravascular thrombus on non-
enhanced computed tomography Emerg Radiol 21 271ndash277
43 Wu X Studer W Skarvan K Seeberger MD 1999 High incidence of intravenous thrombi
after short-term central venous catheterization of the internal jugular vein J Clin Anesth 11
482ndash485
44 Xiang DZ Verbeken EK Van Lommel AT Stas M De Wever I 1998 Composition and
formation of the sleeve enveloping a central venous catheter J Vasc Surg 28 260ndash271
45 Xiang DZ Verbeken EK Van Lommel AT Stas M De Wever I 2001 Sleeve-related
thrombosis a new form of catheter-related thrombosis Thromb Res 104 7ndash14
91
Acknowledgements
First I thank God for every day in my life for His guide and because He was is
and will be always with me and that certainty gives me strength to take a step
forward
My sincere thanks to Prof Dr Reinhard Mischke for the opportunity to perform
my PhD under his supervision for his support and guidance for all that I learned
with him and for his friendship
I also thank to Prof Dr Manfred Kietzmann and PD Dr Mario von Depka
Prondzinski for being part of my supervision group for their time and beneficial
advices and discussion during my PhD
My gratitude to Prof Hewicker-Trautwein and Mrs Rohn for their valuable work
and availability to help me
I thank the Werlhof Institute Medical Care Center Hannover for the valuable
cooperation especially to Mrs Ober-Cassebaum Anne and Franziska for all their
help
I thank all the staff of the Small Animal Clinic of the University of Veterinary
Medicine Hannover (TiHo) My special gratitude to the internal medicine and
critical care surgery neurology ultrasound and anesthesia departments and to
the TiHo students all their cooperation made possible my research
I thank the haematology clinical chemistry and haemostasis laboratory
department of the Small Animal Clinic TiHo especially to Dirk because he was
always available for me thanks for his guide and friendship all his explanations
in the laboratory were of great value but also the conversations about different
topics of the life were of big value for me
92
I thank the Hannover Graduate School for Veterinary Pathobiology
Neuroinfectology and translational Medicine (HGNI) for the well-organized
PhD program and especially thanks to Prof Dr Beatrice Grummer Dr Tina
Selle and Mrs Tania Czeslik for his guide and help during my studies
My sincere thanks to Mrs Maritta Ledwoch for her guidance at the International
office of the TiHo ldquoI remember you since my externship in Germanyrdquo
I also thank the Student Union Hannover for all the help with the accommodation
for me and my family
I want to thank the Universidad Nacional de Costa Rica and therefore to Costa
Rica (my loved country) for the financial support to perform my PhD studies
but also for my professional formation as a veterinarian Special thanks to the
Veterinary Medicine School from Universidad Nacional de Costa Rica To all the
academic and administrative staff for their support and for believing in me
Sincerely thanks to Dr Mauricio Jimenez Soto my mentor my adviser but most
important my friend Thanks for believing in me and for always being by my side
ldquoAgradezco profundamente a mis papaacutes ldquodon Tito (Papillo) y dontildea Eida (Mami)rdquo
Todo lo bueno que hay en mi proviene de Dios y de ellos Gracias a mis hermanos
(as) cuntildeados (as) sobrinos y a toda la familia relacionada a ellos Siempre han
sido un gran apoyo y los amordquo
ldquoGracias a dontildea Alba Fran y David Gracias por toda la ayuda y por estar siempre
pendientesrdquo
I would like to thank all my friends that during a short part or during my whole
life have been with me For all the good time shared really thanks
To Willy thanks ldquogordordquo
93
I sincerely thank to Adriano and Johanna for their help and hospitality Thanks
Adriano for your guidance and support during my beginning at the TiHo for make
me feel in Costa Rica with your friendship for the tourist walks and for the bike
training I missed the coffee times
I want to thank Ina the first person to give me a smile at the start of my PhD
Thanks for your hugs and friendship
I also thank Franz for his help and for being a very nice partner Thanks for help
me always when I requested you
ldquoFinalmente quiero expresar mi gratitud y amor a Karo Faacutetima y Abigaiacutel A Karo
por tu sacrificio y soporte por las palabras de aliento por estar a mi lado en las
buenas pero en especial en las malas A Faacute por tu sacrificio por tus sonrisas por
tus abrazos y besos por bailar con migo siempre por ser mi modelo e inspiracioacuten
A Abi por ser una nueva razoacuten in mi vida para seguir adelante por todo lo que
deseo compartir con tigo por mostrarme junto con Faacutetima la felicidad en una
sonrisardquo
Parts of the thesis have been published as full paper previously in journals
Pereira JM Rohn K Mischke R 2020 Reference intervals for rotational
thromboelastometry measurements using the ROTEMreg delta device in dogs Res Vet
Sci 130 26ndash32
Parts of the thesis have been presented in abstracts related to the poster and oral
presentations at congresses
Pereira JM Mischke R 2017 Reference intervals for thromboelastometric
measurements using the ROTEMreg delta device in dogs In 10th Graduate School
DaysHannover Graduate School for Veterinary Pathobiology Neuroinfectology and
Translational Medicine 01ndash02122017 Bad Salzdetfurth Germany Stiftung
Tieraumlrztliche Hochschule Hannover P 48 P 08
Pereira JM Mischke R 2018 Reference intervals for thromboelastometric
measurements using the ROTEMreg delta device in dogs In 26 Jahrestagung der DVG-
Fachgruppe Innere Medizin und klinische Labordiagnostik 02ndash030218 Hannover
Germany Tierarztl Prax Ausg K Kleintiere Heimtiere 46(02) P18-A16
(DOI 101055s-0038-1647663)
Pereira JM Hewicker-Trautwein M von Depka Prondzinski M Mischke R 2018
Central venous catheter associated thrombosis in dogs In 11th Graduate School
DaysHannover Graduate School for Veterinary Pathobiology Neuroinfectology and
Translational Medicine 3011ndash021217 Hannover Germany Stiftung Tieraumlrztliche
Hochschule Hannover V07
Pereira JM Hewicker-Trautwein M von Depka Prondzinski M Mischke R 2019
Central venous catheter associated thrombosis in dogs In 27 Jahrestagung der DVG-
Fachgruppe Innere Medizin und klinische Labordiagnostik 01ndash020219 Munich
Germany Tierarztl Prax Ausg K Kleintiere Heimtiere 47(02) P14-139 (DOI
101055s-0039-1679119)
Pereira JM Rohn K Mischke R 2019 Reference intervals for rotational
thromboelastometric measurements using the ROTEMreg delta device in dogs In 35th
World Veterinary Association Congress 27 ndash300319 San Joseacute Costa Rica
Pereira JM Rohn K Mischke R 2019 Reference intervals for rotational
thromboelastometric measurements using the ROTEMreg delta device in dogs Abstracts
of the ESVCP-ECVCP Annual Congress 25ndash280919 Arnhem The Netherlands Vet
Clin Pathol 48 813 P 57
Pereira JM Hewicker-Trautwein M von Depka Prondzinski M Mischke R 2020
Central venous catheter associated thrombosis in dogs In 20 Annual ECVIM-CA
Congress 03ndash050920 Barcelona Spain J Vet Int Med (in press)
Sponsorship Joseacute Mauricio Pereira received a scholarship from the Universidad Nacional
de Costa Rica (reference Code JB-C-1107-2016)
Table of contents
Table of contents
Chapter 1 Introduction 1
Epidemiology of CVC related thrombosis 1
Pathogenesis of CVC related thrombosis 2
Diagnosis of CVC related thrombosis 3
Heparin thromboprophylaxis for CVC related thrombosis 5
Prediction of CVC related thrombosis 7
Aims of the study 8
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs 9
ABSTRACT 11
1 Introduction 12
2 Materials and methods 14
3 Results 20
4 Discussion 26
5 Conclusions 31
6 Acknowledgments 31
7 References 32
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes 37
ABSTRACT 40
1 Introduction 42
2 Material and methods 44
3 Results 54
Table of contents
4 Discussion 62
5 Conclusions 67
6 Acknowledgments 67
7 References 68
Chapter 4 General discussion 72
Incidence of CVC related thrombosis 72
Heparin thromboprophylaxis for CVC related thrombosis 76
Prediction of CVC related thrombosis 77
Conclusion 78
Chapter 5 Summary 79
Chapter 6 Zusammenfassung 82
Chapter 7 References 86
Acknowledgements 91
List of abbreviations
i
List of abbreviations
CV coefficient of variation
CVC central venous catheter
EJV external jugular vein
ETP endogenous thrombin potential
HDL high-density lipoprotein
IV intravenous
LDL low-density lipoprotein
LMWH low molecular weight heparin
ROTEM rotational elastometry
SC subcutaneous
TGA thrombin generation assay
UFH unfractionated heparin
Chapters 2 and 3 have their own abbreviations included
List of figures and tables
ii
List of figures and tables
Chapter 2
Table 1 Coefficients of variation () for rotational thromboelastometry using the
ROTEMreg delta analyser in normal blood samples 20
Table 2 Coefficients of variation () for rotational thromboelastometry using the
ROTEMreg delta analyser in canine blood samples with haemostatic alterations 20
Table 3 Reference values (median reference interval based on 25 - and 975 -
quantiles) for thromboelastometric measurements with the ROTEMreg delta analyser in
dogs (n=125 95 confidence intervals in brackets) 23
Table 4 Median values (minimum and maximum in brackets) for different ROTEMreg
variables using ex-tem reagent stratified by sex and neuter status 24
Table 5 Median values (minimum and maximum in brackets) for different ROTEMreg
variables using the in-tem reagent stratified by sex and neutering status 25
Chapter 3
Fig 1 Definition of different grades of central venous catheter induced thrombosis of the
jugular vein as detected using colour Doppler ultrasound 48
Fig 2 Definition and illustration of different grades of central venous catheter induced
(extraluminal) thrombosis as detected using electron microscopy 53
Fig 1S (suppl material) Definition and illustration of different grades of intraluminal
thrombosis in central venous catheters as detected using electron microscopy 53
Table 1S (Supplementary material) Heparin activities (IUml median values
minimumndashmaximum) measured in 27 dogs receiving 75 or 150 IUkg unfractionated
(standard) heparin TID subcutaneously 2 hours after the first injection on the day
Comparison of samples collected via puncture of the peripheral veins and those collected
from the central venous catheter (CVC) using the three-syringe-technique 56
List of figures and tables
iii
Fig 3ab Heparin activities (box- and whisker plot arithmetic means) in dogs receiving
unfractionated heparin at a dosage of 75 IUkg BW tid (a) or 150 IUkg BW tid (b) two
hours after the first daily heparin administration 57
Table 1 Heparin activities (IUml median values minimumndashmaximum) in 28 dogs
receiving 75 or 150 IUkg unfractionated (standard) heparin TID subcutaneously with
(grades 1ndash3) or without (grade 0) sonographically detectable thrombus Blood collections
were performed 2 hours after the first daily injection 57
Table 2 Heparin activities (IUml median values minimumndashmaximum) in 27 dogs
receiving 75 or 150 IUkg unfractionated (standard) heparin TID subcutaneously with
(score 1ndash3) or without (score 0) detectable thrombus via electron microscopy 58
Table 3 Comparison of initial results of haemostasis parameters (median values
minimumndashmaximum in dogs with (grades 1ndash3) and without (grade 0) sonographically
detectable central venous catheter-induced thromboses 60
Table 4 Comparison of results of haemostasis parameters (median values minimumndash
maximum) on the last day in dogs with (grades 1ndash3) and without (grade 0) central venous
catheter-associated thrombus formation as detected by electron microscopy 61
Chapter 1 Introduction
1
Chapter 1 Introduction
Central venous catheters (CVC) are mainly used at critical care units in human and small
animal patients requiring infusion of large amounts of fluids or blood parenteral nutrition
support routine drug therapy haemodialysis chemotherapy plasmapheresis or
periodical blood sampling (Geerts 2014 Vose et al 2019) CVC related thrombosis is
one of the most severe complications (apart from infections and pneumothorax) of CVCs
in humans (Comerlato et al 2017 Evans and Ratchford 2018 Geerts 2014) leading to
patientrsquos distress CVC dysfunction venous stenosis and in some cases to the
development of thromboembolism (Geerts 2014) In the human literature there are
various studies dealing with epidemiologic and prophylactic aspects which are described
below in detail In contrast to the best of the authoracutes knowledge studies about extra
luminal CVC related thrombosis in dogs are rare in the available veterinary literature
Studies are mainly dealing with development of intraluminal CVC thrombi occurring
frequently in dogs and cats (Langston et al 2014) and leading to CVC malfunction (Vose
et al 2019)
Epidemiology of CVC related thrombosis
CVC related thrombosis represents 70ndash80 of all upper extremity thrombosis cases and
approximately 10 of thromboembolism cases in humans (Kucher 2011) The incidence
of CVC related thrombosis reported in humans based in 25 studies ranged widely from
2 to 67 (median = 30 ) (van Rooden et al 2005) The incidence of CVC related
thrombosis detected by colour Doppler ultrasound of the internal jugular vein reported in
3 studies performed in humans was 2 (Harter et al 2002) 30 (Lordick et al 2003)
and 56 (Wu et al 1999)
Chapter 1 Introduction
2
The incidence of CVC related thrombosis in humans can vary between studies according
to the CVC type used entry site of the CVC (jugular femoral subclavian axillary
cephalic vein) and time period with the CVC in place the diagnostic thrombus detection
technique (venography or ultrasonography) and criteria for diagnosis duration of follow
up diseases and presence of predisposing factors (eg previous venous thrombosis
history presence of cancer) and finally prophylactic anticoagulation treatment (Geerts
2014 Liu et al 2015 van Rooden et al 2005)
To the best of the authoracutes knowledge data about the incidence of CVC related
thrombosis detected by colour Doppler ultrasound into the lumen of the jugular vein in
dogs are rare in the available veterinary literature In one study performed in 81 dogs and
12 cats (Adamantos et al 2010) the incidence of vein thrombosis detected by palpation
or ultrasound was 2 and the incidence of intraluminal CVC thrombosis (presumed by
the inability to draw blood back) was 15 Additionally regarding to intraluminal CVC
thrombosis an incidence of 8 was reported in one case control study performed in 24
dogs receiving CVC flushing with heparinised solution (Vose et al 2019) Data about
incidence in other animal species was also found In another case-control study performed
on 37 pigs thrombosis in the caval vein related to CVC placed in the external jugular
vein (EJV) was evaluated by histological analysis however no ultrasound detection was
performed (Kohler and Kirkman 1998) In this study in pigs 94 of patients with CVC
developed thrombi in the vena cava based on sections at the site of the end of the catheter
tip and 05 and 1 cm proximal of the first section (Kohler and Kirkman 1998)
Pathogenesis of CVC related thrombosis
Two types of CVC related thrombosis are distinguished in the human medicine literature
(Geerts 2014 van Rooden et al 2005) First symptomatic thrombosis is defined as
Chapter 1 Introduction
3
thrombosis detected by diagnostic images (ultrasonography venography) when
symptoms and signs (eg localised swelling pain redness) are present (Lee and
Kamphuisen 2012) Second subclinical thrombosis is defined as thrombosis
demonstrated by the previously mentioned diagnostic images without overt symptoms or
signs (Boersma et al 2008) Additionally the CVC related thrombi can be classified as
occlusive thrombus into the CVC lumen peri-catheter related fibrin sheaths and mural
thrombi (Geerts 2014)
During the CVC insertion the puncture of the skin on the CVC insertion site and the
rupture of the blood vessels integrity activate the coagulation cascade and the CVC acts
as a matrix for adherence and accumulation of fibrin and cellular components (Smith et
al 2012) The attachment of proteins and blood cells to the internal andor external wall
of the CVC can lead to (1) (partial or complete) occlusion of the catheter lumen and (2)
sometimes if a thrombus formation starts into the lumen of the vein (attached to the vein
wall or to the CVC) a partial or complete obstruction of the blood flow andor embolism
can develop (Langston and Eatroff 2018) Additionally a study performed in humans
(Lordick et al 2003) demonstrated a relationship between CVC related thrombosis and
microbial colonisation of the catheter leading to CVC related systemic infections This
relationship was explained among others by the ease of microorganisms like
Staphylococcus aureus and Staphylococcus epidermidis to adhere to CVC related
thrombin sheaths (Boersma et al 2008)
Diagnosis of CVC related thrombosis
Venous thrombosis is commonly diagnosed in human and small animal medicine
(especially in dogs) with vascular ultrasonography with or without use of the colour
Doppler technique which is normally used to evaluate the blood flow through the blood
Chapter 1 Introduction
4
vessels (Evans and Ratchford 2018 Laurenson et al 2010) According to the literature
in humans cited below the CVC related thrombosis detected by colour Doppler
ultrasound is present if any of the following aspects are observed (1) well defined
thrombus visualised in the lumen of the vein (2) impossibility to perform compression
of the vein by slight pressure with the ultrasound probe andor (3) no detectable blood
flow signal by colour Doppler ultrasound (Bonnet et al 1989 Koumlksoy et al 1995)
Similarly in dogs the CVC related thrombosis is usually diagnosed by colour Doppler
ultrasonography through the detection of the thrombus within the lumen of the vein
andor the obstruction (partial or complete) of the normal blood flow (Bliss et al 2002)
Especially in humans the contrast venography technique is used as gold standard to
confirm thrombosis For this purpose a contrast solution is injected through a peripheral
vein to assess its passage through the bloodstream by x-ray However this diagnostic
technique is normally used only in the case of a strong clinical suspicion of venous
thrombosis that cannot be detected by ultrasonography (Laurenson et al 2010 Langston
and Eatroff 2018) In a study performed in 44 human patients requiring a CVC (Koumlksoy
et al 1995) a sensitivity of 94 and a specificity of 96 were determined for the colour
Doppler ultrasound diagnosis when it was compared with the diagnosis performed with
the contrast venography technique That results make the colour Doppler ultrasound an
accurate diagnosis method to detect CVC related thrombosis in humans (Koumlksoy et al
1995 van Rooden et al 2003)
Advanced diagnostic image techniques like computed tomography or magnetic resonance
tomography are also accurate to perform the diagnosis of CVC related thrombosis in
humans (Whitesell and Steenburg 2014) In dogs multidetector computed tomography
venography was used in one study for the diagnosis of CVC related thrombosis allowing
the identification of catheter related sheaths around the CVC external wall (Wang at al
Chapter 1 Introduction
5
2015) However the high costs of these advanced diagnostic imaging procedures and the
requirement to perform at least sedation or general anesthesia are disadvantages when
compared with ultrasonography
Finally electron microscopy (ELMI) is a diagnostic technique that has been used in
humans (Lucas et al 2017) and experimental animals (Xiang et al 1998 Xiang et al
2001) mainly to perform a microstructural evaluation of thrombi developed into the
lumen of the CVC or in the vein containing the CVC ELMI allows the ex vivo diagnosis
of very small thrombi or sleeve related thrombosis that perhaps cannot be detected by
ultrasonography or venography during the evaluation of patients with CVCs (Xiang et al
2001) However one point to consider is that it is possible that compact thrombi
developed on the CVC can be detached during the CVC removal from the vein and
thereby before its analysis by ELMI (Xiang et al 2001)
Heparin thromboprophylaxis for CVC related thrombosis
Application of CVCs in human medicine is commonly accompanied by prophylactic
anticoagulation in order to decrease the risk of development of partial or complete vein
occlusion andor thromboembolism and thereby to improve the patientrsquos comfort
(Baumann-Kreuziger et al 2015) Apart from vitamin K antagonists (Mismetti et al
2003) unfractionated heparin (UFH) (Abdelfeki et al 2004) and low molecular weight
heparin (LMWH) (Karthaus et al 2006) are anticoagulants that have been used on
studies in humans administrated by intravenous (IV) infusion or by subcutaneous (SC)
injection to prevent thrombi development into the lumen of the veins containing the CVC
(review article Baumann-Kreuziger et al 2015) Different heparin dosages have been
used as thromboprophylactic anticoagulation in humans with CVC (review article Lee
and Kamphuisen 2012) For example in five different case-control studies performed in
Chapter 1 Introduction
6
human haemato-oncological patients UFH at a daily dosage of 100 IUkg given by IV
infusion (Abdelfeki et al 2004) and LMWH at SC daily dosages of 40 mg enoxaparin
(Verso et al 2005) 5000 IU AXa dalteparin (Karthaus et al 2006 De Cicco et al 2009)
and 2850 IU AXa nadroparin (Niers et al 2007) were used Only in part of these studies
it is stated that systemic heparin was combined with flushing of the CVC using
heparinised saline solution to reduce the risk of catheter occlusion (Karthaus et al 2006)
In 3 of the 5 previously mentioned studies no statistically significant difference was found
in the incidence of CVC related thrombosis between patients receiving the
anticoagulation prophylaxis compared with the control groups without systemic
anticoagulation (Verso et al 2005 p = 035 Karthaus et al 2006 p gt 005 Niers et al
2007 p = 049) In contrast in the residual two studies a lower incidence of CVC related
thrombosis was found in the group receiving an anticoagulation prophylaxis compared
with the control groups (Abdelfeki et al 2004 150 vs 126 p = 003 De Cicco et
al 2009 400 vs 526 p = 005)
Obviously there is no consensus in the available human literature regarding the beneficial
use of anticoagulation prophylaxis to prevent CVC related thrombosis and this may
reflect great variations for example according to the study design period trial drug
administration route (Lee and Kamphuisen 2012) and the moment of the initial
anticoagulant dosage administration (Decicco et al 2009)
To the best of the authoracutes knowledge studies about thromboprophylaxis for extraluminal
CVC related thrombosis in dogs cannot be found in the available veterinary literature
However similarly to humans prophylactic anticoagulation with heparinised saline
(administrated through CVC flushing) to prevent CVC occlusion by intraluminal thrombi
was used in one case-control study performed in 24 dogs (Vose et al 2019) In the cited
study 12 dogs received dosages of 3 ml of heparinised saline solution (10 IU UFHml) to
Chapter 1 Introduction
7
flush the catheter every 6 hours during 72 hours whereas 12 control dogs received
flushing with the same volume of non-heparinised saline solution administered at the
same frequency and no statistically significant difference was found
Prediction of CVC related thrombosis
Studies regarding the relationship between laboratory biomarkers and CVC related
thrombosis are available in the human literature (Boersma et al 2016 Cheng et al 2013
Liu et al 2015) however to the best of the authoracutes knowledge literature about laboratory
tests used as predictors for CVC related thrombosis in small animals cannot be found at
all Availability of approved blood tests which can predict the risk of thrombosis
development associated to CVC already at the moment of the CVC placement are
important in order to identify an increased risk of CVC related thrombosis (Boersma et
al 2016) In the study performed by Boersma et al (2016) in 168 human patients without
indication of anticoagulation prophylaxis elevated leukocyte counts high levels of
plasminogen activator inhibitor and of coagulation factor VIII (all measured on blood
samples taken after CVC placement) were associated with an increased incidence of
symptomatic CVC-related thrombosis diagnosed by ultrasonography In another study
(Liu et al 2015) high blood levels of lactate dehydrogenase and low concentrations of
HDL and albumin were associated with the incidence for CVC related thrombosis
diagnosed by ultrasound in 324 senile patients which was in total 617 Additionally
dyslipidemia (high triglycerides low HDL and high LDLHDL ratio) was associated
with CVC related thrombosis (diagnosed by ultrasound and computer tomography
angiography) in human patients undergoing haemodialysis (Cheng et al 2013) Studies
about predictive tests for CVC related thrombosis in dogs were not found in the available
literature to the best of the authoracutes knowledge
Chapter 1 Introduction
8
Aims of the study
The aims of the preliminary methodological study of the present scientific project were
(1) to define reference intervals for viscoelastic analyses of canine haemostasis using the
ROTEMreg delta analyser and (2) to determine the precision (repeatability) of this
method And the aims of the main study of the present scientific project were (1) to
determine the incidence of catheter-induced thrombosis in canine intensive care patients
(2) to evaluate the efficacy of a prophylactic heparin regimen routinely used in the Small
Animal Clinic of the University of Veterinary Medicine Hannover and (3) to assess
whether selected haemostasis test results are predictive for an increased risk for
thrombosis formation
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
9
Chapter 2 Reference intervals for rotational thromboelastometry measurements
using the ROTEMreg delta device in dogs
Pereira JM Rohn K Mischke R
Contributions of the authors
Pereira JM performed (1) the patients data and blood samples collection (2)
haematology and ROTEMreg analysis (3) generated the reference intervals and
coefficients of variation included in the study and (4) wrote the manuscript
Rohn K performed the statistical analysis
Mischke R performed the clinical chemistry and coagulation tests and was involved in
designing and supervision of the study analysis and discussion of the data and its
statistical analysis and revision edition and correction of the manuscript
Original source Pereira J M Rohn K amp Mischke R 2020 Reference intervals
for rotational thromboelastometry measurements using the ROTEMreg delta device
in dogs Res Vet Sci 130 26ndash32 httpsdoiorg101016jrvsc202001019
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
10
Reference intervals for rotational thromboelastometry measurements using the
ROTEMreg delta device in dogs
Pereira JMa Rohn Kb Mischke Ra
aSmall Animal Clinic University of Veterinary Medicine Hannover Buumlnteweg 9 D-
30559 Hannover Germany
bInstitute of Biometry Epidemiology and Information Processing University of
Veterinary Medicine Hannover Buumlnteweg 2 30559 Hannover Germany
Corresponding author
E-mail ReinhardMischketiho-hannoverde
Original source Pereira J M Rohn K amp Mischke R 2020 Reference intervals
for rotational thromboelastometry measurements using the ROTEMreg delta device
in dogs Res Vet Sci 130 26ndash32 httpsdoiorg101016jrvsc202001019
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
11
ABSTRACT
Aims of the present study were to define reference intervals for viscoelastic analyses of
canine haemostasis using the ROTEMreg delta analyser and as a secondary aspect to
determine the precision (repeatability) of this method Blood samples from 125 clinically
healthy dogs were included Measurements were performed with commercially available
activating reagents (ex-tem in-tem and kaolin solution) as well as without activation
Additional fourfold measurements were done in 3 of the normal blood samples and in 3
samples with haemostatic alterations to evaluate the precision of the method
Coefficients of Variation (CVs) for most of the ROTEM variables were lt 10 Clot
formation time and maximum clot elasticity showed a wide inter-individual variation in
comparison with alpha angle and maximum clot firmness A multivariate analysis on
various ROTEM parameters revealed particularly a significant influence of neuter status
and a significant interrelationship between the factors sex and neuter status for
measurements with different activating reagents These results reflected the fact that
significant differences occurred only between intact and neutered females but not in
males No or only occasionally significant differences were found between groups of sex
age and size
In conclusion CVs demonstrated that the method delivers repeatable results in canine
citrated whole blood Established reference intervals should deliver valuable orientation
for the evaluation of viscoelastic properties of clotting whole blood in dogs using the
ROTEM delta analyser Neuter status in females appeared to be the most relevant
influencing factor and should be considered for the interpretation of ROTEM delta test
results
Keywords Viscoelastic measurements reference values canine age sex neuter status
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
12
1 Introduction
Heamostasis is a physiologic and dynamic process in which plasma soluble coagulation
factors blood cells and blood vessels are involved Clot formation and possible
subsequent clot lysis as well as vascular tissue repair aim to prevent haemorrhages on the
one hand and thromboses on the other hand (Kol and Borjesson 2010 Versteeg et al
2013) Conventional assays of coagulation such as prothrombin time (PT) activated
partial thromboplastin time (aPTT) and thrombin time TT have limitations especially
cellular elements are not incorporated into the test and thereby these tests reflect only
limitedly in vivo functionality of haemostasis (McMichael and Smith 2011 Crochemore
et al 2018)
The rotational thromboelastometry (ROTEMreg) currently termed as thromboelastometry
(TEM) and thromboelastography (TEG) are methods for the rapid evaluation of the
overall haemostasis capacity through the measurement of viscoelastic properties of
clotting whole blood (Kol and Borjesson 2010 Theusinger et al 2010 Sankarankutty
et al 2012 Versteeg et al 2013) In TEG the cup with the blood sample is rotating
whereas the torsion wire is fixed In ROTEMTEM the cup is fixed whereas the pin is
rotating Changes in torque ie resistance to rotation by the fibrin bridge created between
pin and cup in the clotting blood are detected electromechanically in TEG and optically
in ROTEM (Bollinger et al 2012) and displayed as a graph that indicates the time for the
formation of blood clot and its strength (Bagge et al 2016) The resulting graph reflects
the initiation and propagation of the coagulation system fibrin-platelet interactions and
clot lysis
TEG was first described in 1948 by Hellmut Harten (Hartert 1948) as an in vitro method
to evaluate the clot formation process while resembling the in vivo blood flow The
development of the actually used instrument types TEG and ROTEMregTEM were based
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
13
on that original principle Viscoelastic measurements have been widely used since the
1980s in human medicine to evaluate haemostasis and hypercoagulable states in surgical
patients (eg cardiac and hepatic surgeries) and in general in critical care units (Kol and
Borjesson 2010 Nogami 2016 Wikkelso et al 2016 Crochemore et al 2017) In
veterinary medicine viscoelastic measurements using TEG and ROTEMTEM have been
validated for use in dogs cats and horses and used to study haemostasis in different
diseases (eg infectious diseases liver diseases) and medical conditions (eg
orthopaedic surgery hyperfibrinolysis) and also to investigate the effect of
anticoagulants and other drugs (eg corticosteroids) on haemostasis (Kol and Borjesson
2010 Bruno et al 2015a 2015b Woodman et al 2015 Conversy et al 2017 Sigrist et
al 2018 Turner et al 2019) The methodology seems to be especially suitable to detect
hypercoagulability (Wiinberg et al 2008 Kol and Borjesson 2010 Marschner et al
2018)
In the available veterinary literature information about reference values for TEG were
found using kaolin as activation reagent (Bauer et al 2009) Falco et al (2012)
determined reference values for the ROTEM analyser in dogs using ex-tem in-tem and
fib-tem reagents for selected ROTEM parameters in a study on forty five healthy dogs
However data related to reference intervals obtained in an adequate number of samples
(at least 120) according to the American Society for Veterinary Clinical Pathology
(ASVCP) (Friedrichs et al 2012) and the National Committee for Clinical Laboratory
Standards (NCCLS) (Horn and Pesce 2003) could not be found in the available literature
The aim of this study is to determine reference values for the ROTEMreg delta device in
dogs using citrated whole blood with different clot activator reagents (ex-tem in-tem
kaolin) and without activator
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
14
2 Materials and methods
21 Study design
Reference intervals Single thromboelastometric measurements were performed in 125
clinically healthy dogs using the ROTEMreg analyser and different activating reagents (ex-
tem in-tem and kaolin) as well as without activation Reference values were calculated
and influences of age groups (young adult and senior) sex neuter status and size
(groups small medium and large) were assessed
Precision analysis Additional fourfold measurements using ex-tem or in-tem activating
reagents respectively were performed on 3 of the normal and also in 3 abnormal blood
samples each in order to assess within-run imprecision for these measurements
22 Animals
The clinically healthy dogs with a body weight of at least 5 kg were recruited by launching
a call in the University of Veterinary Medicine Hannover network for students and staff
members Interested dog owners received a free health check of their animals (clinical
examination and blood tests specified below) in combination with the blood collection
In addition individual privately owned patient dogs of the Small Animal Clinic with
undisturbed general health were also included if blood was collected due to another
indication (eg general health check pre-anaesthetic examination test for hereditary
diseases etc) Before taking the blood samples all dogs were fasted for at least 12 h with
free access to water 125 animals for which the examination of the health status (clinical
examination routine haematology and clinical chemistry basis coagulation profile) did
not reveal any deviations from the reference were included in the study whereas 3 of the
128 initially recruited animals had to be excluded due to laboratory abnormalities
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
15
Haematological and clinical chemistry profiles performed in order to confirm the health
status included white and red cell count haematocrit platelet counts and reticulocytes
plasma activities of alanine aminotransferase glutamate dehydrogenase alkaline
phosphatase and pseudocholinesterase as well as concentrations of total bilirubin urea
creatinine glucose total protein albumin cholesterol and ionised calcium A basis
coagulation profile including PT PTT and TT was also performed to exclude significant
abnormalities within the blood coagulation system
The 125 healthy dogs enclosed in the establishment of reference values included 48 mixed
breed dogs and the following pedigree dogs 13 Labrador Retrievers 6 Australian
Shepherds 4 Havanese dogs 3 from each of the following breeds American
Staffordshire Terriers and Cocker Spaniels 2 from each of the following breeds Beagles
Border Collies Dalmatians Elo dogs (breed not recognised by Feacutedeacuteration cynologique
international [FCI]) German Longhaired Pointers German Shorthaired Pointers Giant
Schnauzers and Golden Retrievers 1 from each of the following breeds Belgian
Malinois Bolonka Zwetna Briard Brussels Griffon American English Coonhound
Italian Mastiff Flat Coated Retriever French Bulldog German Shepherd Great Dane
Greater Swiss Mountain Dog Giant Spitz Goldendoodle Hovawart Hungarian Vizsla
Italian Bloodhound Lycanis Wolfdog Miniature American Shepherd Miniature
Schnauzer Old German Shepherd Podenco Poodle Pyrenean Shepherd Rhodesian
Ridgeback Rottweiler Schafpudel (breed not recognised by FCI) Small
Munsterlander Spanish Mastiff Tibet Terrier Weimaraner West Highland White
Terrier and Wirehaired Pointer
These dogs were aged between 6 and 154 months (median = 410 months) and had body
weights ranging from 50 to 707 kg (median = 226 kg) 30 were castrated males 33
intact males 41 spayed females and 21 intact females According to the height at withers
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
16
the dogs were divided into 3 different size groups small (le40 cm n = 22) medium (41ndash
60 cm n = 77) and large (gt60 cm n = 26) Dogs were also divided in three different age
groups young (le 24 months n = 34) adult (25ndash84 months n = 59) and senior dogs (gt84
months n = 32)
The three blood samples with haemostatic abnormalities were taken from two dogs with
complex haemostatic disorders (Disseminated Intravascular Coagulation [DIC])
including reduced platelet count and abnormal PT and APTT and one dog with
thrombocytopenia
Animals were treated in accordance with the German Animal Welfare Law The
experimental design was approved by the Animal Welfare officer of the University of
Veterinary Medicine Hannover Foundation and by the ethics committee of the
responsible authority (Lower Saxony State Office for Consumer Protection and Food
Safety reference number 17A101)
23 Blood sampling
The venipuncture area was shaved and then disinfected with alcohol The saphenous or
cephalic vein was visualised with slight pressure and punctured with disposable cannulas
(diameter 09 or 10 mm) The continuously running blood was directly collected from
the needle into the tubes containing the respective anticoagulants From each dog
approximately 10 ml of blood were collected including approximately 8 ml of citrated
blood for the measurement of haemostasis tests (ROTEM PT aPTT TT) in tubes
containing sodium citrate 313 (Eifelfangoreg GmbH amp Co KG Bad Neuenahr-
Ahrweiler Germany) in a ratio of 1 volume9 volumes of blood In addition
approximately 1 ml of EDTA blood for haematological analysis and approximately 1 ml
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
17
of lithium heparin blood for clinical chemical measurements were collected into
commercial 13 ml tubes (Sarstedt AG amp Co Nuumlmbrecht Germany)
Citrated blood was initially kept at room temperature (20-25 C) in order to avoid errors
within the preanalytical phase (Toulon et al 2017) Two ml of citrated blood were used
for the ROTEMreg analysis the residual citrate blood was centrifuged for 10 min at 3800
rpm and 5 degC then the plasma was separated by careful pipetting and it was centrifuged
a second time under the same conditions to get platelet poor plasma which was stored in
aliquots of 500 microl and 250 microl at -80 degC for coagulation tests
24 Rotational elastometry
Viscoelastic measurements using the ROTEMreg delta for the establishment of reference
intervals were performed approximately 15 min after the blood collection using activating
reagents (liquid reagents) provided by the manufacturer of the instrument (r-ex-temreg in-
temreg) and a kaolin solution (5 g1 as part of the PTT reagent CK Prest [Diagnostica
Stago SAS Asniegraveres sur Seine Cedex France]) In addition non-activated analyses
were performed using isotonic NaCl solution instead of the activating reagent in the fourth
channel of the device Fourfold measurements for precision analysis were performed
simultaneously in the four channels of the device in separate runs
According to the standard test protocol provided by the manufacturer of the ROTEMreg
delta (Werfen GmbHInstrumentation Laboratory Munich Germany) 20 μl of the
reagent for recalcification (star-temreg20) and 20 μl of the activating reagent were pipetted
into a cuvette using the automated pipette provided with the device Immediately
thereafter 300 μl of citrated whole blood (prewarmed to 37 degC for five minutes in the
heating device of the instrument and gently rocked to resuspend possibly sedimented
cells) were added to the test preparation in the cuvette using the automated pipette and
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
18
then the cuvette was laced carefully in the ROTEMreg device whereby the rotating pin
entered into the test approach
Different parameters are provided automatically by the ROTEMreg delta device and the
following variables (described according to the Pentapharm GmbH ROTEMreg delta
Manual [2009]) were used in the study
Clotting time (CT) the time (in s) from the beginning of the test (by adding the clot
activator) until the formation of fibrin starts (defined as the moment when the curve
achieves an amplitude of 2 mm) This Parameter facilitates the decision to replace clotting
factors when it is required
Clot formation time (CFT) describes the next phase of clotting by the formation of a
stable clot through the activation of thrombocytes and fibrin formation It is defined as
the time (in s) between the 2 mm amplitude and 20 mm amplitude of the curve
Alpha angle (α angle) is the angle (in degrees) between the middle axis and the tangent
to the clotting curve through the 2 mm amplitude point
Amplitude after ldquoXXrdquo minutes (eg A30 after 30 min) is the amplitude (in mm) at a
certain time after the CT has finished
Maximum clot firmness (MCF) corresponds to the maximum amplitude (in mm) reached
before the clot is dissolved by fibrinolysis This parameter measures the firmness of the
clot and its quality Like CFT and α angle it facilitates the decision for substitution therapy
with platelet concentrate andor fibrinogen especially in humans
Maximum clot elasticity (MCE) is a parameter calculated from MCF and may be superior
at high amplitudes as compared to the MCF
Lysis index at 30 45 and 60 min (LI30 LI45 LI60) represents the fibrinolysis (in
percentage) 30 45 and 60 min after the CT It may facilitate the decision for or against a
therapy with anti-fibrinolytic drugs
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
19
Maximum lysis (ML) describes the degree of fibrinolysis relative to the MCF achieved
during the measurement (percent loss of clot firmness)
25 Statistical methods
Original data obtained from the ROTEMreg delta device software were transferred into the
Microsoftreg Excelreg formal (Microsoft Office Professional Plus 2013) The Statistical
analyses were performed using the SAS Enterprise Guide Version 71 (SAS Institute Inc
Cary NC USA)
Coefficients of Variation (CVs) for selected relevant parameters of the ROTEM delta
analysis using ex-tem and in-tem regents were calculated for the fourfold measurements
on 3 normal and 3 abnormal blood samples in order to assess within-run imprecision CVs
for the results of each sample were calculated by dividing the standard deviation of the
data set by its arithmetic mean Finally the median of the CVs for each group of 3 samples
was calculated The reference values were calculated according to recommendations of
the American College of Veterinary Clinical Pathology (ACVCP) based on non-
parametric methods (25 and 975 quantiles) supplemented by 95 confidence
intervals (Friedrichs et al 2012)
Data were analysed for Standard normal distribution by Kolmogorov Smirnov test and
assessment of residuals of the analysis of variance (ANOVA) Because these tests
indicated almost normally distributed values for the studied ROTEM variables data were
analysed by a fourway ANOVA stratified by the activating reagent (ex-tem in-tem
kaolin or without activation) to test the influence of size age sex and neuter status on
several parameters and taking all interactions into account Post hoc t-tests were
performed when indicated The significance level was set to P lt 05
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
20
3 Results
31 Precision of the method
For all the parameters evaluated in the study the CVs for the fourfold measurement in
healthy dogs were less than 10 (Table 1) In the case of dogs with haemostatic
abnormalities CVs of the vast majority of ROTEM variables were also less than 10
except the variables CFT and ML irrespective whether ex-tem or in-tem reagents were
used (Table 2)
Table 1 Coefficients of variation () for rotational thromboelastometry using the
ROTEMreg delta analyser in normal blood samples
Activator CT (s) CFT (s) α (deg) A30 (mm) MCF (mm) MCE(mm) LI30 () LI45 () LI60 () ML ()
Ex-temreg 875 629 262 213 179 634 000 020 037 534
In-temreg 245 596 166 117 135 299 000 000 046 216
Abbreviations CT ndash clotting time CFT ndash clot formation time α ndash alpha angle A30 ndash
amplitude after 30 min MCF ndash maximum clot firmness MCE ndash maximum clot elasticity
LI 30 LI 45 LI 60 ndash Lysis index at 30 45 and 60 min ML ndash maximum lysis
Median values of the measurements of 3 samples
Table 2 Coefficients of variation () for rotational thromboelastometry using the
ROTEMreg delta analyser in canine blood samples with haemostatic alterations
Activator CT (s) CFT (s) α (deg) A30 (mm) MCF (mm) MCE(mm) LI30 () LI45 () LI60 () ML ()
Ex-temreg 668 1478 393 218 268 582 0 018 000 848
In-temreg 228 799 461 219 251 371 0 0 017 283
Median values of the measurements of 3 samples from patients with DIC (n = 2)
thrombocytopenia (n = 1) abbreviations please see legend to Table 1
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
21
32 Reference intervals
Activation by ex-tem reagent (tissue factor) resulted in faster CTs when compared to
activation via the intrinsic pathway using ellagic acid (in-tem) or kaolin reagents whereas
the CFT was much less affected by the activating reagent (Table 3) Especially CFT and
MCE showed a wide inter-individual variation in comparison for example with α angle
and MCF regardless of the used activating reagent (Table 3)
33 Influence of age sex neuter status and size
ANOVA did not reveal statistically significant differences in ROTEM delta parameters
between dog groups of variable size or sex (P gt 05) Age had influence only on CT and
only when the in-tem reagent was used (P = 0169 ANOVA) but not when using the ex-
tem reagent (P = 7105) Post hoc comparisons using t-test of the in-tem results between
age groups revealed shorter CTs in the group of senior dogs when compared with young
dogs (P = 0194) but not between adult dogs and these two groups ANOVA showed
particularly influences of the neuter status and a significant interrelationship between the
factors sex and neuter status on different ROTEM delta parameters using ex-tem and in-
tem respectively as activator (Tables 4 and 5) ANOVA revealed also a significant
influence of neuter status on different ROTEM parameters when kaolin was used as
activating reagent (CFT α angle MCF MCE) whereas non-activated measurements
were not subject to any significant influence of the assessed factors (results not shown)
The interrelationship between sex and neuter status mentioned above reflected the fact
that significant differences between neutered and not neutered dogs were only found in
females The statistical analysis using the t-test to compare intact vs neutered dogs
stratified by sex showed statistically significantly (P lt 05) longer CTs and CFTs and
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
22
lower α angle A30 MCF and MCE values in spayed when compared to intact females
(Tables 4 and 5)
Chapter 2 Reference intervals for rotational thromboelastometry measurements using the ROTEMreg delta device in dogs
23
Table 3 Reference values (median reference interval based on 25 - and 975 - quantiles) for thromboelastometric measurements with the ROTEMreg delta analyser in
dogs (n=125 95 confidence intervals in brackets) Abbreviations please see legend to Table 1
Activator
Statistical
para-meter
CT
(s)
CFT
(s)
Α
(deg)
A30
(mm)
MCF
(mm)
MCE
(mm)
LI30
()
LI45
()
LI60
()
ML
()
Ex-temreg
Median 450
[430ndash470]
110
[105ndash115]
680
[670ndash690]
600
[580ndash610]
610
[600ndash620]
154
[147ndash164]
100
[960ndash100]
100
[100ndash100]
970
[960ndash980]
220
[210ndash240]
25 340
[270ndash350]
650
[490ndash710]
540
[420ndash560]
460
[410ndash480]
460
[420-490]
870
[720ndash950]
100
[100-100]
870
[800ndash960]
700
[640ndash820]
800
[100ndash110]
975 830
[790ndash990]
220
[201ndash315]
790
[770ndash800]
770
[690ndash770]
730
[700ndash770]
264
[230-334]
100
[100ndash100]
100
[100ndash100]
100
[990ndash100]
590
[530ndash680]
In-tem
Median 190
[187ndash201]
116
[111ndash125]
680
[670ndash690]
570
[560ndash580]
590
[580ndash600]
143
[135ndash149]
100
[100ndash100]
100
[100ndash100]
980
[980ndash980]
180
[170ndash190]
25 135
[112ndash142]
730
[590ndash800]
510
[460ndash570]
450
[410ndash470]
480
[410ndash500]
920
[710ndash101]
100
[950ndash100]
990
[820ndash990]
930
[740ndash930]
500
[100ndash700]
975 283
[268ndash291]
251
[198ndash227]
750
[750ndash780]
660
[660ndash690]
680
[660ndash690]
210
[195ndash227]
100
[100ndash100]
100
[100ndash100]
100
[100ndash100]
270
[260ndash380]
Activator
Statistical
para-meter
CT
(s)
CFT
(s)
Α
(deg)
A30
(mm)
MCF
(mm)
MCE
(mm)
LI30
()
LI45
()
LI60
()
ML
()
Kaolin
Median 162
[154ndash173]
870
[820ndash910]
740
[720ndash750]
500
[470ndash530]
550
[530ndash560]
121
[115ndash127]
960
[940ndash980]
840
[790ndash880]
710
[640ndash770]
500
[450ndash540]
25
109
[990ndash115]
470
[440ndash580]
560
[490ndash600]
110
[700ndash230]
390
[380ndash400]
640
[600ndash680]
270
[140ndash480]
110
[800ndash260]
800
[400ndash170]
200
[160ndash240]
975 246
[230ndash337]
214
[185ndash290]
810
[790ndash810]
640
[630ndash690]
650
[650ndash690]
187
[182ndash223]
100
[100ndash100]
990
[980ndash100]
940
[930ndash990]
970
[930ndash100]
Without activation
Median 607
[495ndash662]
221
[194ndash265]
510
[460ndash550]
520
[500ndash530]
540
[520ndash550]
115
[109ndash123]
100
[100ndash100]
100
[100ndash100]
980
[980ndash990]
170
[160ndash190]
25 203
[123ndash215]
860
[550ndash930]
190
[120ndash230]
350
[230ndash370]
370
[260ndash400]
600
[350ndash670]
100
[950ndash100]
980
[890ndash990]
930
[840ndash950]
400
[000ndash500]
975 1356
[1305ndash1721]
811
[688ndash1333]
730
[710ndash790]
660
[640ndash720]
660
[640ndash730]
197
[179ndash265]
100
[100ndash100]
100
[100ndash100]
100
[100ndash100]
290
[280ndash320]
Chapter 2 Reference intervals for rotational thromboelastometry measurements using the ROTEMreg delta device in dogs
24
Table 4 Median values (minimum and maximum in brackets) for different ROTEMreg variables using ex-tem reagent stratified by sex and neuter status
Group
CT
(s)
CFT
(s)
Α
(deg)
A30
(mm)
MCF
(mm)
MCE
(mm)
LI30
()
LI45
()
LI60
()
ML
()
Female intact 430
(320ndash530)
960
(590ndash198)
710
(570ndash800)
620
(480ndash730)
640
(510ndash740)
179
(103ndash282)
100
(100ndash100)
100
(980ndash100)
960
(800ndash100)
230
(100ndash640)
Female spayed 460
(270ndash830)
121
(650ndash224)
660
(520ndash770)
580
(430ndash690)
590
(440ndash700)
146
(790ndash230)
100
(960ndash100)
100
(800ndash100)
970
(690ndash100)
240
(100ndash590)
p-value (t-test)
Female
intact vs spayed
00025 00063 00049 00023 00016 00015 na na na na
Male intact 450
(320ndash990)
105
(650ndash315)
690
(420ndash790)
590
(420ndash700)
610
(440ndash710)
154
(770ndash244)
100
(100ndash100)
100
(870ndash100)
970
(650ndash100)
220
(700ndash680)
Male castrated 460
(340ndash930)
111
(490ndash255)
685
(480ndash800)
590
(410ndash770)
600
(420ndash770)
152
(720ndash334)
100
(100ndash100)
100
(830ndash100)
980
(640ndash100)
205
(500ndash590)
p-value (t-test)
Male
Intact vs castrated
08326 05106 04756 01799 01568 02163 na na na na
p-values (ANOVA)
Factor Sex
05428 04847 06612 02206 01887 01630 08489 07893 07689 02758
Factor neuter status 01872 00167 00173 00039 00037 00028 07141 04737 09356 05948
Interrelationship
sexneuter status 00383 00968 00782 00488 00472 00319 04943 08611 05422 00160
na ndash not analysed all significant P-values (lt 005) are highlighted in bold print
Chapter 2 Reference intervals for rotational thromboelastometry measurements using the ROTEMreg delta device in dogs
25
Table 5 Median values (minimum and maximum in brackets) for different ROTEMreg variables using the in-tem reagent stratified by sex and neutering status
Group
CT
(s)
CFT
(s)
Α
(deg)
A30
(mm)
MCF
(mm)
MCE
(mm)
LI30
()
LI45
()
LI60
()
ML
()
Female intact 186
(126ndash238)
940
(590ndash164)
720
(620ndash780)
610
(490ndash690)
620
(530ndash690)
166
(113ndash227)
100
(100ndash100)
100
(990ndash100)
980
(930ndash100)
170
(100ndash300)
Female spayed 201
(149ndash291)
1220
(820ndash262)
670
(500ndash740)
550
(450ndash660)
570
(480ndash680)
133
(910ndash214)
100
(950ndash100)
100
(820ndash100)
980
(740ndash100)
190
(500ndash380)
p-value (t-test)
Female
intact vs spayed
na na lt0001 00009 00005 00005 na na na na
Male intact 199
(117ndash283)
122
(730ndash277)
680
(460ndash750)
570
(430ndash660)
590
(450ndash660)
146
(810ndash192)
100
(100ndash100)
100
(990ndash100)
980
(930ndash100)
180
(600ndash270)
Male castrated 181
(112ndash288)
123
(820ndash222)
670
(540ndash740)
565
(410ndash630)
585
(410ndash640)
138
(710ndash180)
100
(100ndash100)
100
(970ndash100)
980
(890ndash100)
180
(800ndash260)
p-value (t-test)
Male
intact vs castrated
na na 08232 01401 01162 00849 na na na na
p-values (ANOVA)
Factor Sex
02105 03317 03370 00877 01258 00510 09564 07832 08117 03638
Factor neuter status 04289 00660 00619 00107 00088 00054 07152 09795 07056 08299
Interrelationship
sexneuter status
04310 00550 00408 00707 01733 00996 05240 05982 08047 04578
na ndash not analysed all significant P-values (lt 005) are highlighted in bold print
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
26
4 Discussion
41 Coefficients of variation
In accordance with the literature CVs below 10 indicate adequate repeatability in intra-
assays evaluations (Steiner et al 2003 Hua et al 2018) According to our results
obtained from the fourfold measurements in citrated whole blood ROTEM delta
measurements on healthy dogs and dogs with haemostatic alterations fulfil these
requirements with only a few exceptions concerning CFT and ML values in abnormal
samples
Our results regarding repeatability are similar to those obtained for several TEM
parameters in previous studies performed in animals In one study the imprecision of the
ROTEM analyser was evaluated by duplicate measurements in 10 blood samples of
healthy dogs and as result of that evaluation a good precision (CVs below 10 ) was
calculated for CT CFT MCF and α angle but in contrary to our results a low
repeatability was found for ML (CVs of 233 [ex-tem] and 285 [in-tem] own values
534 and 216 ) (Falco et al 2012)
In a previous feline study using ex-tem and in-tem as activating reagents for the ROTEM
delta repeatability was assessed similarly to the present study based on fourfold
measurements of blood samples of two healthy individuals (Doumlderlein and Mischke
2015) The results also revealed CVs below 10 for most of the parameters evaluated
The partially low repeatability of ML values may reflect the vulnerability of this variable
which is not only influenced by the complex clot formation process but also by its
subsequent lysis Reproducibility of ex-tem or in-tem activated measurements of the
ROTEM delta analyser which was not investigated in our study was studied in blood
from healthy and unhealthy horses by two different operators using three different
ROTEM devices Results indicated acceptable reproducibility (Junge et al 2016)
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
27
42 Reference intervals
The number of 125 healthy dogs used in the present study to create reference intervals is
regarded adequate according to the guidelines suggested by the American Society of
Veterinary Clinical Pathology (ASVCP) and the National Committee for Clinical
Laboratory Standards (NCCLS) who recommend to include at least 120 individuals
(Friedrichs et al 2012) To the best of our knowledge reference intervals for the most
relevant ROTEMreg delta parameters obtained in an adequate number of samples of whole
citrated canine blood have not been published so far A previous study was performed on
a smaller number of 45 dogs (Falco et al 2012) The median values for reference
intervals obtained in the present study are partially similar to those calculated in the
mentioned study based on 45 healthy dogs (eg ex- tem CT 610 s450 s [Falco et al
2012own results] CFT 150 s 110 s α angle 620deg680deg MCF 530 m 610 mm ML
180 220 in-tem CT 235 s190 s CFT 136 s116 s α angle 660deg680deg MCF
560 mm590 mm ML 30 180 ) However in comparison to our results the
reference intervals in the mentioned study are slightly wider (eg ex-tem CT 29ndash92
s34ndash83 s [Falco et al 2012own results] CFT 540ndash275 s650ndash220 s α angle 470ndash
790deg540ndash790deg MCF 360ndash730 mm460ndash730 mm ML 000ndash560 800ndash590
in-tem CT 126ndash363 s135ndash283 s CFT 470ndash224 s730ndash251 s aα angle 550ndash
810deg510ndash750deg MCF 500-ndash750 mm480ndash680 mm ML 000ndash400 500ndash270 )
although the previous study used the 5 and 95 quantiles to define limits of the
reference range whereas we calculated the commonly used 25 and 975 quantiles
to capture the central 95 of reference values (Friedrichs et al 2012)
Although it is desirable that each laboratory should specifically determine its own
reference intervals the results obtained in this study may provide an orientation for other
laboratories to interpret ROTEM delta parameters analysed in citrated canine blood In
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
28
order to test transference of the reference ranges to another laboratory a validation
procedure with measurement of 20 samples should be performed (Friedrichs et al 2012)
Although intra-individual control values (subject-based reference values) are regarded
superior to population based reference values (Wiinber et al 2007) the latter have to be
routinely used in clinical patients due to lacking individual controls especially for specific
tests such as ROTEM analyses In addition the calculated reference values cannot be
used for the fully automated device ROTEM sigma because - according to an actual
human study - values of part of the parameters do not correlate well between both
instruments (Gillissen et al 2019)
43 Influence of size age sex and neuter status
In previous studies in humans (Attard et al 2013) and animals (Mischke 1994 Borrelli
et al 2017) age has been described as an influencing factor for different haemostasis
parameters We also found an influence of age on the CT with shorter times in senior
dogs (gt 84 months) compared with young dogs (up to 24 months) which was limited to
measurements activated by the in-tem reagent
This age dependency confirms in principle a previous study on 20 healthy dogs
(Barthelemy et al 2015) which found shorter CTs in elderly dogs and results of a human
study where individuals with an age gt 45 years showed shorter CTs compared with
individuals lt 45 years old (Sucher et al 2011) However in the cited studies this effect
was only seen when ex-tem but not when the in-tem reagent was used
Based on the age distribution of our dogs which were primarily recruited to establish
reference intervals we cannot deliver information regarding very young dogs A human
study focusing on ROTEM reference ranges in children demonstrated significant
differences for nearly all ROTEM parameters with the most striking differences in
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
29
subjects aged 0-3 months (Oswald et al 2010) The limited number of dogs with an age
lt 1 year which did not allow to consider these separately in the present study may also
be one reason for the different results in a feline study (Doumlderlein and Mischke 2015) In
the cited study on healthy cats age showed an influence on CT but contrary to our study
the CT was shorter in young individuals (6-12 months) compared with older ones In
addition this was only the case in measurements using kaolin and in non-activated
measurements When compared to humans and cats uni- or multivariate analyses
considering factors age and breed or size based on do populations including different
breeds have the limitation that they can be biased by the variate life expectancy between
small medium and large dog breeds (Willems et al 2017) Apart from age we found a
significant influence only for the neuter status in bitches reflected by a significant
interaction effect between factors neuter status and sex in the ANOVA analysis
Especially for CFT values (eg activated by ex-tem median values of 121 s [spayed] vs
96 s [intact]) this difference seems to be also clinically relevant with respect to the
interpretation of ROTEM results In contrast although also significantly different
absolute and relative differences of CT values were small (eg activated by ex-tem 46 s
[spayed] vs 43 s [intact]) These differences may not only be considered for the result
interpretation of clinical patients but indicate also the necessity for sex-matched patient
groups in scientific studies
The obviously more active haemostasis system in intact females is probably related to an
influence of sex hormones (specifically estrogens) on the coagulation system
Accordingly ex vivo and in vivo studies demonstrated the activating properties of
endogenous estrogens on the haemostasis system In one ex vivo study addition of
endogenous estrogens (estrone [E1] 17szlig-estradiol [E2] and estriol [E3]) to blood samples
from men resulted in shorter mean values of the reaction time in TEG analyses
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
30
corresponding to CT in ROTEM measurements (452-537 s vs 698 s [control]) and of
the TEG variable kinetics (corresponding to the CFT in ROTEM analyses) (Swanepoel
et al 2017) In a further study subcutaneous injection of E2 into male rats induced a
significant increase in activities of coagulation factors X (by 8 ) and XI (by 10-24 )
however did not show any significant effect on PT aPTT and factor VII activity (Franco-
Murillo and Jaimez 2017)
In the cited studies also the effects of the synthetic estrogen ethinylestradiol [EE] were
studied which interestingly seems to have an inhibiting effect as indicated by prolonged
reaction time and kinetics (TEG) compared with the control in the human ex vivo study
and significantly longer PTs and aPTTs and lower factor VII and X activities in the study
on rats As expected we did not find a significant influence of size on ROTEM parameters
in dogs To the best of the authoracutes knowledge size has not been examined as an
influencing factor on viscoelastic measurements or any other haemostatic variable in
dogs Apart from Greyhounds there is limited literature on breed comparisons of
haemostatic variables (Clemons and Meyers 1984 Mischke 1994 Nielsen et al 2007
Nielsen et al 2010 Boyd et al 2018) indicating very limited breed differences for
example for factor VII (Mischke 1994) However these also do not indicate systematic
influence of size and in addition possible breed-specific effects have to be considered
As already stated one limitation of the study is the limited number of young animals
which does not allow to verify reference values for animals in this stage of life In
addition with respect to the main aim of the study possible influencing factors such as
platelet count and haematocrit were not considered in the statistical analysis Finally the
relatively small number of dogs used for the precision analysis which express the fact
that this was only a secondary aspect of the study may be regarded as a limitation At the
same time these considerations provide space for further investigations
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
31
5 Conclusions
Results of the precision analysis revealed acceptable results and thereby confirm that the
ROTEMreg delta analyser delivers repeatable results to measure overall haemostasis in
canine citrated blood samples The established reference values should offer a reliable
basis for the interpretation of thromboelastometric measurements in whole blood using
the device ROTEMreg delta in dogs with suspected haemostasis disorders Neuter status
in female dogs appeared to be a relevant influencing factor on most important ROTEMreg
parameters and should be considered for result interpretation and with respect to
experimental designs
6 Acknowledgments
Jose Mauricio Pereira received a scholarship from the Universidad Nacional de Costa
Rica (reference code JB-C-1107-2016)
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
32
7 References
1 Attard C van der Straaten T Karlafitis V Monagle P Ignjativic V 2013 Developmental
hemostasis age-specific differences in the levels of hemostatic proteins J Thromb and
Haemost 11 1850ndash1854
2 Bagge A Schoumltt U Kander T 2016 Effects of naturopathic medicines on Multiplate and
ROTEMreg A prospective experimental pilot study in healthy volunteers BMC Complemen
Altern Med 17 (16) 64
3 Bartheacutelemy A Rannou B Forterre M Verwaerde P Bonnet-Garin JM Pouzot-Nevoret
C Goy-Thollot I 2015 Differences between coagulation and cytokine profiles in dogs of
different ages Vet J 205 410ndash412
4 Bauer N Eralp O Moritz A 2009 Establishment of reference intervals for kaolin-activated
thromboelastography in dogs including an assessment of the effects of sex and anticoagulat use
J Vet Diagn Investi 21 641ndash648
5 Bollinger D Seeberger MD Tanaka KA 2012 Principles and practice of
thrromboelastography in clinical coagulation management and transfusio practice Transfus Med
Rev 26 1ndash13
6 Borrelli A Botto A Maurella C Falco S Pagani E Miniscaldo B Tarducci A Bruno
B 2017 Thromboelastometric assesment of hemostasis in newborn Piemontese calves J Vet
Diagn Invest 29 293ndash297
7 Boyd CJ Claus MA Raisis AL Hosqood G Sharp CR SmartL 2018
Hypocoagulability and platelet dysfunction are exacerbated by synthetic colloids in a canine
hemorrhagic shock model Front Vet Sci 5 279
8 Bruno B Maurella C Falco S Tarducci A Zanatta R DrsquoAngelo A Borrelli A 2015a
Thromboelastometric evaluation of hemostasis in dogs infected with Leishmania infantum J Vet
Emerg Crit Care (San Antonio) 25 502ndash511
9 Bruno B Maurella C Falco S Tarducci A Zanatta R Gianella P DrsquoAngelo A Piras L
Di Bella A Borrelli A 2015b Assessment of coagulation utilizing thromboelastometry in dogs
undergoing orthopedic surgery J Vet Emerg Crit Care (San Antonio) 25 358ndash363
10 Clemons RM Meyers KM 1984 Acquisition and aggregation of canine blood platelets Basic
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
33
mechanisms of function and differences because of breed origin Am J Vet Res 45 137ndash144
11 Conversy B Blais MC Dunn M Gara-Boivin C del Castillo JRE 2017 Anticoagulant
activity of oral rivaroxaban in healthy dogs Vet J 223 5ndash11
12 Crochemore T Piza FMT Rodriacuteguez RDR Guerra JCC Ferraz LJR Correcirca TD
2017 A new era of thromboelastometry Einstein (Sao Paulo) 15 380ndash385
13 Crochemore T Correcirca TD Lance MD Solomon C Neto AS Guerra JCC Lellis PS
Bernz LM Nunes N Mancio CM Yokoyama APH Silva E 2018 Thromboelastometry
profile in critically ill patients A single-center retrospective observational study PLoS One 13
e0192965
14 Doumlderlein E Mischke R 2015 Reference intervals for thromboelastometry with the ROTEMreg
delta in cats Res Vet Sci 100 271ndash276
15 Falco S Bruno B Maurella C Bellino C DacuteAngello A Gianella P Tarducci A Zannata
R Borrelli A 2012 In vitro evaluation of canine hemostasis following dilution with
hydroxyethyl starch (13004) via thromboelastometry J Vet Emerg Crit Care (San Antonio)
22 640ndash645
16 Franco-Murillo Y Jaimez R 2017 Inhibitory effect of ethinylestradiol on coagulation factors
in rats Exp Anim 66 107ndash113
17 Friedrichs KR Harr KE Freeman KP Szladovits B Walton RM Barnhart KF Blanco-
Chavez J 2012 ASVCP reference interval guidelines Determination of de novo reference
intervals in veterinary species and other related topics Vet Clin Pathol 41 441ndash453
18 Gillissen A va den Akker T Caram-Deelder C Henriquez DDCA Bloemenkamp
RWM Eikenboom J van der Bom JG de Maat MPM 2019 Comparison of
thromboelastometry by ROTEMreg Delta and ROTEMreg Sigma in women with postpartum
haemorrhage Scand J Clin Lab Invest 79 32ndash38
19 Harttert H 1948 Blutgerinnungstudien mit der Thrombelastographie einem neuen
Untersuchungsverfahren Klin Wochenschr 26 577ndash583
20 Horn P Pesce A 2003 Reference intervals and update Clin Chim Acta 334 5ndash23
21 Hua Yanhun Qiu W Xiao Q Wu Q 2018 Precision (repeatability and reproducibility) of
ocular parameters obtained by the Tomey OA-2000 biometer compared to the IOLMaster in
healthy eyes PLoS One 13 e0193023
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
34
22 Junge H Ringer S Mayer N Schwarzwald C 2016 Assesment of method reliability and
determination of reference intervals for rotational thromboelastometry in horses J Vet Emerg
Crit Care (San Antonio) 26 691ndash703
23 Kol A Borjesson DL 2010 Application of thrombelastographythromboelastometry to
veterinary medicine Vet Clin Pathol 39 405ndash416
24 Marschner CB Wiinberg B Tarnow I Markussen B Kuumlhnel L Bochsen L Kristensen
AT 2018 The influence of inflammation and hematocrit on clot strength in canine
thromboelastographic hypercoagulability J Vet Emerg Crit Care (San Antonio) 28 20ndash30
25 McMichael MA Smith SA 2011 Viscoelastic coagulation testing technology applications
and limitations Vet Clin Pathol 40 140ndash153
26 Mischke R 1994 Activity of coagulation factors II V VII and X in healthy dogs- dependence
on age sex and breed Berl Muumlnch Tieraumlrztl Wochenschr 107 289ndash294
27 Nielsen A Zois N Pedersen H Olse L Tarnow I 2007 Platelet function in dogs bread
differences and effect of acetylsalicylic acid administration Vet Clin Pathol 36 267ndash273
28 Nielsen L Kjelgaard‐Hansen M Jensen A Kristensen A 2010 Breed‐specific variation of
hematologic and biochemical analytes in healthy adult Bernese Mountain dogs Vet Clin Pathol
39 20ndash28
29 Nogami K 2016 The utility of thromboelastography in inherited and acquired bleeding
disorders Br J Haematol 174 503ndash514
30 Oswald E Stalzer B Heitz E Weiss M Schmugge M Strasak A Innerhofer P Haas T
2010 Thromboelastometry (ROTEMreg) in children Age-related reference ranges and correlations
with standard coagulation tests Br J Anaesth 105 827ndash835
31 Sankarankutty A Nascimento B Teodoro da Luz L Rizoli S 2012 TEGreg and ROTEMreg
in trauma similar test but different results World J Emerg Surg 7 (Suppl 1) S3
32 Sigrist NE Schefer RJJ Kutter APN 2018 Characteristics of hyperfibrinolysis in dogs and
cats demonstrated by rotational Thromboelastometry (ROTEM) Vet J 242 67-73
33 Steiner J Teague S Williams D 2003 Development and analytic validation of an enzime-
linked immunosorbent assay for the measurement of canine pancreatic lipase immunoreactivity in
serum Can J Vet Res 68 161ndash168
34 Sucker C Tharra K Lithmate J Scharf RE Zotz RB 2011 Rotation thromboelastography
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
35
(ROTEM) parameters are influenced by age gender and oral contraception Perfusion 26 334ndash
340
35 Swanepoel A Emmerson O Pretorius E 2017 The effect of endogenous and synthetic
estrogens on whole blood clot formation and erythrocyte structure Microsc Microanal 23 599ndash
606
36 Theusinger OM Nuumlrnberg J Asmis LM Seifert B Spahn DR 2010 Rotation
thromboelastometry (ROTEM) stability and reproducibility over time Eur J Cardiothorac Surg
37 677ndash683
37 Toulon P Metge S Hangard M Zwahlen S Piaulenne S Besson V 2017 Impact of
different storage times at room temperature of unspun citrated blood samples on routine
coagulation tests results Results of a bicenter study and review of the literature Int J Lab
Hematol 3 458ndash468
38 Turner JS Kutter APN Sigrist NE 2019 Correlation of rotational thrromboelastometry
(ROTEM) parameters with platelet count and their ability to predict thrombocytopenia in dogs
Res Vet Sci 126 45ndash50
39 Versteeg HH Heemskerk JWM Levi M Reitsma PH 2013 New Fundamentals in
Hemostasis Physiol Rev 93 327ndash358
40 Wiinberg B Jensen AL Kjelgaard-Hansen M Rojkjaer R Johansson PI Gade LP
Gram DX Kristensen AT 2007 Study on biological variation of haemostatic parameters in
clinically healthy dogs Vet J 174 62ndash68
41 Wiinberg B Jensen AL Johansson PL Rozanski E Tranholm M Kristensen AT 2008
Thromboelastographic evaluation of hemostatic function in dogs with disseminated intravascular
coagulation J Vet Intern Med 22 357ndash365
42 Wikkelsoslash A Wetterslev J Moslashller AM Afshari A 2016 Thromboelastography (TEG) or
thromboelastometry (ROTEMreg) to monitor haemostatic treatment versus usual care in adults or
children with bleeding Cochrane Database Syst Rev Art
httpsdoiorg10100214651858CD007871pub3 No CD007871
43 Willems A Paepe D Marynissen S Smets P Van de Maele I Picavet P Duchateau L
Daminet S 2017 Results of screening of apparently healthy senior and geriatric dogs J Vet
Intern Med 31 81ndash92
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
36
44 Woodman J Wagg CR Boysen SR Leguillete R Mizen K Roy MF 2015 Evaluation
of coagulation via thromboelastography in healthy horses administered dexamethasone Can Vet
J 56 1271ndash1274
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
37
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of
standard heparin prophylaxis and correlation to haemostatic changes
Pereira JM Hewicker-Trautwein M Rohn K von Depka Prondzinski M
Mischke R
Contributions of the authors
Pereira JM performed (1) the ultrasound evaluations and blood sampling of the
patients under study (2) haematology analysis (3) injection of the anticoagulation
prophylaxis (partially) to the patients under study (4) analysis of samples (collaboration)
at Werlhof Institute Medical Care Center (5) analysis of some haemostasis test
(collaboration) (6) statistical analysis (partially) (7) data documentation (8) and wrote
the manuscript
Hewicker-Trautwein M performed the electron microscopy analysis
Rohn K performed the statistical analysis
von Depka Prondzinski M performed some haemostasis test (thrombin generation
assay and endogenous thrombin potential)
Mischke R performed the coagulation and haemostasis test was involved in designing
and supervision of the study analysis and discussion of the data and its statistical analysis
and revision edition and correction of the manuscript
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
38
Central venous catheter induced thrombosis in dogs ndash efficacy of standard heparin
prophylaxis and correlation to haemostatic changes
Joseacute Mauricio Pereira1 Marion Hewicker-Trautwein2 Karl Rohn3 Mario von Depka
Prondzinski4 Reinhard Mischke1
1Small Animal Clinic University of Veterinary Medicine Hannover Foundation
Buumlnteweg 9 D-30559 Hannover Germany
2Institute for Pathology University of Veterinary Medicine Hannover Foundation
Buumlnteweg 17 D-30559 Hannover Germany
3Institute of Biometry Epidemiology and Information Processing University of
Veterinary Medicine Hannover Foundation Buumlnteweg 2 30559 Hannover Germany
4Werlhof Institute Schillerstrasse 23 30159 Hannover Germany
Abbreviations anti-Xa anti-factor Xa BW body weight CVC central venous catheter
EJV external jugular vein ELMI electron microscopy IV intravenous JV jugular vein
LMWH low molecular weight heparin ML maximum lysis (ROTEM) PIVC
peripheral intravenous catheter ROTEM rotational elastometry SC subcutaneous
TGA thrombin generation assay TID three times a day TT thrombin time UFH
unfractionated heparin
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
39
Corresponding author
E-mail addresses matiscrgmailcom (JM Pereira) MarionHewicker-
Trautweintiho-hannoverde (M Hewicker-Trautwein) KarlRohntiho-hannoverde
(K Rohn) mailwerlhof-institutde (M von Depka Prondzinski)
ReinhardMischketiho-hannoverde (R Mischke)
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
40
ABSTRACT
Central venous catheters (CVC) are of importance for intensive care management of
canine patients Thrombosis of the jugular vein is a severe complication related to the
CVC use However only little is known about the incidence of CVC-associated
thromboses in dogs and the necessity and efficacy of prophylactic antithrombotic
regimens The objectives of the present study were (1) to determine the incidence of
catheter-induced thrombosis in canine intensive care patients and thereby (2) to evaluate
the efficacy of a routine prophylactic heparin treatment and finally (3) to assess whether
initial changes of selected haemostasis parameters are predictive for an increased risk for
thrombosis formation 29 dogs hospitalised in the Small Animal Clinic University of
Veterinary Medicine Hannover Foundation and receiving a central venous catheter
(CVC) in the external jugular vein (EJV) for medical reasons were included in the final
assessment 17 dogs received the standard antithrombotic dosage regimen of
unfractionated heparin (UFH 150 IUKG BW TID sc) and 12 dogs (usually surgical
patients) the reduced dosage (75 IUKG BW TID sc) A colour Doppler ultrasound of
the respective EJV in order to detect thrombus formation and blood collection for
haemostasis tests were performed immediately before the installation procedure (day 0)
and on days 1 3 5 7 and so on after the CVC insertion Haemostasis tests included
prothrombin time activated partial thromboplastin time thrombin time antithrombin
activity D-dimer concentration thrombin generation and rotational elastometry as well
as the heparin activity using a chromogenic anti-factor Xa test Finally electron
microscopy (ELMI) of sections of the removed CVC was performed In 8 dogs
sonographic examination revealed thrombus formation in the lumen of EJV and in 1927
CVCs assessed using ELMI thrombi on the external CVC surface were detected without
significant correlation between both methods Plasmatic heparin activities showed great
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
41
variability but no significant differences were found between dogs with or without CVC-
associated thrombus formation From the initially performed haemostasis tests high
fibrinogen concentrations and low maximum lysis values as measured by the ROTEM
delta device (ex-tem reagent) were associated with sonographically detectable thrombi
The results of the present study indicate that CVC-associated thromboses occur (eg 28
[829] of the patients as detected by ultrasound) although severe thromboses (high
grades) are relatively rare in intensive care canine patients receiving a routine
anticoagulant treatment A high fibrinogen concentration and low maximum lysis
(ROTEM ex-tem) may be useful indicators for an increased thrombotic risk in individual
patients which may require special anticoagulatory treatment
Keywords CVC complication venal occlusion antithrombotic treatment ultrasound
electron microscopy rotational elastometry
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
42
1 Introduction
Central venous catheters (CVCs) are very important tools for care of patients in critical
care units allowing parenteral nutrition support therapy administration of high volumes
of intravenous fluids safe drugs administration that cannot be given through peripheral
veins plasmapheresis haemodialysis blood transfusions as well as periodical blood
sampling (Geerts 2014 Vose et al 2019) CVC related thrombosis is one of the most
severe complications (apart from infections and pneumothorax) of CVCs in humans
(Geerts 2014 Comerlato et al 2017 Evans and Ratchford 2018) leading to patientrsquos
distress CVC dysfunction venous stenosis and in some cases to the development of
thromboembolism (Geerts 2014)
In the human literature there are various studies dealing with epidemiology with reference
to different anticoagulatory regimens including placebo-controlled studies (Lee and
Kamphuisen 2012) In humans CVC related thromboses represent 70ndash80 of all upper
extremity thrombosis cases and approximately 10 of thromboembolism cases in
humans (Kucher 2011) The incidence of CVC related thrombosis reported in humans
based on 25 studies ranged widely from 2 to 67 (median = 30 ) (van Rooden et al
2005 review article) This wide range of calculated incidences of CVC related
thromboses in humans reflects the use of different CVC types different locations of entry
site of the CVC (jugular femoral subclavian axillary cephalic vein) different thrombus
detection technique (venography or ultrasonography) unequal durations of follow up
varying diseases of patients (eg haematological oncological) and presence of
predisposing factors (eg previous venous thrombosis history presence of cancer) (van
Rooden et al 2005 Geerts 2014 Liu et al 2015)
Part of the authors empirically observed CVC-associated thrombosis of the external
jugular vein (EJV) as a relevant complication in their canine intensive care patients which
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
43
awakened their interest in that topic To the best of the authoracutes knowledge studies about
extra luminal CVC related thromboses in dogs are rare in the available veterinary
literature One study evaluating the complications associated with 100 CVCs placed in
the jugular vein of dogs and cats reported only 2 cases of thrombosis (confirmed by
palpation or ultrasound) but obviously did not assess the appearance of subclinical CVC
related thromboses (Adamantos et al 2010) Thereby the incidence of CVC-induced
thromboses in dogs is unknown and systematic studies regarding an effective
anticoagulatory management are lacking
In this context it is of interest whether a possible development of thrombosis or an
increased risk (despite the prevention by a standard application of unfractionated heparin
[UFH]) can be predicted by means of changes of haemostasis related parameters during
the initial examination In one study on 168 human patients without indication of
anticoagulation prophylaxis for example elevated leukocyte counts high plasminogen
activator inhibitor and high coagulation factor VIII levels (all measured on blood samples
taken after CVC placement) were associated with an increased incidence of symptomatic
CVC-related thrombosis diagnosed by ultrasonography (Boersma et al 2016)
Therefore aims of the present study were (1) to determine the incidence of catheter-
induced thrombosis in canine intensive care patients (as detected by ultrasonography and
electron microscopy ELMI) and thereby (2) to evaluate the efficacy of a standard
antithrombotic regimen with unfractionated heparin (UFH) routinely used in the Small
Animal Clinic of the University of Veterinary Medicine Hannover and (3) to assess
whether selected haemostasis test results are predictive for an increased risk for
thrombosis formation
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
44
2 Material and methods
21 Study design
Canine in-patients of the Small Animal Clinic University of Veterinary Medicine
Hannover receiving a CVC in the EJV for medical reasons from March 2017 to
December 2019 were included Anticoagulatory treatment was performed according to
the standard protocol of the clinic Colour Doppler ultrasonographic controls of the EJV
and blood samplings (for heparin activity basic haematology and haemostasis tests) were
done immediately before the catheter was placed (day 0) and on days 1 3 5 etc (2 hours
after the first daily UFH injection) until the CVC was removed Only patients were
included which could be examined at least until day 3 Haemostasis tests included
prothrombin time activated partial thromboplastin time thrombin time fibrinogen
concentration antithrombin activity fibrin monomers D-dimers thrombin generation
assay (TGA) and rotational elastometry as well as the heparin activity using a
chromogenic anti-factor Xa test Finally ELMI of sections of the CVC after its removal
was performed
22 Animals
Originally 32 patients hospitalised in the Small Animal Clinic and receiving a CVC in the
EJV for medical reasons entered the study Three of them were subsequently excluded
because they did not reach the defined minimum study period of 3 days Two were
euthanised and one referred back to the referring vet before day 3 22 dogs weighing more
than 15 kg were included so that the withdrawal of about 10 ml of blood on several days
was not a significant burden for the patient (lt 1 of the total blood volume) Five dogs
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
45
with a body weight of less than 15 kg but at least 8 kg were included with special
consideration of the amount of blood taken (no more than 6 ml on individual days)
All animals received standard anticoagulatory treatment with UFH 17 dogs with internal
diseases received 150 IUkg BW TID subcutaneously and 12 dogs (post surgery) received
a reduced dosage of 75 IUkg BW TID subcutaneously
The 29 dogs enclosed in the study included 4 mixed breed dogs and the following
pedigree dogs 5 Labrador Retrievers 3 German Shepherds 2 Australian Shepherds and
one from each of the following breeds Akita Inu Asian Shepherd Bavarian Mountain
Hound Beagle Belgian Shepherd Briard Canaan dog Dalmatian Golden Retriever
Havanese dog Hovawart Rhodesian Ridgeback Schnauzer Shar Pei and Wolfspitz
These dogs were aged between 2 and 164 months (median 77 months) and had body
weights ranging from 81 and 63 kg (median 27 kg) 9 were castrated males 8 intact
males 7 spayed females and 5 intact females
The diseases of the 29 patients requiring a CVC included 3 from each of the following
diseases hepatopathy and esophagus dilatation 2 from each of the following diseases
bacterial cystitis diabetes mellitus and 1 from each of the following diseases abdominal
wall perforation aspiration pneumonia cystitis intestinal round cells sarcoma carcinoma
of the nasal cavity chronic renal insufficiency cystic duct rupture heartworm disease
ivermectine intoxication lymphoma multifocal alveolar histiocytosis pancreatitis post-
surgical bleeding stomach dilatation stomach foreign body tetanus thoracic
pyogranulomatous inflammation thrombocytopenia and trigeminal nerve paresis
Animals were treated in accordance with the German Animal Welfare Law The
experimental design was approved by the Animal Welfare officer of the University of
Veterinary Medicine and reported to the responsible authority (Lower Saxony State
Office for Consumer Protection and Food Safety reference number 17A101)
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
46
23 Positioning and handling of the central venous catheter
Two types of single lumen radiopaque polyurethane CVC (different on length and
diameter) were used during the study The CVC type I (n = 2529) was Cavafixreg Certoreg
Splittocanreg CVC 32 cm length 11 x 17 mm diameter16 G (BBraun Melsungen AG)
the CVC type II used (n = 429) was Certofixreg Mono S 215 15 cm length 08 x 14 mm
diameter18G (BBraun Melsungen AG) Each CVC was inserted into the EJV using a
modified Seldinger technique as follows the skin of the neck was shaved and disinfected
with antiseptic soap alcohol and iodine Local anesthesia (Lidocaine HCl 2) was
administrated subcutaneously on the catheter insertion site (a middle point in the lateral
neck over the jugular sulcus) A small skin incision was performed in the insertion site
and one peripheral plastic cannula with a metallic needle inside (14 G for CVC type I
and 20 G for CVC type II) was inserted in the EJV in cranio-caudal orientation (Hundley
et al 2018) Once the EJV was reached the needle was removed and a flexible wire
guide was inserted through the peripheral cannula following an external landmark
(4thintercostal space) (Reminga et al 2018) The peripheral cannula was removed and
the respective CVC was inserted slowly and with gentle pressure into the EJV guided
with the flexible wire and following the same external landmark to reach the right
position After the insertion of the CVC the flexible wire guide was removed The correct
positioning of the CVC was checked with a latero-lateral thorax x-ray ensuring that the
CVC tip was placed at the junction of the cranial cava vein with the right atrium Each
CVC was fixed to the neck skin (adjacent to the insertion point) with a monofilament
non-absorbable suture (gauge 3-0) Finally a bandage elaborated with Povidone-Iodine
gauze (Betaisodonareg) padded synthetic band (Artiflexreg Natur) and elastic band
(Elastomullreg) was used to protect the CVC output (Claude et al 2010) and changed
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
47
daily Several times day the catheter was flushed with isotonic sodium chloride
containing 1 IUml UFH
24 Heparin preparation and administration
Heparin-sodium-25000-ratiopharm (injection solution containing 5000 IUml sodium
heparin [mucosa]) was used SC injection was performed over the lateral thorax wall
using 1 mL syringes with fine graduation marks
25 Ultrasonography
Colour Doppler ultrasonographic examinations were performed in lateral position using
a LO-GIQ E9 ultrasound device (GE Healthcare Chicago Illinois USA) with a 15
Megahertz linear ultrasound probe The development of a thrombus or fibrin debris in the
EJV lumen around the CVC or inside it was assessed at each control Once the
ultrasound was finished a new protective bandage was placed around the neck of the dog
According to the percentage of lumen occlusion of the EJV (evaluated in cross sectional
views during the ultrasound scanning) the thrombi were classified in four different grades
(Fig 1)
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
48
of the residual vein lumen C ndash catheter T ndash thrombus
Fig 1 Definition of different grades of central venous catheter induced thrombosis of the
jugular vein as detected using colour Doppler ultrasound
26 Blood collection
At each time point approx 6ndash10 ml of blood (according to the body weight) was collected
This included approx 1 ml of EDTA blood for hematological analysis and approx 5ndash9
ml of citrated blood (tubes containing one part of 011 molL [38 ] sodium citrate to
nine parts of blood) for the measurement of hemostasis tests in citrated blood (ROTEM)
or citrated plasma Blood samples were collected by puncture of peripheral veins with
disposable cannulas (09 x 40 mm [20G] or 10 x 40mm [21G]) directly into sample tubes
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
49
or from the CVC using the three-syringe-technique (Villalta-Garciacutea et al 2015) in
situations when the peripheral venipuncture was not indicated due to phlebitis or irritation
andor inflammation of the surrounding skin Briefly 2ndash3 ml of blood were taken in a
sterile disposable syringe and immediately afterwards the blood sample was taken in a
new disposable syringe and transferred into the test tubes containing the respective
anticoagulants finally the first 2-3 ml taken were re-infused to the dog through the CVC
followed by flushing with a solution of sodium chlorideUFH (02) Immediately after
blood collection each sample tube was swayed gently until blood and anticoagulant had
been mixed thoroughly To prepare citrated platelet poor plasma citrated blood was
centrifuged at 16000 g for 10 min at room temperature using a microcentrifuge The
plasma was transferred into plastic tubes and the centrifugation procedure was repeated
This platelet poor plasma was then frozen in aliquots at -70 degC until used for analysis
Directly before analysis the plastic tubes were thawed at 37 degC in a water bath
27 Laboratory tests
Haematological analyses were made automatically with Adviareg 2120i (Siemens
Healthcare GmbH Germany)
Prothrombin time activated partial thromboplastin time and thrombin time were
measured with the autoanalyser Amax Destiny Plus (Tcoag Deutschland GmbH
Germany) and commercial reagents (Thromborel S Siemens Healthcare Diagnostics
Products GmbH Marburg Germany CK Prestreg and Test Thrombin Reagent
Diagnostica Stago SAS Asniegraveres sur Seine Cedex France) Whereas the last two tests
were measured according to the manufacturer`s instructions a modified test optimised
for canine plasma was used to measure prothrombin time (25 microl citrated plasma 120 with
imidazole buffer + 25 microl of a human fibrinogen solution 2 gl prewarmed for 120 s at 37
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
50
oC addition of 25 microl of activating reagent Thromborel S) and the results converted into a
percentage activity based on a calibration curve prepared with canine pooled plasma The
fibrinogen concentration was measured with the method according to Clauss using a test
kit from Stago Diagnostica SAS D-dimer concentration was measured with a
quantitative commercial human latex turbidimetric method in the autoanalyser Cobas
c311 using a completely assembled kit and human standards (Roche Diagnostics
Mannheim Germany) Antithrombin activity was also measured with a completely
assembled chromogenic test kit in the autoanalyser Cobas c311 and calibrated with a
canine pool plasma (n = 100)
The thrombin generation assay (Technothrombinreg TGA (Technoclone GbmH Vienna
Austria) determines thrombin generation based on monitoring of the fluorescence
generated by cleavage of a fluorogenic substrate by thrombin over time after activation
of the coagulation cascade by different concentrations of tissue factor and negatively
charged phospholipid in platelet poor plasma From the fluorescence development over
time the concentration of thrombin in the sample can be calculated using a thrombin
calibration curve The increase in thrombin concentration with time then allows to
calculate generation of thrombin in the sample and to plot thrombin values over time for
the whole coagulation process
The rotational thromboelastometry analysis with ROTEM delta device (Werfen Munich
Germany) was started approx 15 minutes after the blood collection To perform the
analysis and according to the automated ROTEM software 20 microl of recalcification reagent
(200 mmoll calcium chloride solution star-tem) and 20 microl of the respective activating
reagent (ex-tem in-tem) were pipetted into and carefully mixed in a pre-warmed cuvette
using the automated pipette provided in the device immediately afterwards 300 microl of
citrated whole blood (previously gently revolved) were added and mixed with the
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
51
reagents previously deposited Finally the cuvette was carefully placed and fixed to an
oscillating pin (oscillation movements on left and right through an angle of 475deg) in the
ROTEM delta device The interaction between blood cells and subsequently the bond
between fibrin and platelets promoted by the biomechanical changes in pH electrolytes
and temperature start the clot formation with its subsequent retraction and lysis The
magnitude of the resistance produced by the clot over the oscillating pin is quantitatively
represented by different parameters measured during the analysis and also qualitatively
in a graphic illustration (Bagge et al 2016 Crochemore et al 2017)
A chromogenic substrate test (Coatest Heparin Chromogenix-Instrumentation
Laboratory Haemochrom Diagnostica Essen Germany) was used for measurements of
UFH in the auto-analyser Cobas c311 The test application was created based on
manufacturers` instructions Different dilutions of the same batch of the commercial
sodium UFH preparation used in the experiment with normal canine pool plasma
(prepared from identical aliquots of 10 healthy adult dogs) served as standards for the
calibration of the measurement
28 Electron microscopy
Selected areas on the last third of the CVC (5ndash10 cm according with the CVC) with
macroscopically detectable thrombi were analysed and some of the CVCs were cut in
two different places The cut samples were preserved in a fixative medium composed of
1 glutaraldehyde and 4 formalin Once the sample was fixed it was prepared
according to the osmium thiocarbohydrazide procedure (OTOTO-procedure) (Malick et
al 1976) The fixed CVCs were dehydrated in seven series as follow first three series of
120 minutes each one on 30 50 and 70 ethanol respectively followed by two series
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
52
of 120 min each one on 90 ethanol and then two series of 24 hours each one on 100
ethanol After the dehydration process the samples were placed two times during 24
hours each one on isoamyl acetate and then placed on in a critical point drier Finally the
samples were coated with gold in a splutter-coater with 25 kV acceleration voltage in an
argon atmosphere with a current of 20 mA for one minute The samples were examined
in an EVO 15 scanning electron microscope (ZEISS International Carl Zeiss Jena
Germany) and photographed with magnifications of 600x and 6000x avoiding mechanical
artifacts (Toskala et al 1995) Thrombi on the external wall of the CVC were analysed
and measured using the software ImageJ (National Institutes of Health and the Laboratory
for Optical and Computational Instrumentation University of Wisconsin USA)
According to their extension (surface size) and thickness (related to the CVC wall
thickness in a cross sectional view) thrombi on the external wall and additionally within
the lumen of the CVC were each classified in four grades (Fig 2 Fig 1S [suppl
material]) In case of two cuts the highest degree of thrombosis detected at one of the two
sites was used for the final assessment
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
53
surface sizepercent of CVC wall thickness
Fig 2 Definition and illustration of different grades of central venous catheter induced
(extraluminal) thrombosis as detected using electron microscopy
Fig 1S (suppl material) Definition and illustration of different grades of intraluminal
thrombosis in central venous catheters as detected using electron microscopy
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
54
29 Statistical methods
Comparison between thrombus graduation either based on sonographic and ELMI
findings respectively was performed using the McNemar test with calculation of the
concordance index (κ) Standard normal distribution was assessed using the
Kolomogorov-Smirnov test
Due to non-normal distribution of part of data sets heparin activities and results of
haemostasis assays are reported using non-parametric variables (median minimumndash
maximum box and whisker plots) Heparin activities and results of haemostasis tests
between dogs with or without thrombosis were compared using Mann-Whitney-U test
3 Results
31 Sonographic detection of thrombus formation
Thrombus formation was sonographically detected in 829 patients during the study
period Based on examination on the final day in half of these eight patients (n = 4) the
thrombus formation was scored grade 1 in 3 dogs grade 2 and only in one grade 3 In all
of these 8 dogs this was the maximum grade during the study period One dog with fibrin
precipitates was excluded from further analysis because it could not be clearly assigned
to one of the groups In 78 cases thrombi were attached mainly to the venous wall
whereas only in one case was attached to the CVC external wall Seven of these 8 dogs
which developed thrombi received the standard heparin dosage (150 IUkg TID 41
[717] of dogs receiving this dosage) and only one the reduced heparin dosage (75 IUkg
TID 8 [112] of dogs receiving this dosage)
The time point of first detection of thrombus formation varied between days 3 and 11
(median day 8) In addition detritus (presumably composed mainly of fibrin) was
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
55
detectable in the lumen of the EJV in one further dog and within the lumen of the CVC
in further 5 dogs
32 Thrombus formation by electron microscopy
27 from 29 CVC catheter tips were analysed by electron microscopy 827 samples were
without detectable thrombus (grade 0) and 1927 with detectable thrombi 72 showed
thrombus formation of grade 1 427 of grade 2 and 827 of grade 3 There was no
concordance between thrombus graduation based on sonographic and the ELMI
graduation respectively The concordance index (McNemar test) was negative (κ = -
00718) indicating that the degree of agreement is less than by chance
Of the in total 19 dogs with detectable thrombus formation by electron microscopy 11
dogs received 150 IUkg TID (in total 16 dogs of these 27 dogs received that heparin
dosage) and 8 received 75 IUkg TID (in total 11 of these 27 dogs received that heparin
dosage 73 811) and Of the 12 patients with grade 2 and 3 thrombus formation 5 dogs
received 75 IUkg TID and 7 dogs 150 IUkg
The supplementary analysis of intraluminal thrombus formation revealed no detectable
thrombi in 7 cases and thrombus formation of grade 1 (lt 25 occlusion) in 11 cases of
grade 2 (25ndash50 ) in 3 cases and of grade 3 (gt 50 ) in 6 cases
33 Heparin activities and relationship to thrombus formation
Heparin activities varied remarkably in both dosage groups (Fig 3 ab) There was no
significant difference of heparin activities between blood samples collected via
venipuncture or from the CVC (Table 1S supplementary material) In addition there was
no significant difference between heparin activities in dogs with or without
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
56
sonographically or electron microscopically detectable thrombosis (Tables 1 and 2)
Table 1 refers to sonographically detectable thromboses and is based on median heparin
activities calculated from the measurements on day 1 and following days per patient
firstly for the total number of patients and secondly for the patients receiving 150 IUkg
TID In addition in the eight dogs heparin activities on the day of first thrombus detection
(023 IUml 019ndash036 IUml) were not significantly different from the median values
(Table 1 P = 05214 Wilcoxon test) Table 2 compares heparin activities (median values
of all days and results on the final day) between dogs with and without electron
microscopically detectable CVC-associated thrombi
Table 1S (Supplementary material) Heparin activities (IUml median values
minimumndashmaximum) measured in 27 dogs receiving 75 or 150 IUkg unfractionated
(standard) heparin TID subcutaneously 2 hours after the first injection on the day
Comparison of samples collected via puncture of the peripheral veins and those collected
from the central venous catheter (CVC) using the three-syringe-technique
Peripheral vein
puncture CVC
Mann-Whitney-U
test
Total number
(75 and 150
IUkg TID)
019
(00ndash108)
(n = 65)
023
(000ndash105)
(n = 65)
P = 03033
75 IUkg TID
010
(000ndash108)
(n = 25)
012
(000ndash023)
(n = 21)
P = 06907
150 IUkg
TID
030
(000ndash096)
(n = 40)
036
(000ndash105)
(n = 44)
P = 01223
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
57
Fig 3ab Heparin activities (box- and whisker plot arithmetic means) in dogs receiving
unfractionated heparin at a dosage of 75 IUkg BW tid (a) or 150 IUkg BW tid (b) two
hours after the first daily heparin administration
day 1 represents the first day after initiation of heparin treatment
only days were considered with at least results from 5 patients
Table 1 Heparin activities (IUml median values minimumndashmaximum) in 28 dogs
receiving 75 or 150 IUkg unfractionated (standard) heparin TID subcutaneously with
(grades 1ndash3) or without (grade 0) sonographically detectable thrombus Blood collections
were performed 2 hours after the first daily injection
Grade 0
(sonography)
Grades 1ndash3
(sonography)
Mann-Whitney U
test
Total number
(75 and 150 IUkg
TID)
020 (000ndash072)
(n = 20)
027 (018ndash059)
(n = 8)
P = 03212
150 IUkg TID 042 (000ndash072)
(n = 10)
031 (018ndash059)
(n = 7)
P = 04593
one dog with fibrin precipitates in the lumen of the jugular vein but without defined thrombus formation
was not considered because it could not be clearly assigned to one of the two groups
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
58
Table 2 Heparin activities (IUml median values minimumndashmaximum) in 27 dogs
receiving 75 or 150 IUkg unfractionated (standard) heparin TID subcutaneously with
(score 1ndash3) or without (score 0) detectable thrombus via electron microscopy
Grade 0
(electron
microscop)
(n = 8)
Grades 1ndash3
(electron
microscop)
(n = 19)
Mann-Whitney U
test
Median value of
different days 028 (005ndash060)
022 (000ndash072)
P = 05770
Final day 020 (001ndash045)
015 (000ndash105)
P = 06706
34 Relationship between haemostasis tests and thrombus formation
From the initially performed haemostasis tests high fibrinogen concentrations and low
maximum lysis values as measured by the ROTEM delta device (using the ex-tem
reagent) were associated with sonographically detectable thrombi (Tab 3) Additional
analyses based on the median values of different days for individual patients revealed that
low maximum lysis values as measured by the ROTEM delta device (using the in-tem
reagent) were associated with sonographically detectable thrombi (maximum lysis score
0 850 [100ndash175 ] score 1ndash3 375 [100ndash900 ] median [minimumndash
maximum] P = 00166) but no other test Comparison of the results of haemostasis tests
on the final day between dogs with and without sonographically detectable thromboses
did not show any significant difference (P gt 005)
Comparison of results of haemostasis tests on the final day in dogs with detectable
thrombosis via ELMI and dogs without significant thrombus formation showed several
significant differences including lower fibrinogen concentrations and longer clotting
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
59
times (ex-tem reagent) and clot formation times (ex-tem and in-tem reagents) using the
ROTEM delta analyser in general expressing a less coagulable status (Tab 4) If values
of dogs graded 0 and 1 (n =15) are compared to those of grades 2 and 3 (n = 12) the last-
mentioned had longer clotting times (ROTEM delta in-tem reagent) as well as longer clot
formation times and lower alpha angles (ROTEM delta in-tem reagent) (P lt 005 results
not shown)
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
60
Table 3 Comparison of initial results of haemostasis parameters (median values
minimumndashmaximum in dogs with (grades 1ndash3) and without (grade 0) sonographically
detectable central venous catheter-induced thromboses
Parameter Unit
Sonographic
thrombus grade
P-value
(Mann-Whitney
U test)
0
(n = 20)
123
(n = 8)
Haematocrit 355 (228ndash641)
382 (245ndash499)
06111
Platelet count x103microl 292 (420ndash483)
218 (860ndash458)
02320
Prothrombin time 944 (359ndash169)
896 (471ndash136)
05758
Activated partial thromboplastin
time s 128
(955ndash249) 140
(117ndash164) 02322
Thrombin time s 129 (121ndash178)
141 (650ndash160)
02028
Fibrinogen gl 448 (153ndash770)
747 (153ndash116)
00235
Antithrombin activity 639 (289ndash107)
670 (464ndash746)
05249
D-dimers microgml 006 (000ndash148)
015 (003ndash372)
03997
Thrombin generation assay nmol 130 (000ndash365)
912 (190ndash277)
08787
Clotting time (ROTEM ex-tem) s 420 (290ndash930)
430 (170ndash159)
07989
Clot formation time (ROTEM ex-
tem) s 665
(310ndash143) 470
(240ndash800) 00633
Alpha angle (ROTEM ex-tem) degrees 795 (620ndash840)
815 (750ndash860)
01667
Max clot firmness (ROTEM ex-
tem) mm 750
(570ndash860) 775
(700ndash830) 01753
Max clot elasticity (ROTEM ex-
tem) - 299
(131ndash619) 347
(235ndash484) 01544
Maximum lysis (ROTEM ex-tem) 135 (300ndash290)
400 (000ndash800)
00037
Clotting time (ROTEM in-tem) s 208 (840ndash810)
249 (720ndash371)
05085
Clot formation time (ROTEM in-
tem) s 835
(370ndash634) 745
(390ndash235) 08988
Alpha angle (ROTEM in-tem) degrees 745 (250ndash820)
750 (500ndash820)
09797
Max clot firmness (ROTEM in-
tem) mm 700
(540ndash810) 740
(610ndash770) 03587
Max clot elasticity (ROTEM in-
tem) - 235
(118ndash424) 287
(156ndash332) 04764
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
61
Maximum lysis (ROTEM in-tem) 850 (000ndash210)
350 (000ndash220)
01327
Table 4 Comparison of results of haemostasis parameters (median values minimumndash
maximum) on the last day in dogs with (grades 1ndash3) and without (grade 0) central venous
catheter-associated thrombus formation as detected by electron microscopy
Parameter Unit
Thrombus grade
(ELMI)
P-value
(Mann Whitney
U test)
0
(n = 8)
123
(n = 19)
Haematocrit 322 (241ndash454)
330 (216ndash510)
05953
Platelet count 103microl 446 (321ndash592)
268 (101ndash675)
00558
Prothrombin time 145 (104ndash264)
120 (561ndash148)
00462
Activated partial thromboplastin
time s 123
(109ndash140) 136
(109ndash188) 00665
Thrombin time s 129 (121ndash178)
141 (650ndash160)
02028
Fibrinogen gl 465 (275ndash866)
347 (189ndash992)
00491
Antithrombin activity 793 (448ndash981)
692 (398ndash889)
04572
D-dimers microgml 018 (000ndash173)
007 (000ndash118)
01920
Thrombin generation assay nmol 501 (000ndash160)
93 (000ndash287)
09562
Clotting time (ROTEM ex-tem) s 360
(270ndash410) 400
(310ndash620) 00238
Clot formation time (ROTEM ex-
tem) s 395
(120ndash740) 650
(240ndash174) 00275
Alpha angle (ROTEM ex-tem) degrees 835 (760ndash880)
790 (580ndash850)
00484
Max clot firmness (ROTEM ex-
tem) mm 780
(700ndash850) 720
(550ndash860) 00586
Max clot elasticity (ROTEM ex-
tem) - 346
(239ndash555) 254
(121ndash637) 00495
Maximum lysis (ROTEM ex-tem) 105 (200ndash190)
110 (000ndash380)
03796
Clotting time (ROTEM in-tem) s 178 (100ndash222)
201 (139ndash554)
00525
Clot formation time (ROTEM in-
tem) s 435
(330ndash111) 790
(350ndash882) 00209
Alpha angle (ROTEM in-tem) degrees 815 (710ndash830)
740 (240ndash830)
00191
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
62
Max clot firmness (ROTEM in-
tem) mm 785
(660ndash810) 690
(270ndash840) 00586
Max clot elasticity (ROTEM in-
tem) - 362
(192ndash434) 218
(370ndash507) 00559
Maximum lysis (ROTEM in-tem) 950 (200ndash190)
800 (100ndash180)
06890
4 Discussion
The main result of our study is that under the study conditions part of the dogs developed
CVC-associated thromboses However only a very low part of these thromboses were
obviously of a clinically significant degree regarding the amount of occlusion of the EJV
and none of the dogs had a symptomatic thrombosis whose definition includes presence
of clinical signs such as localised swelling pain and redness (Boersma et al 2008 Lee
and Kamphuisen 2012) In the present study we particularly refer to the extraluminal
thromboses with regard to the assessment of the antithrombotic management
One major problem of the statistical analysis and result presentation was that the two
methods used to define the occurrence and degree of thrombus formation (ultrasound and
ELMI) revealed discrepant results Therefore we performed a statistical analysis
independently regarding thrombus formation with either of the two methods It is well
known that ELMI which has already been used to detect CVC induced thromboses in
humans (Lucas et al 2017) and experimental animals (Xiang et al 1998 Xiang et al
2001) is a very sensitive diagnostic technique which allows the ex vivo diagnosis of very
small or fibrin rich thrombi that perhaps cannot be detected by ultrasonography or
venography during the in vivo evaluation of patients (Xiang et al 2001) A major reason
for the discrepant results between the two methods used to detect CVC associated
thromboses is that different CVC segments are evaluated Using ultrasonography it was
only possible to evaluate the EJV in the neck area of the patient and thereby only its
proximal third In contrast ELMI assessed mainly the CVC tip and a human study
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
63
revealed that even different positions of the catheter tip are associated with various risks
for development of CVC associated thrombosis (Ballard et al 2016) One additional point
to consider which also may contribute to the discrepancy is that it is possible that thrombi
developed on the CVC surface can be detached during the CVC removal from the vein
and thereby before its analysis by ELMI (Xiang et al 2001)
The contrast venography technique is used as gold standard to confirm thrombosis in
human medicine However this diagnostic technique is normally used only in the case of
a strong clinical suspicion of venous thrombosis that cannot be detected by
ultrasonography (Laurenson et al 2010 Langston and Eatroff 2018) Our study protocol
with privately owned clinic patients did not allow to use this method In a study performed
in 44 human patients requiring a CVC (Koumlksoy et al 1995) a sensitivity of 94 and a
specificity of 96 were determined for the colour Doppler ultrasound diagnosis when it
was compared with the diagnosis performed with the contrast venography technique and
that makes the colour Doppler ultrasound an accurate diagnosis method to detect CVC
related thrombosis even in humans (Koumlksoy et al 1995 van Rooden et al 2003)
The incidences of CVC related thrombosis detected by colour Doppler ultrasound of the
internal jugular vein reported in 3 human studies were 2 30 and 56 (Harter et al
2002 Lordick et al 2003 Wu et al 1999) and although with a wide range the median
value of these rates (30 ) is very well suited to the incidence of 28 (829) which we
calculated for our canine study based on sonographic findings The wide variation of
incidences of CVC related thrombosis in different human studies can in general be
explained by differences regarding the type and entry site of the CVC (jugular femoral
subclavian axillary cephalic vein) the time period with the CVC in place the thrombus
detection technique (venography or ultrasonography comment in the cited 3 studies
consistently the last-mentioned method was used) and criteria for diagnosis of
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
64
thrombosis the duration of follow up diseases and presence of predisposing factors in
these patients (eg previous venous thrombosis history presence of cancer) and finally
prophylactic treatment with anticoagulants (van Rooden et al 2005 Geerts 2014 Liu et
al 2015)
Different pathomechanisms contribute to the development of CVC induced thromboses
During the CVC insertion the puncture of the skin on the CVC insertion site and the
rupture of the blood vessels integrity activate the coagulation cascade and the CVC acts
as a matrix for adherence and accumulation of fibrin and cellular components (Smith et
al 2012) The attachment of proteins and blood cells to the internal andor external wall
of the CVC can lead to a partial or complete occlusion of the catheter lumen and
sometimes to a partial or complete obstruction of the blood flow andor
thromboembolism (Langston and Eatroff 2018) There is a strong interrelationship
between thrombus and biofilm formation (Smith et al 2012) Prevention of thrombus
formation has thereby also relevance regarding the increased risk for microbial
colonisation of the catheter (Boersma et al 2008) leading at least in humans to an
increased number of CVC related systemic infections (Lordick et al 2003)
In this context it has to be considered that CVC produced from polyurethane which are
routinely used in our clinic and thereby in the study are usually associated with a
relatively high thrombosis potential and thereby definitely provide an effective test for
an antithrombotic regimen Silicone catheters have a significantly reduced risk for
thrombotic complications but this advantage has to be purchased by a higher rate of
mechanical failure (eg rupture of the catheter) (Wildgruber et al 2016) Promising
experimental approaches work on surface modifications of the standard catheters For
example one group was able to demonstrate that a coating of the surface of the catheter
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
65
with a zwitterionic polymeric sulfobetaine showed a significant reduction of thrombus
formation in vitro with human blood and in an in vivo canine model (Smith et al 2012)
Due to ethical reasons we did not performed a placebo-controlled study However
interestingly not all human studies report systemic anticoagulation and part of the human
studies was even placebo-controlled (review Lee and Kamphuisen 2012) Obviously
there is no consensus in the available human literature regarding the beneficial use of
antithrombotic prophylaxis to prevent CVC related thrombosis Several studies did not
find a statistically significant difference in the incidence of CVC related thrombosis
between patients receiving the antithrombotic prophylaxis compared with the control
groups without systemic anticoagulation (Verso et al 2005 P = 035 Karthaus et al
2006 P gt 005 Niers et al 2007 P = 049) whereas in other studies a lower incidence
of CVC related thrombosis was found in the group receiving anticoagulants as
prophylaxis compared with the control groups (Abdelfeki et al 2004 150 vs 126
P = 003 De Cicco et al 2009 400 vs 526 P = 005)
Although it is well known that subcutaneously administered UFH has a limited and
variable bioavailability and thereby leads to unpredictable blood activities (Hirsh and
Raschke 2004) the extremely wide range of blood activities after a defined dosage in our
study was relatively unexpected Blood was collected 2 hours after the first UFH
injection and thereby at the time of suspected maximum heparin activity considering the
relatively low UGH dosage (Mischke and Jacobs 2001) The cited study was performed
on healthy experimental dogs Possible additional influencing factors in critical ill
patients include circulatory insufficiency which may reduce and delay absorption from
subcutanenous tissue decreased clearance function of the liver and kidneys as well as
altered blood plasma concentrations of heparin binding plasma proteins such as
fibrinogen and platelet factor 4 and alterations of the heparin binding capacity of
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
66
endothelial cells which altogether are reasons for a great inter- but also intrapatient
variability (Hirsh and Raschke 2004) In individual patients this can lead to heparin
resistance It has also to be considered that antithrombin activities which are necessary
for an adequate heparin effect (Bjoumlrk and Lindahl 1982) were reduced in our patients
Availability of approved blood tests which can predict the risk of development of CVC
associated thrombosis already at the moment of the CVC placement are important
(Boersma et al 2016) One of the most interesting results was that dogs which developed
sonographically detectable thrombi had nearly twofold higher fibrinogen levels Well in
agreement with our results one study performed in anticoagulated human patients
demonstrated that high fibrinogen levels were associated with an increased risk of
perioperative venous thromboembolism (P = 0004) (Kato et al 2016) The cited study
does not exclusively refer to CVC associated thrombosis but 76 (1621) of the venous
thromboses were suggested to be associated with CVCs Fibrinogen measurement a test
which is widely available is therefore a useful and practicable indicator of an increased
risk for CVC induced thromboses and may advice the clinician to treat these patients
especially carefully (eg with low molecular weight heparin) In the study performed by
Boersma et al (2016) in 168 human patients without indication of anticoagulation
prophylaxis elevated leukocyte counts high levels of plasminogen activator inhibitor
and of coagulation factor VIII were associated with an increased incidence of
symptomatic CVC related thrombosis diagnosed by ultrasonography A limitation of that
study is that all measurements were performed on blood samples taken after CVC
placement Interestingly in our study ROTEM results were partly associated with
thrombus formation which reflects the well-known fact that results of viscoelastic
measurements are a good indicator of hypercoagulability and thromboembolic events
(Harahsheh and Ho 2018) It is of further interest and possibly unexpeted that on the final
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
67
day of examination dogs with ELMI signs of thromboses had partly lower coagulation
activities than dogs without thromboses Due to the low number of dogs without
detectable thrombi using ELMI this implausible result has to be interpreted with caution
One possible explanation could be that temporary hypercoagulability in these dogs may
have finally resulted in consumption of haemostasis substrates
Limitations of the present study were as a consequence of the study design based on
clinical patients the relatively low total number of patients inclusion of different UFH
treatment regimens and the lack of a control group
5 Conclusions
The results of the present study indicate that the used UFH regimen was not completely
effective to prevent CVC-associated thromboses which were detectable in 28 [829]
of the patients by ultrasound However severe thromboses (high grades) are relatively
rare in intensive care canine patients receiving a routine anticoagulant treatment Because
heparin plasma activities were not different in dogs with or without CVC-associated
thrombus formation other local and systemic factors must play a significant role as well
A high fibrinogen concentration and low maximum lysis (ROTEM ex-tem) may be
useful indicators for an increased thrombotic risk in individual patients which may
require special anticoagulatory treatment
6 Acknowledgments
Jose Mauricio Pereira received a scholarship from the Universidad Nacional de Costa
Rica (reference code JB-C-1107-2016)
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
68
7 References
1 Abdelkefi A Ben Othman T Kammoun L Chelli M Ben Romdhane N Kriaa A Ladeb S
Torjman L Lakhal A Achour W Ben Hassen A Hsaiumlri M Ladeb F Ben Abdeladhim A
2004 Prevention of central venous line-related thrombosis by continuous infusion of low-dose
unfractionated heparin in patients with haemato-oncological disease A randomized controlled trial
Thromb Haemost 92 654ndash661
2 Adamantos S Brodbelt D Moores A L 2010 Prospective evaluation of complications associated
with jugular venous catheter use in a veterinary hospital J Small Anim Pract 51 254ndash257
3 Bagge A Schoumltt U Kander T 2016 Effects of naturopathic medicines on Multiplate and ROTEM
a prospective experimental pilot study in healthy volunteers BMC Complement Altern Med 16 64
4 Ballard DH Samra NS Gifford KM Roller R Wolfe BM Owings JT 2016 Distance of the
internal central venous catheter tip from the right atrium is positively correlated with central venous
thrombosis Emerg Radiol 23 269ndash273
5 Boersma RS Jie KS Verbon A van Pampus EC Schouten HC 2008 Thrombotic and
infectious complications of central venous catheters in patients with hematological malignancies Ann
Oncol 19 433ndash442
6 Boersma R S Hamulyak K van Oerle R Tuinenburg A Ten Cate-Hoek AJ Schouten HC
2016 Biomarkers for prediction of central venous catheter related-thrombosis in patients with
hematological malignancies Clin Appl Thromb Hemost 22 779ndash784
7 Bjoumlrk I Lindahl U 1982 Mechanism of the anticoagulant action of heparin Mol Cell Biochem
48 161ndash182
8 Claude A Riedesel D Riedesel E 2010 Electrocardiography-guided and retrospective analysis of
central venous catheter placement in the dog Vet Anaesth Analg 37 97ndash105
9 Comerlato PH Rebelatto TF Santiago de Almeida FA Klein LB Boniatti M M Schaan
DB Rados VD 2017 Complications of central venous catheter insertion in a teaching hospital
Rev Assoc Med Bras 63 613ndash620
10 Crochemore T Piza FMT Rodriacutegues RDR Guerra JCC Ferraz LJR Correcirca TD 2017
A new era of thromboelastometry Einstein (Sao Paulo) 15 380ndash385
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
69
11 De Cicco M Matovic M Balestreri L Steffan A Pacenzia R Malafronte M Fantin D
Bertuzzi CA Fabiani F Morassut S Bidoli E Veronesi A 2009 Early and short‐term
acenocumarine or dalteparin for the prevention of central vein catheter‐related thrombosis in cancer
patients a randomized controlled study based on serial venographies Ann Oncol 20 1936ndash1942
12 Evans NS Ratchford EV 2018 Catheter related-venous thrombosis Vasc Med 23 411ndash413
13 Geerts W 2014 Central venous catheterndashrelated thrombosis Hematology Am Soc Hematol Educ
Program 2014 306ndash311
14 Harahsheh Y Ho KM 2018 Use of viscoelastic tests to predict clinical thromboembolic events A
systematic review and meta‐analysis Eur J Haematol 100 113ndash123
15 Harter C Salwender HJ Bach A Egerer G Goldschmidt H Ho AD 2002 Catheter-related
infection and thrombosis of the internal jugular vein in hematologic-oncologic patients undergoing
chemotherapy a prospective comparison of silver-coated and uncoated catheters Cancer 94 245ndash251
16 Hirsh J Raschke R 2004 Heparin and low-molecular-weight heparin The Seventh ACCP
Conference on Antithrombotic and Thrombolytic Therapy Chest 126 (Suppl) 188S-203S
17 Hundley D Brooks A Thomovsky E Jhonson P Freeman L Schafbuch R Heng HG Moore
G 2018 Comparison of ultrasound-guided and landmark-based techniques for central venous
catheterization via the external jugular vein in healthy anesthetized dogs Am J Vet Res 79 628ndash
636
18 Kato F Takeuchi H Matsuda S Kawakubo H Omori T Kitagawa Y 2016 Incidence of and
risk factors for venous thromboembolism during surgical treatment for esophageal cancer a single-
institution study Surg Today 46 445ndash452
19 Karthaus M Kretzschmar A Kroning H Biakhov M Irwin D Marschner N Slabber C
Fountzilas G Garin A Abecasis NG Baronius W Steger GG Sudhoff T Giorgetti C
Reichardt P 2006 Dalteparin for prevention of catheter‐related complications in cancer patients with
central venous catheters final results of a double‐blind placebo‐controlled phase III trial Ann Oncol
17 289ndash296
20 Koumlksoy C Kuzu A Kutlay J Erden I Ozcan H Ergicircn K 1995 The diagnostic value of colour
Doppler ultrasound in central venous catheter related thrombosis Clin Radiol 50 687-689
21 Kucher N 2011 Clinical practice Deep-vein thrombosis of the upper extremities N Engl J Med
364 861ndash869
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
70
22 Langston CE Eatroff AE 2018 Hemodialysis catheter-associated fibrin sheath in a dog J Vet
Emerg Crit Care 28 366ndash371
23 Laurenson MP Hopper K Herrera MA Johnson EG 2010 Concurrent diseases and conditions
in dogs with splenic vein thrombosis J Vet Intern Med 24 1298ndash1304
24 Lee AYY Kamphuisen PW 2012 Epidemiology and prevention of catheter‐related thrombosis in
patients with cancer J Thromb Haemost 10 1491ndash1499
25 Liu G Fu ZQ Zhu P Li SJ 2015 Central venous catheter-related thrombosis in senile male
patients New risk factors and predictors J Huazhong Univ Sci Technolog Med Sci 35 445ndash449
26 Lordick F Hentrich M Decker T Hennig M Pohlmann H Hartenstein R Peschel C 2003
Ultrasound screening for internal jugular vein thrombosis aids the detection of central venous catheter-
related infections in patients with haemato-oncological diseases a prospective observational study Br
J Haematol 120 1073ndash1078
27 Lucas TC Silva EED Souza DOD Santos ARD Lara MO 2017 Microstructural
evaluation by confocal and electron microscopy in thrombi developed in central venous catheters Rev
Esc Enferm USP 51 1ndash9
28 Malick L Wilson R Stetson D 1976 Modified thiocarbo- hydrazide procedure for scanning
electron microscopy routine use for normal pathological and experimental tissue Stain Technol 50
265ndash269
29 Mischke R Jacobs C 2001 The monitoring of heparin administration by screening tests in
experimental dogs Res Vet Sci 70 101ndash108
30 Niers TM Di Nisio M Klerk CP Baarslag HJ Buller HR Biemond BJ 2007 Prevention
of catheter‐related venous thrombosis with nadroparin in patients receiving chemotherapy for
hematologic malignancies a randomized placebo‐controlled study J Thromb Haemost 5 1878ndash
1882
31 Reminga C Silverstein D Drobatz K Clarke D 2018 Evaluation of the placement and
maintenance of central venous jugular catheters in critically ill dogs and cats J Vet Emerg Crit Care
28 232ndash243
32 Smith RS Zhang Z Bouchard M Li J Lapp HS Brotske GR Lucchino DL Weaver D
Roth LA Coury A Biggerstaff J Sukavaneshvar S Langer R Loose C 2012 Vascular
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
71
catheters with a nonleaching poly-sulfobetaine surface modification reduce thrombus formation and
microbial attachment Sci Transl Med 4 153ra132 doi101126scitranslmed3004120
33 Toskala E Nuutinen J Rautiainen M 1995 Scanning electron microscopy findings on human
respiratory cilia in chronic sinusitis and in recurrent respiratory infections J Laryngol Otol 109 509ndash
514
34 Van Rooden CJ Tesselaar ME Osanto S Rosendaal FR Huisman MV 2005 Deep vein
thrombosis associated with central venous catheters - a review J Thromb Haemost 3 2409ndash2419
35 Verso M Agnelli G Bertoglio S Di Somma FC Paoletti F Ageno W Bazzan M Parise P
Quintavalle R Naglieri E Santoro A Imberti D Soraru M Mosca S 2005 Enoxaparin for the
prevention of venous thromboembolism associated with central vein catheter a double‐blind placebo‐
controlled randomized study in cancer patients J Clin Oncol 23 4057ndash4062
36 Villalta-Garciacutea P Loacutepez-Herranz M Mazo-Pascual S Honrubia-Fernaacutendez T Jaacutentildeez-Escalada
L Fernaacutendez-Peacuterez C 2015 Reliability of blood test results in samples obtained using a 2-mL discard
volume from the proximal lumen of a triple-lumen central venous catheter in the critically ill patient
Nurs Crit Care 22 298ndash304
37 Vose J Odunayo A Price JM Daves M Schildt JC Tolbert MK 2019 Comparison of
heparinized saline and 09 sodium chloride for maintaining central venous catheter patency in healthy
dogs PeerJ 7 e7072 httpsdoiorg107717peerj7072
38 Wildgruber M Lueg C Borgmeyer S Karimov I Braun U Kiechle M Meier R Koehler M
Ettl J Berger H 2016 Polyurethane versus silicone catheters for central venous port devices
implanted at the forearm Eur J Cancer 59 113ndash124 doi101016jejca201602011
39 Wu X Studer W Skarvan K Seeberger MD 1999 High incidence of intravenous thrombi after
short-term central venous catheterization of the internal jugular vein J Clin Anesth 11 482ndash485
40 Xiang DZ Verbeken EK Van Lommel AT Stas M De Wever I 1998 Composition and
formation of the sleeve enveloping a central venous catheter J Vasc Surg 28 260ndash271
41 Xiang DZ Verbeken EK Van Lommel AT Stas M De Wever I 2001 Sleeve-related
thrombosis a new form of catheter-related thrombosis Thromb Res 104 7ndash14
Chapter 4 General discussion
72
Chapter 4 General discussion
Incidence of CVC related thrombosis
In our study the incidence of CVC related thrombosis detected by ultrasound in critical
care canine patients was 28 To the best of the authoracutes knowledge similar studies
performed in dogs are rare in the available veterinary literature In one study performed
in 81 dogs and 12 cats (Adamantos et al 2010) an incidence of 2 was found for vein
thrombosis related to CVC This low incidence can be due to a lacking of systematic
ultrasound evaluations of the jugular vein containing the CVC In the mentioned study in
dogs and cats the vein thrombosis was confirmed by palpation or ultrasound presumably
after the presentation of clinical symptoms however in many cases as in our study
thrombus into the jugular vein can be developed without manifestation of clinical
symptoms what makes a routinely ultrasound evaluation of the vein containing the CVC
very important Our results are well aligned with the findings in humans in which a
median incidence of 30 was found for thrombosis related to CVC based on 25 studies
performed in patients requiring a CVC (review article van Rooden et al 2005) In the
mentioned 25 human studies the incidence of CVC related thrombosis varied within a
wide range (2ndash67 ) according to factors related to (1) the CVC (eg material type
entry site [jugular femoral subclavian axillary cephalic vein] time period in place (2)
diagnostic thrombus detection technique (ultrasonography or venography) and criteria
(3) patient collective (diseases and presence of predisposing factors [eg previous venous
thrombosis history presence of cancer] and (4) prophylactic antithrombotic treatment
(Geerts 2014 Liu et al 2015 van Rooden et al 2005)
The incidence of CVC related thrombosis detected by colour Doppler ultrasound in our
study fits also with the median incidence (30 ) found in three studies performed in
humans in which colour Doppler ultrasound evaluations of the jugular vein containing a
Chapter 4 General discussion
73
CVC were performed (Harter et al 2002 Lordick et al 2003 Wu et al 1999) In these
three studies the individual incidence of CVC related thrombosis was 2 (Harter et al
2002) 30 (Lordick et al 2003) and 56 (Wu et al 1999) The high thrombus
incidence reported in the study performed by Wu et al (1999) could be due to the criteria
for diagnosis of thrombosis because in that study both fibrin sleeves and compact
thrombi were considered as cases of thrombosis related to CVC The incidence for
compact thrombi was 25 which is more similar to our results In the study performed
by Harter et al (2002) the CVC related thrombosis incidence (2 ) was very low
compared with the incidence found in our study In that study the jugular veins were
examined just before or less than 24 hours after CVC removal (median period of CVC
use = 1025 days) In our study the ultrasound evaluations were performed before the
CVC placement (day 0) and then on days 1 3 5 etc until the CVC removal (median
period of CVC use = 7 days median interval until thrombus detection = 7 days) In our
study we detected thrombus formation as early as on the 3rd day so it could be possible
that in the cited study possibly only temporarily existing thrombi could have been missed
by ultrasonography in the final stage and thereby for the incidence calculation However
this scenario is not very likely because results of the present study show that
sonographically detectable thrombi during the observation period had in all cases their
maximum grade on the final day
In the study performed by Lordick et al (2003) the CVC related thrombosis incidence
determined for 43 human patients was very similar to our study and in both studies only
compact thrombi detectable by ultrasound were considered for assessment of the
incidence In the mentioned study all the human patients were screened for the presence
of thrombosis every 4 days after CVC insertion with real time B-mode ultrasound
(Lordick et al 2003)
Chapter 4 General discussion
74
In our study 70 of CVCs analysed by ELMI showed thrombi of grade 1ndash3 on the
external CVC wall To the best of the authoracutes knowledge studies about incidence of CVC
related thrombosis on the outside surface of the CVC detected by ELMI cannot be found
in the human and small animal literature In one study performed in humans (Lucas et al
2017) thrombus formation was analysed by ELMI in 78 central venous catheters That
study focussed on microstructural assessment of thrombus formation inside the distal
openings of the CVC which were found in 85 of the evaluated CVCs The percentage
of occurrence of thrombosis in the mentioned study in humans was nearly similar to the
incidence of intraluminal thrombus formation as detected in the presented study by ELMI
(74 ) In general intraluminal thrombi were not specially considered in the present
study because they mainly express the efficacy of the flushing technique rather than the
effectivity of the antithrombotic treatment In one study performed in rats the
microstructure of thrombi developed in the jugular vein containing the CVC was also
evaluated by ELMI (Xiang et al 1998) In the mentioned study pericatheter thrombosis
was detected on the proximal section of the CVC in 100 of rats (n = 12) after 1 and 3
days of catheterisation The analyses of the CVCs placed in rats were performed with the
catheter inside the vein in contrast to our study where the CVC was removed from the
jugular vein for the respective analysis Xiang et al (2001) hypothesised in one study
performed in rabbits and rats in which CVCs were placed in the jugular vein that fibrin
rich thrombi associated to the CVC can be detached during the catheter removal and this
could explain the difference between the thrombosis incidence assessed in our study and
that of the study performed by Xiang et al (1998)
In our study the incidence of CVC related thrombosis detected by ultrasonography in the
lumen of the EJV did not correlate with the incidence of CVC related thrombosis detected
by ELMI on the external wall of the CVC This was mainly due to the fact that the CVC
Chapter 4 General discussion
75
segment evaluated with both methods was different Using ultrasonography it was only
possible to evaluate the EJV in the neck area of the patient and thereby only the proximal
third of the CVC In contrast with the ELMI analysis only the distal section of the CVC
(last third) was evaluated because macroscopically detected thrombi mainly developed
near the CVC tip which was routinely positioned at the junction of the cranial cava vein
with the right atrium In a retrospective review of medical records of 169 human patients
requiring a CVC (Ballard et al 2016) the position of the CVC tip was determined by
radiologic evaluation and it was determined that the catheter tip position can be a
predisposing factor for central venous thrombosis development Thereby it could be of
interest to evaluate in further studies in dogs if a high incidence of thrombosis detected
by ELMI as was shown in our study could be related to the CVC tip position
In our study polyurethane catheters were tested which are widely used and are the
standard CVCs used in the Small Animal Clinic University of Veterinary Medicine
Hannover In vivo and in vitro studies have assessed the thrombogenicity of different
catheter materials (Borow and Crowley 1985 Smith et al 2012) In general terms
polyurethane catheters (central or peripheral) had shown a higher thrombogenicity
compared with coated (eg with hydromer) and silicone catheters Due to this property
of polyurethane catheters they can be supposed as a good test system for antithrombotic
regimens as evaluated in our study On the other hand despite silicone catheters have been
associated with a lower thrombogenicity their mechanical flexibility could be a
disadvantage in small clinical practice due to the increased risk of rupture detected even
in humans (Wildgruber et al 2016)
Chapter 4 General discussion
76
Heparin thromboprophylaxis for CVC related thrombosis
The occurence of CVC related thrombosis detected by colour Doppler ultrasound and
ELMI in our study demonstrates that the UFH in the given dosage was not completely
effective to prevent CVC related thrombosis in canine intensive care patients In human
medicine LMWH is commonly used in prophylactic anticoagulation regimens however
studies performed in human patients requiring a CVC and receiving a prophylactic
anticoagulation (with LMWH or UFH) did not achieve consensus regarding the efficacy
of prophylactic heparin to prevent CVC related thrombosis (Abdelfeki et al 2004
Lordick et al 2003 Wu et al 1999) To the best of the authoracutes knowledge studies
performed in dogs evaluating systemic prophylactic anticoagulation to prevent CVC
related thrombosis cannot be found However according to the evidence found in the
human literature and the results of our study further investigations using comparative
groups with different drugs dosages andor administration routes are required to establish
an effective antithrombotic treatment to prevent CVC related thrombosis
The plasma heparin activities measured in canine patients in our study varied significantly
after a defined subcutaneous dosage The limited and variable bioavailability of SC
administered UFH is well documented in humans (Hirsh and Raschke 2004)
Considerable variations of the circulatory status of the liver- and kidney function and
concentration of heparin-binding proteins in intensive care patients obviously amplify
this phenomenon leading to an extremely high inter-individual and intra-individual
variation of the heparin blood levels in our patients A different application route (IV)
may have reduced the influence of part of these factors and therefore could have led to
more predictable UFH blood levels However continuous infusion requires more
technical equipment and is also associated with a wide range of heparin activities in
canine patients (Scott et al 2009)
Chapter 4 General discussion
77
Prediction of CVC related thrombosis
Of 7 haemostasis tests (including ROTEM with 12 different parameters [6 for each
reagent ex-tem and in-tem]) evaluated as possible predictors of CVC related thrombosis
in our study initial values of high fibrinogen and low maximum lysis (ROTEM ex-tem)
were associated with sonographically detectable thrombus formation Some studies in
humans have established the association of laboratory biomarkers as predictors of risk
thrombosis (Boersma et al 2016 Cheng et al 2013 Kato et al 2016 Liu et al 2015)
One study performed in anticoagulated human patients showed ndash well in agreement with
our results ndash that high fibrinogen levels were associated with an increased risk of
perioperative venous thromboembolism (P = 0004) (Kato et al 2016) The cited study
does not exclusively refer to CVC associated thrombosis but 762 (1621) of the
venous thromboses were suggested to be associated with CVCs Fibrinogen concentration
measurements are normally available in the human and veterinary practice and the
association of this parameter with a high risk of development of CVC related thrombosis
can be useful to choose special antithrombotic treatment in individual critical care patients
to prevent such severe complications
Thrombin generation assay (TGA) abnormalities such as higher velocity and endogenous
thrombin potential (ETP) values measured initially ie before any anticoagulation
therapy have been associated with a higher thromboembolism risk in general (ie without
specific association with CVCs) in one study performed on 105 human patients (Espitia
et al 2017) In contrast in our study the initial TGA assay results did not show an
association with formation of CVC related thrombosis
The ROTEM parameters evaluated in the present study were partially associated with
formation of CVC thrombosis detected by colour Doppler ultrasound This finding
confirms the results obtained in humans in which ROTEM measurements are regarded as
Chapter 4 General discussion
78
a valuable indicator of hypercoagulability (Harahsheh and Ho 2018 Tuumlrk et al 2018)
However in our study individual ROTEM parameters showed partly lower coagulation
activities in dogs with CVC related thrombosis detected by ELMI at the final day of
evaluation Because the number of patients without signs of thrombosis detected by ELMI
was low compared with those who showed signs these implausible results should be
interpreted carefully The usefulness of ROTEM analysis which has also many other
indications demonstrates that is was worth to perform a detailed methodological approval
of the device including the establishment of reference values in the preliminary study
Conclusion
In conclusion the prethrombotic or thrombotic changes detected in our study did not
correlate between ultrasonography and ELMI however both methods show accurately
signs of CVC related thrombosis The UFH in the given dosage was not completely
effective to prevent CVC induced thrombus formation in canine intensive care patients
Plasma heparin activities varied significantly after a defined subcutaneous dosage
However plasma heparin activities were not different between dogs with and without
detectable CVC associated thromboses Initial high fibrinogen values and low maximum
lysis (ROTEM ex-tem) values were associated with sonographically detectable thrombi
and may be useful as indicators for an increased thrombotic risk
Finally our study in canine intensive care patients is to the best of the authoracutes
knowledge the first study in the available veterinary literature performed to evaluate the
efficacy of an antithrombotic treatment with UFH to prevent CVC related thrombosis
Chapter 5 Summary
79
Chapter 5 Summary
Central venous catheter induced thrombosis in dogs ndash occurrence under an
antithrombotic treatment regimen and possible indicators
Joseacute Mauricio Pereira Mora
Central venous catheters (CVC) are of importance for intensive care management of
canine patients Thrombosis of the jugular vein is a severe complication related to the
CVC use However only little is known about the incidence of CVC-associated
thromboses in dogs and the necessity and efficacy of prophylactic antithrombotic
regimens
Therefore the aims of the present study were (1) to determine the incidence of catheter-
induced thrombosis in canine intensive care patients (2) to evaluate the efficacy of a
prophylactic heparin regimen routinely used in the Small Animal Clinic of the University
of Veterinary Medicine Hannover and (3) to assess whether selected haemostasis test
results are predictive for an increased risk for thrombosis formation
The thesis also includes a preliminary methodological study on the use of rotational
elastometry in dogs using the ROTEM delta device In this preliminary study reference
intervals were determined based on 125 healthy dogs for different clot activator reagents
and for measurements without activator Coefficients of variation (CVs) were calculated
based on results of fourfold measurements of 3 normal and 3 abnormal blood samples in
order to assess precision of the method Influence of age groups sex neuter status and
size were also assessed The median CVs for most of the parameters evaluated in the
precision analysis were less than 10 and this confirmed that the ROTEM delta analyser
delivers repeatable results Regarding the influence of age sex neuter status and animal
Chapter 5 Summary
80
size only neuter status in female dogs appeared to be a relevant influencing factor on
most of important ROTEM parameters
The main study of the thesis comprises 29 patients hospitalised at the Small Animal Clinic
requiring a CVC for medical reasons These patients received a prophylactic
anticoagulation treatment with unfractionated heparin (UFH 150 IU [surgical dogs
received 75 IU]kg BW TID subcutaneously) A colour Doppler ultrasound of the
respective external jugular vein (EJV) in order to detect thrombus formation in the lumen
of the EJV and blood collection for haemostasis tests were performed directly before the
installation procedure (day 0) and on days 1 3 5 7 and so on after the CVC insertion
Haemostasis tests included prothrombin time activated partial thromboplastin time
thrombin time antithrombin activity D-dimer concentration thrombin generation and
rotational elastometry as well as the heparin activity using a chromogenic anti-factor Xa
test Finally when the CVC was removed electron microscopy (ELMI) of sections of the
CVC with macroscopically visible thrombi in the last third (5ndash10 cm) of the CVC was
performed 3 different grades were defined for CVC-associated (extraluminal) thrombi
detected by either ultrasound or ELMI respectively
829 patients evaluated by colour Doppler ultrasound during the study developed thrombi
into the lumen of EJV (4 x grade 1 3 x grade 2 and 1 x grade 3) 1927 CVCs assessed
using ELMI showed detectable thrombi on the external CVC surface (7 x grade 1 4 x
grade 2 and 8 x grade 3) There was no significant correlation between thrombus
formation detected by ultrasound or ELMI Thus statistical group comparisons of
patients with or without CVC-associated thrombus formation was performed
independently for both diagnostic procedures The plasmatic heparin activities showed
great variability but no significant differences were found between dogs with or without
CVC-associated thrombus formation regardless whether this was based on sonographic
Chapter 5 Summary
81
or ELMI findings From the initially performed haemostasis tests high fibrinogen
concentrations and low maximum lysis values as measured by the ROTEM delta device
(using the ex-tem reagent) were associated with sonographically detectable thrombi
In conclusion the preliminary methodological study on the ROTEM delta in dogs shows
that the method delivers repeatable results in canine citrated whole blood and the
reference values obtained should offer a reliable basis for the interpretation of test results
of this device in dogs with suspected haemostasis disorders The results of the main study
indicate that CVC-associated thromboses occur (eg 28 [829] of the patients as
detected by ultrasound) although severe thromboses (high grades) are relatively rare in
intensive care canine patients receiving a routine anticoagulant treatment Thereby
prophylactic antithrombotic protocol (unfractionated heparin [UFH]) in the given dosage
was not completely effective to prevent CVC induced thrombus formation in canine
intensive care patients A high fibrinogen concentration and low maximum lysis
(ROTEM ex-tem) may be useful indicators for an increased thrombotic risk in individual
patients which may require special anticoagulatory treatment Further studies are
required to evaluate whether different thromboprophylactic treatment regimens eg with
low molecular weight heparin are more effective in preventing CVC-associated
thromboses in dogs
Chapter 6 Zusammenfassung
82
Chapter 6 Zusammenfassung
Zentralvenenkatheter-induzierte Thrombose bei Hunden - Auftreten unter einem
antithrombotischen Behandlungsschema und moumlgliche Indikatoren
Joseacute Mauricio Pereira Mora
Zentralvenenkatheter (ZVK) sind fuumlr das Intensivmanagement von Hunden von
Bedeutung Die Entstehung einer Thrombose der Vena jugularis ist eine schwerwiegende
Komplikation im Zusammenhang mit der Verwendung eines ZVKs Uumlber die Inzidenz
von ZVK-assoziierten Thrombosen bei Hunden und die Notwendigkeit und Wirksamkeit
prophylaktischer antithrombotischer Protokolle liegen jedoch nur wenige fundierte
Angaben in der Literatur vor
Daher waren die Ziele der vorliegenden Studie (1) die Inzidenz der Katheter-induzierten
Thrombose bei caninen Intensivpatienten zu bestimmen (2) die Wirksamkeit eines
prophylaktischen Heparin-Regimes zu bewerten das routinemaumlszligig in der Klinik fuumlr
Kleintiere der Stiftung Tieraumlrztliche Hochschule Hannover angewendet wird und (3) um
zu beurteilen ob ausgewaumlhlte Haumlmostasetestergebnisse ein erhoumlhtes Risiko fuumlr die
Thrombosebildung vorhersagen koumlnnen
Die PhD-Arbeit enthaumllt zudem eine vorbereitende methodische Studie zur Verwendung
der Rotationselastometrie bei Hunden mit Hilfe des ROTEM delta-Geraumltes In dieser
Vorstudie wurden Referenzintervalle basierend auf 125 gesunden Hunden fuumlr
verschiedene Gerinnungsaktivatorreagenzien und fuumlr Messungen ohne Aktivator
bestimmt Um die Genauigkeit der Methode einzustufen wurden Variationskoeffizienten
(VKs) basierend auf den Ergebnissen von Vierfachmessungen von drei normalen und drei
abnormalen Blutproben berechnet Der Einfluss von Alter Geschlecht Kastration und
Groumlszlige wurde ebenfalls getestet Die Medianwerte der VKs fuumlr die meisten in der
Chapter 6 Zusammenfassung
83
Praumlzisionsanalyse bewerteten Parameter betrugen weniger als 10 und dies bestaumltigte
dass das ROTEM delta-Analysegeraumlt wiederholbare Ergebnisse liefert In Bezug auf den
Einfluss von Alter Geschlecht Kastration und Tiergroumlszlige erwies sich nur eine Kastration
bei Huumlndinnen als relevanter Einflussfaktor fuumlr die meisten wichtigen ROTEM-
Parameter
Die Hauptstudie der Arbeit umfasst 29 Patienten welche zum Zeitpunkt der
Untersuchung stationaumlr in der Kleintierklinik aufgenommen worden waren und aus
medizinischer Indikation einen ZVK benoumltigten Diese Patienten erhielten eine
prophylaktische Antikoagulation mit unfraktioniertem Heparin (UFH 150 IE
[chirurgische Hunde erhielten 75 IE]kg KG 3 x taumlglich subkutan) Bildgebende
Untersuchungen mittels Farbdoppler-Ultraschall der jeweiligen Vena jugularis externa
(VJE) zum Nachweis der Thrombusbildung im Lumen der VJE und Blutentnahmen fuumlr
Haumlmostasetests wurden jeweils direkt vor dem Installationsvorgang (Tag 0) sowie an den
Tagen 1 3 5 und 7 nach Legen des ZVKs durchgefuumlhrt Haumlmostasetests umfassten die
Prothrombinzeit die aktivierte partielle Thromboplastinzeit die Thrombinzeit die
Antithrombinaktivitaumlt die D-Dimer-Konzentration die Thrombingenerierung und die
Rotationselastometrie sowie die Heparinaktivitaumlt unter Verwendung eines chromogenen
Anti-Faktor-Xa-Tests Schlieszliglich wurde nach Entfernung des ZVKs eine
Elektronenmikroskopie (ELMI) von Abschnitten aus dem letzten Drittel (5ndash10 cm) des
ZVKs mit makroskopisch sichtbaren Thromben durchgefuumlhrt Fuumlr ZVK-assoziierte
(extraluminale) Thromben die entweder durch Ultraschall oder ELMI nachgewiesen
wurden wurden drei verschiedene Grade definiert
829 Patienten die waumlhrend der Studie mittels Farbdoppler-Ultraschall untersucht
wurden entwickelten Thromben im Lumen der VJE (4 x Grad 1 3 x Grad 2 und 1 x Grad
3) 1927 ZVKs die unter Verwendung von ELMI bewertet wurden zeigten
Chapter 6 Zusammenfassung
84
nachweisbare Thromben auf der aumluszligeren ZVK-Oberflaumlche (7 x Grad 1 4 x Grad 2 und 8
x Grad 3) Es gab keine signifikante Korrelation zwischen der durch Ultraschall oder
ELMI festgestellten Thrombusbildung Daher wurden statistische Gruppenvergleiche
von Patienten mit oder ohne ZVK-assoziierter Thrombusbildung fuumlr beide diagnostische
Verfahren unabhaumlngig voneinander durchgefuumlhrt Die plasmatischen Heparinaktivitaumlten
zeigten eine groszlige Variabilitaumlt es wurden jedoch keine signifikanten Unterschiede
zwischen Hunden mit oder ohne ZVK-assoziierter Thrombusbildung gefunden
unabhaumlngig davon ob diese auf sonographischen oder ELMI-Befunden beruhte Von den
initial durchgefuumlhrten Haumlmostasetests waren hohe Fibrinogenkonzentrationen und
niedrige maximale Lyse-Werte der Messungen mit ROTEM delta (ex-tem Reagenz) mit
sonographisch nachweisbaren Thromben assoziiert
Zusammenfassend zeigt die vorbereitende methodische Studie zum ROTEM delta bei
Hunden dass die Methode wiederholbare Ergebnisse in mit Zitrat
antikoaguliertemVollblut liefert Die erarbeiteten Referenzwerte sollten eine zuverlaumlssige
Grundlage fuumlr die Interpretation der Testergebnisse dieses Geraumlts bei Hunden mit
Verdacht auf Haumlmostasestoumlrungen bieten Die Ergebnisse der Hauptstudie zeigen dass
ZVK-assoziierte Thrombosen auftreten (z B 28 [829] der Patienten bezogen auf den
Ultraschall-gestuumltzten Nachweis) obwohl schwere Thrombosen (hohe Grade) bei
intensivmedizinisch versorgten Hundepatienten die eine Routineantikoagulation
erhalten relativ selten sind Somit war das prophylaktische antithrombotische Protokoll
(unfraktioniertes Heparin [UFH]) in der angewendeten Dosierung nicht vollstaumlndig
wirksam um die ZVK-induzierte Thrombusbildung bei caninen Intensivpatienten zu
verhindern Eine hohe Fibrinogenkonzentration und eine niedrige maximale Lyse
(ROTEM ex-tem) koumlnnen als Indikatoren eines erhoumlhten Thromboserisikos bei einzelnen
Patienten nuumltzlich sein fuumlr die moumlglicherweise eine spezielle gerinnungshemmende
Chapter 6 Zusammenfassung
85
Behandlung notwendig ist Weitere Studien sind erforderlich um zu untersuchen ob
verschiedene thromboprophylaktische Behandlungsschemata z B mit
niedermolekularem Heparin bezuumlglich der Verhinderung von ZVK-assoziierten
Thrombosen bei Hunden wirksamer sind
Chapter 7 References
86
Chapter 7 References
1 Abdelkefi A Ben Othman T Kammoun L Chelli M Ben Romdhane N Kriaa A Ladeb
S Torjman L Lakhal A Achour W Ben Hassen A Hsaiumlri M Ladeb F Ben Abdeladhim
A 2004 Prevention of central venous line-related thrombosis by continuous infusion of low-dose
unfractionated heparin in patients with haemato-oncological disease A randomized controlled
trial Thromb Haemost 92 654ndash661
2 Adamantos S Brodbelt D Moores A L 2010 Prospective evaluation of complications
associated with jugular venous catheter use in a veterinary hospital J Small Anim Pract 51 254ndash
257
3 Ballard D H Samra N S Gifford K M Roller R Wolfe B M Owings J T 2016
Distance of the internal central venous catheter tip from the right atrium is positively correlated
with central venous thrombosis Emerg Radiol 23 269ndash273
4 Baumann-Kreuziger L Onwuemene O Kolesar E Crowther M Lim W 2015 Systematic
review of anticoagulant treatment of catheter-related thrombosis Thromb Res 136 1103ndash1109
5 Bliss SP Bliss SK Harvey HJ 2002 Use of recombinant tissue‐plasminogen activator in a
dog with chylothorax secondary to catheter‐associated thrombosis of the cranial vena cava J Am
Anim Hosp Assoc 38 431ndash435
6 Boersma RS Hamulyak K van Oerle R Tuinenburg A Ten Cate-Hoek AJ Schouten
HC 2016 Biomarkers for prediction of central venous catheter related-thrombosis in patients
with hematological malignancies Clin Appl Thromb Hemost 22 779ndash784
7 Boersma RS Jie KS Verbon A van Pampus EC Schouten HC 2008 Thrombotic and
infectious complications of central venous catheters in patients with hematological
malignancies Ann Oncol 19 433ndash442
8 Bonnet F Loriferne JF Texier JP Texier M Salvat A Vasile N 1989 Evaluation of
Doppler examination for diagnosis of catheter-related deep vein thrombosis Intensive Care Med
15 238ndash240
9 Borow M Crowley J G 1985 Evaluation of central venous catheter thrombogenicity Acta
Anaesthesiol Scand Suppl 81 59ndash64
10 Cheng Y Cui T Fu P Liu F Zhou L 2013 Dyslipidemia is associated with tunneled-cuffed
catheter-related central venous thrombosis in hemodialysis patients a retrospective multicenter
Chapter 7 References
87
study Artif Organs 37 155ndash161
11 Comerlato PH Rebelatto TF Santiago de Almeida FA Klein LB Boniatti MM
Schaan BD Rados DV 2017 Complications of central venous catheter insertion in a teaching
hospital Rev Assoc Med Bras 63 613ndash620
12 De Cicco M Matovic M Balestreri L Steffan A Pacenzia R Malafronte M Fantin
D Bertuzzi CA Fabiani F Morassut S Bidoli E Veronesi A 2009 Early and short‐term
acenocumarine or dalteparin for the prevention of central vein catheter‐related thrombosis in
cancer patients a randomized controlled study based on serial venographies Ann
Oncol 20 1936ndash1942
13 Espitia O Fouassier M Hardouin JB Pistorius MA Agard C Planchon B Trossaert M
Pottier P 2017 Thrombin generation assay in hospitalized nonsurgical patients A new tool to
assess venous thromboembolism risk Clin Appl Thromb Hemost 23 45ndash51
14 Evans NS Ratchford EV 2018 Catheter related-venous thrombosis Vasc Med 23 411ndash413
15 Geerts W 2014 Central Venous Catheter-related thrombosis Hematology Am Soc Hematol
Educ Program 2014 306ndash311
16 Harahsheh Y Ho KM 2018 Use of viscoelastic tests to predict clinical thromboembolic
events A systematic review and meta‐analysis Eur J Haematol 100 113ndash123
17 Harter C Salwender HJ Bach A Egerer G Goldschmidt H Ho AD 2002 Catheter-
related infection and thrombosis of the internal jugular vein in hematologic-oncologic patients
undergoing chemotherapy a prospective comparison of silver-coated and uncoated
catheters Cancer 94 245ndash251
18 Hirsh J Raschke R 2004 Heparin and low-molecular-weight heparin The Seventh ACCP
Conference on Antithrombotic and Thrombolytic Therapy Chest 126 (Suppl) 188Sndash203S
19 Karthaus M Kretzschmar A Kroning H Biakhov M Irwin D Marschner N Slabber
C Fountzilas G Garin A Abecasis NG Baronius W Steger GG Sudhoff T Giorgetti
C Reichardt P 2006 Dalteparin for prevention of catheter‐related complications in cancer
patients with central venous catheters final results of a double‐blind placebo‐controlled phase III
trial Ann Oncol 17 289ndash296
20 Kato F Takeuchi H Matsuda S Kawakubo H Omori T Kitagawa Y 2016 Incidence of
and risk factors for venous thromboembolism during surgical treatment for esophageal cancer a
Chapter 7 References
88
single-institution study Surg Today 46 445ndash452
21 Kohler TR Kirkman TR 1998 Central venous catheter failure is induced by injury and can
be prevented by stabilizing the catheter tip J Vasc Surg 28 59ndash66
22 Koumlksoy C Kuzu A Kutlay J Erden I Ozcan H Ergicircn K 1995 The diagnostic value of
colour Doppler ultrasound in central venous catheter related thrombosis Clin Radiol 50 687ndash
689
23 Kucher N 2011 Clinical practice Deep-vein thrombosis of the upper extremities N Engl J
Med 364 861ndash869
24 Langston CE Eatroff AE 2018 Hemodialysis catheter-associated fibrin sheath in a dog J
Vet Emerg Crit Care 28 366ndash371
25 Langston CE Eatroff AE Poeppel K 2014 Use of tissue plasminogen activator in catheters
used for extracorporeal renal replacement therapy J Vet Intern Med 28 270ndash276
26 Laurenson MP Hopper K Herrera MA Johnson EG 2010 Concurrent diseases and
conditions in dogs with splenic vein thrombosis J Vet Intern Med 24 1298ndash1304
27 Lee AYY Kamphuisen PW 2012 Epidemiology and prevention of catheter‐related
thrombosis in patients with cancer J Thromb Haemost 10 1491ndash1499
28 Liu G Fu ZQ Zhu P Li SJ 2015 Central venous catheter-related thrombosis in senile male
patients New risk factors and predictors J Huazhong Univ Sci Technolog Med Sci 35 445ndash
449
29 Lordick F Hentrich M Decker T Hennig M Pohlmann H Hartenstein R Peschel C
2003 Ultrasound screening for internal jugular vein thrombosis aids the detection of central
venous catheter-related infections in patients with haemato-oncological diseases a prospective
observational study Br J Haematol 120 1073ndash1078
30 Lucas TC Silva EED Souza DOD Santos ARD Lara MO 2017 Microstructural
evaluation by confocal and electron microscopy in thrombi developed in central venous catheters
Rev Esc Enferm USP 51 1ndash9
31 Mismetti P Mille D Laporte S Charlet V Buchmuller‐Cordier A Jacquin JP Fournel
P Dutrey‐Dupagne C Decousus H 2003 Low‐molecular‐weight heparin (nadroparin) and
very low doses of warfarin in the prevention of upper extremity thrombosis in cancer patients with
indwelling long‐term central venous catheters a pilot randomized trial Haematologica 88 67ndash
Chapter 7 References
89
73
32 Niers TM Di Nisio M Klerk CP Baarslag HJ Buller HR Biemond BJ 2007
Prevention of catheter‐related venous thrombosis with nadroparin in patients receiving
chemotherapy for hematologic malignancies a randomized placebo‐controlled study J Thromb
Haemost 5 1878ndash1882
33 Scott K C Hansen B D DeFrancesco T C (2009) Coagulation effects of low molecular
weight heparin compared with heparin in dogs considered to be at risk for clinically significant
venous thrombosis J Vet Emerg Crit Care 19 74ndash80
34 Smith RS Zhang Z Bouchard M Li J Lapp HS Brotske GR Lucchino DL Weaver
D Roth LA Coury A Biggerstaff J Sukavaneshvar S Langer R Loose C 2012
Vascular catheters with a nonleaching poly-sulfobetaine surface modification reduce thrombus
formation and microbial attachment Sci Transl Med 4 153
35 Tuumlrk S M Cansu D Uuml Teke H Uuml Kaşifoğlu T Meltem Akay O Bilgin M amp Korkmaz
C 2018 Can we predict thrombotic tendency in rheumatoid arthritis A thromboelastographic
analysis (with ROTEM) Clin Rheumatol 37 2341ndash2349
36 van Rooden CJ Rosendaal FR Barge RM van Oostayen JA van der Meer FJM
Meinders AE Huisman MV 2003 Central venous catheter related thrombosis in haematology
patients and prediction of risk by screening with Doppler-ultrasound Br J Haematol 123 507ndash
512
37 van Rooden CJ Tesselaar ME Osanto S Rosendaal FR Huisman MV 2005 Deep vein
thrombosis associated with central venous catheters - a review J Thromb Haemost 3 2409ndash
2419
38 Verso M Agnelli G Bertoglio S Di Somma FC Paoletti F Ageno W Bazzan M Parise
P Quintavalle R Naglieri E Santoro A Imberti D Soraru M Mosca S 2005 Enoxaparin
for the prevention of venous thromboembolism associated with central vein catheter a double‐
blind placebo‐controlled randomized study in cancer patients J Clin Oncol 23 4057ndash4062
39 Vose J Odunayo A Price JM Daves M Schildt JC Tolbert MK 2019 Comparison of
heparinized saline and 09 sodium chloride for maintaining central venous catheter patency in
healthy dogs PeerJ 7e7072 httpdoi107717peerj7072
40 Wang LH Wei F Jia L Zhi Lu Wang B Dong HY Yu HY Sun GJ YangJ Li B
Chapter 7 References
90
Meng J Zhang RN Bi XQ Chen HY Jiang AL 2015 Fibrin sheath formation and
intimal thickening after catheter placement in dog model role of hemodynamic wall shear stress J
Vasc Access 16 275ndash284
41 Wildgruber M Lueg C Borgmeyer S Karimov I Braun U Kiechle M Meier R Koehler
M Ettl J Berger H 2016 Polyurethane versus silicone catheters for central venous port devices
implanted at the forearm Eur J Cancer 59 113ndash124
42 Whitesell RT Steenburg SD 2014 Imaging findings of acute intravascular thrombus on non-
enhanced computed tomography Emerg Radiol 21 271ndash277
43 Wu X Studer W Skarvan K Seeberger MD 1999 High incidence of intravenous thrombi
after short-term central venous catheterization of the internal jugular vein J Clin Anesth 11
482ndash485
44 Xiang DZ Verbeken EK Van Lommel AT Stas M De Wever I 1998 Composition and
formation of the sleeve enveloping a central venous catheter J Vasc Surg 28 260ndash271
45 Xiang DZ Verbeken EK Van Lommel AT Stas M De Wever I 2001 Sleeve-related
thrombosis a new form of catheter-related thrombosis Thromb Res 104 7ndash14
91
Acknowledgements
First I thank God for every day in my life for His guide and because He was is
and will be always with me and that certainty gives me strength to take a step
forward
My sincere thanks to Prof Dr Reinhard Mischke for the opportunity to perform
my PhD under his supervision for his support and guidance for all that I learned
with him and for his friendship
I also thank to Prof Dr Manfred Kietzmann and PD Dr Mario von Depka
Prondzinski for being part of my supervision group for their time and beneficial
advices and discussion during my PhD
My gratitude to Prof Hewicker-Trautwein and Mrs Rohn for their valuable work
and availability to help me
I thank the Werlhof Institute Medical Care Center Hannover for the valuable
cooperation especially to Mrs Ober-Cassebaum Anne and Franziska for all their
help
I thank all the staff of the Small Animal Clinic of the University of Veterinary
Medicine Hannover (TiHo) My special gratitude to the internal medicine and
critical care surgery neurology ultrasound and anesthesia departments and to
the TiHo students all their cooperation made possible my research
I thank the haematology clinical chemistry and haemostasis laboratory
department of the Small Animal Clinic TiHo especially to Dirk because he was
always available for me thanks for his guide and friendship all his explanations
in the laboratory were of great value but also the conversations about different
topics of the life were of big value for me
92
I thank the Hannover Graduate School for Veterinary Pathobiology
Neuroinfectology and translational Medicine (HGNI) for the well-organized
PhD program and especially thanks to Prof Dr Beatrice Grummer Dr Tina
Selle and Mrs Tania Czeslik for his guide and help during my studies
My sincere thanks to Mrs Maritta Ledwoch for her guidance at the International
office of the TiHo ldquoI remember you since my externship in Germanyrdquo
I also thank the Student Union Hannover for all the help with the accommodation
for me and my family
I want to thank the Universidad Nacional de Costa Rica and therefore to Costa
Rica (my loved country) for the financial support to perform my PhD studies
but also for my professional formation as a veterinarian Special thanks to the
Veterinary Medicine School from Universidad Nacional de Costa Rica To all the
academic and administrative staff for their support and for believing in me
Sincerely thanks to Dr Mauricio Jimenez Soto my mentor my adviser but most
important my friend Thanks for believing in me and for always being by my side
ldquoAgradezco profundamente a mis papaacutes ldquodon Tito (Papillo) y dontildea Eida (Mami)rdquo
Todo lo bueno que hay en mi proviene de Dios y de ellos Gracias a mis hermanos
(as) cuntildeados (as) sobrinos y a toda la familia relacionada a ellos Siempre han
sido un gran apoyo y los amordquo
ldquoGracias a dontildea Alba Fran y David Gracias por toda la ayuda y por estar siempre
pendientesrdquo
I would like to thank all my friends that during a short part or during my whole
life have been with me For all the good time shared really thanks
To Willy thanks ldquogordordquo
93
I sincerely thank to Adriano and Johanna for their help and hospitality Thanks
Adriano for your guidance and support during my beginning at the TiHo for make
me feel in Costa Rica with your friendship for the tourist walks and for the bike
training I missed the coffee times
I want to thank Ina the first person to give me a smile at the start of my PhD
Thanks for your hugs and friendship
I also thank Franz for his help and for being a very nice partner Thanks for help
me always when I requested you
ldquoFinalmente quiero expresar mi gratitud y amor a Karo Faacutetima y Abigaiacutel A Karo
por tu sacrificio y soporte por las palabras de aliento por estar a mi lado en las
buenas pero en especial en las malas A Faacute por tu sacrificio por tus sonrisas por
tus abrazos y besos por bailar con migo siempre por ser mi modelo e inspiracioacuten
A Abi por ser una nueva razoacuten in mi vida para seguir adelante por todo lo que
deseo compartir con tigo por mostrarme junto con Faacutetima la felicidad en una
sonrisardquo
Translational Medicine 3011ndash021217 Hannover Germany Stiftung Tieraumlrztliche
Hochschule Hannover V07
Pereira JM Hewicker-Trautwein M von Depka Prondzinski M Mischke R 2019
Central venous catheter associated thrombosis in dogs In 27 Jahrestagung der DVG-
Fachgruppe Innere Medizin und klinische Labordiagnostik 01ndash020219 Munich
Germany Tierarztl Prax Ausg K Kleintiere Heimtiere 47(02) P14-139 (DOI
101055s-0039-1679119)
Pereira JM Rohn K Mischke R 2019 Reference intervals for rotational
thromboelastometric measurements using the ROTEMreg delta device in dogs In 35th
World Veterinary Association Congress 27 ndash300319 San Joseacute Costa Rica
Pereira JM Rohn K Mischke R 2019 Reference intervals for rotational
thromboelastometric measurements using the ROTEMreg delta device in dogs Abstracts
of the ESVCP-ECVCP Annual Congress 25ndash280919 Arnhem The Netherlands Vet
Clin Pathol 48 813 P 57
Pereira JM Hewicker-Trautwein M von Depka Prondzinski M Mischke R 2020
Central venous catheter associated thrombosis in dogs In 20 Annual ECVIM-CA
Congress 03ndash050920 Barcelona Spain J Vet Int Med (in press)
Sponsorship Joseacute Mauricio Pereira received a scholarship from the Universidad Nacional
de Costa Rica (reference Code JB-C-1107-2016)
Table of contents
Table of contents
Chapter 1 Introduction 1
Epidemiology of CVC related thrombosis 1
Pathogenesis of CVC related thrombosis 2
Diagnosis of CVC related thrombosis 3
Heparin thromboprophylaxis for CVC related thrombosis 5
Prediction of CVC related thrombosis 7
Aims of the study 8
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs 9
ABSTRACT 11
1 Introduction 12
2 Materials and methods 14
3 Results 20
4 Discussion 26
5 Conclusions 31
6 Acknowledgments 31
7 References 32
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes 37
ABSTRACT 40
1 Introduction 42
2 Material and methods 44
3 Results 54
Table of contents
4 Discussion 62
5 Conclusions 67
6 Acknowledgments 67
7 References 68
Chapter 4 General discussion 72
Incidence of CVC related thrombosis 72
Heparin thromboprophylaxis for CVC related thrombosis 76
Prediction of CVC related thrombosis 77
Conclusion 78
Chapter 5 Summary 79
Chapter 6 Zusammenfassung 82
Chapter 7 References 86
Acknowledgements 91
List of abbreviations
i
List of abbreviations
CV coefficient of variation
CVC central venous catheter
EJV external jugular vein
ETP endogenous thrombin potential
HDL high-density lipoprotein
IV intravenous
LDL low-density lipoprotein
LMWH low molecular weight heparin
ROTEM rotational elastometry
SC subcutaneous
TGA thrombin generation assay
UFH unfractionated heparin
Chapters 2 and 3 have their own abbreviations included
List of figures and tables
ii
List of figures and tables
Chapter 2
Table 1 Coefficients of variation () for rotational thromboelastometry using the
ROTEMreg delta analyser in normal blood samples 20
Table 2 Coefficients of variation () for rotational thromboelastometry using the
ROTEMreg delta analyser in canine blood samples with haemostatic alterations 20
Table 3 Reference values (median reference interval based on 25 - and 975 -
quantiles) for thromboelastometric measurements with the ROTEMreg delta analyser in
dogs (n=125 95 confidence intervals in brackets) 23
Table 4 Median values (minimum and maximum in brackets) for different ROTEMreg
variables using ex-tem reagent stratified by sex and neuter status 24
Table 5 Median values (minimum and maximum in brackets) for different ROTEMreg
variables using the in-tem reagent stratified by sex and neutering status 25
Chapter 3
Fig 1 Definition of different grades of central venous catheter induced thrombosis of the
jugular vein as detected using colour Doppler ultrasound 48
Fig 2 Definition and illustration of different grades of central venous catheter induced
(extraluminal) thrombosis as detected using electron microscopy 53
Fig 1S (suppl material) Definition and illustration of different grades of intraluminal
thrombosis in central venous catheters as detected using electron microscopy 53
Table 1S (Supplementary material) Heparin activities (IUml median values
minimumndashmaximum) measured in 27 dogs receiving 75 or 150 IUkg unfractionated
(standard) heparin TID subcutaneously 2 hours after the first injection on the day
Comparison of samples collected via puncture of the peripheral veins and those collected
from the central venous catheter (CVC) using the three-syringe-technique 56
List of figures and tables
iii
Fig 3ab Heparin activities (box- and whisker plot arithmetic means) in dogs receiving
unfractionated heparin at a dosage of 75 IUkg BW tid (a) or 150 IUkg BW tid (b) two
hours after the first daily heparin administration 57
Table 1 Heparin activities (IUml median values minimumndashmaximum) in 28 dogs
receiving 75 or 150 IUkg unfractionated (standard) heparin TID subcutaneously with
(grades 1ndash3) or without (grade 0) sonographically detectable thrombus Blood collections
were performed 2 hours after the first daily injection 57
Table 2 Heparin activities (IUml median values minimumndashmaximum) in 27 dogs
receiving 75 or 150 IUkg unfractionated (standard) heparin TID subcutaneously with
(score 1ndash3) or without (score 0) detectable thrombus via electron microscopy 58
Table 3 Comparison of initial results of haemostasis parameters (median values
minimumndashmaximum in dogs with (grades 1ndash3) and without (grade 0) sonographically
detectable central venous catheter-induced thromboses 60
Table 4 Comparison of results of haemostasis parameters (median values minimumndash
maximum) on the last day in dogs with (grades 1ndash3) and without (grade 0) central venous
catheter-associated thrombus formation as detected by electron microscopy 61
Chapter 1 Introduction
1
Chapter 1 Introduction
Central venous catheters (CVC) are mainly used at critical care units in human and small
animal patients requiring infusion of large amounts of fluids or blood parenteral nutrition
support routine drug therapy haemodialysis chemotherapy plasmapheresis or
periodical blood sampling (Geerts 2014 Vose et al 2019) CVC related thrombosis is
one of the most severe complications (apart from infections and pneumothorax) of CVCs
in humans (Comerlato et al 2017 Evans and Ratchford 2018 Geerts 2014) leading to
patientrsquos distress CVC dysfunction venous stenosis and in some cases to the
development of thromboembolism (Geerts 2014) In the human literature there are
various studies dealing with epidemiologic and prophylactic aspects which are described
below in detail In contrast to the best of the authoracutes knowledge studies about extra
luminal CVC related thrombosis in dogs are rare in the available veterinary literature
Studies are mainly dealing with development of intraluminal CVC thrombi occurring
frequently in dogs and cats (Langston et al 2014) and leading to CVC malfunction (Vose
et al 2019)
Epidemiology of CVC related thrombosis
CVC related thrombosis represents 70ndash80 of all upper extremity thrombosis cases and
approximately 10 of thromboembolism cases in humans (Kucher 2011) The incidence
of CVC related thrombosis reported in humans based in 25 studies ranged widely from
2 to 67 (median = 30 ) (van Rooden et al 2005) The incidence of CVC related
thrombosis detected by colour Doppler ultrasound of the internal jugular vein reported in
3 studies performed in humans was 2 (Harter et al 2002) 30 (Lordick et al 2003)
and 56 (Wu et al 1999)
Chapter 1 Introduction
2
The incidence of CVC related thrombosis in humans can vary between studies according
to the CVC type used entry site of the CVC (jugular femoral subclavian axillary
cephalic vein) and time period with the CVC in place the diagnostic thrombus detection
technique (venography or ultrasonography) and criteria for diagnosis duration of follow
up diseases and presence of predisposing factors (eg previous venous thrombosis
history presence of cancer) and finally prophylactic anticoagulation treatment (Geerts
2014 Liu et al 2015 van Rooden et al 2005)
To the best of the authoracutes knowledge data about the incidence of CVC related
thrombosis detected by colour Doppler ultrasound into the lumen of the jugular vein in
dogs are rare in the available veterinary literature In one study performed in 81 dogs and
12 cats (Adamantos et al 2010) the incidence of vein thrombosis detected by palpation
or ultrasound was 2 and the incidence of intraluminal CVC thrombosis (presumed by
the inability to draw blood back) was 15 Additionally regarding to intraluminal CVC
thrombosis an incidence of 8 was reported in one case control study performed in 24
dogs receiving CVC flushing with heparinised solution (Vose et al 2019) Data about
incidence in other animal species was also found In another case-control study performed
on 37 pigs thrombosis in the caval vein related to CVC placed in the external jugular
vein (EJV) was evaluated by histological analysis however no ultrasound detection was
performed (Kohler and Kirkman 1998) In this study in pigs 94 of patients with CVC
developed thrombi in the vena cava based on sections at the site of the end of the catheter
tip and 05 and 1 cm proximal of the first section (Kohler and Kirkman 1998)
Pathogenesis of CVC related thrombosis
Two types of CVC related thrombosis are distinguished in the human medicine literature
(Geerts 2014 van Rooden et al 2005) First symptomatic thrombosis is defined as
Chapter 1 Introduction
3
thrombosis detected by diagnostic images (ultrasonography venography) when
symptoms and signs (eg localised swelling pain redness) are present (Lee and
Kamphuisen 2012) Second subclinical thrombosis is defined as thrombosis
demonstrated by the previously mentioned diagnostic images without overt symptoms or
signs (Boersma et al 2008) Additionally the CVC related thrombi can be classified as
occlusive thrombus into the CVC lumen peri-catheter related fibrin sheaths and mural
thrombi (Geerts 2014)
During the CVC insertion the puncture of the skin on the CVC insertion site and the
rupture of the blood vessels integrity activate the coagulation cascade and the CVC acts
as a matrix for adherence and accumulation of fibrin and cellular components (Smith et
al 2012) The attachment of proteins and blood cells to the internal andor external wall
of the CVC can lead to (1) (partial or complete) occlusion of the catheter lumen and (2)
sometimes if a thrombus formation starts into the lumen of the vein (attached to the vein
wall or to the CVC) a partial or complete obstruction of the blood flow andor embolism
can develop (Langston and Eatroff 2018) Additionally a study performed in humans
(Lordick et al 2003) demonstrated a relationship between CVC related thrombosis and
microbial colonisation of the catheter leading to CVC related systemic infections This
relationship was explained among others by the ease of microorganisms like
Staphylococcus aureus and Staphylococcus epidermidis to adhere to CVC related
thrombin sheaths (Boersma et al 2008)
Diagnosis of CVC related thrombosis
Venous thrombosis is commonly diagnosed in human and small animal medicine
(especially in dogs) with vascular ultrasonography with or without use of the colour
Doppler technique which is normally used to evaluate the blood flow through the blood
Chapter 1 Introduction
4
vessels (Evans and Ratchford 2018 Laurenson et al 2010) According to the literature
in humans cited below the CVC related thrombosis detected by colour Doppler
ultrasound is present if any of the following aspects are observed (1) well defined
thrombus visualised in the lumen of the vein (2) impossibility to perform compression
of the vein by slight pressure with the ultrasound probe andor (3) no detectable blood
flow signal by colour Doppler ultrasound (Bonnet et al 1989 Koumlksoy et al 1995)
Similarly in dogs the CVC related thrombosis is usually diagnosed by colour Doppler
ultrasonography through the detection of the thrombus within the lumen of the vein
andor the obstruction (partial or complete) of the normal blood flow (Bliss et al 2002)
Especially in humans the contrast venography technique is used as gold standard to
confirm thrombosis For this purpose a contrast solution is injected through a peripheral
vein to assess its passage through the bloodstream by x-ray However this diagnostic
technique is normally used only in the case of a strong clinical suspicion of venous
thrombosis that cannot be detected by ultrasonography (Laurenson et al 2010 Langston
and Eatroff 2018) In a study performed in 44 human patients requiring a CVC (Koumlksoy
et al 1995) a sensitivity of 94 and a specificity of 96 were determined for the colour
Doppler ultrasound diagnosis when it was compared with the diagnosis performed with
the contrast venography technique That results make the colour Doppler ultrasound an
accurate diagnosis method to detect CVC related thrombosis in humans (Koumlksoy et al
1995 van Rooden et al 2003)
Advanced diagnostic image techniques like computed tomography or magnetic resonance
tomography are also accurate to perform the diagnosis of CVC related thrombosis in
humans (Whitesell and Steenburg 2014) In dogs multidetector computed tomography
venography was used in one study for the diagnosis of CVC related thrombosis allowing
the identification of catheter related sheaths around the CVC external wall (Wang at al
Chapter 1 Introduction
5
2015) However the high costs of these advanced diagnostic imaging procedures and the
requirement to perform at least sedation or general anesthesia are disadvantages when
compared with ultrasonography
Finally electron microscopy (ELMI) is a diagnostic technique that has been used in
humans (Lucas et al 2017) and experimental animals (Xiang et al 1998 Xiang et al
2001) mainly to perform a microstructural evaluation of thrombi developed into the
lumen of the CVC or in the vein containing the CVC ELMI allows the ex vivo diagnosis
of very small thrombi or sleeve related thrombosis that perhaps cannot be detected by
ultrasonography or venography during the evaluation of patients with CVCs (Xiang et al
2001) However one point to consider is that it is possible that compact thrombi
developed on the CVC can be detached during the CVC removal from the vein and
thereby before its analysis by ELMI (Xiang et al 2001)
Heparin thromboprophylaxis for CVC related thrombosis
Application of CVCs in human medicine is commonly accompanied by prophylactic
anticoagulation in order to decrease the risk of development of partial or complete vein
occlusion andor thromboembolism and thereby to improve the patientrsquos comfort
(Baumann-Kreuziger et al 2015) Apart from vitamin K antagonists (Mismetti et al
2003) unfractionated heparin (UFH) (Abdelfeki et al 2004) and low molecular weight
heparin (LMWH) (Karthaus et al 2006) are anticoagulants that have been used on
studies in humans administrated by intravenous (IV) infusion or by subcutaneous (SC)
injection to prevent thrombi development into the lumen of the veins containing the CVC
(review article Baumann-Kreuziger et al 2015) Different heparin dosages have been
used as thromboprophylactic anticoagulation in humans with CVC (review article Lee
and Kamphuisen 2012) For example in five different case-control studies performed in
Chapter 1 Introduction
6
human haemato-oncological patients UFH at a daily dosage of 100 IUkg given by IV
infusion (Abdelfeki et al 2004) and LMWH at SC daily dosages of 40 mg enoxaparin
(Verso et al 2005) 5000 IU AXa dalteparin (Karthaus et al 2006 De Cicco et al 2009)
and 2850 IU AXa nadroparin (Niers et al 2007) were used Only in part of these studies
it is stated that systemic heparin was combined with flushing of the CVC using
heparinised saline solution to reduce the risk of catheter occlusion (Karthaus et al 2006)
In 3 of the 5 previously mentioned studies no statistically significant difference was found
in the incidence of CVC related thrombosis between patients receiving the
anticoagulation prophylaxis compared with the control groups without systemic
anticoagulation (Verso et al 2005 p = 035 Karthaus et al 2006 p gt 005 Niers et al
2007 p = 049) In contrast in the residual two studies a lower incidence of CVC related
thrombosis was found in the group receiving an anticoagulation prophylaxis compared
with the control groups (Abdelfeki et al 2004 150 vs 126 p = 003 De Cicco et
al 2009 400 vs 526 p = 005)
Obviously there is no consensus in the available human literature regarding the beneficial
use of anticoagulation prophylaxis to prevent CVC related thrombosis and this may
reflect great variations for example according to the study design period trial drug
administration route (Lee and Kamphuisen 2012) and the moment of the initial
anticoagulant dosage administration (Decicco et al 2009)
To the best of the authoracutes knowledge studies about thromboprophylaxis for extraluminal
CVC related thrombosis in dogs cannot be found in the available veterinary literature
However similarly to humans prophylactic anticoagulation with heparinised saline
(administrated through CVC flushing) to prevent CVC occlusion by intraluminal thrombi
was used in one case-control study performed in 24 dogs (Vose et al 2019) In the cited
study 12 dogs received dosages of 3 ml of heparinised saline solution (10 IU UFHml) to
Chapter 1 Introduction
7
flush the catheter every 6 hours during 72 hours whereas 12 control dogs received
flushing with the same volume of non-heparinised saline solution administered at the
same frequency and no statistically significant difference was found
Prediction of CVC related thrombosis
Studies regarding the relationship between laboratory biomarkers and CVC related
thrombosis are available in the human literature (Boersma et al 2016 Cheng et al 2013
Liu et al 2015) however to the best of the authoracutes knowledge literature about laboratory
tests used as predictors for CVC related thrombosis in small animals cannot be found at
all Availability of approved blood tests which can predict the risk of thrombosis
development associated to CVC already at the moment of the CVC placement are
important in order to identify an increased risk of CVC related thrombosis (Boersma et
al 2016) In the study performed by Boersma et al (2016) in 168 human patients without
indication of anticoagulation prophylaxis elevated leukocyte counts high levels of
plasminogen activator inhibitor and of coagulation factor VIII (all measured on blood
samples taken after CVC placement) were associated with an increased incidence of
symptomatic CVC-related thrombosis diagnosed by ultrasonography In another study
(Liu et al 2015) high blood levels of lactate dehydrogenase and low concentrations of
HDL and albumin were associated with the incidence for CVC related thrombosis
diagnosed by ultrasound in 324 senile patients which was in total 617 Additionally
dyslipidemia (high triglycerides low HDL and high LDLHDL ratio) was associated
with CVC related thrombosis (diagnosed by ultrasound and computer tomography
angiography) in human patients undergoing haemodialysis (Cheng et al 2013) Studies
about predictive tests for CVC related thrombosis in dogs were not found in the available
literature to the best of the authoracutes knowledge
Chapter 1 Introduction
8
Aims of the study
The aims of the preliminary methodological study of the present scientific project were
(1) to define reference intervals for viscoelastic analyses of canine haemostasis using the
ROTEMreg delta analyser and (2) to determine the precision (repeatability) of this
method And the aims of the main study of the present scientific project were (1) to
determine the incidence of catheter-induced thrombosis in canine intensive care patients
(2) to evaluate the efficacy of a prophylactic heparin regimen routinely used in the Small
Animal Clinic of the University of Veterinary Medicine Hannover and (3) to assess
whether selected haemostasis test results are predictive for an increased risk for
thrombosis formation
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
9
Chapter 2 Reference intervals for rotational thromboelastometry measurements
using the ROTEMreg delta device in dogs
Pereira JM Rohn K Mischke R
Contributions of the authors
Pereira JM performed (1) the patients data and blood samples collection (2)
haematology and ROTEMreg analysis (3) generated the reference intervals and
coefficients of variation included in the study and (4) wrote the manuscript
Rohn K performed the statistical analysis
Mischke R performed the clinical chemistry and coagulation tests and was involved in
designing and supervision of the study analysis and discussion of the data and its
statistical analysis and revision edition and correction of the manuscript
Original source Pereira J M Rohn K amp Mischke R 2020 Reference intervals
for rotational thromboelastometry measurements using the ROTEMreg delta device
in dogs Res Vet Sci 130 26ndash32 httpsdoiorg101016jrvsc202001019
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
10
Reference intervals for rotational thromboelastometry measurements using the
ROTEMreg delta device in dogs
Pereira JMa Rohn Kb Mischke Ra
aSmall Animal Clinic University of Veterinary Medicine Hannover Buumlnteweg 9 D-
30559 Hannover Germany
bInstitute of Biometry Epidemiology and Information Processing University of
Veterinary Medicine Hannover Buumlnteweg 2 30559 Hannover Germany
Corresponding author
E-mail ReinhardMischketiho-hannoverde
Original source Pereira J M Rohn K amp Mischke R 2020 Reference intervals
for rotational thromboelastometry measurements using the ROTEMreg delta device
in dogs Res Vet Sci 130 26ndash32 httpsdoiorg101016jrvsc202001019
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
11
ABSTRACT
Aims of the present study were to define reference intervals for viscoelastic analyses of
canine haemostasis using the ROTEMreg delta analyser and as a secondary aspect to
determine the precision (repeatability) of this method Blood samples from 125 clinically
healthy dogs were included Measurements were performed with commercially available
activating reagents (ex-tem in-tem and kaolin solution) as well as without activation
Additional fourfold measurements were done in 3 of the normal blood samples and in 3
samples with haemostatic alterations to evaluate the precision of the method
Coefficients of Variation (CVs) for most of the ROTEM variables were lt 10 Clot
formation time and maximum clot elasticity showed a wide inter-individual variation in
comparison with alpha angle and maximum clot firmness A multivariate analysis on
various ROTEM parameters revealed particularly a significant influence of neuter status
and a significant interrelationship between the factors sex and neuter status for
measurements with different activating reagents These results reflected the fact that
significant differences occurred only between intact and neutered females but not in
males No or only occasionally significant differences were found between groups of sex
age and size
In conclusion CVs demonstrated that the method delivers repeatable results in canine
citrated whole blood Established reference intervals should deliver valuable orientation
for the evaluation of viscoelastic properties of clotting whole blood in dogs using the
ROTEM delta analyser Neuter status in females appeared to be the most relevant
influencing factor and should be considered for the interpretation of ROTEM delta test
results
Keywords Viscoelastic measurements reference values canine age sex neuter status
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
12
1 Introduction
Heamostasis is a physiologic and dynamic process in which plasma soluble coagulation
factors blood cells and blood vessels are involved Clot formation and possible
subsequent clot lysis as well as vascular tissue repair aim to prevent haemorrhages on the
one hand and thromboses on the other hand (Kol and Borjesson 2010 Versteeg et al
2013) Conventional assays of coagulation such as prothrombin time (PT) activated
partial thromboplastin time (aPTT) and thrombin time TT have limitations especially
cellular elements are not incorporated into the test and thereby these tests reflect only
limitedly in vivo functionality of haemostasis (McMichael and Smith 2011 Crochemore
et al 2018)
The rotational thromboelastometry (ROTEMreg) currently termed as thromboelastometry
(TEM) and thromboelastography (TEG) are methods for the rapid evaluation of the
overall haemostasis capacity through the measurement of viscoelastic properties of
clotting whole blood (Kol and Borjesson 2010 Theusinger et al 2010 Sankarankutty
et al 2012 Versteeg et al 2013) In TEG the cup with the blood sample is rotating
whereas the torsion wire is fixed In ROTEMTEM the cup is fixed whereas the pin is
rotating Changes in torque ie resistance to rotation by the fibrin bridge created between
pin and cup in the clotting blood are detected electromechanically in TEG and optically
in ROTEM (Bollinger et al 2012) and displayed as a graph that indicates the time for the
formation of blood clot and its strength (Bagge et al 2016) The resulting graph reflects
the initiation and propagation of the coagulation system fibrin-platelet interactions and
clot lysis
TEG was first described in 1948 by Hellmut Harten (Hartert 1948) as an in vitro method
to evaluate the clot formation process while resembling the in vivo blood flow The
development of the actually used instrument types TEG and ROTEMregTEM were based
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
13
on that original principle Viscoelastic measurements have been widely used since the
1980s in human medicine to evaluate haemostasis and hypercoagulable states in surgical
patients (eg cardiac and hepatic surgeries) and in general in critical care units (Kol and
Borjesson 2010 Nogami 2016 Wikkelso et al 2016 Crochemore et al 2017) In
veterinary medicine viscoelastic measurements using TEG and ROTEMTEM have been
validated for use in dogs cats and horses and used to study haemostasis in different
diseases (eg infectious diseases liver diseases) and medical conditions (eg
orthopaedic surgery hyperfibrinolysis) and also to investigate the effect of
anticoagulants and other drugs (eg corticosteroids) on haemostasis (Kol and Borjesson
2010 Bruno et al 2015a 2015b Woodman et al 2015 Conversy et al 2017 Sigrist et
al 2018 Turner et al 2019) The methodology seems to be especially suitable to detect
hypercoagulability (Wiinberg et al 2008 Kol and Borjesson 2010 Marschner et al
2018)
In the available veterinary literature information about reference values for TEG were
found using kaolin as activation reagent (Bauer et al 2009) Falco et al (2012)
determined reference values for the ROTEM analyser in dogs using ex-tem in-tem and
fib-tem reagents for selected ROTEM parameters in a study on forty five healthy dogs
However data related to reference intervals obtained in an adequate number of samples
(at least 120) according to the American Society for Veterinary Clinical Pathology
(ASVCP) (Friedrichs et al 2012) and the National Committee for Clinical Laboratory
Standards (NCCLS) (Horn and Pesce 2003) could not be found in the available literature
The aim of this study is to determine reference values for the ROTEMreg delta device in
dogs using citrated whole blood with different clot activator reagents (ex-tem in-tem
kaolin) and without activator
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
14
2 Materials and methods
21 Study design
Reference intervals Single thromboelastometric measurements were performed in 125
clinically healthy dogs using the ROTEMreg analyser and different activating reagents (ex-
tem in-tem and kaolin) as well as without activation Reference values were calculated
and influences of age groups (young adult and senior) sex neuter status and size
(groups small medium and large) were assessed
Precision analysis Additional fourfold measurements using ex-tem or in-tem activating
reagents respectively were performed on 3 of the normal and also in 3 abnormal blood
samples each in order to assess within-run imprecision for these measurements
22 Animals
The clinically healthy dogs with a body weight of at least 5 kg were recruited by launching
a call in the University of Veterinary Medicine Hannover network for students and staff
members Interested dog owners received a free health check of their animals (clinical
examination and blood tests specified below) in combination with the blood collection
In addition individual privately owned patient dogs of the Small Animal Clinic with
undisturbed general health were also included if blood was collected due to another
indication (eg general health check pre-anaesthetic examination test for hereditary
diseases etc) Before taking the blood samples all dogs were fasted for at least 12 h with
free access to water 125 animals for which the examination of the health status (clinical
examination routine haematology and clinical chemistry basis coagulation profile) did
not reveal any deviations from the reference were included in the study whereas 3 of the
128 initially recruited animals had to be excluded due to laboratory abnormalities
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
15
Haematological and clinical chemistry profiles performed in order to confirm the health
status included white and red cell count haematocrit platelet counts and reticulocytes
plasma activities of alanine aminotransferase glutamate dehydrogenase alkaline
phosphatase and pseudocholinesterase as well as concentrations of total bilirubin urea
creatinine glucose total protein albumin cholesterol and ionised calcium A basis
coagulation profile including PT PTT and TT was also performed to exclude significant
abnormalities within the blood coagulation system
The 125 healthy dogs enclosed in the establishment of reference values included 48 mixed
breed dogs and the following pedigree dogs 13 Labrador Retrievers 6 Australian
Shepherds 4 Havanese dogs 3 from each of the following breeds American
Staffordshire Terriers and Cocker Spaniels 2 from each of the following breeds Beagles
Border Collies Dalmatians Elo dogs (breed not recognised by Feacutedeacuteration cynologique
international [FCI]) German Longhaired Pointers German Shorthaired Pointers Giant
Schnauzers and Golden Retrievers 1 from each of the following breeds Belgian
Malinois Bolonka Zwetna Briard Brussels Griffon American English Coonhound
Italian Mastiff Flat Coated Retriever French Bulldog German Shepherd Great Dane
Greater Swiss Mountain Dog Giant Spitz Goldendoodle Hovawart Hungarian Vizsla
Italian Bloodhound Lycanis Wolfdog Miniature American Shepherd Miniature
Schnauzer Old German Shepherd Podenco Poodle Pyrenean Shepherd Rhodesian
Ridgeback Rottweiler Schafpudel (breed not recognised by FCI) Small
Munsterlander Spanish Mastiff Tibet Terrier Weimaraner West Highland White
Terrier and Wirehaired Pointer
These dogs were aged between 6 and 154 months (median = 410 months) and had body
weights ranging from 50 to 707 kg (median = 226 kg) 30 were castrated males 33
intact males 41 spayed females and 21 intact females According to the height at withers
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
16
the dogs were divided into 3 different size groups small (le40 cm n = 22) medium (41ndash
60 cm n = 77) and large (gt60 cm n = 26) Dogs were also divided in three different age
groups young (le 24 months n = 34) adult (25ndash84 months n = 59) and senior dogs (gt84
months n = 32)
The three blood samples with haemostatic abnormalities were taken from two dogs with
complex haemostatic disorders (Disseminated Intravascular Coagulation [DIC])
including reduced platelet count and abnormal PT and APTT and one dog with
thrombocytopenia
Animals were treated in accordance with the German Animal Welfare Law The
experimental design was approved by the Animal Welfare officer of the University of
Veterinary Medicine Hannover Foundation and by the ethics committee of the
responsible authority (Lower Saxony State Office for Consumer Protection and Food
Safety reference number 17A101)
23 Blood sampling
The venipuncture area was shaved and then disinfected with alcohol The saphenous or
cephalic vein was visualised with slight pressure and punctured with disposable cannulas
(diameter 09 or 10 mm) The continuously running blood was directly collected from
the needle into the tubes containing the respective anticoagulants From each dog
approximately 10 ml of blood were collected including approximately 8 ml of citrated
blood for the measurement of haemostasis tests (ROTEM PT aPTT TT) in tubes
containing sodium citrate 313 (Eifelfangoreg GmbH amp Co KG Bad Neuenahr-
Ahrweiler Germany) in a ratio of 1 volume9 volumes of blood In addition
approximately 1 ml of EDTA blood for haematological analysis and approximately 1 ml
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
17
of lithium heparin blood for clinical chemical measurements were collected into
commercial 13 ml tubes (Sarstedt AG amp Co Nuumlmbrecht Germany)
Citrated blood was initially kept at room temperature (20-25 C) in order to avoid errors
within the preanalytical phase (Toulon et al 2017) Two ml of citrated blood were used
for the ROTEMreg analysis the residual citrate blood was centrifuged for 10 min at 3800
rpm and 5 degC then the plasma was separated by careful pipetting and it was centrifuged
a second time under the same conditions to get platelet poor plasma which was stored in
aliquots of 500 microl and 250 microl at -80 degC for coagulation tests
24 Rotational elastometry
Viscoelastic measurements using the ROTEMreg delta for the establishment of reference
intervals were performed approximately 15 min after the blood collection using activating
reagents (liquid reagents) provided by the manufacturer of the instrument (r-ex-temreg in-
temreg) and a kaolin solution (5 g1 as part of the PTT reagent CK Prest [Diagnostica
Stago SAS Asniegraveres sur Seine Cedex France]) In addition non-activated analyses
were performed using isotonic NaCl solution instead of the activating reagent in the fourth
channel of the device Fourfold measurements for precision analysis were performed
simultaneously in the four channels of the device in separate runs
According to the standard test protocol provided by the manufacturer of the ROTEMreg
delta (Werfen GmbHInstrumentation Laboratory Munich Germany) 20 μl of the
reagent for recalcification (star-temreg20) and 20 μl of the activating reagent were pipetted
into a cuvette using the automated pipette provided with the device Immediately
thereafter 300 μl of citrated whole blood (prewarmed to 37 degC for five minutes in the
heating device of the instrument and gently rocked to resuspend possibly sedimented
cells) were added to the test preparation in the cuvette using the automated pipette and
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
18
then the cuvette was laced carefully in the ROTEMreg device whereby the rotating pin
entered into the test approach
Different parameters are provided automatically by the ROTEMreg delta device and the
following variables (described according to the Pentapharm GmbH ROTEMreg delta
Manual [2009]) were used in the study
Clotting time (CT) the time (in s) from the beginning of the test (by adding the clot
activator) until the formation of fibrin starts (defined as the moment when the curve
achieves an amplitude of 2 mm) This Parameter facilitates the decision to replace clotting
factors when it is required
Clot formation time (CFT) describes the next phase of clotting by the formation of a
stable clot through the activation of thrombocytes and fibrin formation It is defined as
the time (in s) between the 2 mm amplitude and 20 mm amplitude of the curve
Alpha angle (α angle) is the angle (in degrees) between the middle axis and the tangent
to the clotting curve through the 2 mm amplitude point
Amplitude after ldquoXXrdquo minutes (eg A30 after 30 min) is the amplitude (in mm) at a
certain time after the CT has finished
Maximum clot firmness (MCF) corresponds to the maximum amplitude (in mm) reached
before the clot is dissolved by fibrinolysis This parameter measures the firmness of the
clot and its quality Like CFT and α angle it facilitates the decision for substitution therapy
with platelet concentrate andor fibrinogen especially in humans
Maximum clot elasticity (MCE) is a parameter calculated from MCF and may be superior
at high amplitudes as compared to the MCF
Lysis index at 30 45 and 60 min (LI30 LI45 LI60) represents the fibrinolysis (in
percentage) 30 45 and 60 min after the CT It may facilitate the decision for or against a
therapy with anti-fibrinolytic drugs
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
19
Maximum lysis (ML) describes the degree of fibrinolysis relative to the MCF achieved
during the measurement (percent loss of clot firmness)
25 Statistical methods
Original data obtained from the ROTEMreg delta device software were transferred into the
Microsoftreg Excelreg formal (Microsoft Office Professional Plus 2013) The Statistical
analyses were performed using the SAS Enterprise Guide Version 71 (SAS Institute Inc
Cary NC USA)
Coefficients of Variation (CVs) for selected relevant parameters of the ROTEM delta
analysis using ex-tem and in-tem regents were calculated for the fourfold measurements
on 3 normal and 3 abnormal blood samples in order to assess within-run imprecision CVs
for the results of each sample were calculated by dividing the standard deviation of the
data set by its arithmetic mean Finally the median of the CVs for each group of 3 samples
was calculated The reference values were calculated according to recommendations of
the American College of Veterinary Clinical Pathology (ACVCP) based on non-
parametric methods (25 and 975 quantiles) supplemented by 95 confidence
intervals (Friedrichs et al 2012)
Data were analysed for Standard normal distribution by Kolmogorov Smirnov test and
assessment of residuals of the analysis of variance (ANOVA) Because these tests
indicated almost normally distributed values for the studied ROTEM variables data were
analysed by a fourway ANOVA stratified by the activating reagent (ex-tem in-tem
kaolin or without activation) to test the influence of size age sex and neuter status on
several parameters and taking all interactions into account Post hoc t-tests were
performed when indicated The significance level was set to P lt 05
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
20
3 Results
31 Precision of the method
For all the parameters evaluated in the study the CVs for the fourfold measurement in
healthy dogs were less than 10 (Table 1) In the case of dogs with haemostatic
abnormalities CVs of the vast majority of ROTEM variables were also less than 10
except the variables CFT and ML irrespective whether ex-tem or in-tem reagents were
used (Table 2)
Table 1 Coefficients of variation () for rotational thromboelastometry using the
ROTEMreg delta analyser in normal blood samples
Activator CT (s) CFT (s) α (deg) A30 (mm) MCF (mm) MCE(mm) LI30 () LI45 () LI60 () ML ()
Ex-temreg 875 629 262 213 179 634 000 020 037 534
In-temreg 245 596 166 117 135 299 000 000 046 216
Abbreviations CT ndash clotting time CFT ndash clot formation time α ndash alpha angle A30 ndash
amplitude after 30 min MCF ndash maximum clot firmness MCE ndash maximum clot elasticity
LI 30 LI 45 LI 60 ndash Lysis index at 30 45 and 60 min ML ndash maximum lysis
Median values of the measurements of 3 samples
Table 2 Coefficients of variation () for rotational thromboelastometry using the
ROTEMreg delta analyser in canine blood samples with haemostatic alterations
Activator CT (s) CFT (s) α (deg) A30 (mm) MCF (mm) MCE(mm) LI30 () LI45 () LI60 () ML ()
Ex-temreg 668 1478 393 218 268 582 0 018 000 848
In-temreg 228 799 461 219 251 371 0 0 017 283
Median values of the measurements of 3 samples from patients with DIC (n = 2)
thrombocytopenia (n = 1) abbreviations please see legend to Table 1
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
21
32 Reference intervals
Activation by ex-tem reagent (tissue factor) resulted in faster CTs when compared to
activation via the intrinsic pathway using ellagic acid (in-tem) or kaolin reagents whereas
the CFT was much less affected by the activating reagent (Table 3) Especially CFT and
MCE showed a wide inter-individual variation in comparison for example with α angle
and MCF regardless of the used activating reagent (Table 3)
33 Influence of age sex neuter status and size
ANOVA did not reveal statistically significant differences in ROTEM delta parameters
between dog groups of variable size or sex (P gt 05) Age had influence only on CT and
only when the in-tem reagent was used (P = 0169 ANOVA) but not when using the ex-
tem reagent (P = 7105) Post hoc comparisons using t-test of the in-tem results between
age groups revealed shorter CTs in the group of senior dogs when compared with young
dogs (P = 0194) but not between adult dogs and these two groups ANOVA showed
particularly influences of the neuter status and a significant interrelationship between the
factors sex and neuter status on different ROTEM delta parameters using ex-tem and in-
tem respectively as activator (Tables 4 and 5) ANOVA revealed also a significant
influence of neuter status on different ROTEM parameters when kaolin was used as
activating reagent (CFT α angle MCF MCE) whereas non-activated measurements
were not subject to any significant influence of the assessed factors (results not shown)
The interrelationship between sex and neuter status mentioned above reflected the fact
that significant differences between neutered and not neutered dogs were only found in
females The statistical analysis using the t-test to compare intact vs neutered dogs
stratified by sex showed statistically significantly (P lt 05) longer CTs and CFTs and
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
22
lower α angle A30 MCF and MCE values in spayed when compared to intact females
(Tables 4 and 5)
Chapter 2 Reference intervals for rotational thromboelastometry measurements using the ROTEMreg delta device in dogs
23
Table 3 Reference values (median reference interval based on 25 - and 975 - quantiles) for thromboelastometric measurements with the ROTEMreg delta analyser in
dogs (n=125 95 confidence intervals in brackets) Abbreviations please see legend to Table 1
Activator
Statistical
para-meter
CT
(s)
CFT
(s)
Α
(deg)
A30
(mm)
MCF
(mm)
MCE
(mm)
LI30
()
LI45
()
LI60
()
ML
()
Ex-temreg
Median 450
[430ndash470]
110
[105ndash115]
680
[670ndash690]
600
[580ndash610]
610
[600ndash620]
154
[147ndash164]
100
[960ndash100]
100
[100ndash100]
970
[960ndash980]
220
[210ndash240]
25 340
[270ndash350]
650
[490ndash710]
540
[420ndash560]
460
[410ndash480]
460
[420-490]
870
[720ndash950]
100
[100-100]
870
[800ndash960]
700
[640ndash820]
800
[100ndash110]
975 830
[790ndash990]
220
[201ndash315]
790
[770ndash800]
770
[690ndash770]
730
[700ndash770]
264
[230-334]
100
[100ndash100]
100
[100ndash100]
100
[990ndash100]
590
[530ndash680]
In-tem
Median 190
[187ndash201]
116
[111ndash125]
680
[670ndash690]
570
[560ndash580]
590
[580ndash600]
143
[135ndash149]
100
[100ndash100]
100
[100ndash100]
980
[980ndash980]
180
[170ndash190]
25 135
[112ndash142]
730
[590ndash800]
510
[460ndash570]
450
[410ndash470]
480
[410ndash500]
920
[710ndash101]
100
[950ndash100]
990
[820ndash990]
930
[740ndash930]
500
[100ndash700]
975 283
[268ndash291]
251
[198ndash227]
750
[750ndash780]
660
[660ndash690]
680
[660ndash690]
210
[195ndash227]
100
[100ndash100]
100
[100ndash100]
100
[100ndash100]
270
[260ndash380]
Activator
Statistical
para-meter
CT
(s)
CFT
(s)
Α
(deg)
A30
(mm)
MCF
(mm)
MCE
(mm)
LI30
()
LI45
()
LI60
()
ML
()
Kaolin
Median 162
[154ndash173]
870
[820ndash910]
740
[720ndash750]
500
[470ndash530]
550
[530ndash560]
121
[115ndash127]
960
[940ndash980]
840
[790ndash880]
710
[640ndash770]
500
[450ndash540]
25
109
[990ndash115]
470
[440ndash580]
560
[490ndash600]
110
[700ndash230]
390
[380ndash400]
640
[600ndash680]
270
[140ndash480]
110
[800ndash260]
800
[400ndash170]
200
[160ndash240]
975 246
[230ndash337]
214
[185ndash290]
810
[790ndash810]
640
[630ndash690]
650
[650ndash690]
187
[182ndash223]
100
[100ndash100]
990
[980ndash100]
940
[930ndash990]
970
[930ndash100]
Without activation
Median 607
[495ndash662]
221
[194ndash265]
510
[460ndash550]
520
[500ndash530]
540
[520ndash550]
115
[109ndash123]
100
[100ndash100]
100
[100ndash100]
980
[980ndash990]
170
[160ndash190]
25 203
[123ndash215]
860
[550ndash930]
190
[120ndash230]
350
[230ndash370]
370
[260ndash400]
600
[350ndash670]
100
[950ndash100]
980
[890ndash990]
930
[840ndash950]
400
[000ndash500]
975 1356
[1305ndash1721]
811
[688ndash1333]
730
[710ndash790]
660
[640ndash720]
660
[640ndash730]
197
[179ndash265]
100
[100ndash100]
100
[100ndash100]
100
[100ndash100]
290
[280ndash320]
Chapter 2 Reference intervals for rotational thromboelastometry measurements using the ROTEMreg delta device in dogs
24
Table 4 Median values (minimum and maximum in brackets) for different ROTEMreg variables using ex-tem reagent stratified by sex and neuter status
Group
CT
(s)
CFT
(s)
Α
(deg)
A30
(mm)
MCF
(mm)
MCE
(mm)
LI30
()
LI45
()
LI60
()
ML
()
Female intact 430
(320ndash530)
960
(590ndash198)
710
(570ndash800)
620
(480ndash730)
640
(510ndash740)
179
(103ndash282)
100
(100ndash100)
100
(980ndash100)
960
(800ndash100)
230
(100ndash640)
Female spayed 460
(270ndash830)
121
(650ndash224)
660
(520ndash770)
580
(430ndash690)
590
(440ndash700)
146
(790ndash230)
100
(960ndash100)
100
(800ndash100)
970
(690ndash100)
240
(100ndash590)
p-value (t-test)
Female
intact vs spayed
00025 00063 00049 00023 00016 00015 na na na na
Male intact 450
(320ndash990)
105
(650ndash315)
690
(420ndash790)
590
(420ndash700)
610
(440ndash710)
154
(770ndash244)
100
(100ndash100)
100
(870ndash100)
970
(650ndash100)
220
(700ndash680)
Male castrated 460
(340ndash930)
111
(490ndash255)
685
(480ndash800)
590
(410ndash770)
600
(420ndash770)
152
(720ndash334)
100
(100ndash100)
100
(830ndash100)
980
(640ndash100)
205
(500ndash590)
p-value (t-test)
Male
Intact vs castrated
08326 05106 04756 01799 01568 02163 na na na na
p-values (ANOVA)
Factor Sex
05428 04847 06612 02206 01887 01630 08489 07893 07689 02758
Factor neuter status 01872 00167 00173 00039 00037 00028 07141 04737 09356 05948
Interrelationship
sexneuter status 00383 00968 00782 00488 00472 00319 04943 08611 05422 00160
na ndash not analysed all significant P-values (lt 005) are highlighted in bold print
Chapter 2 Reference intervals for rotational thromboelastometry measurements using the ROTEMreg delta device in dogs
25
Table 5 Median values (minimum and maximum in brackets) for different ROTEMreg variables using the in-tem reagent stratified by sex and neutering status
Group
CT
(s)
CFT
(s)
Α
(deg)
A30
(mm)
MCF
(mm)
MCE
(mm)
LI30
()
LI45
()
LI60
()
ML
()
Female intact 186
(126ndash238)
940
(590ndash164)
720
(620ndash780)
610
(490ndash690)
620
(530ndash690)
166
(113ndash227)
100
(100ndash100)
100
(990ndash100)
980
(930ndash100)
170
(100ndash300)
Female spayed 201
(149ndash291)
1220
(820ndash262)
670
(500ndash740)
550
(450ndash660)
570
(480ndash680)
133
(910ndash214)
100
(950ndash100)
100
(820ndash100)
980
(740ndash100)
190
(500ndash380)
p-value (t-test)
Female
intact vs spayed
na na lt0001 00009 00005 00005 na na na na
Male intact 199
(117ndash283)
122
(730ndash277)
680
(460ndash750)
570
(430ndash660)
590
(450ndash660)
146
(810ndash192)
100
(100ndash100)
100
(990ndash100)
980
(930ndash100)
180
(600ndash270)
Male castrated 181
(112ndash288)
123
(820ndash222)
670
(540ndash740)
565
(410ndash630)
585
(410ndash640)
138
(710ndash180)
100
(100ndash100)
100
(970ndash100)
980
(890ndash100)
180
(800ndash260)
p-value (t-test)
Male
intact vs castrated
na na 08232 01401 01162 00849 na na na na
p-values (ANOVA)
Factor Sex
02105 03317 03370 00877 01258 00510 09564 07832 08117 03638
Factor neuter status 04289 00660 00619 00107 00088 00054 07152 09795 07056 08299
Interrelationship
sexneuter status
04310 00550 00408 00707 01733 00996 05240 05982 08047 04578
na ndash not analysed all significant P-values (lt 005) are highlighted in bold print
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
26
4 Discussion
41 Coefficients of variation
In accordance with the literature CVs below 10 indicate adequate repeatability in intra-
assays evaluations (Steiner et al 2003 Hua et al 2018) According to our results
obtained from the fourfold measurements in citrated whole blood ROTEM delta
measurements on healthy dogs and dogs with haemostatic alterations fulfil these
requirements with only a few exceptions concerning CFT and ML values in abnormal
samples
Our results regarding repeatability are similar to those obtained for several TEM
parameters in previous studies performed in animals In one study the imprecision of the
ROTEM analyser was evaluated by duplicate measurements in 10 blood samples of
healthy dogs and as result of that evaluation a good precision (CVs below 10 ) was
calculated for CT CFT MCF and α angle but in contrary to our results a low
repeatability was found for ML (CVs of 233 [ex-tem] and 285 [in-tem] own values
534 and 216 ) (Falco et al 2012)
In a previous feline study using ex-tem and in-tem as activating reagents for the ROTEM
delta repeatability was assessed similarly to the present study based on fourfold
measurements of blood samples of two healthy individuals (Doumlderlein and Mischke
2015) The results also revealed CVs below 10 for most of the parameters evaluated
The partially low repeatability of ML values may reflect the vulnerability of this variable
which is not only influenced by the complex clot formation process but also by its
subsequent lysis Reproducibility of ex-tem or in-tem activated measurements of the
ROTEM delta analyser which was not investigated in our study was studied in blood
from healthy and unhealthy horses by two different operators using three different
ROTEM devices Results indicated acceptable reproducibility (Junge et al 2016)
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
27
42 Reference intervals
The number of 125 healthy dogs used in the present study to create reference intervals is
regarded adequate according to the guidelines suggested by the American Society of
Veterinary Clinical Pathology (ASVCP) and the National Committee for Clinical
Laboratory Standards (NCCLS) who recommend to include at least 120 individuals
(Friedrichs et al 2012) To the best of our knowledge reference intervals for the most
relevant ROTEMreg delta parameters obtained in an adequate number of samples of whole
citrated canine blood have not been published so far A previous study was performed on
a smaller number of 45 dogs (Falco et al 2012) The median values for reference
intervals obtained in the present study are partially similar to those calculated in the
mentioned study based on 45 healthy dogs (eg ex- tem CT 610 s450 s [Falco et al
2012own results] CFT 150 s 110 s α angle 620deg680deg MCF 530 m 610 mm ML
180 220 in-tem CT 235 s190 s CFT 136 s116 s α angle 660deg680deg MCF
560 mm590 mm ML 30 180 ) However in comparison to our results the
reference intervals in the mentioned study are slightly wider (eg ex-tem CT 29ndash92
s34ndash83 s [Falco et al 2012own results] CFT 540ndash275 s650ndash220 s α angle 470ndash
790deg540ndash790deg MCF 360ndash730 mm460ndash730 mm ML 000ndash560 800ndash590
in-tem CT 126ndash363 s135ndash283 s CFT 470ndash224 s730ndash251 s aα angle 550ndash
810deg510ndash750deg MCF 500-ndash750 mm480ndash680 mm ML 000ndash400 500ndash270 )
although the previous study used the 5 and 95 quantiles to define limits of the
reference range whereas we calculated the commonly used 25 and 975 quantiles
to capture the central 95 of reference values (Friedrichs et al 2012)
Although it is desirable that each laboratory should specifically determine its own
reference intervals the results obtained in this study may provide an orientation for other
laboratories to interpret ROTEM delta parameters analysed in citrated canine blood In
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
28
order to test transference of the reference ranges to another laboratory a validation
procedure with measurement of 20 samples should be performed (Friedrichs et al 2012)
Although intra-individual control values (subject-based reference values) are regarded
superior to population based reference values (Wiinber et al 2007) the latter have to be
routinely used in clinical patients due to lacking individual controls especially for specific
tests such as ROTEM analyses In addition the calculated reference values cannot be
used for the fully automated device ROTEM sigma because - according to an actual
human study - values of part of the parameters do not correlate well between both
instruments (Gillissen et al 2019)
43 Influence of size age sex and neuter status
In previous studies in humans (Attard et al 2013) and animals (Mischke 1994 Borrelli
et al 2017) age has been described as an influencing factor for different haemostasis
parameters We also found an influence of age on the CT with shorter times in senior
dogs (gt 84 months) compared with young dogs (up to 24 months) which was limited to
measurements activated by the in-tem reagent
This age dependency confirms in principle a previous study on 20 healthy dogs
(Barthelemy et al 2015) which found shorter CTs in elderly dogs and results of a human
study where individuals with an age gt 45 years showed shorter CTs compared with
individuals lt 45 years old (Sucher et al 2011) However in the cited studies this effect
was only seen when ex-tem but not when the in-tem reagent was used
Based on the age distribution of our dogs which were primarily recruited to establish
reference intervals we cannot deliver information regarding very young dogs A human
study focusing on ROTEM reference ranges in children demonstrated significant
differences for nearly all ROTEM parameters with the most striking differences in
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
29
subjects aged 0-3 months (Oswald et al 2010) The limited number of dogs with an age
lt 1 year which did not allow to consider these separately in the present study may also
be one reason for the different results in a feline study (Doumlderlein and Mischke 2015) In
the cited study on healthy cats age showed an influence on CT but contrary to our study
the CT was shorter in young individuals (6-12 months) compared with older ones In
addition this was only the case in measurements using kaolin and in non-activated
measurements When compared to humans and cats uni- or multivariate analyses
considering factors age and breed or size based on do populations including different
breeds have the limitation that they can be biased by the variate life expectancy between
small medium and large dog breeds (Willems et al 2017) Apart from age we found a
significant influence only for the neuter status in bitches reflected by a significant
interaction effect between factors neuter status and sex in the ANOVA analysis
Especially for CFT values (eg activated by ex-tem median values of 121 s [spayed] vs
96 s [intact]) this difference seems to be also clinically relevant with respect to the
interpretation of ROTEM results In contrast although also significantly different
absolute and relative differences of CT values were small (eg activated by ex-tem 46 s
[spayed] vs 43 s [intact]) These differences may not only be considered for the result
interpretation of clinical patients but indicate also the necessity for sex-matched patient
groups in scientific studies
The obviously more active haemostasis system in intact females is probably related to an
influence of sex hormones (specifically estrogens) on the coagulation system
Accordingly ex vivo and in vivo studies demonstrated the activating properties of
endogenous estrogens on the haemostasis system In one ex vivo study addition of
endogenous estrogens (estrone [E1] 17szlig-estradiol [E2] and estriol [E3]) to blood samples
from men resulted in shorter mean values of the reaction time in TEG analyses
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
30
corresponding to CT in ROTEM measurements (452-537 s vs 698 s [control]) and of
the TEG variable kinetics (corresponding to the CFT in ROTEM analyses) (Swanepoel
et al 2017) In a further study subcutaneous injection of E2 into male rats induced a
significant increase in activities of coagulation factors X (by 8 ) and XI (by 10-24 )
however did not show any significant effect on PT aPTT and factor VII activity (Franco-
Murillo and Jaimez 2017)
In the cited studies also the effects of the synthetic estrogen ethinylestradiol [EE] were
studied which interestingly seems to have an inhibiting effect as indicated by prolonged
reaction time and kinetics (TEG) compared with the control in the human ex vivo study
and significantly longer PTs and aPTTs and lower factor VII and X activities in the study
on rats As expected we did not find a significant influence of size on ROTEM parameters
in dogs To the best of the authoracutes knowledge size has not been examined as an
influencing factor on viscoelastic measurements or any other haemostatic variable in
dogs Apart from Greyhounds there is limited literature on breed comparisons of
haemostatic variables (Clemons and Meyers 1984 Mischke 1994 Nielsen et al 2007
Nielsen et al 2010 Boyd et al 2018) indicating very limited breed differences for
example for factor VII (Mischke 1994) However these also do not indicate systematic
influence of size and in addition possible breed-specific effects have to be considered
As already stated one limitation of the study is the limited number of young animals
which does not allow to verify reference values for animals in this stage of life In
addition with respect to the main aim of the study possible influencing factors such as
platelet count and haematocrit were not considered in the statistical analysis Finally the
relatively small number of dogs used for the precision analysis which express the fact
that this was only a secondary aspect of the study may be regarded as a limitation At the
same time these considerations provide space for further investigations
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
31
5 Conclusions
Results of the precision analysis revealed acceptable results and thereby confirm that the
ROTEMreg delta analyser delivers repeatable results to measure overall haemostasis in
canine citrated blood samples The established reference values should offer a reliable
basis for the interpretation of thromboelastometric measurements in whole blood using
the device ROTEMreg delta in dogs with suspected haemostasis disorders Neuter status
in female dogs appeared to be a relevant influencing factor on most important ROTEMreg
parameters and should be considered for result interpretation and with respect to
experimental designs
6 Acknowledgments
Jose Mauricio Pereira received a scholarship from the Universidad Nacional de Costa
Rica (reference code JB-C-1107-2016)
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
32
7 References
1 Attard C van der Straaten T Karlafitis V Monagle P Ignjativic V 2013 Developmental
hemostasis age-specific differences in the levels of hemostatic proteins J Thromb and
Haemost 11 1850ndash1854
2 Bagge A Schoumltt U Kander T 2016 Effects of naturopathic medicines on Multiplate and
ROTEMreg A prospective experimental pilot study in healthy volunteers BMC Complemen
Altern Med 17 (16) 64
3 Bartheacutelemy A Rannou B Forterre M Verwaerde P Bonnet-Garin JM Pouzot-Nevoret
C Goy-Thollot I 2015 Differences between coagulation and cytokine profiles in dogs of
different ages Vet J 205 410ndash412
4 Bauer N Eralp O Moritz A 2009 Establishment of reference intervals for kaolin-activated
thromboelastography in dogs including an assessment of the effects of sex and anticoagulat use
J Vet Diagn Investi 21 641ndash648
5 Bollinger D Seeberger MD Tanaka KA 2012 Principles and practice of
thrromboelastography in clinical coagulation management and transfusio practice Transfus Med
Rev 26 1ndash13
6 Borrelli A Botto A Maurella C Falco S Pagani E Miniscaldo B Tarducci A Bruno
B 2017 Thromboelastometric assesment of hemostasis in newborn Piemontese calves J Vet
Diagn Invest 29 293ndash297
7 Boyd CJ Claus MA Raisis AL Hosqood G Sharp CR SmartL 2018
Hypocoagulability and platelet dysfunction are exacerbated by synthetic colloids in a canine
hemorrhagic shock model Front Vet Sci 5 279
8 Bruno B Maurella C Falco S Tarducci A Zanatta R DrsquoAngelo A Borrelli A 2015a
Thromboelastometric evaluation of hemostasis in dogs infected with Leishmania infantum J Vet
Emerg Crit Care (San Antonio) 25 502ndash511
9 Bruno B Maurella C Falco S Tarducci A Zanatta R Gianella P DrsquoAngelo A Piras L
Di Bella A Borrelli A 2015b Assessment of coagulation utilizing thromboelastometry in dogs
undergoing orthopedic surgery J Vet Emerg Crit Care (San Antonio) 25 358ndash363
10 Clemons RM Meyers KM 1984 Acquisition and aggregation of canine blood platelets Basic
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
33
mechanisms of function and differences because of breed origin Am J Vet Res 45 137ndash144
11 Conversy B Blais MC Dunn M Gara-Boivin C del Castillo JRE 2017 Anticoagulant
activity of oral rivaroxaban in healthy dogs Vet J 223 5ndash11
12 Crochemore T Piza FMT Rodriacuteguez RDR Guerra JCC Ferraz LJR Correcirca TD
2017 A new era of thromboelastometry Einstein (Sao Paulo) 15 380ndash385
13 Crochemore T Correcirca TD Lance MD Solomon C Neto AS Guerra JCC Lellis PS
Bernz LM Nunes N Mancio CM Yokoyama APH Silva E 2018 Thromboelastometry
profile in critically ill patients A single-center retrospective observational study PLoS One 13
e0192965
14 Doumlderlein E Mischke R 2015 Reference intervals for thromboelastometry with the ROTEMreg
delta in cats Res Vet Sci 100 271ndash276
15 Falco S Bruno B Maurella C Bellino C DacuteAngello A Gianella P Tarducci A Zannata
R Borrelli A 2012 In vitro evaluation of canine hemostasis following dilution with
hydroxyethyl starch (13004) via thromboelastometry J Vet Emerg Crit Care (San Antonio)
22 640ndash645
16 Franco-Murillo Y Jaimez R 2017 Inhibitory effect of ethinylestradiol on coagulation factors
in rats Exp Anim 66 107ndash113
17 Friedrichs KR Harr KE Freeman KP Szladovits B Walton RM Barnhart KF Blanco-
Chavez J 2012 ASVCP reference interval guidelines Determination of de novo reference
intervals in veterinary species and other related topics Vet Clin Pathol 41 441ndash453
18 Gillissen A va den Akker T Caram-Deelder C Henriquez DDCA Bloemenkamp
RWM Eikenboom J van der Bom JG de Maat MPM 2019 Comparison of
thromboelastometry by ROTEMreg Delta and ROTEMreg Sigma in women with postpartum
haemorrhage Scand J Clin Lab Invest 79 32ndash38
19 Harttert H 1948 Blutgerinnungstudien mit der Thrombelastographie einem neuen
Untersuchungsverfahren Klin Wochenschr 26 577ndash583
20 Horn P Pesce A 2003 Reference intervals and update Clin Chim Acta 334 5ndash23
21 Hua Yanhun Qiu W Xiao Q Wu Q 2018 Precision (repeatability and reproducibility) of
ocular parameters obtained by the Tomey OA-2000 biometer compared to the IOLMaster in
healthy eyes PLoS One 13 e0193023
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
34
22 Junge H Ringer S Mayer N Schwarzwald C 2016 Assesment of method reliability and
determination of reference intervals for rotational thromboelastometry in horses J Vet Emerg
Crit Care (San Antonio) 26 691ndash703
23 Kol A Borjesson DL 2010 Application of thrombelastographythromboelastometry to
veterinary medicine Vet Clin Pathol 39 405ndash416
24 Marschner CB Wiinberg B Tarnow I Markussen B Kuumlhnel L Bochsen L Kristensen
AT 2018 The influence of inflammation and hematocrit on clot strength in canine
thromboelastographic hypercoagulability J Vet Emerg Crit Care (San Antonio) 28 20ndash30
25 McMichael MA Smith SA 2011 Viscoelastic coagulation testing technology applications
and limitations Vet Clin Pathol 40 140ndash153
26 Mischke R 1994 Activity of coagulation factors II V VII and X in healthy dogs- dependence
on age sex and breed Berl Muumlnch Tieraumlrztl Wochenschr 107 289ndash294
27 Nielsen A Zois N Pedersen H Olse L Tarnow I 2007 Platelet function in dogs bread
differences and effect of acetylsalicylic acid administration Vet Clin Pathol 36 267ndash273
28 Nielsen L Kjelgaard‐Hansen M Jensen A Kristensen A 2010 Breed‐specific variation of
hematologic and biochemical analytes in healthy adult Bernese Mountain dogs Vet Clin Pathol
39 20ndash28
29 Nogami K 2016 The utility of thromboelastography in inherited and acquired bleeding
disorders Br J Haematol 174 503ndash514
30 Oswald E Stalzer B Heitz E Weiss M Schmugge M Strasak A Innerhofer P Haas T
2010 Thromboelastometry (ROTEMreg) in children Age-related reference ranges and correlations
with standard coagulation tests Br J Anaesth 105 827ndash835
31 Sankarankutty A Nascimento B Teodoro da Luz L Rizoli S 2012 TEGreg and ROTEMreg
in trauma similar test but different results World J Emerg Surg 7 (Suppl 1) S3
32 Sigrist NE Schefer RJJ Kutter APN 2018 Characteristics of hyperfibrinolysis in dogs and
cats demonstrated by rotational Thromboelastometry (ROTEM) Vet J 242 67-73
33 Steiner J Teague S Williams D 2003 Development and analytic validation of an enzime-
linked immunosorbent assay for the measurement of canine pancreatic lipase immunoreactivity in
serum Can J Vet Res 68 161ndash168
34 Sucker C Tharra K Lithmate J Scharf RE Zotz RB 2011 Rotation thromboelastography
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
35
(ROTEM) parameters are influenced by age gender and oral contraception Perfusion 26 334ndash
340
35 Swanepoel A Emmerson O Pretorius E 2017 The effect of endogenous and synthetic
estrogens on whole blood clot formation and erythrocyte structure Microsc Microanal 23 599ndash
606
36 Theusinger OM Nuumlrnberg J Asmis LM Seifert B Spahn DR 2010 Rotation
thromboelastometry (ROTEM) stability and reproducibility over time Eur J Cardiothorac Surg
37 677ndash683
37 Toulon P Metge S Hangard M Zwahlen S Piaulenne S Besson V 2017 Impact of
different storage times at room temperature of unspun citrated blood samples on routine
coagulation tests results Results of a bicenter study and review of the literature Int J Lab
Hematol 3 458ndash468
38 Turner JS Kutter APN Sigrist NE 2019 Correlation of rotational thrromboelastometry
(ROTEM) parameters with platelet count and their ability to predict thrombocytopenia in dogs
Res Vet Sci 126 45ndash50
39 Versteeg HH Heemskerk JWM Levi M Reitsma PH 2013 New Fundamentals in
Hemostasis Physiol Rev 93 327ndash358
40 Wiinberg B Jensen AL Kjelgaard-Hansen M Rojkjaer R Johansson PI Gade LP
Gram DX Kristensen AT 2007 Study on biological variation of haemostatic parameters in
clinically healthy dogs Vet J 174 62ndash68
41 Wiinberg B Jensen AL Johansson PL Rozanski E Tranholm M Kristensen AT 2008
Thromboelastographic evaluation of hemostatic function in dogs with disseminated intravascular
coagulation J Vet Intern Med 22 357ndash365
42 Wikkelsoslash A Wetterslev J Moslashller AM Afshari A 2016 Thromboelastography (TEG) or
thromboelastometry (ROTEMreg) to monitor haemostatic treatment versus usual care in adults or
children with bleeding Cochrane Database Syst Rev Art
httpsdoiorg10100214651858CD007871pub3 No CD007871
43 Willems A Paepe D Marynissen S Smets P Van de Maele I Picavet P Duchateau L
Daminet S 2017 Results of screening of apparently healthy senior and geriatric dogs J Vet
Intern Med 31 81ndash92
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
36
44 Woodman J Wagg CR Boysen SR Leguillete R Mizen K Roy MF 2015 Evaluation
of coagulation via thromboelastography in healthy horses administered dexamethasone Can Vet
J 56 1271ndash1274
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
37
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of
standard heparin prophylaxis and correlation to haemostatic changes
Pereira JM Hewicker-Trautwein M Rohn K von Depka Prondzinski M
Mischke R
Contributions of the authors
Pereira JM performed (1) the ultrasound evaluations and blood sampling of the
patients under study (2) haematology analysis (3) injection of the anticoagulation
prophylaxis (partially) to the patients under study (4) analysis of samples (collaboration)
at Werlhof Institute Medical Care Center (5) analysis of some haemostasis test
(collaboration) (6) statistical analysis (partially) (7) data documentation (8) and wrote
the manuscript
Hewicker-Trautwein M performed the electron microscopy analysis
Rohn K performed the statistical analysis
von Depka Prondzinski M performed some haemostasis test (thrombin generation
assay and endogenous thrombin potential)
Mischke R performed the coagulation and haemostasis test was involved in designing
and supervision of the study analysis and discussion of the data and its statistical analysis
and revision edition and correction of the manuscript
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
38
Central venous catheter induced thrombosis in dogs ndash efficacy of standard heparin
prophylaxis and correlation to haemostatic changes
Joseacute Mauricio Pereira1 Marion Hewicker-Trautwein2 Karl Rohn3 Mario von Depka
Prondzinski4 Reinhard Mischke1
1Small Animal Clinic University of Veterinary Medicine Hannover Foundation
Buumlnteweg 9 D-30559 Hannover Germany
2Institute for Pathology University of Veterinary Medicine Hannover Foundation
Buumlnteweg 17 D-30559 Hannover Germany
3Institute of Biometry Epidemiology and Information Processing University of
Veterinary Medicine Hannover Foundation Buumlnteweg 2 30559 Hannover Germany
4Werlhof Institute Schillerstrasse 23 30159 Hannover Germany
Abbreviations anti-Xa anti-factor Xa BW body weight CVC central venous catheter
EJV external jugular vein ELMI electron microscopy IV intravenous JV jugular vein
LMWH low molecular weight heparin ML maximum lysis (ROTEM) PIVC
peripheral intravenous catheter ROTEM rotational elastometry SC subcutaneous
TGA thrombin generation assay TID three times a day TT thrombin time UFH
unfractionated heparin
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
39
Corresponding author
E-mail addresses matiscrgmailcom (JM Pereira) MarionHewicker-
Trautweintiho-hannoverde (M Hewicker-Trautwein) KarlRohntiho-hannoverde
(K Rohn) mailwerlhof-institutde (M von Depka Prondzinski)
ReinhardMischketiho-hannoverde (R Mischke)
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
40
ABSTRACT
Central venous catheters (CVC) are of importance for intensive care management of
canine patients Thrombosis of the jugular vein is a severe complication related to the
CVC use However only little is known about the incidence of CVC-associated
thromboses in dogs and the necessity and efficacy of prophylactic antithrombotic
regimens The objectives of the present study were (1) to determine the incidence of
catheter-induced thrombosis in canine intensive care patients and thereby (2) to evaluate
the efficacy of a routine prophylactic heparin treatment and finally (3) to assess whether
initial changes of selected haemostasis parameters are predictive for an increased risk for
thrombosis formation 29 dogs hospitalised in the Small Animal Clinic University of
Veterinary Medicine Hannover Foundation and receiving a central venous catheter
(CVC) in the external jugular vein (EJV) for medical reasons were included in the final
assessment 17 dogs received the standard antithrombotic dosage regimen of
unfractionated heparin (UFH 150 IUKG BW TID sc) and 12 dogs (usually surgical
patients) the reduced dosage (75 IUKG BW TID sc) A colour Doppler ultrasound of
the respective EJV in order to detect thrombus formation and blood collection for
haemostasis tests were performed immediately before the installation procedure (day 0)
and on days 1 3 5 7 and so on after the CVC insertion Haemostasis tests included
prothrombin time activated partial thromboplastin time thrombin time antithrombin
activity D-dimer concentration thrombin generation and rotational elastometry as well
as the heparin activity using a chromogenic anti-factor Xa test Finally electron
microscopy (ELMI) of sections of the removed CVC was performed In 8 dogs
sonographic examination revealed thrombus formation in the lumen of EJV and in 1927
CVCs assessed using ELMI thrombi on the external CVC surface were detected without
significant correlation between both methods Plasmatic heparin activities showed great
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
41
variability but no significant differences were found between dogs with or without CVC-
associated thrombus formation From the initially performed haemostasis tests high
fibrinogen concentrations and low maximum lysis values as measured by the ROTEM
delta device (ex-tem reagent) were associated with sonographically detectable thrombi
The results of the present study indicate that CVC-associated thromboses occur (eg 28
[829] of the patients as detected by ultrasound) although severe thromboses (high
grades) are relatively rare in intensive care canine patients receiving a routine
anticoagulant treatment A high fibrinogen concentration and low maximum lysis
(ROTEM ex-tem) may be useful indicators for an increased thrombotic risk in individual
patients which may require special anticoagulatory treatment
Keywords CVC complication venal occlusion antithrombotic treatment ultrasound
electron microscopy rotational elastometry
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
42
1 Introduction
Central venous catheters (CVCs) are very important tools for care of patients in critical
care units allowing parenteral nutrition support therapy administration of high volumes
of intravenous fluids safe drugs administration that cannot be given through peripheral
veins plasmapheresis haemodialysis blood transfusions as well as periodical blood
sampling (Geerts 2014 Vose et al 2019) CVC related thrombosis is one of the most
severe complications (apart from infections and pneumothorax) of CVCs in humans
(Geerts 2014 Comerlato et al 2017 Evans and Ratchford 2018) leading to patientrsquos
distress CVC dysfunction venous stenosis and in some cases to the development of
thromboembolism (Geerts 2014)
In the human literature there are various studies dealing with epidemiology with reference
to different anticoagulatory regimens including placebo-controlled studies (Lee and
Kamphuisen 2012) In humans CVC related thromboses represent 70ndash80 of all upper
extremity thrombosis cases and approximately 10 of thromboembolism cases in
humans (Kucher 2011) The incidence of CVC related thrombosis reported in humans
based on 25 studies ranged widely from 2 to 67 (median = 30 ) (van Rooden et al
2005 review article) This wide range of calculated incidences of CVC related
thromboses in humans reflects the use of different CVC types different locations of entry
site of the CVC (jugular femoral subclavian axillary cephalic vein) different thrombus
detection technique (venography or ultrasonography) unequal durations of follow up
varying diseases of patients (eg haematological oncological) and presence of
predisposing factors (eg previous venous thrombosis history presence of cancer) (van
Rooden et al 2005 Geerts 2014 Liu et al 2015)
Part of the authors empirically observed CVC-associated thrombosis of the external
jugular vein (EJV) as a relevant complication in their canine intensive care patients which
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
43
awakened their interest in that topic To the best of the authoracutes knowledge studies about
extra luminal CVC related thromboses in dogs are rare in the available veterinary
literature One study evaluating the complications associated with 100 CVCs placed in
the jugular vein of dogs and cats reported only 2 cases of thrombosis (confirmed by
palpation or ultrasound) but obviously did not assess the appearance of subclinical CVC
related thromboses (Adamantos et al 2010) Thereby the incidence of CVC-induced
thromboses in dogs is unknown and systematic studies regarding an effective
anticoagulatory management are lacking
In this context it is of interest whether a possible development of thrombosis or an
increased risk (despite the prevention by a standard application of unfractionated heparin
[UFH]) can be predicted by means of changes of haemostasis related parameters during
the initial examination In one study on 168 human patients without indication of
anticoagulation prophylaxis for example elevated leukocyte counts high plasminogen
activator inhibitor and high coagulation factor VIII levels (all measured on blood samples
taken after CVC placement) were associated with an increased incidence of symptomatic
CVC-related thrombosis diagnosed by ultrasonography (Boersma et al 2016)
Therefore aims of the present study were (1) to determine the incidence of catheter-
induced thrombosis in canine intensive care patients (as detected by ultrasonography and
electron microscopy ELMI) and thereby (2) to evaluate the efficacy of a standard
antithrombotic regimen with unfractionated heparin (UFH) routinely used in the Small
Animal Clinic of the University of Veterinary Medicine Hannover and (3) to assess
whether selected haemostasis test results are predictive for an increased risk for
thrombosis formation
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
44
2 Material and methods
21 Study design
Canine in-patients of the Small Animal Clinic University of Veterinary Medicine
Hannover receiving a CVC in the EJV for medical reasons from March 2017 to
December 2019 were included Anticoagulatory treatment was performed according to
the standard protocol of the clinic Colour Doppler ultrasonographic controls of the EJV
and blood samplings (for heparin activity basic haematology and haemostasis tests) were
done immediately before the catheter was placed (day 0) and on days 1 3 5 etc (2 hours
after the first daily UFH injection) until the CVC was removed Only patients were
included which could be examined at least until day 3 Haemostasis tests included
prothrombin time activated partial thromboplastin time thrombin time fibrinogen
concentration antithrombin activity fibrin monomers D-dimers thrombin generation
assay (TGA) and rotational elastometry as well as the heparin activity using a
chromogenic anti-factor Xa test Finally ELMI of sections of the CVC after its removal
was performed
22 Animals
Originally 32 patients hospitalised in the Small Animal Clinic and receiving a CVC in the
EJV for medical reasons entered the study Three of them were subsequently excluded
because they did not reach the defined minimum study period of 3 days Two were
euthanised and one referred back to the referring vet before day 3 22 dogs weighing more
than 15 kg were included so that the withdrawal of about 10 ml of blood on several days
was not a significant burden for the patient (lt 1 of the total blood volume) Five dogs
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
45
with a body weight of less than 15 kg but at least 8 kg were included with special
consideration of the amount of blood taken (no more than 6 ml on individual days)
All animals received standard anticoagulatory treatment with UFH 17 dogs with internal
diseases received 150 IUkg BW TID subcutaneously and 12 dogs (post surgery) received
a reduced dosage of 75 IUkg BW TID subcutaneously
The 29 dogs enclosed in the study included 4 mixed breed dogs and the following
pedigree dogs 5 Labrador Retrievers 3 German Shepherds 2 Australian Shepherds and
one from each of the following breeds Akita Inu Asian Shepherd Bavarian Mountain
Hound Beagle Belgian Shepherd Briard Canaan dog Dalmatian Golden Retriever
Havanese dog Hovawart Rhodesian Ridgeback Schnauzer Shar Pei and Wolfspitz
These dogs were aged between 2 and 164 months (median 77 months) and had body
weights ranging from 81 and 63 kg (median 27 kg) 9 were castrated males 8 intact
males 7 spayed females and 5 intact females
The diseases of the 29 patients requiring a CVC included 3 from each of the following
diseases hepatopathy and esophagus dilatation 2 from each of the following diseases
bacterial cystitis diabetes mellitus and 1 from each of the following diseases abdominal
wall perforation aspiration pneumonia cystitis intestinal round cells sarcoma carcinoma
of the nasal cavity chronic renal insufficiency cystic duct rupture heartworm disease
ivermectine intoxication lymphoma multifocal alveolar histiocytosis pancreatitis post-
surgical bleeding stomach dilatation stomach foreign body tetanus thoracic
pyogranulomatous inflammation thrombocytopenia and trigeminal nerve paresis
Animals were treated in accordance with the German Animal Welfare Law The
experimental design was approved by the Animal Welfare officer of the University of
Veterinary Medicine and reported to the responsible authority (Lower Saxony State
Office for Consumer Protection and Food Safety reference number 17A101)
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
46
23 Positioning and handling of the central venous catheter
Two types of single lumen radiopaque polyurethane CVC (different on length and
diameter) were used during the study The CVC type I (n = 2529) was Cavafixreg Certoreg
Splittocanreg CVC 32 cm length 11 x 17 mm diameter16 G (BBraun Melsungen AG)
the CVC type II used (n = 429) was Certofixreg Mono S 215 15 cm length 08 x 14 mm
diameter18G (BBraun Melsungen AG) Each CVC was inserted into the EJV using a
modified Seldinger technique as follows the skin of the neck was shaved and disinfected
with antiseptic soap alcohol and iodine Local anesthesia (Lidocaine HCl 2) was
administrated subcutaneously on the catheter insertion site (a middle point in the lateral
neck over the jugular sulcus) A small skin incision was performed in the insertion site
and one peripheral plastic cannula with a metallic needle inside (14 G for CVC type I
and 20 G for CVC type II) was inserted in the EJV in cranio-caudal orientation (Hundley
et al 2018) Once the EJV was reached the needle was removed and a flexible wire
guide was inserted through the peripheral cannula following an external landmark
(4thintercostal space) (Reminga et al 2018) The peripheral cannula was removed and
the respective CVC was inserted slowly and with gentle pressure into the EJV guided
with the flexible wire and following the same external landmark to reach the right
position After the insertion of the CVC the flexible wire guide was removed The correct
positioning of the CVC was checked with a latero-lateral thorax x-ray ensuring that the
CVC tip was placed at the junction of the cranial cava vein with the right atrium Each
CVC was fixed to the neck skin (adjacent to the insertion point) with a monofilament
non-absorbable suture (gauge 3-0) Finally a bandage elaborated with Povidone-Iodine
gauze (Betaisodonareg) padded synthetic band (Artiflexreg Natur) and elastic band
(Elastomullreg) was used to protect the CVC output (Claude et al 2010) and changed
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
47
daily Several times day the catheter was flushed with isotonic sodium chloride
containing 1 IUml UFH
24 Heparin preparation and administration
Heparin-sodium-25000-ratiopharm (injection solution containing 5000 IUml sodium
heparin [mucosa]) was used SC injection was performed over the lateral thorax wall
using 1 mL syringes with fine graduation marks
25 Ultrasonography
Colour Doppler ultrasonographic examinations were performed in lateral position using
a LO-GIQ E9 ultrasound device (GE Healthcare Chicago Illinois USA) with a 15
Megahertz linear ultrasound probe The development of a thrombus or fibrin debris in the
EJV lumen around the CVC or inside it was assessed at each control Once the
ultrasound was finished a new protective bandage was placed around the neck of the dog
According to the percentage of lumen occlusion of the EJV (evaluated in cross sectional
views during the ultrasound scanning) the thrombi were classified in four different grades
(Fig 1)
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
48
of the residual vein lumen C ndash catheter T ndash thrombus
Fig 1 Definition of different grades of central venous catheter induced thrombosis of the
jugular vein as detected using colour Doppler ultrasound
26 Blood collection
At each time point approx 6ndash10 ml of blood (according to the body weight) was collected
This included approx 1 ml of EDTA blood for hematological analysis and approx 5ndash9
ml of citrated blood (tubes containing one part of 011 molL [38 ] sodium citrate to
nine parts of blood) for the measurement of hemostasis tests in citrated blood (ROTEM)
or citrated plasma Blood samples were collected by puncture of peripheral veins with
disposable cannulas (09 x 40 mm [20G] or 10 x 40mm [21G]) directly into sample tubes
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
49
or from the CVC using the three-syringe-technique (Villalta-Garciacutea et al 2015) in
situations when the peripheral venipuncture was not indicated due to phlebitis or irritation
andor inflammation of the surrounding skin Briefly 2ndash3 ml of blood were taken in a
sterile disposable syringe and immediately afterwards the blood sample was taken in a
new disposable syringe and transferred into the test tubes containing the respective
anticoagulants finally the first 2-3 ml taken were re-infused to the dog through the CVC
followed by flushing with a solution of sodium chlorideUFH (02) Immediately after
blood collection each sample tube was swayed gently until blood and anticoagulant had
been mixed thoroughly To prepare citrated platelet poor plasma citrated blood was
centrifuged at 16000 g for 10 min at room temperature using a microcentrifuge The
plasma was transferred into plastic tubes and the centrifugation procedure was repeated
This platelet poor plasma was then frozen in aliquots at -70 degC until used for analysis
Directly before analysis the plastic tubes were thawed at 37 degC in a water bath
27 Laboratory tests
Haematological analyses were made automatically with Adviareg 2120i (Siemens
Healthcare GmbH Germany)
Prothrombin time activated partial thromboplastin time and thrombin time were
measured with the autoanalyser Amax Destiny Plus (Tcoag Deutschland GmbH
Germany) and commercial reagents (Thromborel S Siemens Healthcare Diagnostics
Products GmbH Marburg Germany CK Prestreg and Test Thrombin Reagent
Diagnostica Stago SAS Asniegraveres sur Seine Cedex France) Whereas the last two tests
were measured according to the manufacturer`s instructions a modified test optimised
for canine plasma was used to measure prothrombin time (25 microl citrated plasma 120 with
imidazole buffer + 25 microl of a human fibrinogen solution 2 gl prewarmed for 120 s at 37
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
50
oC addition of 25 microl of activating reagent Thromborel S) and the results converted into a
percentage activity based on a calibration curve prepared with canine pooled plasma The
fibrinogen concentration was measured with the method according to Clauss using a test
kit from Stago Diagnostica SAS D-dimer concentration was measured with a
quantitative commercial human latex turbidimetric method in the autoanalyser Cobas
c311 using a completely assembled kit and human standards (Roche Diagnostics
Mannheim Germany) Antithrombin activity was also measured with a completely
assembled chromogenic test kit in the autoanalyser Cobas c311 and calibrated with a
canine pool plasma (n = 100)
The thrombin generation assay (Technothrombinreg TGA (Technoclone GbmH Vienna
Austria) determines thrombin generation based on monitoring of the fluorescence
generated by cleavage of a fluorogenic substrate by thrombin over time after activation
of the coagulation cascade by different concentrations of tissue factor and negatively
charged phospholipid in platelet poor plasma From the fluorescence development over
time the concentration of thrombin in the sample can be calculated using a thrombin
calibration curve The increase in thrombin concentration with time then allows to
calculate generation of thrombin in the sample and to plot thrombin values over time for
the whole coagulation process
The rotational thromboelastometry analysis with ROTEM delta device (Werfen Munich
Germany) was started approx 15 minutes after the blood collection To perform the
analysis and according to the automated ROTEM software 20 microl of recalcification reagent
(200 mmoll calcium chloride solution star-tem) and 20 microl of the respective activating
reagent (ex-tem in-tem) were pipetted into and carefully mixed in a pre-warmed cuvette
using the automated pipette provided in the device immediately afterwards 300 microl of
citrated whole blood (previously gently revolved) were added and mixed with the
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
51
reagents previously deposited Finally the cuvette was carefully placed and fixed to an
oscillating pin (oscillation movements on left and right through an angle of 475deg) in the
ROTEM delta device The interaction between blood cells and subsequently the bond
between fibrin and platelets promoted by the biomechanical changes in pH electrolytes
and temperature start the clot formation with its subsequent retraction and lysis The
magnitude of the resistance produced by the clot over the oscillating pin is quantitatively
represented by different parameters measured during the analysis and also qualitatively
in a graphic illustration (Bagge et al 2016 Crochemore et al 2017)
A chromogenic substrate test (Coatest Heparin Chromogenix-Instrumentation
Laboratory Haemochrom Diagnostica Essen Germany) was used for measurements of
UFH in the auto-analyser Cobas c311 The test application was created based on
manufacturers` instructions Different dilutions of the same batch of the commercial
sodium UFH preparation used in the experiment with normal canine pool plasma
(prepared from identical aliquots of 10 healthy adult dogs) served as standards for the
calibration of the measurement
28 Electron microscopy
Selected areas on the last third of the CVC (5ndash10 cm according with the CVC) with
macroscopically detectable thrombi were analysed and some of the CVCs were cut in
two different places The cut samples were preserved in a fixative medium composed of
1 glutaraldehyde and 4 formalin Once the sample was fixed it was prepared
according to the osmium thiocarbohydrazide procedure (OTOTO-procedure) (Malick et
al 1976) The fixed CVCs were dehydrated in seven series as follow first three series of
120 minutes each one on 30 50 and 70 ethanol respectively followed by two series
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
52
of 120 min each one on 90 ethanol and then two series of 24 hours each one on 100
ethanol After the dehydration process the samples were placed two times during 24
hours each one on isoamyl acetate and then placed on in a critical point drier Finally the
samples were coated with gold in a splutter-coater with 25 kV acceleration voltage in an
argon atmosphere with a current of 20 mA for one minute The samples were examined
in an EVO 15 scanning electron microscope (ZEISS International Carl Zeiss Jena
Germany) and photographed with magnifications of 600x and 6000x avoiding mechanical
artifacts (Toskala et al 1995) Thrombi on the external wall of the CVC were analysed
and measured using the software ImageJ (National Institutes of Health and the Laboratory
for Optical and Computational Instrumentation University of Wisconsin USA)
According to their extension (surface size) and thickness (related to the CVC wall
thickness in a cross sectional view) thrombi on the external wall and additionally within
the lumen of the CVC were each classified in four grades (Fig 2 Fig 1S [suppl
material]) In case of two cuts the highest degree of thrombosis detected at one of the two
sites was used for the final assessment
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
53
surface sizepercent of CVC wall thickness
Fig 2 Definition and illustration of different grades of central venous catheter induced
(extraluminal) thrombosis as detected using electron microscopy
Fig 1S (suppl material) Definition and illustration of different grades of intraluminal
thrombosis in central venous catheters as detected using electron microscopy
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
54
29 Statistical methods
Comparison between thrombus graduation either based on sonographic and ELMI
findings respectively was performed using the McNemar test with calculation of the
concordance index (κ) Standard normal distribution was assessed using the
Kolomogorov-Smirnov test
Due to non-normal distribution of part of data sets heparin activities and results of
haemostasis assays are reported using non-parametric variables (median minimumndash
maximum box and whisker plots) Heparin activities and results of haemostasis tests
between dogs with or without thrombosis were compared using Mann-Whitney-U test
3 Results
31 Sonographic detection of thrombus formation
Thrombus formation was sonographically detected in 829 patients during the study
period Based on examination on the final day in half of these eight patients (n = 4) the
thrombus formation was scored grade 1 in 3 dogs grade 2 and only in one grade 3 In all
of these 8 dogs this was the maximum grade during the study period One dog with fibrin
precipitates was excluded from further analysis because it could not be clearly assigned
to one of the groups In 78 cases thrombi were attached mainly to the venous wall
whereas only in one case was attached to the CVC external wall Seven of these 8 dogs
which developed thrombi received the standard heparin dosage (150 IUkg TID 41
[717] of dogs receiving this dosage) and only one the reduced heparin dosage (75 IUkg
TID 8 [112] of dogs receiving this dosage)
The time point of first detection of thrombus formation varied between days 3 and 11
(median day 8) In addition detritus (presumably composed mainly of fibrin) was
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
55
detectable in the lumen of the EJV in one further dog and within the lumen of the CVC
in further 5 dogs
32 Thrombus formation by electron microscopy
27 from 29 CVC catheter tips were analysed by electron microscopy 827 samples were
without detectable thrombus (grade 0) and 1927 with detectable thrombi 72 showed
thrombus formation of grade 1 427 of grade 2 and 827 of grade 3 There was no
concordance between thrombus graduation based on sonographic and the ELMI
graduation respectively The concordance index (McNemar test) was negative (κ = -
00718) indicating that the degree of agreement is less than by chance
Of the in total 19 dogs with detectable thrombus formation by electron microscopy 11
dogs received 150 IUkg TID (in total 16 dogs of these 27 dogs received that heparin
dosage) and 8 received 75 IUkg TID (in total 11 of these 27 dogs received that heparin
dosage 73 811) and Of the 12 patients with grade 2 and 3 thrombus formation 5 dogs
received 75 IUkg TID and 7 dogs 150 IUkg
The supplementary analysis of intraluminal thrombus formation revealed no detectable
thrombi in 7 cases and thrombus formation of grade 1 (lt 25 occlusion) in 11 cases of
grade 2 (25ndash50 ) in 3 cases and of grade 3 (gt 50 ) in 6 cases
33 Heparin activities and relationship to thrombus formation
Heparin activities varied remarkably in both dosage groups (Fig 3 ab) There was no
significant difference of heparin activities between blood samples collected via
venipuncture or from the CVC (Table 1S supplementary material) In addition there was
no significant difference between heparin activities in dogs with or without
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
56
sonographically or electron microscopically detectable thrombosis (Tables 1 and 2)
Table 1 refers to sonographically detectable thromboses and is based on median heparin
activities calculated from the measurements on day 1 and following days per patient
firstly for the total number of patients and secondly for the patients receiving 150 IUkg
TID In addition in the eight dogs heparin activities on the day of first thrombus detection
(023 IUml 019ndash036 IUml) were not significantly different from the median values
(Table 1 P = 05214 Wilcoxon test) Table 2 compares heparin activities (median values
of all days and results on the final day) between dogs with and without electron
microscopically detectable CVC-associated thrombi
Table 1S (Supplementary material) Heparin activities (IUml median values
minimumndashmaximum) measured in 27 dogs receiving 75 or 150 IUkg unfractionated
(standard) heparin TID subcutaneously 2 hours after the first injection on the day
Comparison of samples collected via puncture of the peripheral veins and those collected
from the central venous catheter (CVC) using the three-syringe-technique
Peripheral vein
puncture CVC
Mann-Whitney-U
test
Total number
(75 and 150
IUkg TID)
019
(00ndash108)
(n = 65)
023
(000ndash105)
(n = 65)
P = 03033
75 IUkg TID
010
(000ndash108)
(n = 25)
012
(000ndash023)
(n = 21)
P = 06907
150 IUkg
TID
030
(000ndash096)
(n = 40)
036
(000ndash105)
(n = 44)
P = 01223
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
57
Fig 3ab Heparin activities (box- and whisker plot arithmetic means) in dogs receiving
unfractionated heparin at a dosage of 75 IUkg BW tid (a) or 150 IUkg BW tid (b) two
hours after the first daily heparin administration
day 1 represents the first day after initiation of heparin treatment
only days were considered with at least results from 5 patients
Table 1 Heparin activities (IUml median values minimumndashmaximum) in 28 dogs
receiving 75 or 150 IUkg unfractionated (standard) heparin TID subcutaneously with
(grades 1ndash3) or without (grade 0) sonographically detectable thrombus Blood collections
were performed 2 hours after the first daily injection
Grade 0
(sonography)
Grades 1ndash3
(sonography)
Mann-Whitney U
test
Total number
(75 and 150 IUkg
TID)
020 (000ndash072)
(n = 20)
027 (018ndash059)
(n = 8)
P = 03212
150 IUkg TID 042 (000ndash072)
(n = 10)
031 (018ndash059)
(n = 7)
P = 04593
one dog with fibrin precipitates in the lumen of the jugular vein but without defined thrombus formation
was not considered because it could not be clearly assigned to one of the two groups
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
58
Table 2 Heparin activities (IUml median values minimumndashmaximum) in 27 dogs
receiving 75 or 150 IUkg unfractionated (standard) heparin TID subcutaneously with
(score 1ndash3) or without (score 0) detectable thrombus via electron microscopy
Grade 0
(electron
microscop)
(n = 8)
Grades 1ndash3
(electron
microscop)
(n = 19)
Mann-Whitney U
test
Median value of
different days 028 (005ndash060)
022 (000ndash072)
P = 05770
Final day 020 (001ndash045)
015 (000ndash105)
P = 06706
34 Relationship between haemostasis tests and thrombus formation
From the initially performed haemostasis tests high fibrinogen concentrations and low
maximum lysis values as measured by the ROTEM delta device (using the ex-tem
reagent) were associated with sonographically detectable thrombi (Tab 3) Additional
analyses based on the median values of different days for individual patients revealed that
low maximum lysis values as measured by the ROTEM delta device (using the in-tem
reagent) were associated with sonographically detectable thrombi (maximum lysis score
0 850 [100ndash175 ] score 1ndash3 375 [100ndash900 ] median [minimumndash
maximum] P = 00166) but no other test Comparison of the results of haemostasis tests
on the final day between dogs with and without sonographically detectable thromboses
did not show any significant difference (P gt 005)
Comparison of results of haemostasis tests on the final day in dogs with detectable
thrombosis via ELMI and dogs without significant thrombus formation showed several
significant differences including lower fibrinogen concentrations and longer clotting
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
59
times (ex-tem reagent) and clot formation times (ex-tem and in-tem reagents) using the
ROTEM delta analyser in general expressing a less coagulable status (Tab 4) If values
of dogs graded 0 and 1 (n =15) are compared to those of grades 2 and 3 (n = 12) the last-
mentioned had longer clotting times (ROTEM delta in-tem reagent) as well as longer clot
formation times and lower alpha angles (ROTEM delta in-tem reagent) (P lt 005 results
not shown)
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
60
Table 3 Comparison of initial results of haemostasis parameters (median values
minimumndashmaximum in dogs with (grades 1ndash3) and without (grade 0) sonographically
detectable central venous catheter-induced thromboses
Parameter Unit
Sonographic
thrombus grade
P-value
(Mann-Whitney
U test)
0
(n = 20)
123
(n = 8)
Haematocrit 355 (228ndash641)
382 (245ndash499)
06111
Platelet count x103microl 292 (420ndash483)
218 (860ndash458)
02320
Prothrombin time 944 (359ndash169)
896 (471ndash136)
05758
Activated partial thromboplastin
time s 128
(955ndash249) 140
(117ndash164) 02322
Thrombin time s 129 (121ndash178)
141 (650ndash160)
02028
Fibrinogen gl 448 (153ndash770)
747 (153ndash116)
00235
Antithrombin activity 639 (289ndash107)
670 (464ndash746)
05249
D-dimers microgml 006 (000ndash148)
015 (003ndash372)
03997
Thrombin generation assay nmol 130 (000ndash365)
912 (190ndash277)
08787
Clotting time (ROTEM ex-tem) s 420 (290ndash930)
430 (170ndash159)
07989
Clot formation time (ROTEM ex-
tem) s 665
(310ndash143) 470
(240ndash800) 00633
Alpha angle (ROTEM ex-tem) degrees 795 (620ndash840)
815 (750ndash860)
01667
Max clot firmness (ROTEM ex-
tem) mm 750
(570ndash860) 775
(700ndash830) 01753
Max clot elasticity (ROTEM ex-
tem) - 299
(131ndash619) 347
(235ndash484) 01544
Maximum lysis (ROTEM ex-tem) 135 (300ndash290)
400 (000ndash800)
00037
Clotting time (ROTEM in-tem) s 208 (840ndash810)
249 (720ndash371)
05085
Clot formation time (ROTEM in-
tem) s 835
(370ndash634) 745
(390ndash235) 08988
Alpha angle (ROTEM in-tem) degrees 745 (250ndash820)
750 (500ndash820)
09797
Max clot firmness (ROTEM in-
tem) mm 700
(540ndash810) 740
(610ndash770) 03587
Max clot elasticity (ROTEM in-
tem) - 235
(118ndash424) 287
(156ndash332) 04764
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
61
Maximum lysis (ROTEM in-tem) 850 (000ndash210)
350 (000ndash220)
01327
Table 4 Comparison of results of haemostasis parameters (median values minimumndash
maximum) on the last day in dogs with (grades 1ndash3) and without (grade 0) central venous
catheter-associated thrombus formation as detected by electron microscopy
Parameter Unit
Thrombus grade
(ELMI)
P-value
(Mann Whitney
U test)
0
(n = 8)
123
(n = 19)
Haematocrit 322 (241ndash454)
330 (216ndash510)
05953
Platelet count 103microl 446 (321ndash592)
268 (101ndash675)
00558
Prothrombin time 145 (104ndash264)
120 (561ndash148)
00462
Activated partial thromboplastin
time s 123
(109ndash140) 136
(109ndash188) 00665
Thrombin time s 129 (121ndash178)
141 (650ndash160)
02028
Fibrinogen gl 465 (275ndash866)
347 (189ndash992)
00491
Antithrombin activity 793 (448ndash981)
692 (398ndash889)
04572
D-dimers microgml 018 (000ndash173)
007 (000ndash118)
01920
Thrombin generation assay nmol 501 (000ndash160)
93 (000ndash287)
09562
Clotting time (ROTEM ex-tem) s 360
(270ndash410) 400
(310ndash620) 00238
Clot formation time (ROTEM ex-
tem) s 395
(120ndash740) 650
(240ndash174) 00275
Alpha angle (ROTEM ex-tem) degrees 835 (760ndash880)
790 (580ndash850)
00484
Max clot firmness (ROTEM ex-
tem) mm 780
(700ndash850) 720
(550ndash860) 00586
Max clot elasticity (ROTEM ex-
tem) - 346
(239ndash555) 254
(121ndash637) 00495
Maximum lysis (ROTEM ex-tem) 105 (200ndash190)
110 (000ndash380)
03796
Clotting time (ROTEM in-tem) s 178 (100ndash222)
201 (139ndash554)
00525
Clot formation time (ROTEM in-
tem) s 435
(330ndash111) 790
(350ndash882) 00209
Alpha angle (ROTEM in-tem) degrees 815 (710ndash830)
740 (240ndash830)
00191
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
62
Max clot firmness (ROTEM in-
tem) mm 785
(660ndash810) 690
(270ndash840) 00586
Max clot elasticity (ROTEM in-
tem) - 362
(192ndash434) 218
(370ndash507) 00559
Maximum lysis (ROTEM in-tem) 950 (200ndash190)
800 (100ndash180)
06890
4 Discussion
The main result of our study is that under the study conditions part of the dogs developed
CVC-associated thromboses However only a very low part of these thromboses were
obviously of a clinically significant degree regarding the amount of occlusion of the EJV
and none of the dogs had a symptomatic thrombosis whose definition includes presence
of clinical signs such as localised swelling pain and redness (Boersma et al 2008 Lee
and Kamphuisen 2012) In the present study we particularly refer to the extraluminal
thromboses with regard to the assessment of the antithrombotic management
One major problem of the statistical analysis and result presentation was that the two
methods used to define the occurrence and degree of thrombus formation (ultrasound and
ELMI) revealed discrepant results Therefore we performed a statistical analysis
independently regarding thrombus formation with either of the two methods It is well
known that ELMI which has already been used to detect CVC induced thromboses in
humans (Lucas et al 2017) and experimental animals (Xiang et al 1998 Xiang et al
2001) is a very sensitive diagnostic technique which allows the ex vivo diagnosis of very
small or fibrin rich thrombi that perhaps cannot be detected by ultrasonography or
venography during the in vivo evaluation of patients (Xiang et al 2001) A major reason
for the discrepant results between the two methods used to detect CVC associated
thromboses is that different CVC segments are evaluated Using ultrasonography it was
only possible to evaluate the EJV in the neck area of the patient and thereby only its
proximal third In contrast ELMI assessed mainly the CVC tip and a human study
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
63
revealed that even different positions of the catheter tip are associated with various risks
for development of CVC associated thrombosis (Ballard et al 2016) One additional point
to consider which also may contribute to the discrepancy is that it is possible that thrombi
developed on the CVC surface can be detached during the CVC removal from the vein
and thereby before its analysis by ELMI (Xiang et al 2001)
The contrast venography technique is used as gold standard to confirm thrombosis in
human medicine However this diagnostic technique is normally used only in the case of
a strong clinical suspicion of venous thrombosis that cannot be detected by
ultrasonography (Laurenson et al 2010 Langston and Eatroff 2018) Our study protocol
with privately owned clinic patients did not allow to use this method In a study performed
in 44 human patients requiring a CVC (Koumlksoy et al 1995) a sensitivity of 94 and a
specificity of 96 were determined for the colour Doppler ultrasound diagnosis when it
was compared with the diagnosis performed with the contrast venography technique and
that makes the colour Doppler ultrasound an accurate diagnosis method to detect CVC
related thrombosis even in humans (Koumlksoy et al 1995 van Rooden et al 2003)
The incidences of CVC related thrombosis detected by colour Doppler ultrasound of the
internal jugular vein reported in 3 human studies were 2 30 and 56 (Harter et al
2002 Lordick et al 2003 Wu et al 1999) and although with a wide range the median
value of these rates (30 ) is very well suited to the incidence of 28 (829) which we
calculated for our canine study based on sonographic findings The wide variation of
incidences of CVC related thrombosis in different human studies can in general be
explained by differences regarding the type and entry site of the CVC (jugular femoral
subclavian axillary cephalic vein) the time period with the CVC in place the thrombus
detection technique (venography or ultrasonography comment in the cited 3 studies
consistently the last-mentioned method was used) and criteria for diagnosis of
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
64
thrombosis the duration of follow up diseases and presence of predisposing factors in
these patients (eg previous venous thrombosis history presence of cancer) and finally
prophylactic treatment with anticoagulants (van Rooden et al 2005 Geerts 2014 Liu et
al 2015)
Different pathomechanisms contribute to the development of CVC induced thromboses
During the CVC insertion the puncture of the skin on the CVC insertion site and the
rupture of the blood vessels integrity activate the coagulation cascade and the CVC acts
as a matrix for adherence and accumulation of fibrin and cellular components (Smith et
al 2012) The attachment of proteins and blood cells to the internal andor external wall
of the CVC can lead to a partial or complete occlusion of the catheter lumen and
sometimes to a partial or complete obstruction of the blood flow andor
thromboembolism (Langston and Eatroff 2018) There is a strong interrelationship
between thrombus and biofilm formation (Smith et al 2012) Prevention of thrombus
formation has thereby also relevance regarding the increased risk for microbial
colonisation of the catheter (Boersma et al 2008) leading at least in humans to an
increased number of CVC related systemic infections (Lordick et al 2003)
In this context it has to be considered that CVC produced from polyurethane which are
routinely used in our clinic and thereby in the study are usually associated with a
relatively high thrombosis potential and thereby definitely provide an effective test for
an antithrombotic regimen Silicone catheters have a significantly reduced risk for
thrombotic complications but this advantage has to be purchased by a higher rate of
mechanical failure (eg rupture of the catheter) (Wildgruber et al 2016) Promising
experimental approaches work on surface modifications of the standard catheters For
example one group was able to demonstrate that a coating of the surface of the catheter
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
65
with a zwitterionic polymeric sulfobetaine showed a significant reduction of thrombus
formation in vitro with human blood and in an in vivo canine model (Smith et al 2012)
Due to ethical reasons we did not performed a placebo-controlled study However
interestingly not all human studies report systemic anticoagulation and part of the human
studies was even placebo-controlled (review Lee and Kamphuisen 2012) Obviously
there is no consensus in the available human literature regarding the beneficial use of
antithrombotic prophylaxis to prevent CVC related thrombosis Several studies did not
find a statistically significant difference in the incidence of CVC related thrombosis
between patients receiving the antithrombotic prophylaxis compared with the control
groups without systemic anticoagulation (Verso et al 2005 P = 035 Karthaus et al
2006 P gt 005 Niers et al 2007 P = 049) whereas in other studies a lower incidence
of CVC related thrombosis was found in the group receiving anticoagulants as
prophylaxis compared with the control groups (Abdelfeki et al 2004 150 vs 126
P = 003 De Cicco et al 2009 400 vs 526 P = 005)
Although it is well known that subcutaneously administered UFH has a limited and
variable bioavailability and thereby leads to unpredictable blood activities (Hirsh and
Raschke 2004) the extremely wide range of blood activities after a defined dosage in our
study was relatively unexpected Blood was collected 2 hours after the first UFH
injection and thereby at the time of suspected maximum heparin activity considering the
relatively low UGH dosage (Mischke and Jacobs 2001) The cited study was performed
on healthy experimental dogs Possible additional influencing factors in critical ill
patients include circulatory insufficiency which may reduce and delay absorption from
subcutanenous tissue decreased clearance function of the liver and kidneys as well as
altered blood plasma concentrations of heparin binding plasma proteins such as
fibrinogen and platelet factor 4 and alterations of the heparin binding capacity of
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
66
endothelial cells which altogether are reasons for a great inter- but also intrapatient
variability (Hirsh and Raschke 2004) In individual patients this can lead to heparin
resistance It has also to be considered that antithrombin activities which are necessary
for an adequate heparin effect (Bjoumlrk and Lindahl 1982) were reduced in our patients
Availability of approved blood tests which can predict the risk of development of CVC
associated thrombosis already at the moment of the CVC placement are important
(Boersma et al 2016) One of the most interesting results was that dogs which developed
sonographically detectable thrombi had nearly twofold higher fibrinogen levels Well in
agreement with our results one study performed in anticoagulated human patients
demonstrated that high fibrinogen levels were associated with an increased risk of
perioperative venous thromboembolism (P = 0004) (Kato et al 2016) The cited study
does not exclusively refer to CVC associated thrombosis but 76 (1621) of the venous
thromboses were suggested to be associated with CVCs Fibrinogen measurement a test
which is widely available is therefore a useful and practicable indicator of an increased
risk for CVC induced thromboses and may advice the clinician to treat these patients
especially carefully (eg with low molecular weight heparin) In the study performed by
Boersma et al (2016) in 168 human patients without indication of anticoagulation
prophylaxis elevated leukocyte counts high levels of plasminogen activator inhibitor
and of coagulation factor VIII were associated with an increased incidence of
symptomatic CVC related thrombosis diagnosed by ultrasonography A limitation of that
study is that all measurements were performed on blood samples taken after CVC
placement Interestingly in our study ROTEM results were partly associated with
thrombus formation which reflects the well-known fact that results of viscoelastic
measurements are a good indicator of hypercoagulability and thromboembolic events
(Harahsheh and Ho 2018) It is of further interest and possibly unexpeted that on the final
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
67
day of examination dogs with ELMI signs of thromboses had partly lower coagulation
activities than dogs without thromboses Due to the low number of dogs without
detectable thrombi using ELMI this implausible result has to be interpreted with caution
One possible explanation could be that temporary hypercoagulability in these dogs may
have finally resulted in consumption of haemostasis substrates
Limitations of the present study were as a consequence of the study design based on
clinical patients the relatively low total number of patients inclusion of different UFH
treatment regimens and the lack of a control group
5 Conclusions
The results of the present study indicate that the used UFH regimen was not completely
effective to prevent CVC-associated thromboses which were detectable in 28 [829]
of the patients by ultrasound However severe thromboses (high grades) are relatively
rare in intensive care canine patients receiving a routine anticoagulant treatment Because
heparin plasma activities were not different in dogs with or without CVC-associated
thrombus formation other local and systemic factors must play a significant role as well
A high fibrinogen concentration and low maximum lysis (ROTEM ex-tem) may be
useful indicators for an increased thrombotic risk in individual patients which may
require special anticoagulatory treatment
6 Acknowledgments
Jose Mauricio Pereira received a scholarship from the Universidad Nacional de Costa
Rica (reference code JB-C-1107-2016)
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
68
7 References
1 Abdelkefi A Ben Othman T Kammoun L Chelli M Ben Romdhane N Kriaa A Ladeb S
Torjman L Lakhal A Achour W Ben Hassen A Hsaiumlri M Ladeb F Ben Abdeladhim A
2004 Prevention of central venous line-related thrombosis by continuous infusion of low-dose
unfractionated heparin in patients with haemato-oncological disease A randomized controlled trial
Thromb Haemost 92 654ndash661
2 Adamantos S Brodbelt D Moores A L 2010 Prospective evaluation of complications associated
with jugular venous catheter use in a veterinary hospital J Small Anim Pract 51 254ndash257
3 Bagge A Schoumltt U Kander T 2016 Effects of naturopathic medicines on Multiplate and ROTEM
a prospective experimental pilot study in healthy volunteers BMC Complement Altern Med 16 64
4 Ballard DH Samra NS Gifford KM Roller R Wolfe BM Owings JT 2016 Distance of the
internal central venous catheter tip from the right atrium is positively correlated with central venous
thrombosis Emerg Radiol 23 269ndash273
5 Boersma RS Jie KS Verbon A van Pampus EC Schouten HC 2008 Thrombotic and
infectious complications of central venous catheters in patients with hematological malignancies Ann
Oncol 19 433ndash442
6 Boersma R S Hamulyak K van Oerle R Tuinenburg A Ten Cate-Hoek AJ Schouten HC
2016 Biomarkers for prediction of central venous catheter related-thrombosis in patients with
hematological malignancies Clin Appl Thromb Hemost 22 779ndash784
7 Bjoumlrk I Lindahl U 1982 Mechanism of the anticoagulant action of heparin Mol Cell Biochem
48 161ndash182
8 Claude A Riedesel D Riedesel E 2010 Electrocardiography-guided and retrospective analysis of
central venous catheter placement in the dog Vet Anaesth Analg 37 97ndash105
9 Comerlato PH Rebelatto TF Santiago de Almeida FA Klein LB Boniatti M M Schaan
DB Rados VD 2017 Complications of central venous catheter insertion in a teaching hospital
Rev Assoc Med Bras 63 613ndash620
10 Crochemore T Piza FMT Rodriacutegues RDR Guerra JCC Ferraz LJR Correcirca TD 2017
A new era of thromboelastometry Einstein (Sao Paulo) 15 380ndash385
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
69
11 De Cicco M Matovic M Balestreri L Steffan A Pacenzia R Malafronte M Fantin D
Bertuzzi CA Fabiani F Morassut S Bidoli E Veronesi A 2009 Early and short‐term
acenocumarine or dalteparin for the prevention of central vein catheter‐related thrombosis in cancer
patients a randomized controlled study based on serial venographies Ann Oncol 20 1936ndash1942
12 Evans NS Ratchford EV 2018 Catheter related-venous thrombosis Vasc Med 23 411ndash413
13 Geerts W 2014 Central venous catheterndashrelated thrombosis Hematology Am Soc Hematol Educ
Program 2014 306ndash311
14 Harahsheh Y Ho KM 2018 Use of viscoelastic tests to predict clinical thromboembolic events A
systematic review and meta‐analysis Eur J Haematol 100 113ndash123
15 Harter C Salwender HJ Bach A Egerer G Goldschmidt H Ho AD 2002 Catheter-related
infection and thrombosis of the internal jugular vein in hematologic-oncologic patients undergoing
chemotherapy a prospective comparison of silver-coated and uncoated catheters Cancer 94 245ndash251
16 Hirsh J Raschke R 2004 Heparin and low-molecular-weight heparin The Seventh ACCP
Conference on Antithrombotic and Thrombolytic Therapy Chest 126 (Suppl) 188S-203S
17 Hundley D Brooks A Thomovsky E Jhonson P Freeman L Schafbuch R Heng HG Moore
G 2018 Comparison of ultrasound-guided and landmark-based techniques for central venous
catheterization via the external jugular vein in healthy anesthetized dogs Am J Vet Res 79 628ndash
636
18 Kato F Takeuchi H Matsuda S Kawakubo H Omori T Kitagawa Y 2016 Incidence of and
risk factors for venous thromboembolism during surgical treatment for esophageal cancer a single-
institution study Surg Today 46 445ndash452
19 Karthaus M Kretzschmar A Kroning H Biakhov M Irwin D Marschner N Slabber C
Fountzilas G Garin A Abecasis NG Baronius W Steger GG Sudhoff T Giorgetti C
Reichardt P 2006 Dalteparin for prevention of catheter‐related complications in cancer patients with
central venous catheters final results of a double‐blind placebo‐controlled phase III trial Ann Oncol
17 289ndash296
20 Koumlksoy C Kuzu A Kutlay J Erden I Ozcan H Ergicircn K 1995 The diagnostic value of colour
Doppler ultrasound in central venous catheter related thrombosis Clin Radiol 50 687-689
21 Kucher N 2011 Clinical practice Deep-vein thrombosis of the upper extremities N Engl J Med
364 861ndash869
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
70
22 Langston CE Eatroff AE 2018 Hemodialysis catheter-associated fibrin sheath in a dog J Vet
Emerg Crit Care 28 366ndash371
23 Laurenson MP Hopper K Herrera MA Johnson EG 2010 Concurrent diseases and conditions
in dogs with splenic vein thrombosis J Vet Intern Med 24 1298ndash1304
24 Lee AYY Kamphuisen PW 2012 Epidemiology and prevention of catheter‐related thrombosis in
patients with cancer J Thromb Haemost 10 1491ndash1499
25 Liu G Fu ZQ Zhu P Li SJ 2015 Central venous catheter-related thrombosis in senile male
patients New risk factors and predictors J Huazhong Univ Sci Technolog Med Sci 35 445ndash449
26 Lordick F Hentrich M Decker T Hennig M Pohlmann H Hartenstein R Peschel C 2003
Ultrasound screening for internal jugular vein thrombosis aids the detection of central venous catheter-
related infections in patients with haemato-oncological diseases a prospective observational study Br
J Haematol 120 1073ndash1078
27 Lucas TC Silva EED Souza DOD Santos ARD Lara MO 2017 Microstructural
evaluation by confocal and electron microscopy in thrombi developed in central venous catheters Rev
Esc Enferm USP 51 1ndash9
28 Malick L Wilson R Stetson D 1976 Modified thiocarbo- hydrazide procedure for scanning
electron microscopy routine use for normal pathological and experimental tissue Stain Technol 50
265ndash269
29 Mischke R Jacobs C 2001 The monitoring of heparin administration by screening tests in
experimental dogs Res Vet Sci 70 101ndash108
30 Niers TM Di Nisio M Klerk CP Baarslag HJ Buller HR Biemond BJ 2007 Prevention
of catheter‐related venous thrombosis with nadroparin in patients receiving chemotherapy for
hematologic malignancies a randomized placebo‐controlled study J Thromb Haemost 5 1878ndash
1882
31 Reminga C Silverstein D Drobatz K Clarke D 2018 Evaluation of the placement and
maintenance of central venous jugular catheters in critically ill dogs and cats J Vet Emerg Crit Care
28 232ndash243
32 Smith RS Zhang Z Bouchard M Li J Lapp HS Brotske GR Lucchino DL Weaver D
Roth LA Coury A Biggerstaff J Sukavaneshvar S Langer R Loose C 2012 Vascular
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
71
catheters with a nonleaching poly-sulfobetaine surface modification reduce thrombus formation and
microbial attachment Sci Transl Med 4 153ra132 doi101126scitranslmed3004120
33 Toskala E Nuutinen J Rautiainen M 1995 Scanning electron microscopy findings on human
respiratory cilia in chronic sinusitis and in recurrent respiratory infections J Laryngol Otol 109 509ndash
514
34 Van Rooden CJ Tesselaar ME Osanto S Rosendaal FR Huisman MV 2005 Deep vein
thrombosis associated with central venous catheters - a review J Thromb Haemost 3 2409ndash2419
35 Verso M Agnelli G Bertoglio S Di Somma FC Paoletti F Ageno W Bazzan M Parise P
Quintavalle R Naglieri E Santoro A Imberti D Soraru M Mosca S 2005 Enoxaparin for the
prevention of venous thromboembolism associated with central vein catheter a double‐blind placebo‐
controlled randomized study in cancer patients J Clin Oncol 23 4057ndash4062
36 Villalta-Garciacutea P Loacutepez-Herranz M Mazo-Pascual S Honrubia-Fernaacutendez T Jaacutentildeez-Escalada
L Fernaacutendez-Peacuterez C 2015 Reliability of blood test results in samples obtained using a 2-mL discard
volume from the proximal lumen of a triple-lumen central venous catheter in the critically ill patient
Nurs Crit Care 22 298ndash304
37 Vose J Odunayo A Price JM Daves M Schildt JC Tolbert MK 2019 Comparison of
heparinized saline and 09 sodium chloride for maintaining central venous catheter patency in healthy
dogs PeerJ 7 e7072 httpsdoiorg107717peerj7072
38 Wildgruber M Lueg C Borgmeyer S Karimov I Braun U Kiechle M Meier R Koehler M
Ettl J Berger H 2016 Polyurethane versus silicone catheters for central venous port devices
implanted at the forearm Eur J Cancer 59 113ndash124 doi101016jejca201602011
39 Wu X Studer W Skarvan K Seeberger MD 1999 High incidence of intravenous thrombi after
short-term central venous catheterization of the internal jugular vein J Clin Anesth 11 482ndash485
40 Xiang DZ Verbeken EK Van Lommel AT Stas M De Wever I 1998 Composition and
formation of the sleeve enveloping a central venous catheter J Vasc Surg 28 260ndash271
41 Xiang DZ Verbeken EK Van Lommel AT Stas M De Wever I 2001 Sleeve-related
thrombosis a new form of catheter-related thrombosis Thromb Res 104 7ndash14
Chapter 4 General discussion
72
Chapter 4 General discussion
Incidence of CVC related thrombosis
In our study the incidence of CVC related thrombosis detected by ultrasound in critical
care canine patients was 28 To the best of the authoracutes knowledge similar studies
performed in dogs are rare in the available veterinary literature In one study performed
in 81 dogs and 12 cats (Adamantos et al 2010) an incidence of 2 was found for vein
thrombosis related to CVC This low incidence can be due to a lacking of systematic
ultrasound evaluations of the jugular vein containing the CVC In the mentioned study in
dogs and cats the vein thrombosis was confirmed by palpation or ultrasound presumably
after the presentation of clinical symptoms however in many cases as in our study
thrombus into the jugular vein can be developed without manifestation of clinical
symptoms what makes a routinely ultrasound evaluation of the vein containing the CVC
very important Our results are well aligned with the findings in humans in which a
median incidence of 30 was found for thrombosis related to CVC based on 25 studies
performed in patients requiring a CVC (review article van Rooden et al 2005) In the
mentioned 25 human studies the incidence of CVC related thrombosis varied within a
wide range (2ndash67 ) according to factors related to (1) the CVC (eg material type
entry site [jugular femoral subclavian axillary cephalic vein] time period in place (2)
diagnostic thrombus detection technique (ultrasonography or venography) and criteria
(3) patient collective (diseases and presence of predisposing factors [eg previous venous
thrombosis history presence of cancer] and (4) prophylactic antithrombotic treatment
(Geerts 2014 Liu et al 2015 van Rooden et al 2005)
The incidence of CVC related thrombosis detected by colour Doppler ultrasound in our
study fits also with the median incidence (30 ) found in three studies performed in
humans in which colour Doppler ultrasound evaluations of the jugular vein containing a
Chapter 4 General discussion
73
CVC were performed (Harter et al 2002 Lordick et al 2003 Wu et al 1999) In these
three studies the individual incidence of CVC related thrombosis was 2 (Harter et al
2002) 30 (Lordick et al 2003) and 56 (Wu et al 1999) The high thrombus
incidence reported in the study performed by Wu et al (1999) could be due to the criteria
for diagnosis of thrombosis because in that study both fibrin sleeves and compact
thrombi were considered as cases of thrombosis related to CVC The incidence for
compact thrombi was 25 which is more similar to our results In the study performed
by Harter et al (2002) the CVC related thrombosis incidence (2 ) was very low
compared with the incidence found in our study In that study the jugular veins were
examined just before or less than 24 hours after CVC removal (median period of CVC
use = 1025 days) In our study the ultrasound evaluations were performed before the
CVC placement (day 0) and then on days 1 3 5 etc until the CVC removal (median
period of CVC use = 7 days median interval until thrombus detection = 7 days) In our
study we detected thrombus formation as early as on the 3rd day so it could be possible
that in the cited study possibly only temporarily existing thrombi could have been missed
by ultrasonography in the final stage and thereby for the incidence calculation However
this scenario is not very likely because results of the present study show that
sonographically detectable thrombi during the observation period had in all cases their
maximum grade on the final day
In the study performed by Lordick et al (2003) the CVC related thrombosis incidence
determined for 43 human patients was very similar to our study and in both studies only
compact thrombi detectable by ultrasound were considered for assessment of the
incidence In the mentioned study all the human patients were screened for the presence
of thrombosis every 4 days after CVC insertion with real time B-mode ultrasound
(Lordick et al 2003)
Chapter 4 General discussion
74
In our study 70 of CVCs analysed by ELMI showed thrombi of grade 1ndash3 on the
external CVC wall To the best of the authoracutes knowledge studies about incidence of CVC
related thrombosis on the outside surface of the CVC detected by ELMI cannot be found
in the human and small animal literature In one study performed in humans (Lucas et al
2017) thrombus formation was analysed by ELMI in 78 central venous catheters That
study focussed on microstructural assessment of thrombus formation inside the distal
openings of the CVC which were found in 85 of the evaluated CVCs The percentage
of occurrence of thrombosis in the mentioned study in humans was nearly similar to the
incidence of intraluminal thrombus formation as detected in the presented study by ELMI
(74 ) In general intraluminal thrombi were not specially considered in the present
study because they mainly express the efficacy of the flushing technique rather than the
effectivity of the antithrombotic treatment In one study performed in rats the
microstructure of thrombi developed in the jugular vein containing the CVC was also
evaluated by ELMI (Xiang et al 1998) In the mentioned study pericatheter thrombosis
was detected on the proximal section of the CVC in 100 of rats (n = 12) after 1 and 3
days of catheterisation The analyses of the CVCs placed in rats were performed with the
catheter inside the vein in contrast to our study where the CVC was removed from the
jugular vein for the respective analysis Xiang et al (2001) hypothesised in one study
performed in rabbits and rats in which CVCs were placed in the jugular vein that fibrin
rich thrombi associated to the CVC can be detached during the catheter removal and this
could explain the difference between the thrombosis incidence assessed in our study and
that of the study performed by Xiang et al (1998)
In our study the incidence of CVC related thrombosis detected by ultrasonography in the
lumen of the EJV did not correlate with the incidence of CVC related thrombosis detected
by ELMI on the external wall of the CVC This was mainly due to the fact that the CVC
Chapter 4 General discussion
75
segment evaluated with both methods was different Using ultrasonography it was only
possible to evaluate the EJV in the neck area of the patient and thereby only the proximal
third of the CVC In contrast with the ELMI analysis only the distal section of the CVC
(last third) was evaluated because macroscopically detected thrombi mainly developed
near the CVC tip which was routinely positioned at the junction of the cranial cava vein
with the right atrium In a retrospective review of medical records of 169 human patients
requiring a CVC (Ballard et al 2016) the position of the CVC tip was determined by
radiologic evaluation and it was determined that the catheter tip position can be a
predisposing factor for central venous thrombosis development Thereby it could be of
interest to evaluate in further studies in dogs if a high incidence of thrombosis detected
by ELMI as was shown in our study could be related to the CVC tip position
In our study polyurethane catheters were tested which are widely used and are the
standard CVCs used in the Small Animal Clinic University of Veterinary Medicine
Hannover In vivo and in vitro studies have assessed the thrombogenicity of different
catheter materials (Borow and Crowley 1985 Smith et al 2012) In general terms
polyurethane catheters (central or peripheral) had shown a higher thrombogenicity
compared with coated (eg with hydromer) and silicone catheters Due to this property
of polyurethane catheters they can be supposed as a good test system for antithrombotic
regimens as evaluated in our study On the other hand despite silicone catheters have been
associated with a lower thrombogenicity their mechanical flexibility could be a
disadvantage in small clinical practice due to the increased risk of rupture detected even
in humans (Wildgruber et al 2016)
Chapter 4 General discussion
76
Heparin thromboprophylaxis for CVC related thrombosis
The occurence of CVC related thrombosis detected by colour Doppler ultrasound and
ELMI in our study demonstrates that the UFH in the given dosage was not completely
effective to prevent CVC related thrombosis in canine intensive care patients In human
medicine LMWH is commonly used in prophylactic anticoagulation regimens however
studies performed in human patients requiring a CVC and receiving a prophylactic
anticoagulation (with LMWH or UFH) did not achieve consensus regarding the efficacy
of prophylactic heparin to prevent CVC related thrombosis (Abdelfeki et al 2004
Lordick et al 2003 Wu et al 1999) To the best of the authoracutes knowledge studies
performed in dogs evaluating systemic prophylactic anticoagulation to prevent CVC
related thrombosis cannot be found However according to the evidence found in the
human literature and the results of our study further investigations using comparative
groups with different drugs dosages andor administration routes are required to establish
an effective antithrombotic treatment to prevent CVC related thrombosis
The plasma heparin activities measured in canine patients in our study varied significantly
after a defined subcutaneous dosage The limited and variable bioavailability of SC
administered UFH is well documented in humans (Hirsh and Raschke 2004)
Considerable variations of the circulatory status of the liver- and kidney function and
concentration of heparin-binding proteins in intensive care patients obviously amplify
this phenomenon leading to an extremely high inter-individual and intra-individual
variation of the heparin blood levels in our patients A different application route (IV)
may have reduced the influence of part of these factors and therefore could have led to
more predictable UFH blood levels However continuous infusion requires more
technical equipment and is also associated with a wide range of heparin activities in
canine patients (Scott et al 2009)
Chapter 4 General discussion
77
Prediction of CVC related thrombosis
Of 7 haemostasis tests (including ROTEM with 12 different parameters [6 for each
reagent ex-tem and in-tem]) evaluated as possible predictors of CVC related thrombosis
in our study initial values of high fibrinogen and low maximum lysis (ROTEM ex-tem)
were associated with sonographically detectable thrombus formation Some studies in
humans have established the association of laboratory biomarkers as predictors of risk
thrombosis (Boersma et al 2016 Cheng et al 2013 Kato et al 2016 Liu et al 2015)
One study performed in anticoagulated human patients showed ndash well in agreement with
our results ndash that high fibrinogen levels were associated with an increased risk of
perioperative venous thromboembolism (P = 0004) (Kato et al 2016) The cited study
does not exclusively refer to CVC associated thrombosis but 762 (1621) of the
venous thromboses were suggested to be associated with CVCs Fibrinogen concentration
measurements are normally available in the human and veterinary practice and the
association of this parameter with a high risk of development of CVC related thrombosis
can be useful to choose special antithrombotic treatment in individual critical care patients
to prevent such severe complications
Thrombin generation assay (TGA) abnormalities such as higher velocity and endogenous
thrombin potential (ETP) values measured initially ie before any anticoagulation
therapy have been associated with a higher thromboembolism risk in general (ie without
specific association with CVCs) in one study performed on 105 human patients (Espitia
et al 2017) In contrast in our study the initial TGA assay results did not show an
association with formation of CVC related thrombosis
The ROTEM parameters evaluated in the present study were partially associated with
formation of CVC thrombosis detected by colour Doppler ultrasound This finding
confirms the results obtained in humans in which ROTEM measurements are regarded as
Chapter 4 General discussion
78
a valuable indicator of hypercoagulability (Harahsheh and Ho 2018 Tuumlrk et al 2018)
However in our study individual ROTEM parameters showed partly lower coagulation
activities in dogs with CVC related thrombosis detected by ELMI at the final day of
evaluation Because the number of patients without signs of thrombosis detected by ELMI
was low compared with those who showed signs these implausible results should be
interpreted carefully The usefulness of ROTEM analysis which has also many other
indications demonstrates that is was worth to perform a detailed methodological approval
of the device including the establishment of reference values in the preliminary study
Conclusion
In conclusion the prethrombotic or thrombotic changes detected in our study did not
correlate between ultrasonography and ELMI however both methods show accurately
signs of CVC related thrombosis The UFH in the given dosage was not completely
effective to prevent CVC induced thrombus formation in canine intensive care patients
Plasma heparin activities varied significantly after a defined subcutaneous dosage
However plasma heparin activities were not different between dogs with and without
detectable CVC associated thromboses Initial high fibrinogen values and low maximum
lysis (ROTEM ex-tem) values were associated with sonographically detectable thrombi
and may be useful as indicators for an increased thrombotic risk
Finally our study in canine intensive care patients is to the best of the authoracutes
knowledge the first study in the available veterinary literature performed to evaluate the
efficacy of an antithrombotic treatment with UFH to prevent CVC related thrombosis
Chapter 5 Summary
79
Chapter 5 Summary
Central venous catheter induced thrombosis in dogs ndash occurrence under an
antithrombotic treatment regimen and possible indicators
Joseacute Mauricio Pereira Mora
Central venous catheters (CVC) are of importance for intensive care management of
canine patients Thrombosis of the jugular vein is a severe complication related to the
CVC use However only little is known about the incidence of CVC-associated
thromboses in dogs and the necessity and efficacy of prophylactic antithrombotic
regimens
Therefore the aims of the present study were (1) to determine the incidence of catheter-
induced thrombosis in canine intensive care patients (2) to evaluate the efficacy of a
prophylactic heparin regimen routinely used in the Small Animal Clinic of the University
of Veterinary Medicine Hannover and (3) to assess whether selected haemostasis test
results are predictive for an increased risk for thrombosis formation
The thesis also includes a preliminary methodological study on the use of rotational
elastometry in dogs using the ROTEM delta device In this preliminary study reference
intervals were determined based on 125 healthy dogs for different clot activator reagents
and for measurements without activator Coefficients of variation (CVs) were calculated
based on results of fourfold measurements of 3 normal and 3 abnormal blood samples in
order to assess precision of the method Influence of age groups sex neuter status and
size were also assessed The median CVs for most of the parameters evaluated in the
precision analysis were less than 10 and this confirmed that the ROTEM delta analyser
delivers repeatable results Regarding the influence of age sex neuter status and animal
Chapter 5 Summary
80
size only neuter status in female dogs appeared to be a relevant influencing factor on
most of important ROTEM parameters
The main study of the thesis comprises 29 patients hospitalised at the Small Animal Clinic
requiring a CVC for medical reasons These patients received a prophylactic
anticoagulation treatment with unfractionated heparin (UFH 150 IU [surgical dogs
received 75 IU]kg BW TID subcutaneously) A colour Doppler ultrasound of the
respective external jugular vein (EJV) in order to detect thrombus formation in the lumen
of the EJV and blood collection for haemostasis tests were performed directly before the
installation procedure (day 0) and on days 1 3 5 7 and so on after the CVC insertion
Haemostasis tests included prothrombin time activated partial thromboplastin time
thrombin time antithrombin activity D-dimer concentration thrombin generation and
rotational elastometry as well as the heparin activity using a chromogenic anti-factor Xa
test Finally when the CVC was removed electron microscopy (ELMI) of sections of the
CVC with macroscopically visible thrombi in the last third (5ndash10 cm) of the CVC was
performed 3 different grades were defined for CVC-associated (extraluminal) thrombi
detected by either ultrasound or ELMI respectively
829 patients evaluated by colour Doppler ultrasound during the study developed thrombi
into the lumen of EJV (4 x grade 1 3 x grade 2 and 1 x grade 3) 1927 CVCs assessed
using ELMI showed detectable thrombi on the external CVC surface (7 x grade 1 4 x
grade 2 and 8 x grade 3) There was no significant correlation between thrombus
formation detected by ultrasound or ELMI Thus statistical group comparisons of
patients with or without CVC-associated thrombus formation was performed
independently for both diagnostic procedures The plasmatic heparin activities showed
great variability but no significant differences were found between dogs with or without
CVC-associated thrombus formation regardless whether this was based on sonographic
Chapter 5 Summary
81
or ELMI findings From the initially performed haemostasis tests high fibrinogen
concentrations and low maximum lysis values as measured by the ROTEM delta device
(using the ex-tem reagent) were associated with sonographically detectable thrombi
In conclusion the preliminary methodological study on the ROTEM delta in dogs shows
that the method delivers repeatable results in canine citrated whole blood and the
reference values obtained should offer a reliable basis for the interpretation of test results
of this device in dogs with suspected haemostasis disorders The results of the main study
indicate that CVC-associated thromboses occur (eg 28 [829] of the patients as
detected by ultrasound) although severe thromboses (high grades) are relatively rare in
intensive care canine patients receiving a routine anticoagulant treatment Thereby
prophylactic antithrombotic protocol (unfractionated heparin [UFH]) in the given dosage
was not completely effective to prevent CVC induced thrombus formation in canine
intensive care patients A high fibrinogen concentration and low maximum lysis
(ROTEM ex-tem) may be useful indicators for an increased thrombotic risk in individual
patients which may require special anticoagulatory treatment Further studies are
required to evaluate whether different thromboprophylactic treatment regimens eg with
low molecular weight heparin are more effective in preventing CVC-associated
thromboses in dogs
Chapter 6 Zusammenfassung
82
Chapter 6 Zusammenfassung
Zentralvenenkatheter-induzierte Thrombose bei Hunden - Auftreten unter einem
antithrombotischen Behandlungsschema und moumlgliche Indikatoren
Joseacute Mauricio Pereira Mora
Zentralvenenkatheter (ZVK) sind fuumlr das Intensivmanagement von Hunden von
Bedeutung Die Entstehung einer Thrombose der Vena jugularis ist eine schwerwiegende
Komplikation im Zusammenhang mit der Verwendung eines ZVKs Uumlber die Inzidenz
von ZVK-assoziierten Thrombosen bei Hunden und die Notwendigkeit und Wirksamkeit
prophylaktischer antithrombotischer Protokolle liegen jedoch nur wenige fundierte
Angaben in der Literatur vor
Daher waren die Ziele der vorliegenden Studie (1) die Inzidenz der Katheter-induzierten
Thrombose bei caninen Intensivpatienten zu bestimmen (2) die Wirksamkeit eines
prophylaktischen Heparin-Regimes zu bewerten das routinemaumlszligig in der Klinik fuumlr
Kleintiere der Stiftung Tieraumlrztliche Hochschule Hannover angewendet wird und (3) um
zu beurteilen ob ausgewaumlhlte Haumlmostasetestergebnisse ein erhoumlhtes Risiko fuumlr die
Thrombosebildung vorhersagen koumlnnen
Die PhD-Arbeit enthaumllt zudem eine vorbereitende methodische Studie zur Verwendung
der Rotationselastometrie bei Hunden mit Hilfe des ROTEM delta-Geraumltes In dieser
Vorstudie wurden Referenzintervalle basierend auf 125 gesunden Hunden fuumlr
verschiedene Gerinnungsaktivatorreagenzien und fuumlr Messungen ohne Aktivator
bestimmt Um die Genauigkeit der Methode einzustufen wurden Variationskoeffizienten
(VKs) basierend auf den Ergebnissen von Vierfachmessungen von drei normalen und drei
abnormalen Blutproben berechnet Der Einfluss von Alter Geschlecht Kastration und
Groumlszlige wurde ebenfalls getestet Die Medianwerte der VKs fuumlr die meisten in der
Chapter 6 Zusammenfassung
83
Praumlzisionsanalyse bewerteten Parameter betrugen weniger als 10 und dies bestaumltigte
dass das ROTEM delta-Analysegeraumlt wiederholbare Ergebnisse liefert In Bezug auf den
Einfluss von Alter Geschlecht Kastration und Tiergroumlszlige erwies sich nur eine Kastration
bei Huumlndinnen als relevanter Einflussfaktor fuumlr die meisten wichtigen ROTEM-
Parameter
Die Hauptstudie der Arbeit umfasst 29 Patienten welche zum Zeitpunkt der
Untersuchung stationaumlr in der Kleintierklinik aufgenommen worden waren und aus
medizinischer Indikation einen ZVK benoumltigten Diese Patienten erhielten eine
prophylaktische Antikoagulation mit unfraktioniertem Heparin (UFH 150 IE
[chirurgische Hunde erhielten 75 IE]kg KG 3 x taumlglich subkutan) Bildgebende
Untersuchungen mittels Farbdoppler-Ultraschall der jeweiligen Vena jugularis externa
(VJE) zum Nachweis der Thrombusbildung im Lumen der VJE und Blutentnahmen fuumlr
Haumlmostasetests wurden jeweils direkt vor dem Installationsvorgang (Tag 0) sowie an den
Tagen 1 3 5 und 7 nach Legen des ZVKs durchgefuumlhrt Haumlmostasetests umfassten die
Prothrombinzeit die aktivierte partielle Thromboplastinzeit die Thrombinzeit die
Antithrombinaktivitaumlt die D-Dimer-Konzentration die Thrombingenerierung und die
Rotationselastometrie sowie die Heparinaktivitaumlt unter Verwendung eines chromogenen
Anti-Faktor-Xa-Tests Schlieszliglich wurde nach Entfernung des ZVKs eine
Elektronenmikroskopie (ELMI) von Abschnitten aus dem letzten Drittel (5ndash10 cm) des
ZVKs mit makroskopisch sichtbaren Thromben durchgefuumlhrt Fuumlr ZVK-assoziierte
(extraluminale) Thromben die entweder durch Ultraschall oder ELMI nachgewiesen
wurden wurden drei verschiedene Grade definiert
829 Patienten die waumlhrend der Studie mittels Farbdoppler-Ultraschall untersucht
wurden entwickelten Thromben im Lumen der VJE (4 x Grad 1 3 x Grad 2 und 1 x Grad
3) 1927 ZVKs die unter Verwendung von ELMI bewertet wurden zeigten
Chapter 6 Zusammenfassung
84
nachweisbare Thromben auf der aumluszligeren ZVK-Oberflaumlche (7 x Grad 1 4 x Grad 2 und 8
x Grad 3) Es gab keine signifikante Korrelation zwischen der durch Ultraschall oder
ELMI festgestellten Thrombusbildung Daher wurden statistische Gruppenvergleiche
von Patienten mit oder ohne ZVK-assoziierter Thrombusbildung fuumlr beide diagnostische
Verfahren unabhaumlngig voneinander durchgefuumlhrt Die plasmatischen Heparinaktivitaumlten
zeigten eine groszlige Variabilitaumlt es wurden jedoch keine signifikanten Unterschiede
zwischen Hunden mit oder ohne ZVK-assoziierter Thrombusbildung gefunden
unabhaumlngig davon ob diese auf sonographischen oder ELMI-Befunden beruhte Von den
initial durchgefuumlhrten Haumlmostasetests waren hohe Fibrinogenkonzentrationen und
niedrige maximale Lyse-Werte der Messungen mit ROTEM delta (ex-tem Reagenz) mit
sonographisch nachweisbaren Thromben assoziiert
Zusammenfassend zeigt die vorbereitende methodische Studie zum ROTEM delta bei
Hunden dass die Methode wiederholbare Ergebnisse in mit Zitrat
antikoaguliertemVollblut liefert Die erarbeiteten Referenzwerte sollten eine zuverlaumlssige
Grundlage fuumlr die Interpretation der Testergebnisse dieses Geraumlts bei Hunden mit
Verdacht auf Haumlmostasestoumlrungen bieten Die Ergebnisse der Hauptstudie zeigen dass
ZVK-assoziierte Thrombosen auftreten (z B 28 [829] der Patienten bezogen auf den
Ultraschall-gestuumltzten Nachweis) obwohl schwere Thrombosen (hohe Grade) bei
intensivmedizinisch versorgten Hundepatienten die eine Routineantikoagulation
erhalten relativ selten sind Somit war das prophylaktische antithrombotische Protokoll
(unfraktioniertes Heparin [UFH]) in der angewendeten Dosierung nicht vollstaumlndig
wirksam um die ZVK-induzierte Thrombusbildung bei caninen Intensivpatienten zu
verhindern Eine hohe Fibrinogenkonzentration und eine niedrige maximale Lyse
(ROTEM ex-tem) koumlnnen als Indikatoren eines erhoumlhten Thromboserisikos bei einzelnen
Patienten nuumltzlich sein fuumlr die moumlglicherweise eine spezielle gerinnungshemmende
Chapter 6 Zusammenfassung
85
Behandlung notwendig ist Weitere Studien sind erforderlich um zu untersuchen ob
verschiedene thromboprophylaktische Behandlungsschemata z B mit
niedermolekularem Heparin bezuumlglich der Verhinderung von ZVK-assoziierten
Thrombosen bei Hunden wirksamer sind
Chapter 7 References
86
Chapter 7 References
1 Abdelkefi A Ben Othman T Kammoun L Chelli M Ben Romdhane N Kriaa A Ladeb
S Torjman L Lakhal A Achour W Ben Hassen A Hsaiumlri M Ladeb F Ben Abdeladhim
A 2004 Prevention of central venous line-related thrombosis by continuous infusion of low-dose
unfractionated heparin in patients with haemato-oncological disease A randomized controlled
trial Thromb Haemost 92 654ndash661
2 Adamantos S Brodbelt D Moores A L 2010 Prospective evaluation of complications
associated with jugular venous catheter use in a veterinary hospital J Small Anim Pract 51 254ndash
257
3 Ballard D H Samra N S Gifford K M Roller R Wolfe B M Owings J T 2016
Distance of the internal central venous catheter tip from the right atrium is positively correlated
with central venous thrombosis Emerg Radiol 23 269ndash273
4 Baumann-Kreuziger L Onwuemene O Kolesar E Crowther M Lim W 2015 Systematic
review of anticoagulant treatment of catheter-related thrombosis Thromb Res 136 1103ndash1109
5 Bliss SP Bliss SK Harvey HJ 2002 Use of recombinant tissue‐plasminogen activator in a
dog with chylothorax secondary to catheter‐associated thrombosis of the cranial vena cava J Am
Anim Hosp Assoc 38 431ndash435
6 Boersma RS Hamulyak K van Oerle R Tuinenburg A Ten Cate-Hoek AJ Schouten
HC 2016 Biomarkers for prediction of central venous catheter related-thrombosis in patients
with hematological malignancies Clin Appl Thromb Hemost 22 779ndash784
7 Boersma RS Jie KS Verbon A van Pampus EC Schouten HC 2008 Thrombotic and
infectious complications of central venous catheters in patients with hematological
malignancies Ann Oncol 19 433ndash442
8 Bonnet F Loriferne JF Texier JP Texier M Salvat A Vasile N 1989 Evaluation of
Doppler examination for diagnosis of catheter-related deep vein thrombosis Intensive Care Med
15 238ndash240
9 Borow M Crowley J G 1985 Evaluation of central venous catheter thrombogenicity Acta
Anaesthesiol Scand Suppl 81 59ndash64
10 Cheng Y Cui T Fu P Liu F Zhou L 2013 Dyslipidemia is associated with tunneled-cuffed
catheter-related central venous thrombosis in hemodialysis patients a retrospective multicenter
Chapter 7 References
87
study Artif Organs 37 155ndash161
11 Comerlato PH Rebelatto TF Santiago de Almeida FA Klein LB Boniatti MM
Schaan BD Rados DV 2017 Complications of central venous catheter insertion in a teaching
hospital Rev Assoc Med Bras 63 613ndash620
12 De Cicco M Matovic M Balestreri L Steffan A Pacenzia R Malafronte M Fantin
D Bertuzzi CA Fabiani F Morassut S Bidoli E Veronesi A 2009 Early and short‐term
acenocumarine or dalteparin for the prevention of central vein catheter‐related thrombosis in
cancer patients a randomized controlled study based on serial venographies Ann
Oncol 20 1936ndash1942
13 Espitia O Fouassier M Hardouin JB Pistorius MA Agard C Planchon B Trossaert M
Pottier P 2017 Thrombin generation assay in hospitalized nonsurgical patients A new tool to
assess venous thromboembolism risk Clin Appl Thromb Hemost 23 45ndash51
14 Evans NS Ratchford EV 2018 Catheter related-venous thrombosis Vasc Med 23 411ndash413
15 Geerts W 2014 Central Venous Catheter-related thrombosis Hematology Am Soc Hematol
Educ Program 2014 306ndash311
16 Harahsheh Y Ho KM 2018 Use of viscoelastic tests to predict clinical thromboembolic
events A systematic review and meta‐analysis Eur J Haematol 100 113ndash123
17 Harter C Salwender HJ Bach A Egerer G Goldschmidt H Ho AD 2002 Catheter-
related infection and thrombosis of the internal jugular vein in hematologic-oncologic patients
undergoing chemotherapy a prospective comparison of silver-coated and uncoated
catheters Cancer 94 245ndash251
18 Hirsh J Raschke R 2004 Heparin and low-molecular-weight heparin The Seventh ACCP
Conference on Antithrombotic and Thrombolytic Therapy Chest 126 (Suppl) 188Sndash203S
19 Karthaus M Kretzschmar A Kroning H Biakhov M Irwin D Marschner N Slabber
C Fountzilas G Garin A Abecasis NG Baronius W Steger GG Sudhoff T Giorgetti
C Reichardt P 2006 Dalteparin for prevention of catheter‐related complications in cancer
patients with central venous catheters final results of a double‐blind placebo‐controlled phase III
trial Ann Oncol 17 289ndash296
20 Kato F Takeuchi H Matsuda S Kawakubo H Omori T Kitagawa Y 2016 Incidence of
and risk factors for venous thromboembolism during surgical treatment for esophageal cancer a
Chapter 7 References
88
single-institution study Surg Today 46 445ndash452
21 Kohler TR Kirkman TR 1998 Central venous catheter failure is induced by injury and can
be prevented by stabilizing the catheter tip J Vasc Surg 28 59ndash66
22 Koumlksoy C Kuzu A Kutlay J Erden I Ozcan H Ergicircn K 1995 The diagnostic value of
colour Doppler ultrasound in central venous catheter related thrombosis Clin Radiol 50 687ndash
689
23 Kucher N 2011 Clinical practice Deep-vein thrombosis of the upper extremities N Engl J
Med 364 861ndash869
24 Langston CE Eatroff AE 2018 Hemodialysis catheter-associated fibrin sheath in a dog J
Vet Emerg Crit Care 28 366ndash371
25 Langston CE Eatroff AE Poeppel K 2014 Use of tissue plasminogen activator in catheters
used for extracorporeal renal replacement therapy J Vet Intern Med 28 270ndash276
26 Laurenson MP Hopper K Herrera MA Johnson EG 2010 Concurrent diseases and
conditions in dogs with splenic vein thrombosis J Vet Intern Med 24 1298ndash1304
27 Lee AYY Kamphuisen PW 2012 Epidemiology and prevention of catheter‐related
thrombosis in patients with cancer J Thromb Haemost 10 1491ndash1499
28 Liu G Fu ZQ Zhu P Li SJ 2015 Central venous catheter-related thrombosis in senile male
patients New risk factors and predictors J Huazhong Univ Sci Technolog Med Sci 35 445ndash
449
29 Lordick F Hentrich M Decker T Hennig M Pohlmann H Hartenstein R Peschel C
2003 Ultrasound screening for internal jugular vein thrombosis aids the detection of central
venous catheter-related infections in patients with haemato-oncological diseases a prospective
observational study Br J Haematol 120 1073ndash1078
30 Lucas TC Silva EED Souza DOD Santos ARD Lara MO 2017 Microstructural
evaluation by confocal and electron microscopy in thrombi developed in central venous catheters
Rev Esc Enferm USP 51 1ndash9
31 Mismetti P Mille D Laporte S Charlet V Buchmuller‐Cordier A Jacquin JP Fournel
P Dutrey‐Dupagne C Decousus H 2003 Low‐molecular‐weight heparin (nadroparin) and
very low doses of warfarin in the prevention of upper extremity thrombosis in cancer patients with
indwelling long‐term central venous catheters a pilot randomized trial Haematologica 88 67ndash
Chapter 7 References
89
73
32 Niers TM Di Nisio M Klerk CP Baarslag HJ Buller HR Biemond BJ 2007
Prevention of catheter‐related venous thrombosis with nadroparin in patients receiving
chemotherapy for hematologic malignancies a randomized placebo‐controlled study J Thromb
Haemost 5 1878ndash1882
33 Scott K C Hansen B D DeFrancesco T C (2009) Coagulation effects of low molecular
weight heparin compared with heparin in dogs considered to be at risk for clinically significant
venous thrombosis J Vet Emerg Crit Care 19 74ndash80
34 Smith RS Zhang Z Bouchard M Li J Lapp HS Brotske GR Lucchino DL Weaver
D Roth LA Coury A Biggerstaff J Sukavaneshvar S Langer R Loose C 2012
Vascular catheters with a nonleaching poly-sulfobetaine surface modification reduce thrombus
formation and microbial attachment Sci Transl Med 4 153
35 Tuumlrk S M Cansu D Uuml Teke H Uuml Kaşifoğlu T Meltem Akay O Bilgin M amp Korkmaz
C 2018 Can we predict thrombotic tendency in rheumatoid arthritis A thromboelastographic
analysis (with ROTEM) Clin Rheumatol 37 2341ndash2349
36 van Rooden CJ Rosendaal FR Barge RM van Oostayen JA van der Meer FJM
Meinders AE Huisman MV 2003 Central venous catheter related thrombosis in haematology
patients and prediction of risk by screening with Doppler-ultrasound Br J Haematol 123 507ndash
512
37 van Rooden CJ Tesselaar ME Osanto S Rosendaal FR Huisman MV 2005 Deep vein
thrombosis associated with central venous catheters - a review J Thromb Haemost 3 2409ndash
2419
38 Verso M Agnelli G Bertoglio S Di Somma FC Paoletti F Ageno W Bazzan M Parise
P Quintavalle R Naglieri E Santoro A Imberti D Soraru M Mosca S 2005 Enoxaparin
for the prevention of venous thromboembolism associated with central vein catheter a double‐
blind placebo‐controlled randomized study in cancer patients J Clin Oncol 23 4057ndash4062
39 Vose J Odunayo A Price JM Daves M Schildt JC Tolbert MK 2019 Comparison of
heparinized saline and 09 sodium chloride for maintaining central venous catheter patency in
healthy dogs PeerJ 7e7072 httpdoi107717peerj7072
40 Wang LH Wei F Jia L Zhi Lu Wang B Dong HY Yu HY Sun GJ YangJ Li B
Chapter 7 References
90
Meng J Zhang RN Bi XQ Chen HY Jiang AL 2015 Fibrin sheath formation and
intimal thickening after catheter placement in dog model role of hemodynamic wall shear stress J
Vasc Access 16 275ndash284
41 Wildgruber M Lueg C Borgmeyer S Karimov I Braun U Kiechle M Meier R Koehler
M Ettl J Berger H 2016 Polyurethane versus silicone catheters for central venous port devices
implanted at the forearm Eur J Cancer 59 113ndash124
42 Whitesell RT Steenburg SD 2014 Imaging findings of acute intravascular thrombus on non-
enhanced computed tomography Emerg Radiol 21 271ndash277
43 Wu X Studer W Skarvan K Seeberger MD 1999 High incidence of intravenous thrombi
after short-term central venous catheterization of the internal jugular vein J Clin Anesth 11
482ndash485
44 Xiang DZ Verbeken EK Van Lommel AT Stas M De Wever I 1998 Composition and
formation of the sleeve enveloping a central venous catheter J Vasc Surg 28 260ndash271
45 Xiang DZ Verbeken EK Van Lommel AT Stas M De Wever I 2001 Sleeve-related
thrombosis a new form of catheter-related thrombosis Thromb Res 104 7ndash14
91
Acknowledgements
First I thank God for every day in my life for His guide and because He was is
and will be always with me and that certainty gives me strength to take a step
forward
My sincere thanks to Prof Dr Reinhard Mischke for the opportunity to perform
my PhD under his supervision for his support and guidance for all that I learned
with him and for his friendship
I also thank to Prof Dr Manfred Kietzmann and PD Dr Mario von Depka
Prondzinski for being part of my supervision group for their time and beneficial
advices and discussion during my PhD
My gratitude to Prof Hewicker-Trautwein and Mrs Rohn for their valuable work
and availability to help me
I thank the Werlhof Institute Medical Care Center Hannover for the valuable
cooperation especially to Mrs Ober-Cassebaum Anne and Franziska for all their
help
I thank all the staff of the Small Animal Clinic of the University of Veterinary
Medicine Hannover (TiHo) My special gratitude to the internal medicine and
critical care surgery neurology ultrasound and anesthesia departments and to
the TiHo students all their cooperation made possible my research
I thank the haematology clinical chemistry and haemostasis laboratory
department of the Small Animal Clinic TiHo especially to Dirk because he was
always available for me thanks for his guide and friendship all his explanations
in the laboratory were of great value but also the conversations about different
topics of the life were of big value for me
92
I thank the Hannover Graduate School for Veterinary Pathobiology
Neuroinfectology and translational Medicine (HGNI) for the well-organized
PhD program and especially thanks to Prof Dr Beatrice Grummer Dr Tina
Selle and Mrs Tania Czeslik for his guide and help during my studies
My sincere thanks to Mrs Maritta Ledwoch for her guidance at the International
office of the TiHo ldquoI remember you since my externship in Germanyrdquo
I also thank the Student Union Hannover for all the help with the accommodation
for me and my family
I want to thank the Universidad Nacional de Costa Rica and therefore to Costa
Rica (my loved country) for the financial support to perform my PhD studies
but also for my professional formation as a veterinarian Special thanks to the
Veterinary Medicine School from Universidad Nacional de Costa Rica To all the
academic and administrative staff for their support and for believing in me
Sincerely thanks to Dr Mauricio Jimenez Soto my mentor my adviser but most
important my friend Thanks for believing in me and for always being by my side
ldquoAgradezco profundamente a mis papaacutes ldquodon Tito (Papillo) y dontildea Eida (Mami)rdquo
Todo lo bueno que hay en mi proviene de Dios y de ellos Gracias a mis hermanos
(as) cuntildeados (as) sobrinos y a toda la familia relacionada a ellos Siempre han
sido un gran apoyo y los amordquo
ldquoGracias a dontildea Alba Fran y David Gracias por toda la ayuda y por estar siempre
pendientesrdquo
I would like to thank all my friends that during a short part or during my whole
life have been with me For all the good time shared really thanks
To Willy thanks ldquogordordquo
93
I sincerely thank to Adriano and Johanna for their help and hospitality Thanks
Adriano for your guidance and support during my beginning at the TiHo for make
me feel in Costa Rica with your friendship for the tourist walks and for the bike
training I missed the coffee times
I want to thank Ina the first person to give me a smile at the start of my PhD
Thanks for your hugs and friendship
I also thank Franz for his help and for being a very nice partner Thanks for help
me always when I requested you
ldquoFinalmente quiero expresar mi gratitud y amor a Karo Faacutetima y Abigaiacutel A Karo
por tu sacrificio y soporte por las palabras de aliento por estar a mi lado en las
buenas pero en especial en las malas A Faacute por tu sacrificio por tus sonrisas por
tus abrazos y besos por bailar con migo siempre por ser mi modelo e inspiracioacuten
A Abi por ser una nueva razoacuten in mi vida para seguir adelante por todo lo que
deseo compartir con tigo por mostrarme junto con Faacutetima la felicidad en una
sonrisardquo
Table of contents
Table of contents
Chapter 1 Introduction 1
Epidemiology of CVC related thrombosis 1
Pathogenesis of CVC related thrombosis 2
Diagnosis of CVC related thrombosis 3
Heparin thromboprophylaxis for CVC related thrombosis 5
Prediction of CVC related thrombosis 7
Aims of the study 8
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs 9
ABSTRACT 11
1 Introduction 12
2 Materials and methods 14
3 Results 20
4 Discussion 26
5 Conclusions 31
6 Acknowledgments 31
7 References 32
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes 37
ABSTRACT 40
1 Introduction 42
2 Material and methods 44
3 Results 54
Table of contents
4 Discussion 62
5 Conclusions 67
6 Acknowledgments 67
7 References 68
Chapter 4 General discussion 72
Incidence of CVC related thrombosis 72
Heparin thromboprophylaxis for CVC related thrombosis 76
Prediction of CVC related thrombosis 77
Conclusion 78
Chapter 5 Summary 79
Chapter 6 Zusammenfassung 82
Chapter 7 References 86
Acknowledgements 91
List of abbreviations
i
List of abbreviations
CV coefficient of variation
CVC central venous catheter
EJV external jugular vein
ETP endogenous thrombin potential
HDL high-density lipoprotein
IV intravenous
LDL low-density lipoprotein
LMWH low molecular weight heparin
ROTEM rotational elastometry
SC subcutaneous
TGA thrombin generation assay
UFH unfractionated heparin
Chapters 2 and 3 have their own abbreviations included
List of figures and tables
ii
List of figures and tables
Chapter 2
Table 1 Coefficients of variation () for rotational thromboelastometry using the
ROTEMreg delta analyser in normal blood samples 20
Table 2 Coefficients of variation () for rotational thromboelastometry using the
ROTEMreg delta analyser in canine blood samples with haemostatic alterations 20
Table 3 Reference values (median reference interval based on 25 - and 975 -
quantiles) for thromboelastometric measurements with the ROTEMreg delta analyser in
dogs (n=125 95 confidence intervals in brackets) 23
Table 4 Median values (minimum and maximum in brackets) for different ROTEMreg
variables using ex-tem reagent stratified by sex and neuter status 24
Table 5 Median values (minimum and maximum in brackets) for different ROTEMreg
variables using the in-tem reagent stratified by sex and neutering status 25
Chapter 3
Fig 1 Definition of different grades of central venous catheter induced thrombosis of the
jugular vein as detected using colour Doppler ultrasound 48
Fig 2 Definition and illustration of different grades of central venous catheter induced
(extraluminal) thrombosis as detected using electron microscopy 53
Fig 1S (suppl material) Definition and illustration of different grades of intraluminal
thrombosis in central venous catheters as detected using electron microscopy 53
Table 1S (Supplementary material) Heparin activities (IUml median values
minimumndashmaximum) measured in 27 dogs receiving 75 or 150 IUkg unfractionated
(standard) heparin TID subcutaneously 2 hours after the first injection on the day
Comparison of samples collected via puncture of the peripheral veins and those collected
from the central venous catheter (CVC) using the three-syringe-technique 56
List of figures and tables
iii
Fig 3ab Heparin activities (box- and whisker plot arithmetic means) in dogs receiving
unfractionated heparin at a dosage of 75 IUkg BW tid (a) or 150 IUkg BW tid (b) two
hours after the first daily heparin administration 57
Table 1 Heparin activities (IUml median values minimumndashmaximum) in 28 dogs
receiving 75 or 150 IUkg unfractionated (standard) heparin TID subcutaneously with
(grades 1ndash3) or without (grade 0) sonographically detectable thrombus Blood collections
were performed 2 hours after the first daily injection 57
Table 2 Heparin activities (IUml median values minimumndashmaximum) in 27 dogs
receiving 75 or 150 IUkg unfractionated (standard) heparin TID subcutaneously with
(score 1ndash3) or without (score 0) detectable thrombus via electron microscopy 58
Table 3 Comparison of initial results of haemostasis parameters (median values
minimumndashmaximum in dogs with (grades 1ndash3) and without (grade 0) sonographically
detectable central venous catheter-induced thromboses 60
Table 4 Comparison of results of haemostasis parameters (median values minimumndash
maximum) on the last day in dogs with (grades 1ndash3) and without (grade 0) central venous
catheter-associated thrombus formation as detected by electron microscopy 61
Chapter 1 Introduction
1
Chapter 1 Introduction
Central venous catheters (CVC) are mainly used at critical care units in human and small
animal patients requiring infusion of large amounts of fluids or blood parenteral nutrition
support routine drug therapy haemodialysis chemotherapy plasmapheresis or
periodical blood sampling (Geerts 2014 Vose et al 2019) CVC related thrombosis is
one of the most severe complications (apart from infections and pneumothorax) of CVCs
in humans (Comerlato et al 2017 Evans and Ratchford 2018 Geerts 2014) leading to
patientrsquos distress CVC dysfunction venous stenosis and in some cases to the
development of thromboembolism (Geerts 2014) In the human literature there are
various studies dealing with epidemiologic and prophylactic aspects which are described
below in detail In contrast to the best of the authoracutes knowledge studies about extra
luminal CVC related thrombosis in dogs are rare in the available veterinary literature
Studies are mainly dealing with development of intraluminal CVC thrombi occurring
frequently in dogs and cats (Langston et al 2014) and leading to CVC malfunction (Vose
et al 2019)
Epidemiology of CVC related thrombosis
CVC related thrombosis represents 70ndash80 of all upper extremity thrombosis cases and
approximately 10 of thromboembolism cases in humans (Kucher 2011) The incidence
of CVC related thrombosis reported in humans based in 25 studies ranged widely from
2 to 67 (median = 30 ) (van Rooden et al 2005) The incidence of CVC related
thrombosis detected by colour Doppler ultrasound of the internal jugular vein reported in
3 studies performed in humans was 2 (Harter et al 2002) 30 (Lordick et al 2003)
and 56 (Wu et al 1999)
Chapter 1 Introduction
2
The incidence of CVC related thrombosis in humans can vary between studies according
to the CVC type used entry site of the CVC (jugular femoral subclavian axillary
cephalic vein) and time period with the CVC in place the diagnostic thrombus detection
technique (venography or ultrasonography) and criteria for diagnosis duration of follow
up diseases and presence of predisposing factors (eg previous venous thrombosis
history presence of cancer) and finally prophylactic anticoagulation treatment (Geerts
2014 Liu et al 2015 van Rooden et al 2005)
To the best of the authoracutes knowledge data about the incidence of CVC related
thrombosis detected by colour Doppler ultrasound into the lumen of the jugular vein in
dogs are rare in the available veterinary literature In one study performed in 81 dogs and
12 cats (Adamantos et al 2010) the incidence of vein thrombosis detected by palpation
or ultrasound was 2 and the incidence of intraluminal CVC thrombosis (presumed by
the inability to draw blood back) was 15 Additionally regarding to intraluminal CVC
thrombosis an incidence of 8 was reported in one case control study performed in 24
dogs receiving CVC flushing with heparinised solution (Vose et al 2019) Data about
incidence in other animal species was also found In another case-control study performed
on 37 pigs thrombosis in the caval vein related to CVC placed in the external jugular
vein (EJV) was evaluated by histological analysis however no ultrasound detection was
performed (Kohler and Kirkman 1998) In this study in pigs 94 of patients with CVC
developed thrombi in the vena cava based on sections at the site of the end of the catheter
tip and 05 and 1 cm proximal of the first section (Kohler and Kirkman 1998)
Pathogenesis of CVC related thrombosis
Two types of CVC related thrombosis are distinguished in the human medicine literature
(Geerts 2014 van Rooden et al 2005) First symptomatic thrombosis is defined as
Chapter 1 Introduction
3
thrombosis detected by diagnostic images (ultrasonography venography) when
symptoms and signs (eg localised swelling pain redness) are present (Lee and
Kamphuisen 2012) Second subclinical thrombosis is defined as thrombosis
demonstrated by the previously mentioned diagnostic images without overt symptoms or
signs (Boersma et al 2008) Additionally the CVC related thrombi can be classified as
occlusive thrombus into the CVC lumen peri-catheter related fibrin sheaths and mural
thrombi (Geerts 2014)
During the CVC insertion the puncture of the skin on the CVC insertion site and the
rupture of the blood vessels integrity activate the coagulation cascade and the CVC acts
as a matrix for adherence and accumulation of fibrin and cellular components (Smith et
al 2012) The attachment of proteins and blood cells to the internal andor external wall
of the CVC can lead to (1) (partial or complete) occlusion of the catheter lumen and (2)
sometimes if a thrombus formation starts into the lumen of the vein (attached to the vein
wall or to the CVC) a partial or complete obstruction of the blood flow andor embolism
can develop (Langston and Eatroff 2018) Additionally a study performed in humans
(Lordick et al 2003) demonstrated a relationship between CVC related thrombosis and
microbial colonisation of the catheter leading to CVC related systemic infections This
relationship was explained among others by the ease of microorganisms like
Staphylococcus aureus and Staphylococcus epidermidis to adhere to CVC related
thrombin sheaths (Boersma et al 2008)
Diagnosis of CVC related thrombosis
Venous thrombosis is commonly diagnosed in human and small animal medicine
(especially in dogs) with vascular ultrasonography with or without use of the colour
Doppler technique which is normally used to evaluate the blood flow through the blood
Chapter 1 Introduction
4
vessels (Evans and Ratchford 2018 Laurenson et al 2010) According to the literature
in humans cited below the CVC related thrombosis detected by colour Doppler
ultrasound is present if any of the following aspects are observed (1) well defined
thrombus visualised in the lumen of the vein (2) impossibility to perform compression
of the vein by slight pressure with the ultrasound probe andor (3) no detectable blood
flow signal by colour Doppler ultrasound (Bonnet et al 1989 Koumlksoy et al 1995)
Similarly in dogs the CVC related thrombosis is usually diagnosed by colour Doppler
ultrasonography through the detection of the thrombus within the lumen of the vein
andor the obstruction (partial or complete) of the normal blood flow (Bliss et al 2002)
Especially in humans the contrast venography technique is used as gold standard to
confirm thrombosis For this purpose a contrast solution is injected through a peripheral
vein to assess its passage through the bloodstream by x-ray However this diagnostic
technique is normally used only in the case of a strong clinical suspicion of venous
thrombosis that cannot be detected by ultrasonography (Laurenson et al 2010 Langston
and Eatroff 2018) In a study performed in 44 human patients requiring a CVC (Koumlksoy
et al 1995) a sensitivity of 94 and a specificity of 96 were determined for the colour
Doppler ultrasound diagnosis when it was compared with the diagnosis performed with
the contrast venography technique That results make the colour Doppler ultrasound an
accurate diagnosis method to detect CVC related thrombosis in humans (Koumlksoy et al
1995 van Rooden et al 2003)
Advanced diagnostic image techniques like computed tomography or magnetic resonance
tomography are also accurate to perform the diagnosis of CVC related thrombosis in
humans (Whitesell and Steenburg 2014) In dogs multidetector computed tomography
venography was used in one study for the diagnosis of CVC related thrombosis allowing
the identification of catheter related sheaths around the CVC external wall (Wang at al
Chapter 1 Introduction
5
2015) However the high costs of these advanced diagnostic imaging procedures and the
requirement to perform at least sedation or general anesthesia are disadvantages when
compared with ultrasonography
Finally electron microscopy (ELMI) is a diagnostic technique that has been used in
humans (Lucas et al 2017) and experimental animals (Xiang et al 1998 Xiang et al
2001) mainly to perform a microstructural evaluation of thrombi developed into the
lumen of the CVC or in the vein containing the CVC ELMI allows the ex vivo diagnosis
of very small thrombi or sleeve related thrombosis that perhaps cannot be detected by
ultrasonography or venography during the evaluation of patients with CVCs (Xiang et al
2001) However one point to consider is that it is possible that compact thrombi
developed on the CVC can be detached during the CVC removal from the vein and
thereby before its analysis by ELMI (Xiang et al 2001)
Heparin thromboprophylaxis for CVC related thrombosis
Application of CVCs in human medicine is commonly accompanied by prophylactic
anticoagulation in order to decrease the risk of development of partial or complete vein
occlusion andor thromboembolism and thereby to improve the patientrsquos comfort
(Baumann-Kreuziger et al 2015) Apart from vitamin K antagonists (Mismetti et al
2003) unfractionated heparin (UFH) (Abdelfeki et al 2004) and low molecular weight
heparin (LMWH) (Karthaus et al 2006) are anticoagulants that have been used on
studies in humans administrated by intravenous (IV) infusion or by subcutaneous (SC)
injection to prevent thrombi development into the lumen of the veins containing the CVC
(review article Baumann-Kreuziger et al 2015) Different heparin dosages have been
used as thromboprophylactic anticoagulation in humans with CVC (review article Lee
and Kamphuisen 2012) For example in five different case-control studies performed in
Chapter 1 Introduction
6
human haemato-oncological patients UFH at a daily dosage of 100 IUkg given by IV
infusion (Abdelfeki et al 2004) and LMWH at SC daily dosages of 40 mg enoxaparin
(Verso et al 2005) 5000 IU AXa dalteparin (Karthaus et al 2006 De Cicco et al 2009)
and 2850 IU AXa nadroparin (Niers et al 2007) were used Only in part of these studies
it is stated that systemic heparin was combined with flushing of the CVC using
heparinised saline solution to reduce the risk of catheter occlusion (Karthaus et al 2006)
In 3 of the 5 previously mentioned studies no statistically significant difference was found
in the incidence of CVC related thrombosis between patients receiving the
anticoagulation prophylaxis compared with the control groups without systemic
anticoagulation (Verso et al 2005 p = 035 Karthaus et al 2006 p gt 005 Niers et al
2007 p = 049) In contrast in the residual two studies a lower incidence of CVC related
thrombosis was found in the group receiving an anticoagulation prophylaxis compared
with the control groups (Abdelfeki et al 2004 150 vs 126 p = 003 De Cicco et
al 2009 400 vs 526 p = 005)
Obviously there is no consensus in the available human literature regarding the beneficial
use of anticoagulation prophylaxis to prevent CVC related thrombosis and this may
reflect great variations for example according to the study design period trial drug
administration route (Lee and Kamphuisen 2012) and the moment of the initial
anticoagulant dosage administration (Decicco et al 2009)
To the best of the authoracutes knowledge studies about thromboprophylaxis for extraluminal
CVC related thrombosis in dogs cannot be found in the available veterinary literature
However similarly to humans prophylactic anticoagulation with heparinised saline
(administrated through CVC flushing) to prevent CVC occlusion by intraluminal thrombi
was used in one case-control study performed in 24 dogs (Vose et al 2019) In the cited
study 12 dogs received dosages of 3 ml of heparinised saline solution (10 IU UFHml) to
Chapter 1 Introduction
7
flush the catheter every 6 hours during 72 hours whereas 12 control dogs received
flushing with the same volume of non-heparinised saline solution administered at the
same frequency and no statistically significant difference was found
Prediction of CVC related thrombosis
Studies regarding the relationship between laboratory biomarkers and CVC related
thrombosis are available in the human literature (Boersma et al 2016 Cheng et al 2013
Liu et al 2015) however to the best of the authoracutes knowledge literature about laboratory
tests used as predictors for CVC related thrombosis in small animals cannot be found at
all Availability of approved blood tests which can predict the risk of thrombosis
development associated to CVC already at the moment of the CVC placement are
important in order to identify an increased risk of CVC related thrombosis (Boersma et
al 2016) In the study performed by Boersma et al (2016) in 168 human patients without
indication of anticoagulation prophylaxis elevated leukocyte counts high levels of
plasminogen activator inhibitor and of coagulation factor VIII (all measured on blood
samples taken after CVC placement) were associated with an increased incidence of
symptomatic CVC-related thrombosis diagnosed by ultrasonography In another study
(Liu et al 2015) high blood levels of lactate dehydrogenase and low concentrations of
HDL and albumin were associated with the incidence for CVC related thrombosis
diagnosed by ultrasound in 324 senile patients which was in total 617 Additionally
dyslipidemia (high triglycerides low HDL and high LDLHDL ratio) was associated
with CVC related thrombosis (diagnosed by ultrasound and computer tomography
angiography) in human patients undergoing haemodialysis (Cheng et al 2013) Studies
about predictive tests for CVC related thrombosis in dogs were not found in the available
literature to the best of the authoracutes knowledge
Chapter 1 Introduction
8
Aims of the study
The aims of the preliminary methodological study of the present scientific project were
(1) to define reference intervals for viscoelastic analyses of canine haemostasis using the
ROTEMreg delta analyser and (2) to determine the precision (repeatability) of this
method And the aims of the main study of the present scientific project were (1) to
determine the incidence of catheter-induced thrombosis in canine intensive care patients
(2) to evaluate the efficacy of a prophylactic heparin regimen routinely used in the Small
Animal Clinic of the University of Veterinary Medicine Hannover and (3) to assess
whether selected haemostasis test results are predictive for an increased risk for
thrombosis formation
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
9
Chapter 2 Reference intervals for rotational thromboelastometry measurements
using the ROTEMreg delta device in dogs
Pereira JM Rohn K Mischke R
Contributions of the authors
Pereira JM performed (1) the patients data and blood samples collection (2)
haematology and ROTEMreg analysis (3) generated the reference intervals and
coefficients of variation included in the study and (4) wrote the manuscript
Rohn K performed the statistical analysis
Mischke R performed the clinical chemistry and coagulation tests and was involved in
designing and supervision of the study analysis and discussion of the data and its
statistical analysis and revision edition and correction of the manuscript
Original source Pereira J M Rohn K amp Mischke R 2020 Reference intervals
for rotational thromboelastometry measurements using the ROTEMreg delta device
in dogs Res Vet Sci 130 26ndash32 httpsdoiorg101016jrvsc202001019
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
10
Reference intervals for rotational thromboelastometry measurements using the
ROTEMreg delta device in dogs
Pereira JMa Rohn Kb Mischke Ra
aSmall Animal Clinic University of Veterinary Medicine Hannover Buumlnteweg 9 D-
30559 Hannover Germany
bInstitute of Biometry Epidemiology and Information Processing University of
Veterinary Medicine Hannover Buumlnteweg 2 30559 Hannover Germany
Corresponding author
E-mail ReinhardMischketiho-hannoverde
Original source Pereira J M Rohn K amp Mischke R 2020 Reference intervals
for rotational thromboelastometry measurements using the ROTEMreg delta device
in dogs Res Vet Sci 130 26ndash32 httpsdoiorg101016jrvsc202001019
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
11
ABSTRACT
Aims of the present study were to define reference intervals for viscoelastic analyses of
canine haemostasis using the ROTEMreg delta analyser and as a secondary aspect to
determine the precision (repeatability) of this method Blood samples from 125 clinically
healthy dogs were included Measurements were performed with commercially available
activating reagents (ex-tem in-tem and kaolin solution) as well as without activation
Additional fourfold measurements were done in 3 of the normal blood samples and in 3
samples with haemostatic alterations to evaluate the precision of the method
Coefficients of Variation (CVs) for most of the ROTEM variables were lt 10 Clot
formation time and maximum clot elasticity showed a wide inter-individual variation in
comparison with alpha angle and maximum clot firmness A multivariate analysis on
various ROTEM parameters revealed particularly a significant influence of neuter status
and a significant interrelationship between the factors sex and neuter status for
measurements with different activating reagents These results reflected the fact that
significant differences occurred only between intact and neutered females but not in
males No or only occasionally significant differences were found between groups of sex
age and size
In conclusion CVs demonstrated that the method delivers repeatable results in canine
citrated whole blood Established reference intervals should deliver valuable orientation
for the evaluation of viscoelastic properties of clotting whole blood in dogs using the
ROTEM delta analyser Neuter status in females appeared to be the most relevant
influencing factor and should be considered for the interpretation of ROTEM delta test
results
Keywords Viscoelastic measurements reference values canine age sex neuter status
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
12
1 Introduction
Heamostasis is a physiologic and dynamic process in which plasma soluble coagulation
factors blood cells and blood vessels are involved Clot formation and possible
subsequent clot lysis as well as vascular tissue repair aim to prevent haemorrhages on the
one hand and thromboses on the other hand (Kol and Borjesson 2010 Versteeg et al
2013) Conventional assays of coagulation such as prothrombin time (PT) activated
partial thromboplastin time (aPTT) and thrombin time TT have limitations especially
cellular elements are not incorporated into the test and thereby these tests reflect only
limitedly in vivo functionality of haemostasis (McMichael and Smith 2011 Crochemore
et al 2018)
The rotational thromboelastometry (ROTEMreg) currently termed as thromboelastometry
(TEM) and thromboelastography (TEG) are methods for the rapid evaluation of the
overall haemostasis capacity through the measurement of viscoelastic properties of
clotting whole blood (Kol and Borjesson 2010 Theusinger et al 2010 Sankarankutty
et al 2012 Versteeg et al 2013) In TEG the cup with the blood sample is rotating
whereas the torsion wire is fixed In ROTEMTEM the cup is fixed whereas the pin is
rotating Changes in torque ie resistance to rotation by the fibrin bridge created between
pin and cup in the clotting blood are detected electromechanically in TEG and optically
in ROTEM (Bollinger et al 2012) and displayed as a graph that indicates the time for the
formation of blood clot and its strength (Bagge et al 2016) The resulting graph reflects
the initiation and propagation of the coagulation system fibrin-platelet interactions and
clot lysis
TEG was first described in 1948 by Hellmut Harten (Hartert 1948) as an in vitro method
to evaluate the clot formation process while resembling the in vivo blood flow The
development of the actually used instrument types TEG and ROTEMregTEM were based
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
13
on that original principle Viscoelastic measurements have been widely used since the
1980s in human medicine to evaluate haemostasis and hypercoagulable states in surgical
patients (eg cardiac and hepatic surgeries) and in general in critical care units (Kol and
Borjesson 2010 Nogami 2016 Wikkelso et al 2016 Crochemore et al 2017) In
veterinary medicine viscoelastic measurements using TEG and ROTEMTEM have been
validated for use in dogs cats and horses and used to study haemostasis in different
diseases (eg infectious diseases liver diseases) and medical conditions (eg
orthopaedic surgery hyperfibrinolysis) and also to investigate the effect of
anticoagulants and other drugs (eg corticosteroids) on haemostasis (Kol and Borjesson
2010 Bruno et al 2015a 2015b Woodman et al 2015 Conversy et al 2017 Sigrist et
al 2018 Turner et al 2019) The methodology seems to be especially suitable to detect
hypercoagulability (Wiinberg et al 2008 Kol and Borjesson 2010 Marschner et al
2018)
In the available veterinary literature information about reference values for TEG were
found using kaolin as activation reagent (Bauer et al 2009) Falco et al (2012)
determined reference values for the ROTEM analyser in dogs using ex-tem in-tem and
fib-tem reagents for selected ROTEM parameters in a study on forty five healthy dogs
However data related to reference intervals obtained in an adequate number of samples
(at least 120) according to the American Society for Veterinary Clinical Pathology
(ASVCP) (Friedrichs et al 2012) and the National Committee for Clinical Laboratory
Standards (NCCLS) (Horn and Pesce 2003) could not be found in the available literature
The aim of this study is to determine reference values for the ROTEMreg delta device in
dogs using citrated whole blood with different clot activator reagents (ex-tem in-tem
kaolin) and without activator
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
14
2 Materials and methods
21 Study design
Reference intervals Single thromboelastometric measurements were performed in 125
clinically healthy dogs using the ROTEMreg analyser and different activating reagents (ex-
tem in-tem and kaolin) as well as without activation Reference values were calculated
and influences of age groups (young adult and senior) sex neuter status and size
(groups small medium and large) were assessed
Precision analysis Additional fourfold measurements using ex-tem or in-tem activating
reagents respectively were performed on 3 of the normal and also in 3 abnormal blood
samples each in order to assess within-run imprecision for these measurements
22 Animals
The clinically healthy dogs with a body weight of at least 5 kg were recruited by launching
a call in the University of Veterinary Medicine Hannover network for students and staff
members Interested dog owners received a free health check of their animals (clinical
examination and blood tests specified below) in combination with the blood collection
In addition individual privately owned patient dogs of the Small Animal Clinic with
undisturbed general health were also included if blood was collected due to another
indication (eg general health check pre-anaesthetic examination test for hereditary
diseases etc) Before taking the blood samples all dogs were fasted for at least 12 h with
free access to water 125 animals for which the examination of the health status (clinical
examination routine haematology and clinical chemistry basis coagulation profile) did
not reveal any deviations from the reference were included in the study whereas 3 of the
128 initially recruited animals had to be excluded due to laboratory abnormalities
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
15
Haematological and clinical chemistry profiles performed in order to confirm the health
status included white and red cell count haematocrit platelet counts and reticulocytes
plasma activities of alanine aminotransferase glutamate dehydrogenase alkaline
phosphatase and pseudocholinesterase as well as concentrations of total bilirubin urea
creatinine glucose total protein albumin cholesterol and ionised calcium A basis
coagulation profile including PT PTT and TT was also performed to exclude significant
abnormalities within the blood coagulation system
The 125 healthy dogs enclosed in the establishment of reference values included 48 mixed
breed dogs and the following pedigree dogs 13 Labrador Retrievers 6 Australian
Shepherds 4 Havanese dogs 3 from each of the following breeds American
Staffordshire Terriers and Cocker Spaniels 2 from each of the following breeds Beagles
Border Collies Dalmatians Elo dogs (breed not recognised by Feacutedeacuteration cynologique
international [FCI]) German Longhaired Pointers German Shorthaired Pointers Giant
Schnauzers and Golden Retrievers 1 from each of the following breeds Belgian
Malinois Bolonka Zwetna Briard Brussels Griffon American English Coonhound
Italian Mastiff Flat Coated Retriever French Bulldog German Shepherd Great Dane
Greater Swiss Mountain Dog Giant Spitz Goldendoodle Hovawart Hungarian Vizsla
Italian Bloodhound Lycanis Wolfdog Miniature American Shepherd Miniature
Schnauzer Old German Shepherd Podenco Poodle Pyrenean Shepherd Rhodesian
Ridgeback Rottweiler Schafpudel (breed not recognised by FCI) Small
Munsterlander Spanish Mastiff Tibet Terrier Weimaraner West Highland White
Terrier and Wirehaired Pointer
These dogs were aged between 6 and 154 months (median = 410 months) and had body
weights ranging from 50 to 707 kg (median = 226 kg) 30 were castrated males 33
intact males 41 spayed females and 21 intact females According to the height at withers
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
16
the dogs were divided into 3 different size groups small (le40 cm n = 22) medium (41ndash
60 cm n = 77) and large (gt60 cm n = 26) Dogs were also divided in three different age
groups young (le 24 months n = 34) adult (25ndash84 months n = 59) and senior dogs (gt84
months n = 32)
The three blood samples with haemostatic abnormalities were taken from two dogs with
complex haemostatic disorders (Disseminated Intravascular Coagulation [DIC])
including reduced platelet count and abnormal PT and APTT and one dog with
thrombocytopenia
Animals were treated in accordance with the German Animal Welfare Law The
experimental design was approved by the Animal Welfare officer of the University of
Veterinary Medicine Hannover Foundation and by the ethics committee of the
responsible authority (Lower Saxony State Office for Consumer Protection and Food
Safety reference number 17A101)
23 Blood sampling
The venipuncture area was shaved and then disinfected with alcohol The saphenous or
cephalic vein was visualised with slight pressure and punctured with disposable cannulas
(diameter 09 or 10 mm) The continuously running blood was directly collected from
the needle into the tubes containing the respective anticoagulants From each dog
approximately 10 ml of blood were collected including approximately 8 ml of citrated
blood for the measurement of haemostasis tests (ROTEM PT aPTT TT) in tubes
containing sodium citrate 313 (Eifelfangoreg GmbH amp Co KG Bad Neuenahr-
Ahrweiler Germany) in a ratio of 1 volume9 volumes of blood In addition
approximately 1 ml of EDTA blood for haematological analysis and approximately 1 ml
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
17
of lithium heparin blood for clinical chemical measurements were collected into
commercial 13 ml tubes (Sarstedt AG amp Co Nuumlmbrecht Germany)
Citrated blood was initially kept at room temperature (20-25 C) in order to avoid errors
within the preanalytical phase (Toulon et al 2017) Two ml of citrated blood were used
for the ROTEMreg analysis the residual citrate blood was centrifuged for 10 min at 3800
rpm and 5 degC then the plasma was separated by careful pipetting and it was centrifuged
a second time under the same conditions to get platelet poor plasma which was stored in
aliquots of 500 microl and 250 microl at -80 degC for coagulation tests
24 Rotational elastometry
Viscoelastic measurements using the ROTEMreg delta for the establishment of reference
intervals were performed approximately 15 min after the blood collection using activating
reagents (liquid reagents) provided by the manufacturer of the instrument (r-ex-temreg in-
temreg) and a kaolin solution (5 g1 as part of the PTT reagent CK Prest [Diagnostica
Stago SAS Asniegraveres sur Seine Cedex France]) In addition non-activated analyses
were performed using isotonic NaCl solution instead of the activating reagent in the fourth
channel of the device Fourfold measurements for precision analysis were performed
simultaneously in the four channels of the device in separate runs
According to the standard test protocol provided by the manufacturer of the ROTEMreg
delta (Werfen GmbHInstrumentation Laboratory Munich Germany) 20 μl of the
reagent for recalcification (star-temreg20) and 20 μl of the activating reagent were pipetted
into a cuvette using the automated pipette provided with the device Immediately
thereafter 300 μl of citrated whole blood (prewarmed to 37 degC for five minutes in the
heating device of the instrument and gently rocked to resuspend possibly sedimented
cells) were added to the test preparation in the cuvette using the automated pipette and
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
18
then the cuvette was laced carefully in the ROTEMreg device whereby the rotating pin
entered into the test approach
Different parameters are provided automatically by the ROTEMreg delta device and the
following variables (described according to the Pentapharm GmbH ROTEMreg delta
Manual [2009]) were used in the study
Clotting time (CT) the time (in s) from the beginning of the test (by adding the clot
activator) until the formation of fibrin starts (defined as the moment when the curve
achieves an amplitude of 2 mm) This Parameter facilitates the decision to replace clotting
factors when it is required
Clot formation time (CFT) describes the next phase of clotting by the formation of a
stable clot through the activation of thrombocytes and fibrin formation It is defined as
the time (in s) between the 2 mm amplitude and 20 mm amplitude of the curve
Alpha angle (α angle) is the angle (in degrees) between the middle axis and the tangent
to the clotting curve through the 2 mm amplitude point
Amplitude after ldquoXXrdquo minutes (eg A30 after 30 min) is the amplitude (in mm) at a
certain time after the CT has finished
Maximum clot firmness (MCF) corresponds to the maximum amplitude (in mm) reached
before the clot is dissolved by fibrinolysis This parameter measures the firmness of the
clot and its quality Like CFT and α angle it facilitates the decision for substitution therapy
with platelet concentrate andor fibrinogen especially in humans
Maximum clot elasticity (MCE) is a parameter calculated from MCF and may be superior
at high amplitudes as compared to the MCF
Lysis index at 30 45 and 60 min (LI30 LI45 LI60) represents the fibrinolysis (in
percentage) 30 45 and 60 min after the CT It may facilitate the decision for or against a
therapy with anti-fibrinolytic drugs
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
19
Maximum lysis (ML) describes the degree of fibrinolysis relative to the MCF achieved
during the measurement (percent loss of clot firmness)
25 Statistical methods
Original data obtained from the ROTEMreg delta device software were transferred into the
Microsoftreg Excelreg formal (Microsoft Office Professional Plus 2013) The Statistical
analyses were performed using the SAS Enterprise Guide Version 71 (SAS Institute Inc
Cary NC USA)
Coefficients of Variation (CVs) for selected relevant parameters of the ROTEM delta
analysis using ex-tem and in-tem regents were calculated for the fourfold measurements
on 3 normal and 3 abnormal blood samples in order to assess within-run imprecision CVs
for the results of each sample were calculated by dividing the standard deviation of the
data set by its arithmetic mean Finally the median of the CVs for each group of 3 samples
was calculated The reference values were calculated according to recommendations of
the American College of Veterinary Clinical Pathology (ACVCP) based on non-
parametric methods (25 and 975 quantiles) supplemented by 95 confidence
intervals (Friedrichs et al 2012)
Data were analysed for Standard normal distribution by Kolmogorov Smirnov test and
assessment of residuals of the analysis of variance (ANOVA) Because these tests
indicated almost normally distributed values for the studied ROTEM variables data were
analysed by a fourway ANOVA stratified by the activating reagent (ex-tem in-tem
kaolin or without activation) to test the influence of size age sex and neuter status on
several parameters and taking all interactions into account Post hoc t-tests were
performed when indicated The significance level was set to P lt 05
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
20
3 Results
31 Precision of the method
For all the parameters evaluated in the study the CVs for the fourfold measurement in
healthy dogs were less than 10 (Table 1) In the case of dogs with haemostatic
abnormalities CVs of the vast majority of ROTEM variables were also less than 10
except the variables CFT and ML irrespective whether ex-tem or in-tem reagents were
used (Table 2)
Table 1 Coefficients of variation () for rotational thromboelastometry using the
ROTEMreg delta analyser in normal blood samples
Activator CT (s) CFT (s) α (deg) A30 (mm) MCF (mm) MCE(mm) LI30 () LI45 () LI60 () ML ()
Ex-temreg 875 629 262 213 179 634 000 020 037 534
In-temreg 245 596 166 117 135 299 000 000 046 216
Abbreviations CT ndash clotting time CFT ndash clot formation time α ndash alpha angle A30 ndash
amplitude after 30 min MCF ndash maximum clot firmness MCE ndash maximum clot elasticity
LI 30 LI 45 LI 60 ndash Lysis index at 30 45 and 60 min ML ndash maximum lysis
Median values of the measurements of 3 samples
Table 2 Coefficients of variation () for rotational thromboelastometry using the
ROTEMreg delta analyser in canine blood samples with haemostatic alterations
Activator CT (s) CFT (s) α (deg) A30 (mm) MCF (mm) MCE(mm) LI30 () LI45 () LI60 () ML ()
Ex-temreg 668 1478 393 218 268 582 0 018 000 848
In-temreg 228 799 461 219 251 371 0 0 017 283
Median values of the measurements of 3 samples from patients with DIC (n = 2)
thrombocytopenia (n = 1) abbreviations please see legend to Table 1
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
21
32 Reference intervals
Activation by ex-tem reagent (tissue factor) resulted in faster CTs when compared to
activation via the intrinsic pathway using ellagic acid (in-tem) or kaolin reagents whereas
the CFT was much less affected by the activating reagent (Table 3) Especially CFT and
MCE showed a wide inter-individual variation in comparison for example with α angle
and MCF regardless of the used activating reagent (Table 3)
33 Influence of age sex neuter status and size
ANOVA did not reveal statistically significant differences in ROTEM delta parameters
between dog groups of variable size or sex (P gt 05) Age had influence only on CT and
only when the in-tem reagent was used (P = 0169 ANOVA) but not when using the ex-
tem reagent (P = 7105) Post hoc comparisons using t-test of the in-tem results between
age groups revealed shorter CTs in the group of senior dogs when compared with young
dogs (P = 0194) but not between adult dogs and these two groups ANOVA showed
particularly influences of the neuter status and a significant interrelationship between the
factors sex and neuter status on different ROTEM delta parameters using ex-tem and in-
tem respectively as activator (Tables 4 and 5) ANOVA revealed also a significant
influence of neuter status on different ROTEM parameters when kaolin was used as
activating reagent (CFT α angle MCF MCE) whereas non-activated measurements
were not subject to any significant influence of the assessed factors (results not shown)
The interrelationship between sex and neuter status mentioned above reflected the fact
that significant differences between neutered and not neutered dogs were only found in
females The statistical analysis using the t-test to compare intact vs neutered dogs
stratified by sex showed statistically significantly (P lt 05) longer CTs and CFTs and
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
22
lower α angle A30 MCF and MCE values in spayed when compared to intact females
(Tables 4 and 5)
Chapter 2 Reference intervals for rotational thromboelastometry measurements using the ROTEMreg delta device in dogs
23
Table 3 Reference values (median reference interval based on 25 - and 975 - quantiles) for thromboelastometric measurements with the ROTEMreg delta analyser in
dogs (n=125 95 confidence intervals in brackets) Abbreviations please see legend to Table 1
Activator
Statistical
para-meter
CT
(s)
CFT
(s)
Α
(deg)
A30
(mm)
MCF
(mm)
MCE
(mm)
LI30
()
LI45
()
LI60
()
ML
()
Ex-temreg
Median 450
[430ndash470]
110
[105ndash115]
680
[670ndash690]
600
[580ndash610]
610
[600ndash620]
154
[147ndash164]
100
[960ndash100]
100
[100ndash100]
970
[960ndash980]
220
[210ndash240]
25 340
[270ndash350]
650
[490ndash710]
540
[420ndash560]
460
[410ndash480]
460
[420-490]
870
[720ndash950]
100
[100-100]
870
[800ndash960]
700
[640ndash820]
800
[100ndash110]
975 830
[790ndash990]
220
[201ndash315]
790
[770ndash800]
770
[690ndash770]
730
[700ndash770]
264
[230-334]
100
[100ndash100]
100
[100ndash100]
100
[990ndash100]
590
[530ndash680]
In-tem
Median 190
[187ndash201]
116
[111ndash125]
680
[670ndash690]
570
[560ndash580]
590
[580ndash600]
143
[135ndash149]
100
[100ndash100]
100
[100ndash100]
980
[980ndash980]
180
[170ndash190]
25 135
[112ndash142]
730
[590ndash800]
510
[460ndash570]
450
[410ndash470]
480
[410ndash500]
920
[710ndash101]
100
[950ndash100]
990
[820ndash990]
930
[740ndash930]
500
[100ndash700]
975 283
[268ndash291]
251
[198ndash227]
750
[750ndash780]
660
[660ndash690]
680
[660ndash690]
210
[195ndash227]
100
[100ndash100]
100
[100ndash100]
100
[100ndash100]
270
[260ndash380]
Activator
Statistical
para-meter
CT
(s)
CFT
(s)
Α
(deg)
A30
(mm)
MCF
(mm)
MCE
(mm)
LI30
()
LI45
()
LI60
()
ML
()
Kaolin
Median 162
[154ndash173]
870
[820ndash910]
740
[720ndash750]
500
[470ndash530]
550
[530ndash560]
121
[115ndash127]
960
[940ndash980]
840
[790ndash880]
710
[640ndash770]
500
[450ndash540]
25
109
[990ndash115]
470
[440ndash580]
560
[490ndash600]
110
[700ndash230]
390
[380ndash400]
640
[600ndash680]
270
[140ndash480]
110
[800ndash260]
800
[400ndash170]
200
[160ndash240]
975 246
[230ndash337]
214
[185ndash290]
810
[790ndash810]
640
[630ndash690]
650
[650ndash690]
187
[182ndash223]
100
[100ndash100]
990
[980ndash100]
940
[930ndash990]
970
[930ndash100]
Without activation
Median 607
[495ndash662]
221
[194ndash265]
510
[460ndash550]
520
[500ndash530]
540
[520ndash550]
115
[109ndash123]
100
[100ndash100]
100
[100ndash100]
980
[980ndash990]
170
[160ndash190]
25 203
[123ndash215]
860
[550ndash930]
190
[120ndash230]
350
[230ndash370]
370
[260ndash400]
600
[350ndash670]
100
[950ndash100]
980
[890ndash990]
930
[840ndash950]
400
[000ndash500]
975 1356
[1305ndash1721]
811
[688ndash1333]
730
[710ndash790]
660
[640ndash720]
660
[640ndash730]
197
[179ndash265]
100
[100ndash100]
100
[100ndash100]
100
[100ndash100]
290
[280ndash320]
Chapter 2 Reference intervals for rotational thromboelastometry measurements using the ROTEMreg delta device in dogs
24
Table 4 Median values (minimum and maximum in brackets) for different ROTEMreg variables using ex-tem reagent stratified by sex and neuter status
Group
CT
(s)
CFT
(s)
Α
(deg)
A30
(mm)
MCF
(mm)
MCE
(mm)
LI30
()
LI45
()
LI60
()
ML
()
Female intact 430
(320ndash530)
960
(590ndash198)
710
(570ndash800)
620
(480ndash730)
640
(510ndash740)
179
(103ndash282)
100
(100ndash100)
100
(980ndash100)
960
(800ndash100)
230
(100ndash640)
Female spayed 460
(270ndash830)
121
(650ndash224)
660
(520ndash770)
580
(430ndash690)
590
(440ndash700)
146
(790ndash230)
100
(960ndash100)
100
(800ndash100)
970
(690ndash100)
240
(100ndash590)
p-value (t-test)
Female
intact vs spayed
00025 00063 00049 00023 00016 00015 na na na na
Male intact 450
(320ndash990)
105
(650ndash315)
690
(420ndash790)
590
(420ndash700)
610
(440ndash710)
154
(770ndash244)
100
(100ndash100)
100
(870ndash100)
970
(650ndash100)
220
(700ndash680)
Male castrated 460
(340ndash930)
111
(490ndash255)
685
(480ndash800)
590
(410ndash770)
600
(420ndash770)
152
(720ndash334)
100
(100ndash100)
100
(830ndash100)
980
(640ndash100)
205
(500ndash590)
p-value (t-test)
Male
Intact vs castrated
08326 05106 04756 01799 01568 02163 na na na na
p-values (ANOVA)
Factor Sex
05428 04847 06612 02206 01887 01630 08489 07893 07689 02758
Factor neuter status 01872 00167 00173 00039 00037 00028 07141 04737 09356 05948
Interrelationship
sexneuter status 00383 00968 00782 00488 00472 00319 04943 08611 05422 00160
na ndash not analysed all significant P-values (lt 005) are highlighted in bold print
Chapter 2 Reference intervals for rotational thromboelastometry measurements using the ROTEMreg delta device in dogs
25
Table 5 Median values (minimum and maximum in brackets) for different ROTEMreg variables using the in-tem reagent stratified by sex and neutering status
Group
CT
(s)
CFT
(s)
Α
(deg)
A30
(mm)
MCF
(mm)
MCE
(mm)
LI30
()
LI45
()
LI60
()
ML
()
Female intact 186
(126ndash238)
940
(590ndash164)
720
(620ndash780)
610
(490ndash690)
620
(530ndash690)
166
(113ndash227)
100
(100ndash100)
100
(990ndash100)
980
(930ndash100)
170
(100ndash300)
Female spayed 201
(149ndash291)
1220
(820ndash262)
670
(500ndash740)
550
(450ndash660)
570
(480ndash680)
133
(910ndash214)
100
(950ndash100)
100
(820ndash100)
980
(740ndash100)
190
(500ndash380)
p-value (t-test)
Female
intact vs spayed
na na lt0001 00009 00005 00005 na na na na
Male intact 199
(117ndash283)
122
(730ndash277)
680
(460ndash750)
570
(430ndash660)
590
(450ndash660)
146
(810ndash192)
100
(100ndash100)
100
(990ndash100)
980
(930ndash100)
180
(600ndash270)
Male castrated 181
(112ndash288)
123
(820ndash222)
670
(540ndash740)
565
(410ndash630)
585
(410ndash640)
138
(710ndash180)
100
(100ndash100)
100
(970ndash100)
980
(890ndash100)
180
(800ndash260)
p-value (t-test)
Male
intact vs castrated
na na 08232 01401 01162 00849 na na na na
p-values (ANOVA)
Factor Sex
02105 03317 03370 00877 01258 00510 09564 07832 08117 03638
Factor neuter status 04289 00660 00619 00107 00088 00054 07152 09795 07056 08299
Interrelationship
sexneuter status
04310 00550 00408 00707 01733 00996 05240 05982 08047 04578
na ndash not analysed all significant P-values (lt 005) are highlighted in bold print
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
26
4 Discussion
41 Coefficients of variation
In accordance with the literature CVs below 10 indicate adequate repeatability in intra-
assays evaluations (Steiner et al 2003 Hua et al 2018) According to our results
obtained from the fourfold measurements in citrated whole blood ROTEM delta
measurements on healthy dogs and dogs with haemostatic alterations fulfil these
requirements with only a few exceptions concerning CFT and ML values in abnormal
samples
Our results regarding repeatability are similar to those obtained for several TEM
parameters in previous studies performed in animals In one study the imprecision of the
ROTEM analyser was evaluated by duplicate measurements in 10 blood samples of
healthy dogs and as result of that evaluation a good precision (CVs below 10 ) was
calculated for CT CFT MCF and α angle but in contrary to our results a low
repeatability was found for ML (CVs of 233 [ex-tem] and 285 [in-tem] own values
534 and 216 ) (Falco et al 2012)
In a previous feline study using ex-tem and in-tem as activating reagents for the ROTEM
delta repeatability was assessed similarly to the present study based on fourfold
measurements of blood samples of two healthy individuals (Doumlderlein and Mischke
2015) The results also revealed CVs below 10 for most of the parameters evaluated
The partially low repeatability of ML values may reflect the vulnerability of this variable
which is not only influenced by the complex clot formation process but also by its
subsequent lysis Reproducibility of ex-tem or in-tem activated measurements of the
ROTEM delta analyser which was not investigated in our study was studied in blood
from healthy and unhealthy horses by two different operators using three different
ROTEM devices Results indicated acceptable reproducibility (Junge et al 2016)
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
27
42 Reference intervals
The number of 125 healthy dogs used in the present study to create reference intervals is
regarded adequate according to the guidelines suggested by the American Society of
Veterinary Clinical Pathology (ASVCP) and the National Committee for Clinical
Laboratory Standards (NCCLS) who recommend to include at least 120 individuals
(Friedrichs et al 2012) To the best of our knowledge reference intervals for the most
relevant ROTEMreg delta parameters obtained in an adequate number of samples of whole
citrated canine blood have not been published so far A previous study was performed on
a smaller number of 45 dogs (Falco et al 2012) The median values for reference
intervals obtained in the present study are partially similar to those calculated in the
mentioned study based on 45 healthy dogs (eg ex- tem CT 610 s450 s [Falco et al
2012own results] CFT 150 s 110 s α angle 620deg680deg MCF 530 m 610 mm ML
180 220 in-tem CT 235 s190 s CFT 136 s116 s α angle 660deg680deg MCF
560 mm590 mm ML 30 180 ) However in comparison to our results the
reference intervals in the mentioned study are slightly wider (eg ex-tem CT 29ndash92
s34ndash83 s [Falco et al 2012own results] CFT 540ndash275 s650ndash220 s α angle 470ndash
790deg540ndash790deg MCF 360ndash730 mm460ndash730 mm ML 000ndash560 800ndash590
in-tem CT 126ndash363 s135ndash283 s CFT 470ndash224 s730ndash251 s aα angle 550ndash
810deg510ndash750deg MCF 500-ndash750 mm480ndash680 mm ML 000ndash400 500ndash270 )
although the previous study used the 5 and 95 quantiles to define limits of the
reference range whereas we calculated the commonly used 25 and 975 quantiles
to capture the central 95 of reference values (Friedrichs et al 2012)
Although it is desirable that each laboratory should specifically determine its own
reference intervals the results obtained in this study may provide an orientation for other
laboratories to interpret ROTEM delta parameters analysed in citrated canine blood In
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
28
order to test transference of the reference ranges to another laboratory a validation
procedure with measurement of 20 samples should be performed (Friedrichs et al 2012)
Although intra-individual control values (subject-based reference values) are regarded
superior to population based reference values (Wiinber et al 2007) the latter have to be
routinely used in clinical patients due to lacking individual controls especially for specific
tests such as ROTEM analyses In addition the calculated reference values cannot be
used for the fully automated device ROTEM sigma because - according to an actual
human study - values of part of the parameters do not correlate well between both
instruments (Gillissen et al 2019)
43 Influence of size age sex and neuter status
In previous studies in humans (Attard et al 2013) and animals (Mischke 1994 Borrelli
et al 2017) age has been described as an influencing factor for different haemostasis
parameters We also found an influence of age on the CT with shorter times in senior
dogs (gt 84 months) compared with young dogs (up to 24 months) which was limited to
measurements activated by the in-tem reagent
This age dependency confirms in principle a previous study on 20 healthy dogs
(Barthelemy et al 2015) which found shorter CTs in elderly dogs and results of a human
study where individuals with an age gt 45 years showed shorter CTs compared with
individuals lt 45 years old (Sucher et al 2011) However in the cited studies this effect
was only seen when ex-tem but not when the in-tem reagent was used
Based on the age distribution of our dogs which were primarily recruited to establish
reference intervals we cannot deliver information regarding very young dogs A human
study focusing on ROTEM reference ranges in children demonstrated significant
differences for nearly all ROTEM parameters with the most striking differences in
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
29
subjects aged 0-3 months (Oswald et al 2010) The limited number of dogs with an age
lt 1 year which did not allow to consider these separately in the present study may also
be one reason for the different results in a feline study (Doumlderlein and Mischke 2015) In
the cited study on healthy cats age showed an influence on CT but contrary to our study
the CT was shorter in young individuals (6-12 months) compared with older ones In
addition this was only the case in measurements using kaolin and in non-activated
measurements When compared to humans and cats uni- or multivariate analyses
considering factors age and breed or size based on do populations including different
breeds have the limitation that they can be biased by the variate life expectancy between
small medium and large dog breeds (Willems et al 2017) Apart from age we found a
significant influence only for the neuter status in bitches reflected by a significant
interaction effect between factors neuter status and sex in the ANOVA analysis
Especially for CFT values (eg activated by ex-tem median values of 121 s [spayed] vs
96 s [intact]) this difference seems to be also clinically relevant with respect to the
interpretation of ROTEM results In contrast although also significantly different
absolute and relative differences of CT values were small (eg activated by ex-tem 46 s
[spayed] vs 43 s [intact]) These differences may not only be considered for the result
interpretation of clinical patients but indicate also the necessity for sex-matched patient
groups in scientific studies
The obviously more active haemostasis system in intact females is probably related to an
influence of sex hormones (specifically estrogens) on the coagulation system
Accordingly ex vivo and in vivo studies demonstrated the activating properties of
endogenous estrogens on the haemostasis system In one ex vivo study addition of
endogenous estrogens (estrone [E1] 17szlig-estradiol [E2] and estriol [E3]) to blood samples
from men resulted in shorter mean values of the reaction time in TEG analyses
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
30
corresponding to CT in ROTEM measurements (452-537 s vs 698 s [control]) and of
the TEG variable kinetics (corresponding to the CFT in ROTEM analyses) (Swanepoel
et al 2017) In a further study subcutaneous injection of E2 into male rats induced a
significant increase in activities of coagulation factors X (by 8 ) and XI (by 10-24 )
however did not show any significant effect on PT aPTT and factor VII activity (Franco-
Murillo and Jaimez 2017)
In the cited studies also the effects of the synthetic estrogen ethinylestradiol [EE] were
studied which interestingly seems to have an inhibiting effect as indicated by prolonged
reaction time and kinetics (TEG) compared with the control in the human ex vivo study
and significantly longer PTs and aPTTs and lower factor VII and X activities in the study
on rats As expected we did not find a significant influence of size on ROTEM parameters
in dogs To the best of the authoracutes knowledge size has not been examined as an
influencing factor on viscoelastic measurements or any other haemostatic variable in
dogs Apart from Greyhounds there is limited literature on breed comparisons of
haemostatic variables (Clemons and Meyers 1984 Mischke 1994 Nielsen et al 2007
Nielsen et al 2010 Boyd et al 2018) indicating very limited breed differences for
example for factor VII (Mischke 1994) However these also do not indicate systematic
influence of size and in addition possible breed-specific effects have to be considered
As already stated one limitation of the study is the limited number of young animals
which does not allow to verify reference values for animals in this stage of life In
addition with respect to the main aim of the study possible influencing factors such as
platelet count and haematocrit were not considered in the statistical analysis Finally the
relatively small number of dogs used for the precision analysis which express the fact
that this was only a secondary aspect of the study may be regarded as a limitation At the
same time these considerations provide space for further investigations
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
31
5 Conclusions
Results of the precision analysis revealed acceptable results and thereby confirm that the
ROTEMreg delta analyser delivers repeatable results to measure overall haemostasis in
canine citrated blood samples The established reference values should offer a reliable
basis for the interpretation of thromboelastometric measurements in whole blood using
the device ROTEMreg delta in dogs with suspected haemostasis disorders Neuter status
in female dogs appeared to be a relevant influencing factor on most important ROTEMreg
parameters and should be considered for result interpretation and with respect to
experimental designs
6 Acknowledgments
Jose Mauricio Pereira received a scholarship from the Universidad Nacional de Costa
Rica (reference code JB-C-1107-2016)
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
32
7 References
1 Attard C van der Straaten T Karlafitis V Monagle P Ignjativic V 2013 Developmental
hemostasis age-specific differences in the levels of hemostatic proteins J Thromb and
Haemost 11 1850ndash1854
2 Bagge A Schoumltt U Kander T 2016 Effects of naturopathic medicines on Multiplate and
ROTEMreg A prospective experimental pilot study in healthy volunteers BMC Complemen
Altern Med 17 (16) 64
3 Bartheacutelemy A Rannou B Forterre M Verwaerde P Bonnet-Garin JM Pouzot-Nevoret
C Goy-Thollot I 2015 Differences between coagulation and cytokine profiles in dogs of
different ages Vet J 205 410ndash412
4 Bauer N Eralp O Moritz A 2009 Establishment of reference intervals for kaolin-activated
thromboelastography in dogs including an assessment of the effects of sex and anticoagulat use
J Vet Diagn Investi 21 641ndash648
5 Bollinger D Seeberger MD Tanaka KA 2012 Principles and practice of
thrromboelastography in clinical coagulation management and transfusio practice Transfus Med
Rev 26 1ndash13
6 Borrelli A Botto A Maurella C Falco S Pagani E Miniscaldo B Tarducci A Bruno
B 2017 Thromboelastometric assesment of hemostasis in newborn Piemontese calves J Vet
Diagn Invest 29 293ndash297
7 Boyd CJ Claus MA Raisis AL Hosqood G Sharp CR SmartL 2018
Hypocoagulability and platelet dysfunction are exacerbated by synthetic colloids in a canine
hemorrhagic shock model Front Vet Sci 5 279
8 Bruno B Maurella C Falco S Tarducci A Zanatta R DrsquoAngelo A Borrelli A 2015a
Thromboelastometric evaluation of hemostasis in dogs infected with Leishmania infantum J Vet
Emerg Crit Care (San Antonio) 25 502ndash511
9 Bruno B Maurella C Falco S Tarducci A Zanatta R Gianella P DrsquoAngelo A Piras L
Di Bella A Borrelli A 2015b Assessment of coagulation utilizing thromboelastometry in dogs
undergoing orthopedic surgery J Vet Emerg Crit Care (San Antonio) 25 358ndash363
10 Clemons RM Meyers KM 1984 Acquisition and aggregation of canine blood platelets Basic
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
33
mechanisms of function and differences because of breed origin Am J Vet Res 45 137ndash144
11 Conversy B Blais MC Dunn M Gara-Boivin C del Castillo JRE 2017 Anticoagulant
activity of oral rivaroxaban in healthy dogs Vet J 223 5ndash11
12 Crochemore T Piza FMT Rodriacuteguez RDR Guerra JCC Ferraz LJR Correcirca TD
2017 A new era of thromboelastometry Einstein (Sao Paulo) 15 380ndash385
13 Crochemore T Correcirca TD Lance MD Solomon C Neto AS Guerra JCC Lellis PS
Bernz LM Nunes N Mancio CM Yokoyama APH Silva E 2018 Thromboelastometry
profile in critically ill patients A single-center retrospective observational study PLoS One 13
e0192965
14 Doumlderlein E Mischke R 2015 Reference intervals for thromboelastometry with the ROTEMreg
delta in cats Res Vet Sci 100 271ndash276
15 Falco S Bruno B Maurella C Bellino C DacuteAngello A Gianella P Tarducci A Zannata
R Borrelli A 2012 In vitro evaluation of canine hemostasis following dilution with
hydroxyethyl starch (13004) via thromboelastometry J Vet Emerg Crit Care (San Antonio)
22 640ndash645
16 Franco-Murillo Y Jaimez R 2017 Inhibitory effect of ethinylestradiol on coagulation factors
in rats Exp Anim 66 107ndash113
17 Friedrichs KR Harr KE Freeman KP Szladovits B Walton RM Barnhart KF Blanco-
Chavez J 2012 ASVCP reference interval guidelines Determination of de novo reference
intervals in veterinary species and other related topics Vet Clin Pathol 41 441ndash453
18 Gillissen A va den Akker T Caram-Deelder C Henriquez DDCA Bloemenkamp
RWM Eikenboom J van der Bom JG de Maat MPM 2019 Comparison of
thromboelastometry by ROTEMreg Delta and ROTEMreg Sigma in women with postpartum
haemorrhage Scand J Clin Lab Invest 79 32ndash38
19 Harttert H 1948 Blutgerinnungstudien mit der Thrombelastographie einem neuen
Untersuchungsverfahren Klin Wochenschr 26 577ndash583
20 Horn P Pesce A 2003 Reference intervals and update Clin Chim Acta 334 5ndash23
21 Hua Yanhun Qiu W Xiao Q Wu Q 2018 Precision (repeatability and reproducibility) of
ocular parameters obtained by the Tomey OA-2000 biometer compared to the IOLMaster in
healthy eyes PLoS One 13 e0193023
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
34
22 Junge H Ringer S Mayer N Schwarzwald C 2016 Assesment of method reliability and
determination of reference intervals for rotational thromboelastometry in horses J Vet Emerg
Crit Care (San Antonio) 26 691ndash703
23 Kol A Borjesson DL 2010 Application of thrombelastographythromboelastometry to
veterinary medicine Vet Clin Pathol 39 405ndash416
24 Marschner CB Wiinberg B Tarnow I Markussen B Kuumlhnel L Bochsen L Kristensen
AT 2018 The influence of inflammation and hematocrit on clot strength in canine
thromboelastographic hypercoagulability J Vet Emerg Crit Care (San Antonio) 28 20ndash30
25 McMichael MA Smith SA 2011 Viscoelastic coagulation testing technology applications
and limitations Vet Clin Pathol 40 140ndash153
26 Mischke R 1994 Activity of coagulation factors II V VII and X in healthy dogs- dependence
on age sex and breed Berl Muumlnch Tieraumlrztl Wochenschr 107 289ndash294
27 Nielsen A Zois N Pedersen H Olse L Tarnow I 2007 Platelet function in dogs bread
differences and effect of acetylsalicylic acid administration Vet Clin Pathol 36 267ndash273
28 Nielsen L Kjelgaard‐Hansen M Jensen A Kristensen A 2010 Breed‐specific variation of
hematologic and biochemical analytes in healthy adult Bernese Mountain dogs Vet Clin Pathol
39 20ndash28
29 Nogami K 2016 The utility of thromboelastography in inherited and acquired bleeding
disorders Br J Haematol 174 503ndash514
30 Oswald E Stalzer B Heitz E Weiss M Schmugge M Strasak A Innerhofer P Haas T
2010 Thromboelastometry (ROTEMreg) in children Age-related reference ranges and correlations
with standard coagulation tests Br J Anaesth 105 827ndash835
31 Sankarankutty A Nascimento B Teodoro da Luz L Rizoli S 2012 TEGreg and ROTEMreg
in trauma similar test but different results World J Emerg Surg 7 (Suppl 1) S3
32 Sigrist NE Schefer RJJ Kutter APN 2018 Characteristics of hyperfibrinolysis in dogs and
cats demonstrated by rotational Thromboelastometry (ROTEM) Vet J 242 67-73
33 Steiner J Teague S Williams D 2003 Development and analytic validation of an enzime-
linked immunosorbent assay for the measurement of canine pancreatic lipase immunoreactivity in
serum Can J Vet Res 68 161ndash168
34 Sucker C Tharra K Lithmate J Scharf RE Zotz RB 2011 Rotation thromboelastography
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
35
(ROTEM) parameters are influenced by age gender and oral contraception Perfusion 26 334ndash
340
35 Swanepoel A Emmerson O Pretorius E 2017 The effect of endogenous and synthetic
estrogens on whole blood clot formation and erythrocyte structure Microsc Microanal 23 599ndash
606
36 Theusinger OM Nuumlrnberg J Asmis LM Seifert B Spahn DR 2010 Rotation
thromboelastometry (ROTEM) stability and reproducibility over time Eur J Cardiothorac Surg
37 677ndash683
37 Toulon P Metge S Hangard M Zwahlen S Piaulenne S Besson V 2017 Impact of
different storage times at room temperature of unspun citrated blood samples on routine
coagulation tests results Results of a bicenter study and review of the literature Int J Lab
Hematol 3 458ndash468
38 Turner JS Kutter APN Sigrist NE 2019 Correlation of rotational thrromboelastometry
(ROTEM) parameters with platelet count and their ability to predict thrombocytopenia in dogs
Res Vet Sci 126 45ndash50
39 Versteeg HH Heemskerk JWM Levi M Reitsma PH 2013 New Fundamentals in
Hemostasis Physiol Rev 93 327ndash358
40 Wiinberg B Jensen AL Kjelgaard-Hansen M Rojkjaer R Johansson PI Gade LP
Gram DX Kristensen AT 2007 Study on biological variation of haemostatic parameters in
clinically healthy dogs Vet J 174 62ndash68
41 Wiinberg B Jensen AL Johansson PL Rozanski E Tranholm M Kristensen AT 2008
Thromboelastographic evaluation of hemostatic function in dogs with disseminated intravascular
coagulation J Vet Intern Med 22 357ndash365
42 Wikkelsoslash A Wetterslev J Moslashller AM Afshari A 2016 Thromboelastography (TEG) or
thromboelastometry (ROTEMreg) to monitor haemostatic treatment versus usual care in adults or
children with bleeding Cochrane Database Syst Rev Art
httpsdoiorg10100214651858CD007871pub3 No CD007871
43 Willems A Paepe D Marynissen S Smets P Van de Maele I Picavet P Duchateau L
Daminet S 2017 Results of screening of apparently healthy senior and geriatric dogs J Vet
Intern Med 31 81ndash92
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
36
44 Woodman J Wagg CR Boysen SR Leguillete R Mizen K Roy MF 2015 Evaluation
of coagulation via thromboelastography in healthy horses administered dexamethasone Can Vet
J 56 1271ndash1274
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
37
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of
standard heparin prophylaxis and correlation to haemostatic changes
Pereira JM Hewicker-Trautwein M Rohn K von Depka Prondzinski M
Mischke R
Contributions of the authors
Pereira JM performed (1) the ultrasound evaluations and blood sampling of the
patients under study (2) haematology analysis (3) injection of the anticoagulation
prophylaxis (partially) to the patients under study (4) analysis of samples (collaboration)
at Werlhof Institute Medical Care Center (5) analysis of some haemostasis test
(collaboration) (6) statistical analysis (partially) (7) data documentation (8) and wrote
the manuscript
Hewicker-Trautwein M performed the electron microscopy analysis
Rohn K performed the statistical analysis
von Depka Prondzinski M performed some haemostasis test (thrombin generation
assay and endogenous thrombin potential)
Mischke R performed the coagulation and haemostasis test was involved in designing
and supervision of the study analysis and discussion of the data and its statistical analysis
and revision edition and correction of the manuscript
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
38
Central venous catheter induced thrombosis in dogs ndash efficacy of standard heparin
prophylaxis and correlation to haemostatic changes
Joseacute Mauricio Pereira1 Marion Hewicker-Trautwein2 Karl Rohn3 Mario von Depka
Prondzinski4 Reinhard Mischke1
1Small Animal Clinic University of Veterinary Medicine Hannover Foundation
Buumlnteweg 9 D-30559 Hannover Germany
2Institute for Pathology University of Veterinary Medicine Hannover Foundation
Buumlnteweg 17 D-30559 Hannover Germany
3Institute of Biometry Epidemiology and Information Processing University of
Veterinary Medicine Hannover Foundation Buumlnteweg 2 30559 Hannover Germany
4Werlhof Institute Schillerstrasse 23 30159 Hannover Germany
Abbreviations anti-Xa anti-factor Xa BW body weight CVC central venous catheter
EJV external jugular vein ELMI electron microscopy IV intravenous JV jugular vein
LMWH low molecular weight heparin ML maximum lysis (ROTEM) PIVC
peripheral intravenous catheter ROTEM rotational elastometry SC subcutaneous
TGA thrombin generation assay TID three times a day TT thrombin time UFH
unfractionated heparin
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
39
Corresponding author
E-mail addresses matiscrgmailcom (JM Pereira) MarionHewicker-
Trautweintiho-hannoverde (M Hewicker-Trautwein) KarlRohntiho-hannoverde
(K Rohn) mailwerlhof-institutde (M von Depka Prondzinski)
ReinhardMischketiho-hannoverde (R Mischke)
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
40
ABSTRACT
Central venous catheters (CVC) are of importance for intensive care management of
canine patients Thrombosis of the jugular vein is a severe complication related to the
CVC use However only little is known about the incidence of CVC-associated
thromboses in dogs and the necessity and efficacy of prophylactic antithrombotic
regimens The objectives of the present study were (1) to determine the incidence of
catheter-induced thrombosis in canine intensive care patients and thereby (2) to evaluate
the efficacy of a routine prophylactic heparin treatment and finally (3) to assess whether
initial changes of selected haemostasis parameters are predictive for an increased risk for
thrombosis formation 29 dogs hospitalised in the Small Animal Clinic University of
Veterinary Medicine Hannover Foundation and receiving a central venous catheter
(CVC) in the external jugular vein (EJV) for medical reasons were included in the final
assessment 17 dogs received the standard antithrombotic dosage regimen of
unfractionated heparin (UFH 150 IUKG BW TID sc) and 12 dogs (usually surgical
patients) the reduced dosage (75 IUKG BW TID sc) A colour Doppler ultrasound of
the respective EJV in order to detect thrombus formation and blood collection for
haemostasis tests were performed immediately before the installation procedure (day 0)
and on days 1 3 5 7 and so on after the CVC insertion Haemostasis tests included
prothrombin time activated partial thromboplastin time thrombin time antithrombin
activity D-dimer concentration thrombin generation and rotational elastometry as well
as the heparin activity using a chromogenic anti-factor Xa test Finally electron
microscopy (ELMI) of sections of the removed CVC was performed In 8 dogs
sonographic examination revealed thrombus formation in the lumen of EJV and in 1927
CVCs assessed using ELMI thrombi on the external CVC surface were detected without
significant correlation between both methods Plasmatic heparin activities showed great
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
41
variability but no significant differences were found between dogs with or without CVC-
associated thrombus formation From the initially performed haemostasis tests high
fibrinogen concentrations and low maximum lysis values as measured by the ROTEM
delta device (ex-tem reagent) were associated with sonographically detectable thrombi
The results of the present study indicate that CVC-associated thromboses occur (eg 28
[829] of the patients as detected by ultrasound) although severe thromboses (high
grades) are relatively rare in intensive care canine patients receiving a routine
anticoagulant treatment A high fibrinogen concentration and low maximum lysis
(ROTEM ex-tem) may be useful indicators for an increased thrombotic risk in individual
patients which may require special anticoagulatory treatment
Keywords CVC complication venal occlusion antithrombotic treatment ultrasound
electron microscopy rotational elastometry
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
42
1 Introduction
Central venous catheters (CVCs) are very important tools for care of patients in critical
care units allowing parenteral nutrition support therapy administration of high volumes
of intravenous fluids safe drugs administration that cannot be given through peripheral
veins plasmapheresis haemodialysis blood transfusions as well as periodical blood
sampling (Geerts 2014 Vose et al 2019) CVC related thrombosis is one of the most
severe complications (apart from infections and pneumothorax) of CVCs in humans
(Geerts 2014 Comerlato et al 2017 Evans and Ratchford 2018) leading to patientrsquos
distress CVC dysfunction venous stenosis and in some cases to the development of
thromboembolism (Geerts 2014)
In the human literature there are various studies dealing with epidemiology with reference
to different anticoagulatory regimens including placebo-controlled studies (Lee and
Kamphuisen 2012) In humans CVC related thromboses represent 70ndash80 of all upper
extremity thrombosis cases and approximately 10 of thromboembolism cases in
humans (Kucher 2011) The incidence of CVC related thrombosis reported in humans
based on 25 studies ranged widely from 2 to 67 (median = 30 ) (van Rooden et al
2005 review article) This wide range of calculated incidences of CVC related
thromboses in humans reflects the use of different CVC types different locations of entry
site of the CVC (jugular femoral subclavian axillary cephalic vein) different thrombus
detection technique (venography or ultrasonography) unequal durations of follow up
varying diseases of patients (eg haematological oncological) and presence of
predisposing factors (eg previous venous thrombosis history presence of cancer) (van
Rooden et al 2005 Geerts 2014 Liu et al 2015)
Part of the authors empirically observed CVC-associated thrombosis of the external
jugular vein (EJV) as a relevant complication in their canine intensive care patients which
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
43
awakened their interest in that topic To the best of the authoracutes knowledge studies about
extra luminal CVC related thromboses in dogs are rare in the available veterinary
literature One study evaluating the complications associated with 100 CVCs placed in
the jugular vein of dogs and cats reported only 2 cases of thrombosis (confirmed by
palpation or ultrasound) but obviously did not assess the appearance of subclinical CVC
related thromboses (Adamantos et al 2010) Thereby the incidence of CVC-induced
thromboses in dogs is unknown and systematic studies regarding an effective
anticoagulatory management are lacking
In this context it is of interest whether a possible development of thrombosis or an
increased risk (despite the prevention by a standard application of unfractionated heparin
[UFH]) can be predicted by means of changes of haemostasis related parameters during
the initial examination In one study on 168 human patients without indication of
anticoagulation prophylaxis for example elevated leukocyte counts high plasminogen
activator inhibitor and high coagulation factor VIII levels (all measured on blood samples
taken after CVC placement) were associated with an increased incidence of symptomatic
CVC-related thrombosis diagnosed by ultrasonography (Boersma et al 2016)
Therefore aims of the present study were (1) to determine the incidence of catheter-
induced thrombosis in canine intensive care patients (as detected by ultrasonography and
electron microscopy ELMI) and thereby (2) to evaluate the efficacy of a standard
antithrombotic regimen with unfractionated heparin (UFH) routinely used in the Small
Animal Clinic of the University of Veterinary Medicine Hannover and (3) to assess
whether selected haemostasis test results are predictive for an increased risk for
thrombosis formation
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
44
2 Material and methods
21 Study design
Canine in-patients of the Small Animal Clinic University of Veterinary Medicine
Hannover receiving a CVC in the EJV for medical reasons from March 2017 to
December 2019 were included Anticoagulatory treatment was performed according to
the standard protocol of the clinic Colour Doppler ultrasonographic controls of the EJV
and blood samplings (for heparin activity basic haematology and haemostasis tests) were
done immediately before the catheter was placed (day 0) and on days 1 3 5 etc (2 hours
after the first daily UFH injection) until the CVC was removed Only patients were
included which could be examined at least until day 3 Haemostasis tests included
prothrombin time activated partial thromboplastin time thrombin time fibrinogen
concentration antithrombin activity fibrin monomers D-dimers thrombin generation
assay (TGA) and rotational elastometry as well as the heparin activity using a
chromogenic anti-factor Xa test Finally ELMI of sections of the CVC after its removal
was performed
22 Animals
Originally 32 patients hospitalised in the Small Animal Clinic and receiving a CVC in the
EJV for medical reasons entered the study Three of them were subsequently excluded
because they did not reach the defined minimum study period of 3 days Two were
euthanised and one referred back to the referring vet before day 3 22 dogs weighing more
than 15 kg were included so that the withdrawal of about 10 ml of blood on several days
was not a significant burden for the patient (lt 1 of the total blood volume) Five dogs
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
45
with a body weight of less than 15 kg but at least 8 kg were included with special
consideration of the amount of blood taken (no more than 6 ml on individual days)
All animals received standard anticoagulatory treatment with UFH 17 dogs with internal
diseases received 150 IUkg BW TID subcutaneously and 12 dogs (post surgery) received
a reduced dosage of 75 IUkg BW TID subcutaneously
The 29 dogs enclosed in the study included 4 mixed breed dogs and the following
pedigree dogs 5 Labrador Retrievers 3 German Shepherds 2 Australian Shepherds and
one from each of the following breeds Akita Inu Asian Shepherd Bavarian Mountain
Hound Beagle Belgian Shepherd Briard Canaan dog Dalmatian Golden Retriever
Havanese dog Hovawart Rhodesian Ridgeback Schnauzer Shar Pei and Wolfspitz
These dogs were aged between 2 and 164 months (median 77 months) and had body
weights ranging from 81 and 63 kg (median 27 kg) 9 were castrated males 8 intact
males 7 spayed females and 5 intact females
The diseases of the 29 patients requiring a CVC included 3 from each of the following
diseases hepatopathy and esophagus dilatation 2 from each of the following diseases
bacterial cystitis diabetes mellitus and 1 from each of the following diseases abdominal
wall perforation aspiration pneumonia cystitis intestinal round cells sarcoma carcinoma
of the nasal cavity chronic renal insufficiency cystic duct rupture heartworm disease
ivermectine intoxication lymphoma multifocal alveolar histiocytosis pancreatitis post-
surgical bleeding stomach dilatation stomach foreign body tetanus thoracic
pyogranulomatous inflammation thrombocytopenia and trigeminal nerve paresis
Animals were treated in accordance with the German Animal Welfare Law The
experimental design was approved by the Animal Welfare officer of the University of
Veterinary Medicine and reported to the responsible authority (Lower Saxony State
Office for Consumer Protection and Food Safety reference number 17A101)
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
46
23 Positioning and handling of the central venous catheter
Two types of single lumen radiopaque polyurethane CVC (different on length and
diameter) were used during the study The CVC type I (n = 2529) was Cavafixreg Certoreg
Splittocanreg CVC 32 cm length 11 x 17 mm diameter16 G (BBraun Melsungen AG)
the CVC type II used (n = 429) was Certofixreg Mono S 215 15 cm length 08 x 14 mm
diameter18G (BBraun Melsungen AG) Each CVC was inserted into the EJV using a
modified Seldinger technique as follows the skin of the neck was shaved and disinfected
with antiseptic soap alcohol and iodine Local anesthesia (Lidocaine HCl 2) was
administrated subcutaneously on the catheter insertion site (a middle point in the lateral
neck over the jugular sulcus) A small skin incision was performed in the insertion site
and one peripheral plastic cannula with a metallic needle inside (14 G for CVC type I
and 20 G for CVC type II) was inserted in the EJV in cranio-caudal orientation (Hundley
et al 2018) Once the EJV was reached the needle was removed and a flexible wire
guide was inserted through the peripheral cannula following an external landmark
(4thintercostal space) (Reminga et al 2018) The peripheral cannula was removed and
the respective CVC was inserted slowly and with gentle pressure into the EJV guided
with the flexible wire and following the same external landmark to reach the right
position After the insertion of the CVC the flexible wire guide was removed The correct
positioning of the CVC was checked with a latero-lateral thorax x-ray ensuring that the
CVC tip was placed at the junction of the cranial cava vein with the right atrium Each
CVC was fixed to the neck skin (adjacent to the insertion point) with a monofilament
non-absorbable suture (gauge 3-0) Finally a bandage elaborated with Povidone-Iodine
gauze (Betaisodonareg) padded synthetic band (Artiflexreg Natur) and elastic band
(Elastomullreg) was used to protect the CVC output (Claude et al 2010) and changed
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
47
daily Several times day the catheter was flushed with isotonic sodium chloride
containing 1 IUml UFH
24 Heparin preparation and administration
Heparin-sodium-25000-ratiopharm (injection solution containing 5000 IUml sodium
heparin [mucosa]) was used SC injection was performed over the lateral thorax wall
using 1 mL syringes with fine graduation marks
25 Ultrasonography
Colour Doppler ultrasonographic examinations were performed in lateral position using
a LO-GIQ E9 ultrasound device (GE Healthcare Chicago Illinois USA) with a 15
Megahertz linear ultrasound probe The development of a thrombus or fibrin debris in the
EJV lumen around the CVC or inside it was assessed at each control Once the
ultrasound was finished a new protective bandage was placed around the neck of the dog
According to the percentage of lumen occlusion of the EJV (evaluated in cross sectional
views during the ultrasound scanning) the thrombi were classified in four different grades
(Fig 1)
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
48
of the residual vein lumen C ndash catheter T ndash thrombus
Fig 1 Definition of different grades of central venous catheter induced thrombosis of the
jugular vein as detected using colour Doppler ultrasound
26 Blood collection
At each time point approx 6ndash10 ml of blood (according to the body weight) was collected
This included approx 1 ml of EDTA blood for hematological analysis and approx 5ndash9
ml of citrated blood (tubes containing one part of 011 molL [38 ] sodium citrate to
nine parts of blood) for the measurement of hemostasis tests in citrated blood (ROTEM)
or citrated plasma Blood samples were collected by puncture of peripheral veins with
disposable cannulas (09 x 40 mm [20G] or 10 x 40mm [21G]) directly into sample tubes
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
49
or from the CVC using the three-syringe-technique (Villalta-Garciacutea et al 2015) in
situations when the peripheral venipuncture was not indicated due to phlebitis or irritation
andor inflammation of the surrounding skin Briefly 2ndash3 ml of blood were taken in a
sterile disposable syringe and immediately afterwards the blood sample was taken in a
new disposable syringe and transferred into the test tubes containing the respective
anticoagulants finally the first 2-3 ml taken were re-infused to the dog through the CVC
followed by flushing with a solution of sodium chlorideUFH (02) Immediately after
blood collection each sample tube was swayed gently until blood and anticoagulant had
been mixed thoroughly To prepare citrated platelet poor plasma citrated blood was
centrifuged at 16000 g for 10 min at room temperature using a microcentrifuge The
plasma was transferred into plastic tubes and the centrifugation procedure was repeated
This platelet poor plasma was then frozen in aliquots at -70 degC until used for analysis
Directly before analysis the plastic tubes were thawed at 37 degC in a water bath
27 Laboratory tests
Haematological analyses were made automatically with Adviareg 2120i (Siemens
Healthcare GmbH Germany)
Prothrombin time activated partial thromboplastin time and thrombin time were
measured with the autoanalyser Amax Destiny Plus (Tcoag Deutschland GmbH
Germany) and commercial reagents (Thromborel S Siemens Healthcare Diagnostics
Products GmbH Marburg Germany CK Prestreg and Test Thrombin Reagent
Diagnostica Stago SAS Asniegraveres sur Seine Cedex France) Whereas the last two tests
were measured according to the manufacturer`s instructions a modified test optimised
for canine plasma was used to measure prothrombin time (25 microl citrated plasma 120 with
imidazole buffer + 25 microl of a human fibrinogen solution 2 gl prewarmed for 120 s at 37
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
50
oC addition of 25 microl of activating reagent Thromborel S) and the results converted into a
percentage activity based on a calibration curve prepared with canine pooled plasma The
fibrinogen concentration was measured with the method according to Clauss using a test
kit from Stago Diagnostica SAS D-dimer concentration was measured with a
quantitative commercial human latex turbidimetric method in the autoanalyser Cobas
c311 using a completely assembled kit and human standards (Roche Diagnostics
Mannheim Germany) Antithrombin activity was also measured with a completely
assembled chromogenic test kit in the autoanalyser Cobas c311 and calibrated with a
canine pool plasma (n = 100)
The thrombin generation assay (Technothrombinreg TGA (Technoclone GbmH Vienna
Austria) determines thrombin generation based on monitoring of the fluorescence
generated by cleavage of a fluorogenic substrate by thrombin over time after activation
of the coagulation cascade by different concentrations of tissue factor and negatively
charged phospholipid in platelet poor plasma From the fluorescence development over
time the concentration of thrombin in the sample can be calculated using a thrombin
calibration curve The increase in thrombin concentration with time then allows to
calculate generation of thrombin in the sample and to plot thrombin values over time for
the whole coagulation process
The rotational thromboelastometry analysis with ROTEM delta device (Werfen Munich
Germany) was started approx 15 minutes after the blood collection To perform the
analysis and according to the automated ROTEM software 20 microl of recalcification reagent
(200 mmoll calcium chloride solution star-tem) and 20 microl of the respective activating
reagent (ex-tem in-tem) were pipetted into and carefully mixed in a pre-warmed cuvette
using the automated pipette provided in the device immediately afterwards 300 microl of
citrated whole blood (previously gently revolved) were added and mixed with the
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
51
reagents previously deposited Finally the cuvette was carefully placed and fixed to an
oscillating pin (oscillation movements on left and right through an angle of 475deg) in the
ROTEM delta device The interaction between blood cells and subsequently the bond
between fibrin and platelets promoted by the biomechanical changes in pH electrolytes
and temperature start the clot formation with its subsequent retraction and lysis The
magnitude of the resistance produced by the clot over the oscillating pin is quantitatively
represented by different parameters measured during the analysis and also qualitatively
in a graphic illustration (Bagge et al 2016 Crochemore et al 2017)
A chromogenic substrate test (Coatest Heparin Chromogenix-Instrumentation
Laboratory Haemochrom Diagnostica Essen Germany) was used for measurements of
UFH in the auto-analyser Cobas c311 The test application was created based on
manufacturers` instructions Different dilutions of the same batch of the commercial
sodium UFH preparation used in the experiment with normal canine pool plasma
(prepared from identical aliquots of 10 healthy adult dogs) served as standards for the
calibration of the measurement
28 Electron microscopy
Selected areas on the last third of the CVC (5ndash10 cm according with the CVC) with
macroscopically detectable thrombi were analysed and some of the CVCs were cut in
two different places The cut samples were preserved in a fixative medium composed of
1 glutaraldehyde and 4 formalin Once the sample was fixed it was prepared
according to the osmium thiocarbohydrazide procedure (OTOTO-procedure) (Malick et
al 1976) The fixed CVCs were dehydrated in seven series as follow first three series of
120 minutes each one on 30 50 and 70 ethanol respectively followed by two series
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
52
of 120 min each one on 90 ethanol and then two series of 24 hours each one on 100
ethanol After the dehydration process the samples were placed two times during 24
hours each one on isoamyl acetate and then placed on in a critical point drier Finally the
samples were coated with gold in a splutter-coater with 25 kV acceleration voltage in an
argon atmosphere with a current of 20 mA for one minute The samples were examined
in an EVO 15 scanning electron microscope (ZEISS International Carl Zeiss Jena
Germany) and photographed with magnifications of 600x and 6000x avoiding mechanical
artifacts (Toskala et al 1995) Thrombi on the external wall of the CVC were analysed
and measured using the software ImageJ (National Institutes of Health and the Laboratory
for Optical and Computational Instrumentation University of Wisconsin USA)
According to their extension (surface size) and thickness (related to the CVC wall
thickness in a cross sectional view) thrombi on the external wall and additionally within
the lumen of the CVC were each classified in four grades (Fig 2 Fig 1S [suppl
material]) In case of two cuts the highest degree of thrombosis detected at one of the two
sites was used for the final assessment
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
53
surface sizepercent of CVC wall thickness
Fig 2 Definition and illustration of different grades of central venous catheter induced
(extraluminal) thrombosis as detected using electron microscopy
Fig 1S (suppl material) Definition and illustration of different grades of intraluminal
thrombosis in central venous catheters as detected using electron microscopy
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
54
29 Statistical methods
Comparison between thrombus graduation either based on sonographic and ELMI
findings respectively was performed using the McNemar test with calculation of the
concordance index (κ) Standard normal distribution was assessed using the
Kolomogorov-Smirnov test
Due to non-normal distribution of part of data sets heparin activities and results of
haemostasis assays are reported using non-parametric variables (median minimumndash
maximum box and whisker plots) Heparin activities and results of haemostasis tests
between dogs with or without thrombosis were compared using Mann-Whitney-U test
3 Results
31 Sonographic detection of thrombus formation
Thrombus formation was sonographically detected in 829 patients during the study
period Based on examination on the final day in half of these eight patients (n = 4) the
thrombus formation was scored grade 1 in 3 dogs grade 2 and only in one grade 3 In all
of these 8 dogs this was the maximum grade during the study period One dog with fibrin
precipitates was excluded from further analysis because it could not be clearly assigned
to one of the groups In 78 cases thrombi were attached mainly to the venous wall
whereas only in one case was attached to the CVC external wall Seven of these 8 dogs
which developed thrombi received the standard heparin dosage (150 IUkg TID 41
[717] of dogs receiving this dosage) and only one the reduced heparin dosage (75 IUkg
TID 8 [112] of dogs receiving this dosage)
The time point of first detection of thrombus formation varied between days 3 and 11
(median day 8) In addition detritus (presumably composed mainly of fibrin) was
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
55
detectable in the lumen of the EJV in one further dog and within the lumen of the CVC
in further 5 dogs
32 Thrombus formation by electron microscopy
27 from 29 CVC catheter tips were analysed by electron microscopy 827 samples were
without detectable thrombus (grade 0) and 1927 with detectable thrombi 72 showed
thrombus formation of grade 1 427 of grade 2 and 827 of grade 3 There was no
concordance between thrombus graduation based on sonographic and the ELMI
graduation respectively The concordance index (McNemar test) was negative (κ = -
00718) indicating that the degree of agreement is less than by chance
Of the in total 19 dogs with detectable thrombus formation by electron microscopy 11
dogs received 150 IUkg TID (in total 16 dogs of these 27 dogs received that heparin
dosage) and 8 received 75 IUkg TID (in total 11 of these 27 dogs received that heparin
dosage 73 811) and Of the 12 patients with grade 2 and 3 thrombus formation 5 dogs
received 75 IUkg TID and 7 dogs 150 IUkg
The supplementary analysis of intraluminal thrombus formation revealed no detectable
thrombi in 7 cases and thrombus formation of grade 1 (lt 25 occlusion) in 11 cases of
grade 2 (25ndash50 ) in 3 cases and of grade 3 (gt 50 ) in 6 cases
33 Heparin activities and relationship to thrombus formation
Heparin activities varied remarkably in both dosage groups (Fig 3 ab) There was no
significant difference of heparin activities between blood samples collected via
venipuncture or from the CVC (Table 1S supplementary material) In addition there was
no significant difference between heparin activities in dogs with or without
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
56
sonographically or electron microscopically detectable thrombosis (Tables 1 and 2)
Table 1 refers to sonographically detectable thromboses and is based on median heparin
activities calculated from the measurements on day 1 and following days per patient
firstly for the total number of patients and secondly for the patients receiving 150 IUkg
TID In addition in the eight dogs heparin activities on the day of first thrombus detection
(023 IUml 019ndash036 IUml) were not significantly different from the median values
(Table 1 P = 05214 Wilcoxon test) Table 2 compares heparin activities (median values
of all days and results on the final day) between dogs with and without electron
microscopically detectable CVC-associated thrombi
Table 1S (Supplementary material) Heparin activities (IUml median values
minimumndashmaximum) measured in 27 dogs receiving 75 or 150 IUkg unfractionated
(standard) heparin TID subcutaneously 2 hours after the first injection on the day
Comparison of samples collected via puncture of the peripheral veins and those collected
from the central venous catheter (CVC) using the three-syringe-technique
Peripheral vein
puncture CVC
Mann-Whitney-U
test
Total number
(75 and 150
IUkg TID)
019
(00ndash108)
(n = 65)
023
(000ndash105)
(n = 65)
P = 03033
75 IUkg TID
010
(000ndash108)
(n = 25)
012
(000ndash023)
(n = 21)
P = 06907
150 IUkg
TID
030
(000ndash096)
(n = 40)
036
(000ndash105)
(n = 44)
P = 01223
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
57
Fig 3ab Heparin activities (box- and whisker plot arithmetic means) in dogs receiving
unfractionated heparin at a dosage of 75 IUkg BW tid (a) or 150 IUkg BW tid (b) two
hours after the first daily heparin administration
day 1 represents the first day after initiation of heparin treatment
only days were considered with at least results from 5 patients
Table 1 Heparin activities (IUml median values minimumndashmaximum) in 28 dogs
receiving 75 or 150 IUkg unfractionated (standard) heparin TID subcutaneously with
(grades 1ndash3) or without (grade 0) sonographically detectable thrombus Blood collections
were performed 2 hours after the first daily injection
Grade 0
(sonography)
Grades 1ndash3
(sonography)
Mann-Whitney U
test
Total number
(75 and 150 IUkg
TID)
020 (000ndash072)
(n = 20)
027 (018ndash059)
(n = 8)
P = 03212
150 IUkg TID 042 (000ndash072)
(n = 10)
031 (018ndash059)
(n = 7)
P = 04593
one dog with fibrin precipitates in the lumen of the jugular vein but without defined thrombus formation
was not considered because it could not be clearly assigned to one of the two groups
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
58
Table 2 Heparin activities (IUml median values minimumndashmaximum) in 27 dogs
receiving 75 or 150 IUkg unfractionated (standard) heparin TID subcutaneously with
(score 1ndash3) or without (score 0) detectable thrombus via electron microscopy
Grade 0
(electron
microscop)
(n = 8)
Grades 1ndash3
(electron
microscop)
(n = 19)
Mann-Whitney U
test
Median value of
different days 028 (005ndash060)
022 (000ndash072)
P = 05770
Final day 020 (001ndash045)
015 (000ndash105)
P = 06706
34 Relationship between haemostasis tests and thrombus formation
From the initially performed haemostasis tests high fibrinogen concentrations and low
maximum lysis values as measured by the ROTEM delta device (using the ex-tem
reagent) were associated with sonographically detectable thrombi (Tab 3) Additional
analyses based on the median values of different days for individual patients revealed that
low maximum lysis values as measured by the ROTEM delta device (using the in-tem
reagent) were associated with sonographically detectable thrombi (maximum lysis score
0 850 [100ndash175 ] score 1ndash3 375 [100ndash900 ] median [minimumndash
maximum] P = 00166) but no other test Comparison of the results of haemostasis tests
on the final day between dogs with and without sonographically detectable thromboses
did not show any significant difference (P gt 005)
Comparison of results of haemostasis tests on the final day in dogs with detectable
thrombosis via ELMI and dogs without significant thrombus formation showed several
significant differences including lower fibrinogen concentrations and longer clotting
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
59
times (ex-tem reagent) and clot formation times (ex-tem and in-tem reagents) using the
ROTEM delta analyser in general expressing a less coagulable status (Tab 4) If values
of dogs graded 0 and 1 (n =15) are compared to those of grades 2 and 3 (n = 12) the last-
mentioned had longer clotting times (ROTEM delta in-tem reagent) as well as longer clot
formation times and lower alpha angles (ROTEM delta in-tem reagent) (P lt 005 results
not shown)
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
60
Table 3 Comparison of initial results of haemostasis parameters (median values
minimumndashmaximum in dogs with (grades 1ndash3) and without (grade 0) sonographically
detectable central venous catheter-induced thromboses
Parameter Unit
Sonographic
thrombus grade
P-value
(Mann-Whitney
U test)
0
(n = 20)
123
(n = 8)
Haematocrit 355 (228ndash641)
382 (245ndash499)
06111
Platelet count x103microl 292 (420ndash483)
218 (860ndash458)
02320
Prothrombin time 944 (359ndash169)
896 (471ndash136)
05758
Activated partial thromboplastin
time s 128
(955ndash249) 140
(117ndash164) 02322
Thrombin time s 129 (121ndash178)
141 (650ndash160)
02028
Fibrinogen gl 448 (153ndash770)
747 (153ndash116)
00235
Antithrombin activity 639 (289ndash107)
670 (464ndash746)
05249
D-dimers microgml 006 (000ndash148)
015 (003ndash372)
03997
Thrombin generation assay nmol 130 (000ndash365)
912 (190ndash277)
08787
Clotting time (ROTEM ex-tem) s 420 (290ndash930)
430 (170ndash159)
07989
Clot formation time (ROTEM ex-
tem) s 665
(310ndash143) 470
(240ndash800) 00633
Alpha angle (ROTEM ex-tem) degrees 795 (620ndash840)
815 (750ndash860)
01667
Max clot firmness (ROTEM ex-
tem) mm 750
(570ndash860) 775
(700ndash830) 01753
Max clot elasticity (ROTEM ex-
tem) - 299
(131ndash619) 347
(235ndash484) 01544
Maximum lysis (ROTEM ex-tem) 135 (300ndash290)
400 (000ndash800)
00037
Clotting time (ROTEM in-tem) s 208 (840ndash810)
249 (720ndash371)
05085
Clot formation time (ROTEM in-
tem) s 835
(370ndash634) 745
(390ndash235) 08988
Alpha angle (ROTEM in-tem) degrees 745 (250ndash820)
750 (500ndash820)
09797
Max clot firmness (ROTEM in-
tem) mm 700
(540ndash810) 740
(610ndash770) 03587
Max clot elasticity (ROTEM in-
tem) - 235
(118ndash424) 287
(156ndash332) 04764
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
61
Maximum lysis (ROTEM in-tem) 850 (000ndash210)
350 (000ndash220)
01327
Table 4 Comparison of results of haemostasis parameters (median values minimumndash
maximum) on the last day in dogs with (grades 1ndash3) and without (grade 0) central venous
catheter-associated thrombus formation as detected by electron microscopy
Parameter Unit
Thrombus grade
(ELMI)
P-value
(Mann Whitney
U test)
0
(n = 8)
123
(n = 19)
Haematocrit 322 (241ndash454)
330 (216ndash510)
05953
Platelet count 103microl 446 (321ndash592)
268 (101ndash675)
00558
Prothrombin time 145 (104ndash264)
120 (561ndash148)
00462
Activated partial thromboplastin
time s 123
(109ndash140) 136
(109ndash188) 00665
Thrombin time s 129 (121ndash178)
141 (650ndash160)
02028
Fibrinogen gl 465 (275ndash866)
347 (189ndash992)
00491
Antithrombin activity 793 (448ndash981)
692 (398ndash889)
04572
D-dimers microgml 018 (000ndash173)
007 (000ndash118)
01920
Thrombin generation assay nmol 501 (000ndash160)
93 (000ndash287)
09562
Clotting time (ROTEM ex-tem) s 360
(270ndash410) 400
(310ndash620) 00238
Clot formation time (ROTEM ex-
tem) s 395
(120ndash740) 650
(240ndash174) 00275
Alpha angle (ROTEM ex-tem) degrees 835 (760ndash880)
790 (580ndash850)
00484
Max clot firmness (ROTEM ex-
tem) mm 780
(700ndash850) 720
(550ndash860) 00586
Max clot elasticity (ROTEM ex-
tem) - 346
(239ndash555) 254
(121ndash637) 00495
Maximum lysis (ROTEM ex-tem) 105 (200ndash190)
110 (000ndash380)
03796
Clotting time (ROTEM in-tem) s 178 (100ndash222)
201 (139ndash554)
00525
Clot formation time (ROTEM in-
tem) s 435
(330ndash111) 790
(350ndash882) 00209
Alpha angle (ROTEM in-tem) degrees 815 (710ndash830)
740 (240ndash830)
00191
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
62
Max clot firmness (ROTEM in-
tem) mm 785
(660ndash810) 690
(270ndash840) 00586
Max clot elasticity (ROTEM in-
tem) - 362
(192ndash434) 218
(370ndash507) 00559
Maximum lysis (ROTEM in-tem) 950 (200ndash190)
800 (100ndash180)
06890
4 Discussion
The main result of our study is that under the study conditions part of the dogs developed
CVC-associated thromboses However only a very low part of these thromboses were
obviously of a clinically significant degree regarding the amount of occlusion of the EJV
and none of the dogs had a symptomatic thrombosis whose definition includes presence
of clinical signs such as localised swelling pain and redness (Boersma et al 2008 Lee
and Kamphuisen 2012) In the present study we particularly refer to the extraluminal
thromboses with regard to the assessment of the antithrombotic management
One major problem of the statistical analysis and result presentation was that the two
methods used to define the occurrence and degree of thrombus formation (ultrasound and
ELMI) revealed discrepant results Therefore we performed a statistical analysis
independently regarding thrombus formation with either of the two methods It is well
known that ELMI which has already been used to detect CVC induced thromboses in
humans (Lucas et al 2017) and experimental animals (Xiang et al 1998 Xiang et al
2001) is a very sensitive diagnostic technique which allows the ex vivo diagnosis of very
small or fibrin rich thrombi that perhaps cannot be detected by ultrasonography or
venography during the in vivo evaluation of patients (Xiang et al 2001) A major reason
for the discrepant results between the two methods used to detect CVC associated
thromboses is that different CVC segments are evaluated Using ultrasonography it was
only possible to evaluate the EJV in the neck area of the patient and thereby only its
proximal third In contrast ELMI assessed mainly the CVC tip and a human study
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
63
revealed that even different positions of the catheter tip are associated with various risks
for development of CVC associated thrombosis (Ballard et al 2016) One additional point
to consider which also may contribute to the discrepancy is that it is possible that thrombi
developed on the CVC surface can be detached during the CVC removal from the vein
and thereby before its analysis by ELMI (Xiang et al 2001)
The contrast venography technique is used as gold standard to confirm thrombosis in
human medicine However this diagnostic technique is normally used only in the case of
a strong clinical suspicion of venous thrombosis that cannot be detected by
ultrasonography (Laurenson et al 2010 Langston and Eatroff 2018) Our study protocol
with privately owned clinic patients did not allow to use this method In a study performed
in 44 human patients requiring a CVC (Koumlksoy et al 1995) a sensitivity of 94 and a
specificity of 96 were determined for the colour Doppler ultrasound diagnosis when it
was compared with the diagnosis performed with the contrast venography technique and
that makes the colour Doppler ultrasound an accurate diagnosis method to detect CVC
related thrombosis even in humans (Koumlksoy et al 1995 van Rooden et al 2003)
The incidences of CVC related thrombosis detected by colour Doppler ultrasound of the
internal jugular vein reported in 3 human studies were 2 30 and 56 (Harter et al
2002 Lordick et al 2003 Wu et al 1999) and although with a wide range the median
value of these rates (30 ) is very well suited to the incidence of 28 (829) which we
calculated for our canine study based on sonographic findings The wide variation of
incidences of CVC related thrombosis in different human studies can in general be
explained by differences regarding the type and entry site of the CVC (jugular femoral
subclavian axillary cephalic vein) the time period with the CVC in place the thrombus
detection technique (venography or ultrasonography comment in the cited 3 studies
consistently the last-mentioned method was used) and criteria for diagnosis of
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
64
thrombosis the duration of follow up diseases and presence of predisposing factors in
these patients (eg previous venous thrombosis history presence of cancer) and finally
prophylactic treatment with anticoagulants (van Rooden et al 2005 Geerts 2014 Liu et
al 2015)
Different pathomechanisms contribute to the development of CVC induced thromboses
During the CVC insertion the puncture of the skin on the CVC insertion site and the
rupture of the blood vessels integrity activate the coagulation cascade and the CVC acts
as a matrix for adherence and accumulation of fibrin and cellular components (Smith et
al 2012) The attachment of proteins and blood cells to the internal andor external wall
of the CVC can lead to a partial or complete occlusion of the catheter lumen and
sometimes to a partial or complete obstruction of the blood flow andor
thromboembolism (Langston and Eatroff 2018) There is a strong interrelationship
between thrombus and biofilm formation (Smith et al 2012) Prevention of thrombus
formation has thereby also relevance regarding the increased risk for microbial
colonisation of the catheter (Boersma et al 2008) leading at least in humans to an
increased number of CVC related systemic infections (Lordick et al 2003)
In this context it has to be considered that CVC produced from polyurethane which are
routinely used in our clinic and thereby in the study are usually associated with a
relatively high thrombosis potential and thereby definitely provide an effective test for
an antithrombotic regimen Silicone catheters have a significantly reduced risk for
thrombotic complications but this advantage has to be purchased by a higher rate of
mechanical failure (eg rupture of the catheter) (Wildgruber et al 2016) Promising
experimental approaches work on surface modifications of the standard catheters For
example one group was able to demonstrate that a coating of the surface of the catheter
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
65
with a zwitterionic polymeric sulfobetaine showed a significant reduction of thrombus
formation in vitro with human blood and in an in vivo canine model (Smith et al 2012)
Due to ethical reasons we did not performed a placebo-controlled study However
interestingly not all human studies report systemic anticoagulation and part of the human
studies was even placebo-controlled (review Lee and Kamphuisen 2012) Obviously
there is no consensus in the available human literature regarding the beneficial use of
antithrombotic prophylaxis to prevent CVC related thrombosis Several studies did not
find a statistically significant difference in the incidence of CVC related thrombosis
between patients receiving the antithrombotic prophylaxis compared with the control
groups without systemic anticoagulation (Verso et al 2005 P = 035 Karthaus et al
2006 P gt 005 Niers et al 2007 P = 049) whereas in other studies a lower incidence
of CVC related thrombosis was found in the group receiving anticoagulants as
prophylaxis compared with the control groups (Abdelfeki et al 2004 150 vs 126
P = 003 De Cicco et al 2009 400 vs 526 P = 005)
Although it is well known that subcutaneously administered UFH has a limited and
variable bioavailability and thereby leads to unpredictable blood activities (Hirsh and
Raschke 2004) the extremely wide range of blood activities after a defined dosage in our
study was relatively unexpected Blood was collected 2 hours after the first UFH
injection and thereby at the time of suspected maximum heparin activity considering the
relatively low UGH dosage (Mischke and Jacobs 2001) The cited study was performed
on healthy experimental dogs Possible additional influencing factors in critical ill
patients include circulatory insufficiency which may reduce and delay absorption from
subcutanenous tissue decreased clearance function of the liver and kidneys as well as
altered blood plasma concentrations of heparin binding plasma proteins such as
fibrinogen and platelet factor 4 and alterations of the heparin binding capacity of
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
66
endothelial cells which altogether are reasons for a great inter- but also intrapatient
variability (Hirsh and Raschke 2004) In individual patients this can lead to heparin
resistance It has also to be considered that antithrombin activities which are necessary
for an adequate heparin effect (Bjoumlrk and Lindahl 1982) were reduced in our patients
Availability of approved blood tests which can predict the risk of development of CVC
associated thrombosis already at the moment of the CVC placement are important
(Boersma et al 2016) One of the most interesting results was that dogs which developed
sonographically detectable thrombi had nearly twofold higher fibrinogen levels Well in
agreement with our results one study performed in anticoagulated human patients
demonstrated that high fibrinogen levels were associated with an increased risk of
perioperative venous thromboembolism (P = 0004) (Kato et al 2016) The cited study
does not exclusively refer to CVC associated thrombosis but 76 (1621) of the venous
thromboses were suggested to be associated with CVCs Fibrinogen measurement a test
which is widely available is therefore a useful and practicable indicator of an increased
risk for CVC induced thromboses and may advice the clinician to treat these patients
especially carefully (eg with low molecular weight heparin) In the study performed by
Boersma et al (2016) in 168 human patients without indication of anticoagulation
prophylaxis elevated leukocyte counts high levels of plasminogen activator inhibitor
and of coagulation factor VIII were associated with an increased incidence of
symptomatic CVC related thrombosis diagnosed by ultrasonography A limitation of that
study is that all measurements were performed on blood samples taken after CVC
placement Interestingly in our study ROTEM results were partly associated with
thrombus formation which reflects the well-known fact that results of viscoelastic
measurements are a good indicator of hypercoagulability and thromboembolic events
(Harahsheh and Ho 2018) It is of further interest and possibly unexpeted that on the final
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
67
day of examination dogs with ELMI signs of thromboses had partly lower coagulation
activities than dogs without thromboses Due to the low number of dogs without
detectable thrombi using ELMI this implausible result has to be interpreted with caution
One possible explanation could be that temporary hypercoagulability in these dogs may
have finally resulted in consumption of haemostasis substrates
Limitations of the present study were as a consequence of the study design based on
clinical patients the relatively low total number of patients inclusion of different UFH
treatment regimens and the lack of a control group
5 Conclusions
The results of the present study indicate that the used UFH regimen was not completely
effective to prevent CVC-associated thromboses which were detectable in 28 [829]
of the patients by ultrasound However severe thromboses (high grades) are relatively
rare in intensive care canine patients receiving a routine anticoagulant treatment Because
heparin plasma activities were not different in dogs with or without CVC-associated
thrombus formation other local and systemic factors must play a significant role as well
A high fibrinogen concentration and low maximum lysis (ROTEM ex-tem) may be
useful indicators for an increased thrombotic risk in individual patients which may
require special anticoagulatory treatment
6 Acknowledgments
Jose Mauricio Pereira received a scholarship from the Universidad Nacional de Costa
Rica (reference code JB-C-1107-2016)
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
68
7 References
1 Abdelkefi A Ben Othman T Kammoun L Chelli M Ben Romdhane N Kriaa A Ladeb S
Torjman L Lakhal A Achour W Ben Hassen A Hsaiumlri M Ladeb F Ben Abdeladhim A
2004 Prevention of central venous line-related thrombosis by continuous infusion of low-dose
unfractionated heparin in patients with haemato-oncological disease A randomized controlled trial
Thromb Haemost 92 654ndash661
2 Adamantos S Brodbelt D Moores A L 2010 Prospective evaluation of complications associated
with jugular venous catheter use in a veterinary hospital J Small Anim Pract 51 254ndash257
3 Bagge A Schoumltt U Kander T 2016 Effects of naturopathic medicines on Multiplate and ROTEM
a prospective experimental pilot study in healthy volunteers BMC Complement Altern Med 16 64
4 Ballard DH Samra NS Gifford KM Roller R Wolfe BM Owings JT 2016 Distance of the
internal central venous catheter tip from the right atrium is positively correlated with central venous
thrombosis Emerg Radiol 23 269ndash273
5 Boersma RS Jie KS Verbon A van Pampus EC Schouten HC 2008 Thrombotic and
infectious complications of central venous catheters in patients with hematological malignancies Ann
Oncol 19 433ndash442
6 Boersma R S Hamulyak K van Oerle R Tuinenburg A Ten Cate-Hoek AJ Schouten HC
2016 Biomarkers for prediction of central venous catheter related-thrombosis in patients with
hematological malignancies Clin Appl Thromb Hemost 22 779ndash784
7 Bjoumlrk I Lindahl U 1982 Mechanism of the anticoagulant action of heparin Mol Cell Biochem
48 161ndash182
8 Claude A Riedesel D Riedesel E 2010 Electrocardiography-guided and retrospective analysis of
central venous catheter placement in the dog Vet Anaesth Analg 37 97ndash105
9 Comerlato PH Rebelatto TF Santiago de Almeida FA Klein LB Boniatti M M Schaan
DB Rados VD 2017 Complications of central venous catheter insertion in a teaching hospital
Rev Assoc Med Bras 63 613ndash620
10 Crochemore T Piza FMT Rodriacutegues RDR Guerra JCC Ferraz LJR Correcirca TD 2017
A new era of thromboelastometry Einstein (Sao Paulo) 15 380ndash385
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
69
11 De Cicco M Matovic M Balestreri L Steffan A Pacenzia R Malafronte M Fantin D
Bertuzzi CA Fabiani F Morassut S Bidoli E Veronesi A 2009 Early and short‐term
acenocumarine or dalteparin for the prevention of central vein catheter‐related thrombosis in cancer
patients a randomized controlled study based on serial venographies Ann Oncol 20 1936ndash1942
12 Evans NS Ratchford EV 2018 Catheter related-venous thrombosis Vasc Med 23 411ndash413
13 Geerts W 2014 Central venous catheterndashrelated thrombosis Hematology Am Soc Hematol Educ
Program 2014 306ndash311
14 Harahsheh Y Ho KM 2018 Use of viscoelastic tests to predict clinical thromboembolic events A
systematic review and meta‐analysis Eur J Haematol 100 113ndash123
15 Harter C Salwender HJ Bach A Egerer G Goldschmidt H Ho AD 2002 Catheter-related
infection and thrombosis of the internal jugular vein in hematologic-oncologic patients undergoing
chemotherapy a prospective comparison of silver-coated and uncoated catheters Cancer 94 245ndash251
16 Hirsh J Raschke R 2004 Heparin and low-molecular-weight heparin The Seventh ACCP
Conference on Antithrombotic and Thrombolytic Therapy Chest 126 (Suppl) 188S-203S
17 Hundley D Brooks A Thomovsky E Jhonson P Freeman L Schafbuch R Heng HG Moore
G 2018 Comparison of ultrasound-guided and landmark-based techniques for central venous
catheterization via the external jugular vein in healthy anesthetized dogs Am J Vet Res 79 628ndash
636
18 Kato F Takeuchi H Matsuda S Kawakubo H Omori T Kitagawa Y 2016 Incidence of and
risk factors for venous thromboembolism during surgical treatment for esophageal cancer a single-
institution study Surg Today 46 445ndash452
19 Karthaus M Kretzschmar A Kroning H Biakhov M Irwin D Marschner N Slabber C
Fountzilas G Garin A Abecasis NG Baronius W Steger GG Sudhoff T Giorgetti C
Reichardt P 2006 Dalteparin for prevention of catheter‐related complications in cancer patients with
central venous catheters final results of a double‐blind placebo‐controlled phase III trial Ann Oncol
17 289ndash296
20 Koumlksoy C Kuzu A Kutlay J Erden I Ozcan H Ergicircn K 1995 The diagnostic value of colour
Doppler ultrasound in central venous catheter related thrombosis Clin Radiol 50 687-689
21 Kucher N 2011 Clinical practice Deep-vein thrombosis of the upper extremities N Engl J Med
364 861ndash869
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
70
22 Langston CE Eatroff AE 2018 Hemodialysis catheter-associated fibrin sheath in a dog J Vet
Emerg Crit Care 28 366ndash371
23 Laurenson MP Hopper K Herrera MA Johnson EG 2010 Concurrent diseases and conditions
in dogs with splenic vein thrombosis J Vet Intern Med 24 1298ndash1304
24 Lee AYY Kamphuisen PW 2012 Epidemiology and prevention of catheter‐related thrombosis in
patients with cancer J Thromb Haemost 10 1491ndash1499
25 Liu G Fu ZQ Zhu P Li SJ 2015 Central venous catheter-related thrombosis in senile male
patients New risk factors and predictors J Huazhong Univ Sci Technolog Med Sci 35 445ndash449
26 Lordick F Hentrich M Decker T Hennig M Pohlmann H Hartenstein R Peschel C 2003
Ultrasound screening for internal jugular vein thrombosis aids the detection of central venous catheter-
related infections in patients with haemato-oncological diseases a prospective observational study Br
J Haematol 120 1073ndash1078
27 Lucas TC Silva EED Souza DOD Santos ARD Lara MO 2017 Microstructural
evaluation by confocal and electron microscopy in thrombi developed in central venous catheters Rev
Esc Enferm USP 51 1ndash9
28 Malick L Wilson R Stetson D 1976 Modified thiocarbo- hydrazide procedure for scanning
electron microscopy routine use for normal pathological and experimental tissue Stain Technol 50
265ndash269
29 Mischke R Jacobs C 2001 The monitoring of heparin administration by screening tests in
experimental dogs Res Vet Sci 70 101ndash108
30 Niers TM Di Nisio M Klerk CP Baarslag HJ Buller HR Biemond BJ 2007 Prevention
of catheter‐related venous thrombosis with nadroparin in patients receiving chemotherapy for
hematologic malignancies a randomized placebo‐controlled study J Thromb Haemost 5 1878ndash
1882
31 Reminga C Silverstein D Drobatz K Clarke D 2018 Evaluation of the placement and
maintenance of central venous jugular catheters in critically ill dogs and cats J Vet Emerg Crit Care
28 232ndash243
32 Smith RS Zhang Z Bouchard M Li J Lapp HS Brotske GR Lucchino DL Weaver D
Roth LA Coury A Biggerstaff J Sukavaneshvar S Langer R Loose C 2012 Vascular
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
71
catheters with a nonleaching poly-sulfobetaine surface modification reduce thrombus formation and
microbial attachment Sci Transl Med 4 153ra132 doi101126scitranslmed3004120
33 Toskala E Nuutinen J Rautiainen M 1995 Scanning electron microscopy findings on human
respiratory cilia in chronic sinusitis and in recurrent respiratory infections J Laryngol Otol 109 509ndash
514
34 Van Rooden CJ Tesselaar ME Osanto S Rosendaal FR Huisman MV 2005 Deep vein
thrombosis associated with central venous catheters - a review J Thromb Haemost 3 2409ndash2419
35 Verso M Agnelli G Bertoglio S Di Somma FC Paoletti F Ageno W Bazzan M Parise P
Quintavalle R Naglieri E Santoro A Imberti D Soraru M Mosca S 2005 Enoxaparin for the
prevention of venous thromboembolism associated with central vein catheter a double‐blind placebo‐
controlled randomized study in cancer patients J Clin Oncol 23 4057ndash4062
36 Villalta-Garciacutea P Loacutepez-Herranz M Mazo-Pascual S Honrubia-Fernaacutendez T Jaacutentildeez-Escalada
L Fernaacutendez-Peacuterez C 2015 Reliability of blood test results in samples obtained using a 2-mL discard
volume from the proximal lumen of a triple-lumen central venous catheter in the critically ill patient
Nurs Crit Care 22 298ndash304
37 Vose J Odunayo A Price JM Daves M Schildt JC Tolbert MK 2019 Comparison of
heparinized saline and 09 sodium chloride for maintaining central venous catheter patency in healthy
dogs PeerJ 7 e7072 httpsdoiorg107717peerj7072
38 Wildgruber M Lueg C Borgmeyer S Karimov I Braun U Kiechle M Meier R Koehler M
Ettl J Berger H 2016 Polyurethane versus silicone catheters for central venous port devices
implanted at the forearm Eur J Cancer 59 113ndash124 doi101016jejca201602011
39 Wu X Studer W Skarvan K Seeberger MD 1999 High incidence of intravenous thrombi after
short-term central venous catheterization of the internal jugular vein J Clin Anesth 11 482ndash485
40 Xiang DZ Verbeken EK Van Lommel AT Stas M De Wever I 1998 Composition and
formation of the sleeve enveloping a central venous catheter J Vasc Surg 28 260ndash271
41 Xiang DZ Verbeken EK Van Lommel AT Stas M De Wever I 2001 Sleeve-related
thrombosis a new form of catheter-related thrombosis Thromb Res 104 7ndash14
Chapter 4 General discussion
72
Chapter 4 General discussion
Incidence of CVC related thrombosis
In our study the incidence of CVC related thrombosis detected by ultrasound in critical
care canine patients was 28 To the best of the authoracutes knowledge similar studies
performed in dogs are rare in the available veterinary literature In one study performed
in 81 dogs and 12 cats (Adamantos et al 2010) an incidence of 2 was found for vein
thrombosis related to CVC This low incidence can be due to a lacking of systematic
ultrasound evaluations of the jugular vein containing the CVC In the mentioned study in
dogs and cats the vein thrombosis was confirmed by palpation or ultrasound presumably
after the presentation of clinical symptoms however in many cases as in our study
thrombus into the jugular vein can be developed without manifestation of clinical
symptoms what makes a routinely ultrasound evaluation of the vein containing the CVC
very important Our results are well aligned with the findings in humans in which a
median incidence of 30 was found for thrombosis related to CVC based on 25 studies
performed in patients requiring a CVC (review article van Rooden et al 2005) In the
mentioned 25 human studies the incidence of CVC related thrombosis varied within a
wide range (2ndash67 ) according to factors related to (1) the CVC (eg material type
entry site [jugular femoral subclavian axillary cephalic vein] time period in place (2)
diagnostic thrombus detection technique (ultrasonography or venography) and criteria
(3) patient collective (diseases and presence of predisposing factors [eg previous venous
thrombosis history presence of cancer] and (4) prophylactic antithrombotic treatment
(Geerts 2014 Liu et al 2015 van Rooden et al 2005)
The incidence of CVC related thrombosis detected by colour Doppler ultrasound in our
study fits also with the median incidence (30 ) found in three studies performed in
humans in which colour Doppler ultrasound evaluations of the jugular vein containing a
Chapter 4 General discussion
73
CVC were performed (Harter et al 2002 Lordick et al 2003 Wu et al 1999) In these
three studies the individual incidence of CVC related thrombosis was 2 (Harter et al
2002) 30 (Lordick et al 2003) and 56 (Wu et al 1999) The high thrombus
incidence reported in the study performed by Wu et al (1999) could be due to the criteria
for diagnosis of thrombosis because in that study both fibrin sleeves and compact
thrombi were considered as cases of thrombosis related to CVC The incidence for
compact thrombi was 25 which is more similar to our results In the study performed
by Harter et al (2002) the CVC related thrombosis incidence (2 ) was very low
compared with the incidence found in our study In that study the jugular veins were
examined just before or less than 24 hours after CVC removal (median period of CVC
use = 1025 days) In our study the ultrasound evaluations were performed before the
CVC placement (day 0) and then on days 1 3 5 etc until the CVC removal (median
period of CVC use = 7 days median interval until thrombus detection = 7 days) In our
study we detected thrombus formation as early as on the 3rd day so it could be possible
that in the cited study possibly only temporarily existing thrombi could have been missed
by ultrasonography in the final stage and thereby for the incidence calculation However
this scenario is not very likely because results of the present study show that
sonographically detectable thrombi during the observation period had in all cases their
maximum grade on the final day
In the study performed by Lordick et al (2003) the CVC related thrombosis incidence
determined for 43 human patients was very similar to our study and in both studies only
compact thrombi detectable by ultrasound were considered for assessment of the
incidence In the mentioned study all the human patients were screened for the presence
of thrombosis every 4 days after CVC insertion with real time B-mode ultrasound
(Lordick et al 2003)
Chapter 4 General discussion
74
In our study 70 of CVCs analysed by ELMI showed thrombi of grade 1ndash3 on the
external CVC wall To the best of the authoracutes knowledge studies about incidence of CVC
related thrombosis on the outside surface of the CVC detected by ELMI cannot be found
in the human and small animal literature In one study performed in humans (Lucas et al
2017) thrombus formation was analysed by ELMI in 78 central venous catheters That
study focussed on microstructural assessment of thrombus formation inside the distal
openings of the CVC which were found in 85 of the evaluated CVCs The percentage
of occurrence of thrombosis in the mentioned study in humans was nearly similar to the
incidence of intraluminal thrombus formation as detected in the presented study by ELMI
(74 ) In general intraluminal thrombi were not specially considered in the present
study because they mainly express the efficacy of the flushing technique rather than the
effectivity of the antithrombotic treatment In one study performed in rats the
microstructure of thrombi developed in the jugular vein containing the CVC was also
evaluated by ELMI (Xiang et al 1998) In the mentioned study pericatheter thrombosis
was detected on the proximal section of the CVC in 100 of rats (n = 12) after 1 and 3
days of catheterisation The analyses of the CVCs placed in rats were performed with the
catheter inside the vein in contrast to our study where the CVC was removed from the
jugular vein for the respective analysis Xiang et al (2001) hypothesised in one study
performed in rabbits and rats in which CVCs were placed in the jugular vein that fibrin
rich thrombi associated to the CVC can be detached during the catheter removal and this
could explain the difference between the thrombosis incidence assessed in our study and
that of the study performed by Xiang et al (1998)
In our study the incidence of CVC related thrombosis detected by ultrasonography in the
lumen of the EJV did not correlate with the incidence of CVC related thrombosis detected
by ELMI on the external wall of the CVC This was mainly due to the fact that the CVC
Chapter 4 General discussion
75
segment evaluated with both methods was different Using ultrasonography it was only
possible to evaluate the EJV in the neck area of the patient and thereby only the proximal
third of the CVC In contrast with the ELMI analysis only the distal section of the CVC
(last third) was evaluated because macroscopically detected thrombi mainly developed
near the CVC tip which was routinely positioned at the junction of the cranial cava vein
with the right atrium In a retrospective review of medical records of 169 human patients
requiring a CVC (Ballard et al 2016) the position of the CVC tip was determined by
radiologic evaluation and it was determined that the catheter tip position can be a
predisposing factor for central venous thrombosis development Thereby it could be of
interest to evaluate in further studies in dogs if a high incidence of thrombosis detected
by ELMI as was shown in our study could be related to the CVC tip position
In our study polyurethane catheters were tested which are widely used and are the
standard CVCs used in the Small Animal Clinic University of Veterinary Medicine
Hannover In vivo and in vitro studies have assessed the thrombogenicity of different
catheter materials (Borow and Crowley 1985 Smith et al 2012) In general terms
polyurethane catheters (central or peripheral) had shown a higher thrombogenicity
compared with coated (eg with hydromer) and silicone catheters Due to this property
of polyurethane catheters they can be supposed as a good test system for antithrombotic
regimens as evaluated in our study On the other hand despite silicone catheters have been
associated with a lower thrombogenicity their mechanical flexibility could be a
disadvantage in small clinical practice due to the increased risk of rupture detected even
in humans (Wildgruber et al 2016)
Chapter 4 General discussion
76
Heparin thromboprophylaxis for CVC related thrombosis
The occurence of CVC related thrombosis detected by colour Doppler ultrasound and
ELMI in our study demonstrates that the UFH in the given dosage was not completely
effective to prevent CVC related thrombosis in canine intensive care patients In human
medicine LMWH is commonly used in prophylactic anticoagulation regimens however
studies performed in human patients requiring a CVC and receiving a prophylactic
anticoagulation (with LMWH or UFH) did not achieve consensus regarding the efficacy
of prophylactic heparin to prevent CVC related thrombosis (Abdelfeki et al 2004
Lordick et al 2003 Wu et al 1999) To the best of the authoracutes knowledge studies
performed in dogs evaluating systemic prophylactic anticoagulation to prevent CVC
related thrombosis cannot be found However according to the evidence found in the
human literature and the results of our study further investigations using comparative
groups with different drugs dosages andor administration routes are required to establish
an effective antithrombotic treatment to prevent CVC related thrombosis
The plasma heparin activities measured in canine patients in our study varied significantly
after a defined subcutaneous dosage The limited and variable bioavailability of SC
administered UFH is well documented in humans (Hirsh and Raschke 2004)
Considerable variations of the circulatory status of the liver- and kidney function and
concentration of heparin-binding proteins in intensive care patients obviously amplify
this phenomenon leading to an extremely high inter-individual and intra-individual
variation of the heparin blood levels in our patients A different application route (IV)
may have reduced the influence of part of these factors and therefore could have led to
more predictable UFH blood levels However continuous infusion requires more
technical equipment and is also associated with a wide range of heparin activities in
canine patients (Scott et al 2009)
Chapter 4 General discussion
77
Prediction of CVC related thrombosis
Of 7 haemostasis tests (including ROTEM with 12 different parameters [6 for each
reagent ex-tem and in-tem]) evaluated as possible predictors of CVC related thrombosis
in our study initial values of high fibrinogen and low maximum lysis (ROTEM ex-tem)
were associated with sonographically detectable thrombus formation Some studies in
humans have established the association of laboratory biomarkers as predictors of risk
thrombosis (Boersma et al 2016 Cheng et al 2013 Kato et al 2016 Liu et al 2015)
One study performed in anticoagulated human patients showed ndash well in agreement with
our results ndash that high fibrinogen levels were associated with an increased risk of
perioperative venous thromboembolism (P = 0004) (Kato et al 2016) The cited study
does not exclusively refer to CVC associated thrombosis but 762 (1621) of the
venous thromboses were suggested to be associated with CVCs Fibrinogen concentration
measurements are normally available in the human and veterinary practice and the
association of this parameter with a high risk of development of CVC related thrombosis
can be useful to choose special antithrombotic treatment in individual critical care patients
to prevent such severe complications
Thrombin generation assay (TGA) abnormalities such as higher velocity and endogenous
thrombin potential (ETP) values measured initially ie before any anticoagulation
therapy have been associated with a higher thromboembolism risk in general (ie without
specific association with CVCs) in one study performed on 105 human patients (Espitia
et al 2017) In contrast in our study the initial TGA assay results did not show an
association with formation of CVC related thrombosis
The ROTEM parameters evaluated in the present study were partially associated with
formation of CVC thrombosis detected by colour Doppler ultrasound This finding
confirms the results obtained in humans in which ROTEM measurements are regarded as
Chapter 4 General discussion
78
a valuable indicator of hypercoagulability (Harahsheh and Ho 2018 Tuumlrk et al 2018)
However in our study individual ROTEM parameters showed partly lower coagulation
activities in dogs with CVC related thrombosis detected by ELMI at the final day of
evaluation Because the number of patients without signs of thrombosis detected by ELMI
was low compared with those who showed signs these implausible results should be
interpreted carefully The usefulness of ROTEM analysis which has also many other
indications demonstrates that is was worth to perform a detailed methodological approval
of the device including the establishment of reference values in the preliminary study
Conclusion
In conclusion the prethrombotic or thrombotic changes detected in our study did not
correlate between ultrasonography and ELMI however both methods show accurately
signs of CVC related thrombosis The UFH in the given dosage was not completely
effective to prevent CVC induced thrombus formation in canine intensive care patients
Plasma heparin activities varied significantly after a defined subcutaneous dosage
However plasma heparin activities were not different between dogs with and without
detectable CVC associated thromboses Initial high fibrinogen values and low maximum
lysis (ROTEM ex-tem) values were associated with sonographically detectable thrombi
and may be useful as indicators for an increased thrombotic risk
Finally our study in canine intensive care patients is to the best of the authoracutes
knowledge the first study in the available veterinary literature performed to evaluate the
efficacy of an antithrombotic treatment with UFH to prevent CVC related thrombosis
Chapter 5 Summary
79
Chapter 5 Summary
Central venous catheter induced thrombosis in dogs ndash occurrence under an
antithrombotic treatment regimen and possible indicators
Joseacute Mauricio Pereira Mora
Central venous catheters (CVC) are of importance for intensive care management of
canine patients Thrombosis of the jugular vein is a severe complication related to the
CVC use However only little is known about the incidence of CVC-associated
thromboses in dogs and the necessity and efficacy of prophylactic antithrombotic
regimens
Therefore the aims of the present study were (1) to determine the incidence of catheter-
induced thrombosis in canine intensive care patients (2) to evaluate the efficacy of a
prophylactic heparin regimen routinely used in the Small Animal Clinic of the University
of Veterinary Medicine Hannover and (3) to assess whether selected haemostasis test
results are predictive for an increased risk for thrombosis formation
The thesis also includes a preliminary methodological study on the use of rotational
elastometry in dogs using the ROTEM delta device In this preliminary study reference
intervals were determined based on 125 healthy dogs for different clot activator reagents
and for measurements without activator Coefficients of variation (CVs) were calculated
based on results of fourfold measurements of 3 normal and 3 abnormal blood samples in
order to assess precision of the method Influence of age groups sex neuter status and
size were also assessed The median CVs for most of the parameters evaluated in the
precision analysis were less than 10 and this confirmed that the ROTEM delta analyser
delivers repeatable results Regarding the influence of age sex neuter status and animal
Chapter 5 Summary
80
size only neuter status in female dogs appeared to be a relevant influencing factor on
most of important ROTEM parameters
The main study of the thesis comprises 29 patients hospitalised at the Small Animal Clinic
requiring a CVC for medical reasons These patients received a prophylactic
anticoagulation treatment with unfractionated heparin (UFH 150 IU [surgical dogs
received 75 IU]kg BW TID subcutaneously) A colour Doppler ultrasound of the
respective external jugular vein (EJV) in order to detect thrombus formation in the lumen
of the EJV and blood collection for haemostasis tests were performed directly before the
installation procedure (day 0) and on days 1 3 5 7 and so on after the CVC insertion
Haemostasis tests included prothrombin time activated partial thromboplastin time
thrombin time antithrombin activity D-dimer concentration thrombin generation and
rotational elastometry as well as the heparin activity using a chromogenic anti-factor Xa
test Finally when the CVC was removed electron microscopy (ELMI) of sections of the
CVC with macroscopically visible thrombi in the last third (5ndash10 cm) of the CVC was
performed 3 different grades were defined for CVC-associated (extraluminal) thrombi
detected by either ultrasound or ELMI respectively
829 patients evaluated by colour Doppler ultrasound during the study developed thrombi
into the lumen of EJV (4 x grade 1 3 x grade 2 and 1 x grade 3) 1927 CVCs assessed
using ELMI showed detectable thrombi on the external CVC surface (7 x grade 1 4 x
grade 2 and 8 x grade 3) There was no significant correlation between thrombus
formation detected by ultrasound or ELMI Thus statistical group comparisons of
patients with or without CVC-associated thrombus formation was performed
independently for both diagnostic procedures The plasmatic heparin activities showed
great variability but no significant differences were found between dogs with or without
CVC-associated thrombus formation regardless whether this was based on sonographic
Chapter 5 Summary
81
or ELMI findings From the initially performed haemostasis tests high fibrinogen
concentrations and low maximum lysis values as measured by the ROTEM delta device
(using the ex-tem reagent) were associated with sonographically detectable thrombi
In conclusion the preliminary methodological study on the ROTEM delta in dogs shows
that the method delivers repeatable results in canine citrated whole blood and the
reference values obtained should offer a reliable basis for the interpretation of test results
of this device in dogs with suspected haemostasis disorders The results of the main study
indicate that CVC-associated thromboses occur (eg 28 [829] of the patients as
detected by ultrasound) although severe thromboses (high grades) are relatively rare in
intensive care canine patients receiving a routine anticoagulant treatment Thereby
prophylactic antithrombotic protocol (unfractionated heparin [UFH]) in the given dosage
was not completely effective to prevent CVC induced thrombus formation in canine
intensive care patients A high fibrinogen concentration and low maximum lysis
(ROTEM ex-tem) may be useful indicators for an increased thrombotic risk in individual
patients which may require special anticoagulatory treatment Further studies are
required to evaluate whether different thromboprophylactic treatment regimens eg with
low molecular weight heparin are more effective in preventing CVC-associated
thromboses in dogs
Chapter 6 Zusammenfassung
82
Chapter 6 Zusammenfassung
Zentralvenenkatheter-induzierte Thrombose bei Hunden - Auftreten unter einem
antithrombotischen Behandlungsschema und moumlgliche Indikatoren
Joseacute Mauricio Pereira Mora
Zentralvenenkatheter (ZVK) sind fuumlr das Intensivmanagement von Hunden von
Bedeutung Die Entstehung einer Thrombose der Vena jugularis ist eine schwerwiegende
Komplikation im Zusammenhang mit der Verwendung eines ZVKs Uumlber die Inzidenz
von ZVK-assoziierten Thrombosen bei Hunden und die Notwendigkeit und Wirksamkeit
prophylaktischer antithrombotischer Protokolle liegen jedoch nur wenige fundierte
Angaben in der Literatur vor
Daher waren die Ziele der vorliegenden Studie (1) die Inzidenz der Katheter-induzierten
Thrombose bei caninen Intensivpatienten zu bestimmen (2) die Wirksamkeit eines
prophylaktischen Heparin-Regimes zu bewerten das routinemaumlszligig in der Klinik fuumlr
Kleintiere der Stiftung Tieraumlrztliche Hochschule Hannover angewendet wird und (3) um
zu beurteilen ob ausgewaumlhlte Haumlmostasetestergebnisse ein erhoumlhtes Risiko fuumlr die
Thrombosebildung vorhersagen koumlnnen
Die PhD-Arbeit enthaumllt zudem eine vorbereitende methodische Studie zur Verwendung
der Rotationselastometrie bei Hunden mit Hilfe des ROTEM delta-Geraumltes In dieser
Vorstudie wurden Referenzintervalle basierend auf 125 gesunden Hunden fuumlr
verschiedene Gerinnungsaktivatorreagenzien und fuumlr Messungen ohne Aktivator
bestimmt Um die Genauigkeit der Methode einzustufen wurden Variationskoeffizienten
(VKs) basierend auf den Ergebnissen von Vierfachmessungen von drei normalen und drei
abnormalen Blutproben berechnet Der Einfluss von Alter Geschlecht Kastration und
Groumlszlige wurde ebenfalls getestet Die Medianwerte der VKs fuumlr die meisten in der
Chapter 6 Zusammenfassung
83
Praumlzisionsanalyse bewerteten Parameter betrugen weniger als 10 und dies bestaumltigte
dass das ROTEM delta-Analysegeraumlt wiederholbare Ergebnisse liefert In Bezug auf den
Einfluss von Alter Geschlecht Kastration und Tiergroumlszlige erwies sich nur eine Kastration
bei Huumlndinnen als relevanter Einflussfaktor fuumlr die meisten wichtigen ROTEM-
Parameter
Die Hauptstudie der Arbeit umfasst 29 Patienten welche zum Zeitpunkt der
Untersuchung stationaumlr in der Kleintierklinik aufgenommen worden waren und aus
medizinischer Indikation einen ZVK benoumltigten Diese Patienten erhielten eine
prophylaktische Antikoagulation mit unfraktioniertem Heparin (UFH 150 IE
[chirurgische Hunde erhielten 75 IE]kg KG 3 x taumlglich subkutan) Bildgebende
Untersuchungen mittels Farbdoppler-Ultraschall der jeweiligen Vena jugularis externa
(VJE) zum Nachweis der Thrombusbildung im Lumen der VJE und Blutentnahmen fuumlr
Haumlmostasetests wurden jeweils direkt vor dem Installationsvorgang (Tag 0) sowie an den
Tagen 1 3 5 und 7 nach Legen des ZVKs durchgefuumlhrt Haumlmostasetests umfassten die
Prothrombinzeit die aktivierte partielle Thromboplastinzeit die Thrombinzeit die
Antithrombinaktivitaumlt die D-Dimer-Konzentration die Thrombingenerierung und die
Rotationselastometrie sowie die Heparinaktivitaumlt unter Verwendung eines chromogenen
Anti-Faktor-Xa-Tests Schlieszliglich wurde nach Entfernung des ZVKs eine
Elektronenmikroskopie (ELMI) von Abschnitten aus dem letzten Drittel (5ndash10 cm) des
ZVKs mit makroskopisch sichtbaren Thromben durchgefuumlhrt Fuumlr ZVK-assoziierte
(extraluminale) Thromben die entweder durch Ultraschall oder ELMI nachgewiesen
wurden wurden drei verschiedene Grade definiert
829 Patienten die waumlhrend der Studie mittels Farbdoppler-Ultraschall untersucht
wurden entwickelten Thromben im Lumen der VJE (4 x Grad 1 3 x Grad 2 und 1 x Grad
3) 1927 ZVKs die unter Verwendung von ELMI bewertet wurden zeigten
Chapter 6 Zusammenfassung
84
nachweisbare Thromben auf der aumluszligeren ZVK-Oberflaumlche (7 x Grad 1 4 x Grad 2 und 8
x Grad 3) Es gab keine signifikante Korrelation zwischen der durch Ultraschall oder
ELMI festgestellten Thrombusbildung Daher wurden statistische Gruppenvergleiche
von Patienten mit oder ohne ZVK-assoziierter Thrombusbildung fuumlr beide diagnostische
Verfahren unabhaumlngig voneinander durchgefuumlhrt Die plasmatischen Heparinaktivitaumlten
zeigten eine groszlige Variabilitaumlt es wurden jedoch keine signifikanten Unterschiede
zwischen Hunden mit oder ohne ZVK-assoziierter Thrombusbildung gefunden
unabhaumlngig davon ob diese auf sonographischen oder ELMI-Befunden beruhte Von den
initial durchgefuumlhrten Haumlmostasetests waren hohe Fibrinogenkonzentrationen und
niedrige maximale Lyse-Werte der Messungen mit ROTEM delta (ex-tem Reagenz) mit
sonographisch nachweisbaren Thromben assoziiert
Zusammenfassend zeigt die vorbereitende methodische Studie zum ROTEM delta bei
Hunden dass die Methode wiederholbare Ergebnisse in mit Zitrat
antikoaguliertemVollblut liefert Die erarbeiteten Referenzwerte sollten eine zuverlaumlssige
Grundlage fuumlr die Interpretation der Testergebnisse dieses Geraumlts bei Hunden mit
Verdacht auf Haumlmostasestoumlrungen bieten Die Ergebnisse der Hauptstudie zeigen dass
ZVK-assoziierte Thrombosen auftreten (z B 28 [829] der Patienten bezogen auf den
Ultraschall-gestuumltzten Nachweis) obwohl schwere Thrombosen (hohe Grade) bei
intensivmedizinisch versorgten Hundepatienten die eine Routineantikoagulation
erhalten relativ selten sind Somit war das prophylaktische antithrombotische Protokoll
(unfraktioniertes Heparin [UFH]) in der angewendeten Dosierung nicht vollstaumlndig
wirksam um die ZVK-induzierte Thrombusbildung bei caninen Intensivpatienten zu
verhindern Eine hohe Fibrinogenkonzentration und eine niedrige maximale Lyse
(ROTEM ex-tem) koumlnnen als Indikatoren eines erhoumlhten Thromboserisikos bei einzelnen
Patienten nuumltzlich sein fuumlr die moumlglicherweise eine spezielle gerinnungshemmende
Chapter 6 Zusammenfassung
85
Behandlung notwendig ist Weitere Studien sind erforderlich um zu untersuchen ob
verschiedene thromboprophylaktische Behandlungsschemata z B mit
niedermolekularem Heparin bezuumlglich der Verhinderung von ZVK-assoziierten
Thrombosen bei Hunden wirksamer sind
Chapter 7 References
86
Chapter 7 References
1 Abdelkefi A Ben Othman T Kammoun L Chelli M Ben Romdhane N Kriaa A Ladeb
S Torjman L Lakhal A Achour W Ben Hassen A Hsaiumlri M Ladeb F Ben Abdeladhim
A 2004 Prevention of central venous line-related thrombosis by continuous infusion of low-dose
unfractionated heparin in patients with haemato-oncological disease A randomized controlled
trial Thromb Haemost 92 654ndash661
2 Adamantos S Brodbelt D Moores A L 2010 Prospective evaluation of complications
associated with jugular venous catheter use in a veterinary hospital J Small Anim Pract 51 254ndash
257
3 Ballard D H Samra N S Gifford K M Roller R Wolfe B M Owings J T 2016
Distance of the internal central venous catheter tip from the right atrium is positively correlated
with central venous thrombosis Emerg Radiol 23 269ndash273
4 Baumann-Kreuziger L Onwuemene O Kolesar E Crowther M Lim W 2015 Systematic
review of anticoagulant treatment of catheter-related thrombosis Thromb Res 136 1103ndash1109
5 Bliss SP Bliss SK Harvey HJ 2002 Use of recombinant tissue‐plasminogen activator in a
dog with chylothorax secondary to catheter‐associated thrombosis of the cranial vena cava J Am
Anim Hosp Assoc 38 431ndash435
6 Boersma RS Hamulyak K van Oerle R Tuinenburg A Ten Cate-Hoek AJ Schouten
HC 2016 Biomarkers for prediction of central venous catheter related-thrombosis in patients
with hematological malignancies Clin Appl Thromb Hemost 22 779ndash784
7 Boersma RS Jie KS Verbon A van Pampus EC Schouten HC 2008 Thrombotic and
infectious complications of central venous catheters in patients with hematological
malignancies Ann Oncol 19 433ndash442
8 Bonnet F Loriferne JF Texier JP Texier M Salvat A Vasile N 1989 Evaluation of
Doppler examination for diagnosis of catheter-related deep vein thrombosis Intensive Care Med
15 238ndash240
9 Borow M Crowley J G 1985 Evaluation of central venous catheter thrombogenicity Acta
Anaesthesiol Scand Suppl 81 59ndash64
10 Cheng Y Cui T Fu P Liu F Zhou L 2013 Dyslipidemia is associated with tunneled-cuffed
catheter-related central venous thrombosis in hemodialysis patients a retrospective multicenter
Chapter 7 References
87
study Artif Organs 37 155ndash161
11 Comerlato PH Rebelatto TF Santiago de Almeida FA Klein LB Boniatti MM
Schaan BD Rados DV 2017 Complications of central venous catheter insertion in a teaching
hospital Rev Assoc Med Bras 63 613ndash620
12 De Cicco M Matovic M Balestreri L Steffan A Pacenzia R Malafronte M Fantin
D Bertuzzi CA Fabiani F Morassut S Bidoli E Veronesi A 2009 Early and short‐term
acenocumarine or dalteparin for the prevention of central vein catheter‐related thrombosis in
cancer patients a randomized controlled study based on serial venographies Ann
Oncol 20 1936ndash1942
13 Espitia O Fouassier M Hardouin JB Pistorius MA Agard C Planchon B Trossaert M
Pottier P 2017 Thrombin generation assay in hospitalized nonsurgical patients A new tool to
assess venous thromboembolism risk Clin Appl Thromb Hemost 23 45ndash51
14 Evans NS Ratchford EV 2018 Catheter related-venous thrombosis Vasc Med 23 411ndash413
15 Geerts W 2014 Central Venous Catheter-related thrombosis Hematology Am Soc Hematol
Educ Program 2014 306ndash311
16 Harahsheh Y Ho KM 2018 Use of viscoelastic tests to predict clinical thromboembolic
events A systematic review and meta‐analysis Eur J Haematol 100 113ndash123
17 Harter C Salwender HJ Bach A Egerer G Goldschmidt H Ho AD 2002 Catheter-
related infection and thrombosis of the internal jugular vein in hematologic-oncologic patients
undergoing chemotherapy a prospective comparison of silver-coated and uncoated
catheters Cancer 94 245ndash251
18 Hirsh J Raschke R 2004 Heparin and low-molecular-weight heparin The Seventh ACCP
Conference on Antithrombotic and Thrombolytic Therapy Chest 126 (Suppl) 188Sndash203S
19 Karthaus M Kretzschmar A Kroning H Biakhov M Irwin D Marschner N Slabber
C Fountzilas G Garin A Abecasis NG Baronius W Steger GG Sudhoff T Giorgetti
C Reichardt P 2006 Dalteparin for prevention of catheter‐related complications in cancer
patients with central venous catheters final results of a double‐blind placebo‐controlled phase III
trial Ann Oncol 17 289ndash296
20 Kato F Takeuchi H Matsuda S Kawakubo H Omori T Kitagawa Y 2016 Incidence of
and risk factors for venous thromboembolism during surgical treatment for esophageal cancer a
Chapter 7 References
88
single-institution study Surg Today 46 445ndash452
21 Kohler TR Kirkman TR 1998 Central venous catheter failure is induced by injury and can
be prevented by stabilizing the catheter tip J Vasc Surg 28 59ndash66
22 Koumlksoy C Kuzu A Kutlay J Erden I Ozcan H Ergicircn K 1995 The diagnostic value of
colour Doppler ultrasound in central venous catheter related thrombosis Clin Radiol 50 687ndash
689
23 Kucher N 2011 Clinical practice Deep-vein thrombosis of the upper extremities N Engl J
Med 364 861ndash869
24 Langston CE Eatroff AE 2018 Hemodialysis catheter-associated fibrin sheath in a dog J
Vet Emerg Crit Care 28 366ndash371
25 Langston CE Eatroff AE Poeppel K 2014 Use of tissue plasminogen activator in catheters
used for extracorporeal renal replacement therapy J Vet Intern Med 28 270ndash276
26 Laurenson MP Hopper K Herrera MA Johnson EG 2010 Concurrent diseases and
conditions in dogs with splenic vein thrombosis J Vet Intern Med 24 1298ndash1304
27 Lee AYY Kamphuisen PW 2012 Epidemiology and prevention of catheter‐related
thrombosis in patients with cancer J Thromb Haemost 10 1491ndash1499
28 Liu G Fu ZQ Zhu P Li SJ 2015 Central venous catheter-related thrombosis in senile male
patients New risk factors and predictors J Huazhong Univ Sci Technolog Med Sci 35 445ndash
449
29 Lordick F Hentrich M Decker T Hennig M Pohlmann H Hartenstein R Peschel C
2003 Ultrasound screening for internal jugular vein thrombosis aids the detection of central
venous catheter-related infections in patients with haemato-oncological diseases a prospective
observational study Br J Haematol 120 1073ndash1078
30 Lucas TC Silva EED Souza DOD Santos ARD Lara MO 2017 Microstructural
evaluation by confocal and electron microscopy in thrombi developed in central venous catheters
Rev Esc Enferm USP 51 1ndash9
31 Mismetti P Mille D Laporte S Charlet V Buchmuller‐Cordier A Jacquin JP Fournel
P Dutrey‐Dupagne C Decousus H 2003 Low‐molecular‐weight heparin (nadroparin) and
very low doses of warfarin in the prevention of upper extremity thrombosis in cancer patients with
indwelling long‐term central venous catheters a pilot randomized trial Haematologica 88 67ndash
Chapter 7 References
89
73
32 Niers TM Di Nisio M Klerk CP Baarslag HJ Buller HR Biemond BJ 2007
Prevention of catheter‐related venous thrombosis with nadroparin in patients receiving
chemotherapy for hematologic malignancies a randomized placebo‐controlled study J Thromb
Haemost 5 1878ndash1882
33 Scott K C Hansen B D DeFrancesco T C (2009) Coagulation effects of low molecular
weight heparin compared with heparin in dogs considered to be at risk for clinically significant
venous thrombosis J Vet Emerg Crit Care 19 74ndash80
34 Smith RS Zhang Z Bouchard M Li J Lapp HS Brotske GR Lucchino DL Weaver
D Roth LA Coury A Biggerstaff J Sukavaneshvar S Langer R Loose C 2012
Vascular catheters with a nonleaching poly-sulfobetaine surface modification reduce thrombus
formation and microbial attachment Sci Transl Med 4 153
35 Tuumlrk S M Cansu D Uuml Teke H Uuml Kaşifoğlu T Meltem Akay O Bilgin M amp Korkmaz
C 2018 Can we predict thrombotic tendency in rheumatoid arthritis A thromboelastographic
analysis (with ROTEM) Clin Rheumatol 37 2341ndash2349
36 van Rooden CJ Rosendaal FR Barge RM van Oostayen JA van der Meer FJM
Meinders AE Huisman MV 2003 Central venous catheter related thrombosis in haematology
patients and prediction of risk by screening with Doppler-ultrasound Br J Haematol 123 507ndash
512
37 van Rooden CJ Tesselaar ME Osanto S Rosendaal FR Huisman MV 2005 Deep vein
thrombosis associated with central venous catheters - a review J Thromb Haemost 3 2409ndash
2419
38 Verso M Agnelli G Bertoglio S Di Somma FC Paoletti F Ageno W Bazzan M Parise
P Quintavalle R Naglieri E Santoro A Imberti D Soraru M Mosca S 2005 Enoxaparin
for the prevention of venous thromboembolism associated with central vein catheter a double‐
blind placebo‐controlled randomized study in cancer patients J Clin Oncol 23 4057ndash4062
39 Vose J Odunayo A Price JM Daves M Schildt JC Tolbert MK 2019 Comparison of
heparinized saline and 09 sodium chloride for maintaining central venous catheter patency in
healthy dogs PeerJ 7e7072 httpdoi107717peerj7072
40 Wang LH Wei F Jia L Zhi Lu Wang B Dong HY Yu HY Sun GJ YangJ Li B
Chapter 7 References
90
Meng J Zhang RN Bi XQ Chen HY Jiang AL 2015 Fibrin sheath formation and
intimal thickening after catheter placement in dog model role of hemodynamic wall shear stress J
Vasc Access 16 275ndash284
41 Wildgruber M Lueg C Borgmeyer S Karimov I Braun U Kiechle M Meier R Koehler
M Ettl J Berger H 2016 Polyurethane versus silicone catheters for central venous port devices
implanted at the forearm Eur J Cancer 59 113ndash124
42 Whitesell RT Steenburg SD 2014 Imaging findings of acute intravascular thrombus on non-
enhanced computed tomography Emerg Radiol 21 271ndash277
43 Wu X Studer W Skarvan K Seeberger MD 1999 High incidence of intravenous thrombi
after short-term central venous catheterization of the internal jugular vein J Clin Anesth 11
482ndash485
44 Xiang DZ Verbeken EK Van Lommel AT Stas M De Wever I 1998 Composition and
formation of the sleeve enveloping a central venous catheter J Vasc Surg 28 260ndash271
45 Xiang DZ Verbeken EK Van Lommel AT Stas M De Wever I 2001 Sleeve-related
thrombosis a new form of catheter-related thrombosis Thromb Res 104 7ndash14
91
Acknowledgements
First I thank God for every day in my life for His guide and because He was is
and will be always with me and that certainty gives me strength to take a step
forward
My sincere thanks to Prof Dr Reinhard Mischke for the opportunity to perform
my PhD under his supervision for his support and guidance for all that I learned
with him and for his friendship
I also thank to Prof Dr Manfred Kietzmann and PD Dr Mario von Depka
Prondzinski for being part of my supervision group for their time and beneficial
advices and discussion during my PhD
My gratitude to Prof Hewicker-Trautwein and Mrs Rohn for their valuable work
and availability to help me
I thank the Werlhof Institute Medical Care Center Hannover for the valuable
cooperation especially to Mrs Ober-Cassebaum Anne and Franziska for all their
help
I thank all the staff of the Small Animal Clinic of the University of Veterinary
Medicine Hannover (TiHo) My special gratitude to the internal medicine and
critical care surgery neurology ultrasound and anesthesia departments and to
the TiHo students all their cooperation made possible my research
I thank the haematology clinical chemistry and haemostasis laboratory
department of the Small Animal Clinic TiHo especially to Dirk because he was
always available for me thanks for his guide and friendship all his explanations
in the laboratory were of great value but also the conversations about different
topics of the life were of big value for me
92
I thank the Hannover Graduate School for Veterinary Pathobiology
Neuroinfectology and translational Medicine (HGNI) for the well-organized
PhD program and especially thanks to Prof Dr Beatrice Grummer Dr Tina
Selle and Mrs Tania Czeslik for his guide and help during my studies
My sincere thanks to Mrs Maritta Ledwoch for her guidance at the International
office of the TiHo ldquoI remember you since my externship in Germanyrdquo
I also thank the Student Union Hannover for all the help with the accommodation
for me and my family
I want to thank the Universidad Nacional de Costa Rica and therefore to Costa
Rica (my loved country) for the financial support to perform my PhD studies
but also for my professional formation as a veterinarian Special thanks to the
Veterinary Medicine School from Universidad Nacional de Costa Rica To all the
academic and administrative staff for their support and for believing in me
Sincerely thanks to Dr Mauricio Jimenez Soto my mentor my adviser but most
important my friend Thanks for believing in me and for always being by my side
ldquoAgradezco profundamente a mis papaacutes ldquodon Tito (Papillo) y dontildea Eida (Mami)rdquo
Todo lo bueno que hay en mi proviene de Dios y de ellos Gracias a mis hermanos
(as) cuntildeados (as) sobrinos y a toda la familia relacionada a ellos Siempre han
sido un gran apoyo y los amordquo
ldquoGracias a dontildea Alba Fran y David Gracias por toda la ayuda y por estar siempre
pendientesrdquo
I would like to thank all my friends that during a short part or during my whole
life have been with me For all the good time shared really thanks
To Willy thanks ldquogordordquo
93
I sincerely thank to Adriano and Johanna for their help and hospitality Thanks
Adriano for your guidance and support during my beginning at the TiHo for make
me feel in Costa Rica with your friendship for the tourist walks and for the bike
training I missed the coffee times
I want to thank Ina the first person to give me a smile at the start of my PhD
Thanks for your hugs and friendship
I also thank Franz for his help and for being a very nice partner Thanks for help
me always when I requested you
ldquoFinalmente quiero expresar mi gratitud y amor a Karo Faacutetima y Abigaiacutel A Karo
por tu sacrificio y soporte por las palabras de aliento por estar a mi lado en las
buenas pero en especial en las malas A Faacute por tu sacrificio por tus sonrisas por
tus abrazos y besos por bailar con migo siempre por ser mi modelo e inspiracioacuten
A Abi por ser una nueva razoacuten in mi vida para seguir adelante por todo lo que
deseo compartir con tigo por mostrarme junto con Faacutetima la felicidad en una
sonrisardquo
Table of contents
4 Discussion 62
5 Conclusions 67
6 Acknowledgments 67
7 References 68
Chapter 4 General discussion 72
Incidence of CVC related thrombosis 72
Heparin thromboprophylaxis for CVC related thrombosis 76
Prediction of CVC related thrombosis 77
Conclusion 78
Chapter 5 Summary 79
Chapter 6 Zusammenfassung 82
Chapter 7 References 86
Acknowledgements 91
List of abbreviations
i
List of abbreviations
CV coefficient of variation
CVC central venous catheter
EJV external jugular vein
ETP endogenous thrombin potential
HDL high-density lipoprotein
IV intravenous
LDL low-density lipoprotein
LMWH low molecular weight heparin
ROTEM rotational elastometry
SC subcutaneous
TGA thrombin generation assay
UFH unfractionated heparin
Chapters 2 and 3 have their own abbreviations included
List of figures and tables
ii
List of figures and tables
Chapter 2
Table 1 Coefficients of variation () for rotational thromboelastometry using the
ROTEMreg delta analyser in normal blood samples 20
Table 2 Coefficients of variation () for rotational thromboelastometry using the
ROTEMreg delta analyser in canine blood samples with haemostatic alterations 20
Table 3 Reference values (median reference interval based on 25 - and 975 -
quantiles) for thromboelastometric measurements with the ROTEMreg delta analyser in
dogs (n=125 95 confidence intervals in brackets) 23
Table 4 Median values (minimum and maximum in brackets) for different ROTEMreg
variables using ex-tem reagent stratified by sex and neuter status 24
Table 5 Median values (minimum and maximum in brackets) for different ROTEMreg
variables using the in-tem reagent stratified by sex and neutering status 25
Chapter 3
Fig 1 Definition of different grades of central venous catheter induced thrombosis of the
jugular vein as detected using colour Doppler ultrasound 48
Fig 2 Definition and illustration of different grades of central venous catheter induced
(extraluminal) thrombosis as detected using electron microscopy 53
Fig 1S (suppl material) Definition and illustration of different grades of intraluminal
thrombosis in central venous catheters as detected using electron microscopy 53
Table 1S (Supplementary material) Heparin activities (IUml median values
minimumndashmaximum) measured in 27 dogs receiving 75 or 150 IUkg unfractionated
(standard) heparin TID subcutaneously 2 hours after the first injection on the day
Comparison of samples collected via puncture of the peripheral veins and those collected
from the central venous catheter (CVC) using the three-syringe-technique 56
List of figures and tables
iii
Fig 3ab Heparin activities (box- and whisker plot arithmetic means) in dogs receiving
unfractionated heparin at a dosage of 75 IUkg BW tid (a) or 150 IUkg BW tid (b) two
hours after the first daily heparin administration 57
Table 1 Heparin activities (IUml median values minimumndashmaximum) in 28 dogs
receiving 75 or 150 IUkg unfractionated (standard) heparin TID subcutaneously with
(grades 1ndash3) or without (grade 0) sonographically detectable thrombus Blood collections
were performed 2 hours after the first daily injection 57
Table 2 Heparin activities (IUml median values minimumndashmaximum) in 27 dogs
receiving 75 or 150 IUkg unfractionated (standard) heparin TID subcutaneously with
(score 1ndash3) or without (score 0) detectable thrombus via electron microscopy 58
Table 3 Comparison of initial results of haemostasis parameters (median values
minimumndashmaximum in dogs with (grades 1ndash3) and without (grade 0) sonographically
detectable central venous catheter-induced thromboses 60
Table 4 Comparison of results of haemostasis parameters (median values minimumndash
maximum) on the last day in dogs with (grades 1ndash3) and without (grade 0) central venous
catheter-associated thrombus formation as detected by electron microscopy 61
Chapter 1 Introduction
1
Chapter 1 Introduction
Central venous catheters (CVC) are mainly used at critical care units in human and small
animal patients requiring infusion of large amounts of fluids or blood parenteral nutrition
support routine drug therapy haemodialysis chemotherapy plasmapheresis or
periodical blood sampling (Geerts 2014 Vose et al 2019) CVC related thrombosis is
one of the most severe complications (apart from infections and pneumothorax) of CVCs
in humans (Comerlato et al 2017 Evans and Ratchford 2018 Geerts 2014) leading to
patientrsquos distress CVC dysfunction venous stenosis and in some cases to the
development of thromboembolism (Geerts 2014) In the human literature there are
various studies dealing with epidemiologic and prophylactic aspects which are described
below in detail In contrast to the best of the authoracutes knowledge studies about extra
luminal CVC related thrombosis in dogs are rare in the available veterinary literature
Studies are mainly dealing with development of intraluminal CVC thrombi occurring
frequently in dogs and cats (Langston et al 2014) and leading to CVC malfunction (Vose
et al 2019)
Epidemiology of CVC related thrombosis
CVC related thrombosis represents 70ndash80 of all upper extremity thrombosis cases and
approximately 10 of thromboembolism cases in humans (Kucher 2011) The incidence
of CVC related thrombosis reported in humans based in 25 studies ranged widely from
2 to 67 (median = 30 ) (van Rooden et al 2005) The incidence of CVC related
thrombosis detected by colour Doppler ultrasound of the internal jugular vein reported in
3 studies performed in humans was 2 (Harter et al 2002) 30 (Lordick et al 2003)
and 56 (Wu et al 1999)
Chapter 1 Introduction
2
The incidence of CVC related thrombosis in humans can vary between studies according
to the CVC type used entry site of the CVC (jugular femoral subclavian axillary
cephalic vein) and time period with the CVC in place the diagnostic thrombus detection
technique (venography or ultrasonography) and criteria for diagnosis duration of follow
up diseases and presence of predisposing factors (eg previous venous thrombosis
history presence of cancer) and finally prophylactic anticoagulation treatment (Geerts
2014 Liu et al 2015 van Rooden et al 2005)
To the best of the authoracutes knowledge data about the incidence of CVC related
thrombosis detected by colour Doppler ultrasound into the lumen of the jugular vein in
dogs are rare in the available veterinary literature In one study performed in 81 dogs and
12 cats (Adamantos et al 2010) the incidence of vein thrombosis detected by palpation
or ultrasound was 2 and the incidence of intraluminal CVC thrombosis (presumed by
the inability to draw blood back) was 15 Additionally regarding to intraluminal CVC
thrombosis an incidence of 8 was reported in one case control study performed in 24
dogs receiving CVC flushing with heparinised solution (Vose et al 2019) Data about
incidence in other animal species was also found In another case-control study performed
on 37 pigs thrombosis in the caval vein related to CVC placed in the external jugular
vein (EJV) was evaluated by histological analysis however no ultrasound detection was
performed (Kohler and Kirkman 1998) In this study in pigs 94 of patients with CVC
developed thrombi in the vena cava based on sections at the site of the end of the catheter
tip and 05 and 1 cm proximal of the first section (Kohler and Kirkman 1998)
Pathogenesis of CVC related thrombosis
Two types of CVC related thrombosis are distinguished in the human medicine literature
(Geerts 2014 van Rooden et al 2005) First symptomatic thrombosis is defined as
Chapter 1 Introduction
3
thrombosis detected by diagnostic images (ultrasonography venography) when
symptoms and signs (eg localised swelling pain redness) are present (Lee and
Kamphuisen 2012) Second subclinical thrombosis is defined as thrombosis
demonstrated by the previously mentioned diagnostic images without overt symptoms or
signs (Boersma et al 2008) Additionally the CVC related thrombi can be classified as
occlusive thrombus into the CVC lumen peri-catheter related fibrin sheaths and mural
thrombi (Geerts 2014)
During the CVC insertion the puncture of the skin on the CVC insertion site and the
rupture of the blood vessels integrity activate the coagulation cascade and the CVC acts
as a matrix for adherence and accumulation of fibrin and cellular components (Smith et
al 2012) The attachment of proteins and blood cells to the internal andor external wall
of the CVC can lead to (1) (partial or complete) occlusion of the catheter lumen and (2)
sometimes if a thrombus formation starts into the lumen of the vein (attached to the vein
wall or to the CVC) a partial or complete obstruction of the blood flow andor embolism
can develop (Langston and Eatroff 2018) Additionally a study performed in humans
(Lordick et al 2003) demonstrated a relationship between CVC related thrombosis and
microbial colonisation of the catheter leading to CVC related systemic infections This
relationship was explained among others by the ease of microorganisms like
Staphylococcus aureus and Staphylococcus epidermidis to adhere to CVC related
thrombin sheaths (Boersma et al 2008)
Diagnosis of CVC related thrombosis
Venous thrombosis is commonly diagnosed in human and small animal medicine
(especially in dogs) with vascular ultrasonography with or without use of the colour
Doppler technique which is normally used to evaluate the blood flow through the blood
Chapter 1 Introduction
4
vessels (Evans and Ratchford 2018 Laurenson et al 2010) According to the literature
in humans cited below the CVC related thrombosis detected by colour Doppler
ultrasound is present if any of the following aspects are observed (1) well defined
thrombus visualised in the lumen of the vein (2) impossibility to perform compression
of the vein by slight pressure with the ultrasound probe andor (3) no detectable blood
flow signal by colour Doppler ultrasound (Bonnet et al 1989 Koumlksoy et al 1995)
Similarly in dogs the CVC related thrombosis is usually diagnosed by colour Doppler
ultrasonography through the detection of the thrombus within the lumen of the vein
andor the obstruction (partial or complete) of the normal blood flow (Bliss et al 2002)
Especially in humans the contrast venography technique is used as gold standard to
confirm thrombosis For this purpose a contrast solution is injected through a peripheral
vein to assess its passage through the bloodstream by x-ray However this diagnostic
technique is normally used only in the case of a strong clinical suspicion of venous
thrombosis that cannot be detected by ultrasonography (Laurenson et al 2010 Langston
and Eatroff 2018) In a study performed in 44 human patients requiring a CVC (Koumlksoy
et al 1995) a sensitivity of 94 and a specificity of 96 were determined for the colour
Doppler ultrasound diagnosis when it was compared with the diagnosis performed with
the contrast venography technique That results make the colour Doppler ultrasound an
accurate diagnosis method to detect CVC related thrombosis in humans (Koumlksoy et al
1995 van Rooden et al 2003)
Advanced diagnostic image techniques like computed tomography or magnetic resonance
tomography are also accurate to perform the diagnosis of CVC related thrombosis in
humans (Whitesell and Steenburg 2014) In dogs multidetector computed tomography
venography was used in one study for the diagnosis of CVC related thrombosis allowing
the identification of catheter related sheaths around the CVC external wall (Wang at al
Chapter 1 Introduction
5
2015) However the high costs of these advanced diagnostic imaging procedures and the
requirement to perform at least sedation or general anesthesia are disadvantages when
compared with ultrasonography
Finally electron microscopy (ELMI) is a diagnostic technique that has been used in
humans (Lucas et al 2017) and experimental animals (Xiang et al 1998 Xiang et al
2001) mainly to perform a microstructural evaluation of thrombi developed into the
lumen of the CVC or in the vein containing the CVC ELMI allows the ex vivo diagnosis
of very small thrombi or sleeve related thrombosis that perhaps cannot be detected by
ultrasonography or venography during the evaluation of patients with CVCs (Xiang et al
2001) However one point to consider is that it is possible that compact thrombi
developed on the CVC can be detached during the CVC removal from the vein and
thereby before its analysis by ELMI (Xiang et al 2001)
Heparin thromboprophylaxis for CVC related thrombosis
Application of CVCs in human medicine is commonly accompanied by prophylactic
anticoagulation in order to decrease the risk of development of partial or complete vein
occlusion andor thromboembolism and thereby to improve the patientrsquos comfort
(Baumann-Kreuziger et al 2015) Apart from vitamin K antagonists (Mismetti et al
2003) unfractionated heparin (UFH) (Abdelfeki et al 2004) and low molecular weight
heparin (LMWH) (Karthaus et al 2006) are anticoagulants that have been used on
studies in humans administrated by intravenous (IV) infusion or by subcutaneous (SC)
injection to prevent thrombi development into the lumen of the veins containing the CVC
(review article Baumann-Kreuziger et al 2015) Different heparin dosages have been
used as thromboprophylactic anticoagulation in humans with CVC (review article Lee
and Kamphuisen 2012) For example in five different case-control studies performed in
Chapter 1 Introduction
6
human haemato-oncological patients UFH at a daily dosage of 100 IUkg given by IV
infusion (Abdelfeki et al 2004) and LMWH at SC daily dosages of 40 mg enoxaparin
(Verso et al 2005) 5000 IU AXa dalteparin (Karthaus et al 2006 De Cicco et al 2009)
and 2850 IU AXa nadroparin (Niers et al 2007) were used Only in part of these studies
it is stated that systemic heparin was combined with flushing of the CVC using
heparinised saline solution to reduce the risk of catheter occlusion (Karthaus et al 2006)
In 3 of the 5 previously mentioned studies no statistically significant difference was found
in the incidence of CVC related thrombosis between patients receiving the
anticoagulation prophylaxis compared with the control groups without systemic
anticoagulation (Verso et al 2005 p = 035 Karthaus et al 2006 p gt 005 Niers et al
2007 p = 049) In contrast in the residual two studies a lower incidence of CVC related
thrombosis was found in the group receiving an anticoagulation prophylaxis compared
with the control groups (Abdelfeki et al 2004 150 vs 126 p = 003 De Cicco et
al 2009 400 vs 526 p = 005)
Obviously there is no consensus in the available human literature regarding the beneficial
use of anticoagulation prophylaxis to prevent CVC related thrombosis and this may
reflect great variations for example according to the study design period trial drug
administration route (Lee and Kamphuisen 2012) and the moment of the initial
anticoagulant dosage administration (Decicco et al 2009)
To the best of the authoracutes knowledge studies about thromboprophylaxis for extraluminal
CVC related thrombosis in dogs cannot be found in the available veterinary literature
However similarly to humans prophylactic anticoagulation with heparinised saline
(administrated through CVC flushing) to prevent CVC occlusion by intraluminal thrombi
was used in one case-control study performed in 24 dogs (Vose et al 2019) In the cited
study 12 dogs received dosages of 3 ml of heparinised saline solution (10 IU UFHml) to
Chapter 1 Introduction
7
flush the catheter every 6 hours during 72 hours whereas 12 control dogs received
flushing with the same volume of non-heparinised saline solution administered at the
same frequency and no statistically significant difference was found
Prediction of CVC related thrombosis
Studies regarding the relationship between laboratory biomarkers and CVC related
thrombosis are available in the human literature (Boersma et al 2016 Cheng et al 2013
Liu et al 2015) however to the best of the authoracutes knowledge literature about laboratory
tests used as predictors for CVC related thrombosis in small animals cannot be found at
all Availability of approved blood tests which can predict the risk of thrombosis
development associated to CVC already at the moment of the CVC placement are
important in order to identify an increased risk of CVC related thrombosis (Boersma et
al 2016) In the study performed by Boersma et al (2016) in 168 human patients without
indication of anticoagulation prophylaxis elevated leukocyte counts high levels of
plasminogen activator inhibitor and of coagulation factor VIII (all measured on blood
samples taken after CVC placement) were associated with an increased incidence of
symptomatic CVC-related thrombosis diagnosed by ultrasonography In another study
(Liu et al 2015) high blood levels of lactate dehydrogenase and low concentrations of
HDL and albumin were associated with the incidence for CVC related thrombosis
diagnosed by ultrasound in 324 senile patients which was in total 617 Additionally
dyslipidemia (high triglycerides low HDL and high LDLHDL ratio) was associated
with CVC related thrombosis (diagnosed by ultrasound and computer tomography
angiography) in human patients undergoing haemodialysis (Cheng et al 2013) Studies
about predictive tests for CVC related thrombosis in dogs were not found in the available
literature to the best of the authoracutes knowledge
Chapter 1 Introduction
8
Aims of the study
The aims of the preliminary methodological study of the present scientific project were
(1) to define reference intervals for viscoelastic analyses of canine haemostasis using the
ROTEMreg delta analyser and (2) to determine the precision (repeatability) of this
method And the aims of the main study of the present scientific project were (1) to
determine the incidence of catheter-induced thrombosis in canine intensive care patients
(2) to evaluate the efficacy of a prophylactic heparin regimen routinely used in the Small
Animal Clinic of the University of Veterinary Medicine Hannover and (3) to assess
whether selected haemostasis test results are predictive for an increased risk for
thrombosis formation
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
9
Chapter 2 Reference intervals for rotational thromboelastometry measurements
using the ROTEMreg delta device in dogs
Pereira JM Rohn K Mischke R
Contributions of the authors
Pereira JM performed (1) the patients data and blood samples collection (2)
haematology and ROTEMreg analysis (3) generated the reference intervals and
coefficients of variation included in the study and (4) wrote the manuscript
Rohn K performed the statistical analysis
Mischke R performed the clinical chemistry and coagulation tests and was involved in
designing and supervision of the study analysis and discussion of the data and its
statistical analysis and revision edition and correction of the manuscript
Original source Pereira J M Rohn K amp Mischke R 2020 Reference intervals
for rotational thromboelastometry measurements using the ROTEMreg delta device
in dogs Res Vet Sci 130 26ndash32 httpsdoiorg101016jrvsc202001019
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
10
Reference intervals for rotational thromboelastometry measurements using the
ROTEMreg delta device in dogs
Pereira JMa Rohn Kb Mischke Ra
aSmall Animal Clinic University of Veterinary Medicine Hannover Buumlnteweg 9 D-
30559 Hannover Germany
bInstitute of Biometry Epidemiology and Information Processing University of
Veterinary Medicine Hannover Buumlnteweg 2 30559 Hannover Germany
Corresponding author
E-mail ReinhardMischketiho-hannoverde
Original source Pereira J M Rohn K amp Mischke R 2020 Reference intervals
for rotational thromboelastometry measurements using the ROTEMreg delta device
in dogs Res Vet Sci 130 26ndash32 httpsdoiorg101016jrvsc202001019
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
11
ABSTRACT
Aims of the present study were to define reference intervals for viscoelastic analyses of
canine haemostasis using the ROTEMreg delta analyser and as a secondary aspect to
determine the precision (repeatability) of this method Blood samples from 125 clinically
healthy dogs were included Measurements were performed with commercially available
activating reagents (ex-tem in-tem and kaolin solution) as well as without activation
Additional fourfold measurements were done in 3 of the normal blood samples and in 3
samples with haemostatic alterations to evaluate the precision of the method
Coefficients of Variation (CVs) for most of the ROTEM variables were lt 10 Clot
formation time and maximum clot elasticity showed a wide inter-individual variation in
comparison with alpha angle and maximum clot firmness A multivariate analysis on
various ROTEM parameters revealed particularly a significant influence of neuter status
and a significant interrelationship between the factors sex and neuter status for
measurements with different activating reagents These results reflected the fact that
significant differences occurred only between intact and neutered females but not in
males No or only occasionally significant differences were found between groups of sex
age and size
In conclusion CVs demonstrated that the method delivers repeatable results in canine
citrated whole blood Established reference intervals should deliver valuable orientation
for the evaluation of viscoelastic properties of clotting whole blood in dogs using the
ROTEM delta analyser Neuter status in females appeared to be the most relevant
influencing factor and should be considered for the interpretation of ROTEM delta test
results
Keywords Viscoelastic measurements reference values canine age sex neuter status
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
12
1 Introduction
Heamostasis is a physiologic and dynamic process in which plasma soluble coagulation
factors blood cells and blood vessels are involved Clot formation and possible
subsequent clot lysis as well as vascular tissue repair aim to prevent haemorrhages on the
one hand and thromboses on the other hand (Kol and Borjesson 2010 Versteeg et al
2013) Conventional assays of coagulation such as prothrombin time (PT) activated
partial thromboplastin time (aPTT) and thrombin time TT have limitations especially
cellular elements are not incorporated into the test and thereby these tests reflect only
limitedly in vivo functionality of haemostasis (McMichael and Smith 2011 Crochemore
et al 2018)
The rotational thromboelastometry (ROTEMreg) currently termed as thromboelastometry
(TEM) and thromboelastography (TEG) are methods for the rapid evaluation of the
overall haemostasis capacity through the measurement of viscoelastic properties of
clotting whole blood (Kol and Borjesson 2010 Theusinger et al 2010 Sankarankutty
et al 2012 Versteeg et al 2013) In TEG the cup with the blood sample is rotating
whereas the torsion wire is fixed In ROTEMTEM the cup is fixed whereas the pin is
rotating Changes in torque ie resistance to rotation by the fibrin bridge created between
pin and cup in the clotting blood are detected electromechanically in TEG and optically
in ROTEM (Bollinger et al 2012) and displayed as a graph that indicates the time for the
formation of blood clot and its strength (Bagge et al 2016) The resulting graph reflects
the initiation and propagation of the coagulation system fibrin-platelet interactions and
clot lysis
TEG was first described in 1948 by Hellmut Harten (Hartert 1948) as an in vitro method
to evaluate the clot formation process while resembling the in vivo blood flow The
development of the actually used instrument types TEG and ROTEMregTEM were based
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
13
on that original principle Viscoelastic measurements have been widely used since the
1980s in human medicine to evaluate haemostasis and hypercoagulable states in surgical
patients (eg cardiac and hepatic surgeries) and in general in critical care units (Kol and
Borjesson 2010 Nogami 2016 Wikkelso et al 2016 Crochemore et al 2017) In
veterinary medicine viscoelastic measurements using TEG and ROTEMTEM have been
validated for use in dogs cats and horses and used to study haemostasis in different
diseases (eg infectious diseases liver diseases) and medical conditions (eg
orthopaedic surgery hyperfibrinolysis) and also to investigate the effect of
anticoagulants and other drugs (eg corticosteroids) on haemostasis (Kol and Borjesson
2010 Bruno et al 2015a 2015b Woodman et al 2015 Conversy et al 2017 Sigrist et
al 2018 Turner et al 2019) The methodology seems to be especially suitable to detect
hypercoagulability (Wiinberg et al 2008 Kol and Borjesson 2010 Marschner et al
2018)
In the available veterinary literature information about reference values for TEG were
found using kaolin as activation reagent (Bauer et al 2009) Falco et al (2012)
determined reference values for the ROTEM analyser in dogs using ex-tem in-tem and
fib-tem reagents for selected ROTEM parameters in a study on forty five healthy dogs
However data related to reference intervals obtained in an adequate number of samples
(at least 120) according to the American Society for Veterinary Clinical Pathology
(ASVCP) (Friedrichs et al 2012) and the National Committee for Clinical Laboratory
Standards (NCCLS) (Horn and Pesce 2003) could not be found in the available literature
The aim of this study is to determine reference values for the ROTEMreg delta device in
dogs using citrated whole blood with different clot activator reagents (ex-tem in-tem
kaolin) and without activator
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
14
2 Materials and methods
21 Study design
Reference intervals Single thromboelastometric measurements were performed in 125
clinically healthy dogs using the ROTEMreg analyser and different activating reagents (ex-
tem in-tem and kaolin) as well as without activation Reference values were calculated
and influences of age groups (young adult and senior) sex neuter status and size
(groups small medium and large) were assessed
Precision analysis Additional fourfold measurements using ex-tem or in-tem activating
reagents respectively were performed on 3 of the normal and also in 3 abnormal blood
samples each in order to assess within-run imprecision for these measurements
22 Animals
The clinically healthy dogs with a body weight of at least 5 kg were recruited by launching
a call in the University of Veterinary Medicine Hannover network for students and staff
members Interested dog owners received a free health check of their animals (clinical
examination and blood tests specified below) in combination with the blood collection
In addition individual privately owned patient dogs of the Small Animal Clinic with
undisturbed general health were also included if blood was collected due to another
indication (eg general health check pre-anaesthetic examination test for hereditary
diseases etc) Before taking the blood samples all dogs were fasted for at least 12 h with
free access to water 125 animals for which the examination of the health status (clinical
examination routine haematology and clinical chemistry basis coagulation profile) did
not reveal any deviations from the reference were included in the study whereas 3 of the
128 initially recruited animals had to be excluded due to laboratory abnormalities
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
15
Haematological and clinical chemistry profiles performed in order to confirm the health
status included white and red cell count haematocrit platelet counts and reticulocytes
plasma activities of alanine aminotransferase glutamate dehydrogenase alkaline
phosphatase and pseudocholinesterase as well as concentrations of total bilirubin urea
creatinine glucose total protein albumin cholesterol and ionised calcium A basis
coagulation profile including PT PTT and TT was also performed to exclude significant
abnormalities within the blood coagulation system
The 125 healthy dogs enclosed in the establishment of reference values included 48 mixed
breed dogs and the following pedigree dogs 13 Labrador Retrievers 6 Australian
Shepherds 4 Havanese dogs 3 from each of the following breeds American
Staffordshire Terriers and Cocker Spaniels 2 from each of the following breeds Beagles
Border Collies Dalmatians Elo dogs (breed not recognised by Feacutedeacuteration cynologique
international [FCI]) German Longhaired Pointers German Shorthaired Pointers Giant
Schnauzers and Golden Retrievers 1 from each of the following breeds Belgian
Malinois Bolonka Zwetna Briard Brussels Griffon American English Coonhound
Italian Mastiff Flat Coated Retriever French Bulldog German Shepherd Great Dane
Greater Swiss Mountain Dog Giant Spitz Goldendoodle Hovawart Hungarian Vizsla
Italian Bloodhound Lycanis Wolfdog Miniature American Shepherd Miniature
Schnauzer Old German Shepherd Podenco Poodle Pyrenean Shepherd Rhodesian
Ridgeback Rottweiler Schafpudel (breed not recognised by FCI) Small
Munsterlander Spanish Mastiff Tibet Terrier Weimaraner West Highland White
Terrier and Wirehaired Pointer
These dogs were aged between 6 and 154 months (median = 410 months) and had body
weights ranging from 50 to 707 kg (median = 226 kg) 30 were castrated males 33
intact males 41 spayed females and 21 intact females According to the height at withers
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
16
the dogs were divided into 3 different size groups small (le40 cm n = 22) medium (41ndash
60 cm n = 77) and large (gt60 cm n = 26) Dogs were also divided in three different age
groups young (le 24 months n = 34) adult (25ndash84 months n = 59) and senior dogs (gt84
months n = 32)
The three blood samples with haemostatic abnormalities were taken from two dogs with
complex haemostatic disorders (Disseminated Intravascular Coagulation [DIC])
including reduced platelet count and abnormal PT and APTT and one dog with
thrombocytopenia
Animals were treated in accordance with the German Animal Welfare Law The
experimental design was approved by the Animal Welfare officer of the University of
Veterinary Medicine Hannover Foundation and by the ethics committee of the
responsible authority (Lower Saxony State Office for Consumer Protection and Food
Safety reference number 17A101)
23 Blood sampling
The venipuncture area was shaved and then disinfected with alcohol The saphenous or
cephalic vein was visualised with slight pressure and punctured with disposable cannulas
(diameter 09 or 10 mm) The continuously running blood was directly collected from
the needle into the tubes containing the respective anticoagulants From each dog
approximately 10 ml of blood were collected including approximately 8 ml of citrated
blood for the measurement of haemostasis tests (ROTEM PT aPTT TT) in tubes
containing sodium citrate 313 (Eifelfangoreg GmbH amp Co KG Bad Neuenahr-
Ahrweiler Germany) in a ratio of 1 volume9 volumes of blood In addition
approximately 1 ml of EDTA blood for haematological analysis and approximately 1 ml
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
17
of lithium heparin blood for clinical chemical measurements were collected into
commercial 13 ml tubes (Sarstedt AG amp Co Nuumlmbrecht Germany)
Citrated blood was initially kept at room temperature (20-25 C) in order to avoid errors
within the preanalytical phase (Toulon et al 2017) Two ml of citrated blood were used
for the ROTEMreg analysis the residual citrate blood was centrifuged for 10 min at 3800
rpm and 5 degC then the plasma was separated by careful pipetting and it was centrifuged
a second time under the same conditions to get platelet poor plasma which was stored in
aliquots of 500 microl and 250 microl at -80 degC for coagulation tests
24 Rotational elastometry
Viscoelastic measurements using the ROTEMreg delta for the establishment of reference
intervals were performed approximately 15 min after the blood collection using activating
reagents (liquid reagents) provided by the manufacturer of the instrument (r-ex-temreg in-
temreg) and a kaolin solution (5 g1 as part of the PTT reagent CK Prest [Diagnostica
Stago SAS Asniegraveres sur Seine Cedex France]) In addition non-activated analyses
were performed using isotonic NaCl solution instead of the activating reagent in the fourth
channel of the device Fourfold measurements for precision analysis were performed
simultaneously in the four channels of the device in separate runs
According to the standard test protocol provided by the manufacturer of the ROTEMreg
delta (Werfen GmbHInstrumentation Laboratory Munich Germany) 20 μl of the
reagent for recalcification (star-temreg20) and 20 μl of the activating reagent were pipetted
into a cuvette using the automated pipette provided with the device Immediately
thereafter 300 μl of citrated whole blood (prewarmed to 37 degC for five minutes in the
heating device of the instrument and gently rocked to resuspend possibly sedimented
cells) were added to the test preparation in the cuvette using the automated pipette and
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
18
then the cuvette was laced carefully in the ROTEMreg device whereby the rotating pin
entered into the test approach
Different parameters are provided automatically by the ROTEMreg delta device and the
following variables (described according to the Pentapharm GmbH ROTEMreg delta
Manual [2009]) were used in the study
Clotting time (CT) the time (in s) from the beginning of the test (by adding the clot
activator) until the formation of fibrin starts (defined as the moment when the curve
achieves an amplitude of 2 mm) This Parameter facilitates the decision to replace clotting
factors when it is required
Clot formation time (CFT) describes the next phase of clotting by the formation of a
stable clot through the activation of thrombocytes and fibrin formation It is defined as
the time (in s) between the 2 mm amplitude and 20 mm amplitude of the curve
Alpha angle (α angle) is the angle (in degrees) between the middle axis and the tangent
to the clotting curve through the 2 mm amplitude point
Amplitude after ldquoXXrdquo minutes (eg A30 after 30 min) is the amplitude (in mm) at a
certain time after the CT has finished
Maximum clot firmness (MCF) corresponds to the maximum amplitude (in mm) reached
before the clot is dissolved by fibrinolysis This parameter measures the firmness of the
clot and its quality Like CFT and α angle it facilitates the decision for substitution therapy
with platelet concentrate andor fibrinogen especially in humans
Maximum clot elasticity (MCE) is a parameter calculated from MCF and may be superior
at high amplitudes as compared to the MCF
Lysis index at 30 45 and 60 min (LI30 LI45 LI60) represents the fibrinolysis (in
percentage) 30 45 and 60 min after the CT It may facilitate the decision for or against a
therapy with anti-fibrinolytic drugs
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
19
Maximum lysis (ML) describes the degree of fibrinolysis relative to the MCF achieved
during the measurement (percent loss of clot firmness)
25 Statistical methods
Original data obtained from the ROTEMreg delta device software were transferred into the
Microsoftreg Excelreg formal (Microsoft Office Professional Plus 2013) The Statistical
analyses were performed using the SAS Enterprise Guide Version 71 (SAS Institute Inc
Cary NC USA)
Coefficients of Variation (CVs) for selected relevant parameters of the ROTEM delta
analysis using ex-tem and in-tem regents were calculated for the fourfold measurements
on 3 normal and 3 abnormal blood samples in order to assess within-run imprecision CVs
for the results of each sample were calculated by dividing the standard deviation of the
data set by its arithmetic mean Finally the median of the CVs for each group of 3 samples
was calculated The reference values were calculated according to recommendations of
the American College of Veterinary Clinical Pathology (ACVCP) based on non-
parametric methods (25 and 975 quantiles) supplemented by 95 confidence
intervals (Friedrichs et al 2012)
Data were analysed for Standard normal distribution by Kolmogorov Smirnov test and
assessment of residuals of the analysis of variance (ANOVA) Because these tests
indicated almost normally distributed values for the studied ROTEM variables data were
analysed by a fourway ANOVA stratified by the activating reagent (ex-tem in-tem
kaolin or without activation) to test the influence of size age sex and neuter status on
several parameters and taking all interactions into account Post hoc t-tests were
performed when indicated The significance level was set to P lt 05
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
20
3 Results
31 Precision of the method
For all the parameters evaluated in the study the CVs for the fourfold measurement in
healthy dogs were less than 10 (Table 1) In the case of dogs with haemostatic
abnormalities CVs of the vast majority of ROTEM variables were also less than 10
except the variables CFT and ML irrespective whether ex-tem or in-tem reagents were
used (Table 2)
Table 1 Coefficients of variation () for rotational thromboelastometry using the
ROTEMreg delta analyser in normal blood samples
Activator CT (s) CFT (s) α (deg) A30 (mm) MCF (mm) MCE(mm) LI30 () LI45 () LI60 () ML ()
Ex-temreg 875 629 262 213 179 634 000 020 037 534
In-temreg 245 596 166 117 135 299 000 000 046 216
Abbreviations CT ndash clotting time CFT ndash clot formation time α ndash alpha angle A30 ndash
amplitude after 30 min MCF ndash maximum clot firmness MCE ndash maximum clot elasticity
LI 30 LI 45 LI 60 ndash Lysis index at 30 45 and 60 min ML ndash maximum lysis
Median values of the measurements of 3 samples
Table 2 Coefficients of variation () for rotational thromboelastometry using the
ROTEMreg delta analyser in canine blood samples with haemostatic alterations
Activator CT (s) CFT (s) α (deg) A30 (mm) MCF (mm) MCE(mm) LI30 () LI45 () LI60 () ML ()
Ex-temreg 668 1478 393 218 268 582 0 018 000 848
In-temreg 228 799 461 219 251 371 0 0 017 283
Median values of the measurements of 3 samples from patients with DIC (n = 2)
thrombocytopenia (n = 1) abbreviations please see legend to Table 1
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
21
32 Reference intervals
Activation by ex-tem reagent (tissue factor) resulted in faster CTs when compared to
activation via the intrinsic pathway using ellagic acid (in-tem) or kaolin reagents whereas
the CFT was much less affected by the activating reagent (Table 3) Especially CFT and
MCE showed a wide inter-individual variation in comparison for example with α angle
and MCF regardless of the used activating reagent (Table 3)
33 Influence of age sex neuter status and size
ANOVA did not reveal statistically significant differences in ROTEM delta parameters
between dog groups of variable size or sex (P gt 05) Age had influence only on CT and
only when the in-tem reagent was used (P = 0169 ANOVA) but not when using the ex-
tem reagent (P = 7105) Post hoc comparisons using t-test of the in-tem results between
age groups revealed shorter CTs in the group of senior dogs when compared with young
dogs (P = 0194) but not between adult dogs and these two groups ANOVA showed
particularly influences of the neuter status and a significant interrelationship between the
factors sex and neuter status on different ROTEM delta parameters using ex-tem and in-
tem respectively as activator (Tables 4 and 5) ANOVA revealed also a significant
influence of neuter status on different ROTEM parameters when kaolin was used as
activating reagent (CFT α angle MCF MCE) whereas non-activated measurements
were not subject to any significant influence of the assessed factors (results not shown)
The interrelationship between sex and neuter status mentioned above reflected the fact
that significant differences between neutered and not neutered dogs were only found in
females The statistical analysis using the t-test to compare intact vs neutered dogs
stratified by sex showed statistically significantly (P lt 05) longer CTs and CFTs and
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
22
lower α angle A30 MCF and MCE values in spayed when compared to intact females
(Tables 4 and 5)
Chapter 2 Reference intervals for rotational thromboelastometry measurements using the ROTEMreg delta device in dogs
23
Table 3 Reference values (median reference interval based on 25 - and 975 - quantiles) for thromboelastometric measurements with the ROTEMreg delta analyser in
dogs (n=125 95 confidence intervals in brackets) Abbreviations please see legend to Table 1
Activator
Statistical
para-meter
CT
(s)
CFT
(s)
Α
(deg)
A30
(mm)
MCF
(mm)
MCE
(mm)
LI30
()
LI45
()
LI60
()
ML
()
Ex-temreg
Median 450
[430ndash470]
110
[105ndash115]
680
[670ndash690]
600
[580ndash610]
610
[600ndash620]
154
[147ndash164]
100
[960ndash100]
100
[100ndash100]
970
[960ndash980]
220
[210ndash240]
25 340
[270ndash350]
650
[490ndash710]
540
[420ndash560]
460
[410ndash480]
460
[420-490]
870
[720ndash950]
100
[100-100]
870
[800ndash960]
700
[640ndash820]
800
[100ndash110]
975 830
[790ndash990]
220
[201ndash315]
790
[770ndash800]
770
[690ndash770]
730
[700ndash770]
264
[230-334]
100
[100ndash100]
100
[100ndash100]
100
[990ndash100]
590
[530ndash680]
In-tem
Median 190
[187ndash201]
116
[111ndash125]
680
[670ndash690]
570
[560ndash580]
590
[580ndash600]
143
[135ndash149]
100
[100ndash100]
100
[100ndash100]
980
[980ndash980]
180
[170ndash190]
25 135
[112ndash142]
730
[590ndash800]
510
[460ndash570]
450
[410ndash470]
480
[410ndash500]
920
[710ndash101]
100
[950ndash100]
990
[820ndash990]
930
[740ndash930]
500
[100ndash700]
975 283
[268ndash291]
251
[198ndash227]
750
[750ndash780]
660
[660ndash690]
680
[660ndash690]
210
[195ndash227]
100
[100ndash100]
100
[100ndash100]
100
[100ndash100]
270
[260ndash380]
Activator
Statistical
para-meter
CT
(s)
CFT
(s)
Α
(deg)
A30
(mm)
MCF
(mm)
MCE
(mm)
LI30
()
LI45
()
LI60
()
ML
()
Kaolin
Median 162
[154ndash173]
870
[820ndash910]
740
[720ndash750]
500
[470ndash530]
550
[530ndash560]
121
[115ndash127]
960
[940ndash980]
840
[790ndash880]
710
[640ndash770]
500
[450ndash540]
25
109
[990ndash115]
470
[440ndash580]
560
[490ndash600]
110
[700ndash230]
390
[380ndash400]
640
[600ndash680]
270
[140ndash480]
110
[800ndash260]
800
[400ndash170]
200
[160ndash240]
975 246
[230ndash337]
214
[185ndash290]
810
[790ndash810]
640
[630ndash690]
650
[650ndash690]
187
[182ndash223]
100
[100ndash100]
990
[980ndash100]
940
[930ndash990]
970
[930ndash100]
Without activation
Median 607
[495ndash662]
221
[194ndash265]
510
[460ndash550]
520
[500ndash530]
540
[520ndash550]
115
[109ndash123]
100
[100ndash100]
100
[100ndash100]
980
[980ndash990]
170
[160ndash190]
25 203
[123ndash215]
860
[550ndash930]
190
[120ndash230]
350
[230ndash370]
370
[260ndash400]
600
[350ndash670]
100
[950ndash100]
980
[890ndash990]
930
[840ndash950]
400
[000ndash500]
975 1356
[1305ndash1721]
811
[688ndash1333]
730
[710ndash790]
660
[640ndash720]
660
[640ndash730]
197
[179ndash265]
100
[100ndash100]
100
[100ndash100]
100
[100ndash100]
290
[280ndash320]
Chapter 2 Reference intervals for rotational thromboelastometry measurements using the ROTEMreg delta device in dogs
24
Table 4 Median values (minimum and maximum in brackets) for different ROTEMreg variables using ex-tem reagent stratified by sex and neuter status
Group
CT
(s)
CFT
(s)
Α
(deg)
A30
(mm)
MCF
(mm)
MCE
(mm)
LI30
()
LI45
()
LI60
()
ML
()
Female intact 430
(320ndash530)
960
(590ndash198)
710
(570ndash800)
620
(480ndash730)
640
(510ndash740)
179
(103ndash282)
100
(100ndash100)
100
(980ndash100)
960
(800ndash100)
230
(100ndash640)
Female spayed 460
(270ndash830)
121
(650ndash224)
660
(520ndash770)
580
(430ndash690)
590
(440ndash700)
146
(790ndash230)
100
(960ndash100)
100
(800ndash100)
970
(690ndash100)
240
(100ndash590)
p-value (t-test)
Female
intact vs spayed
00025 00063 00049 00023 00016 00015 na na na na
Male intact 450
(320ndash990)
105
(650ndash315)
690
(420ndash790)
590
(420ndash700)
610
(440ndash710)
154
(770ndash244)
100
(100ndash100)
100
(870ndash100)
970
(650ndash100)
220
(700ndash680)
Male castrated 460
(340ndash930)
111
(490ndash255)
685
(480ndash800)
590
(410ndash770)
600
(420ndash770)
152
(720ndash334)
100
(100ndash100)
100
(830ndash100)
980
(640ndash100)
205
(500ndash590)
p-value (t-test)
Male
Intact vs castrated
08326 05106 04756 01799 01568 02163 na na na na
p-values (ANOVA)
Factor Sex
05428 04847 06612 02206 01887 01630 08489 07893 07689 02758
Factor neuter status 01872 00167 00173 00039 00037 00028 07141 04737 09356 05948
Interrelationship
sexneuter status 00383 00968 00782 00488 00472 00319 04943 08611 05422 00160
na ndash not analysed all significant P-values (lt 005) are highlighted in bold print
Chapter 2 Reference intervals for rotational thromboelastometry measurements using the ROTEMreg delta device in dogs
25
Table 5 Median values (minimum and maximum in brackets) for different ROTEMreg variables using the in-tem reagent stratified by sex and neutering status
Group
CT
(s)
CFT
(s)
Α
(deg)
A30
(mm)
MCF
(mm)
MCE
(mm)
LI30
()
LI45
()
LI60
()
ML
()
Female intact 186
(126ndash238)
940
(590ndash164)
720
(620ndash780)
610
(490ndash690)
620
(530ndash690)
166
(113ndash227)
100
(100ndash100)
100
(990ndash100)
980
(930ndash100)
170
(100ndash300)
Female spayed 201
(149ndash291)
1220
(820ndash262)
670
(500ndash740)
550
(450ndash660)
570
(480ndash680)
133
(910ndash214)
100
(950ndash100)
100
(820ndash100)
980
(740ndash100)
190
(500ndash380)
p-value (t-test)
Female
intact vs spayed
na na lt0001 00009 00005 00005 na na na na
Male intact 199
(117ndash283)
122
(730ndash277)
680
(460ndash750)
570
(430ndash660)
590
(450ndash660)
146
(810ndash192)
100
(100ndash100)
100
(990ndash100)
980
(930ndash100)
180
(600ndash270)
Male castrated 181
(112ndash288)
123
(820ndash222)
670
(540ndash740)
565
(410ndash630)
585
(410ndash640)
138
(710ndash180)
100
(100ndash100)
100
(970ndash100)
980
(890ndash100)
180
(800ndash260)
p-value (t-test)
Male
intact vs castrated
na na 08232 01401 01162 00849 na na na na
p-values (ANOVA)
Factor Sex
02105 03317 03370 00877 01258 00510 09564 07832 08117 03638
Factor neuter status 04289 00660 00619 00107 00088 00054 07152 09795 07056 08299
Interrelationship
sexneuter status
04310 00550 00408 00707 01733 00996 05240 05982 08047 04578
na ndash not analysed all significant P-values (lt 005) are highlighted in bold print
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
26
4 Discussion
41 Coefficients of variation
In accordance with the literature CVs below 10 indicate adequate repeatability in intra-
assays evaluations (Steiner et al 2003 Hua et al 2018) According to our results
obtained from the fourfold measurements in citrated whole blood ROTEM delta
measurements on healthy dogs and dogs with haemostatic alterations fulfil these
requirements with only a few exceptions concerning CFT and ML values in abnormal
samples
Our results regarding repeatability are similar to those obtained for several TEM
parameters in previous studies performed in animals In one study the imprecision of the
ROTEM analyser was evaluated by duplicate measurements in 10 blood samples of
healthy dogs and as result of that evaluation a good precision (CVs below 10 ) was
calculated for CT CFT MCF and α angle but in contrary to our results a low
repeatability was found for ML (CVs of 233 [ex-tem] and 285 [in-tem] own values
534 and 216 ) (Falco et al 2012)
In a previous feline study using ex-tem and in-tem as activating reagents for the ROTEM
delta repeatability was assessed similarly to the present study based on fourfold
measurements of blood samples of two healthy individuals (Doumlderlein and Mischke
2015) The results also revealed CVs below 10 for most of the parameters evaluated
The partially low repeatability of ML values may reflect the vulnerability of this variable
which is not only influenced by the complex clot formation process but also by its
subsequent lysis Reproducibility of ex-tem or in-tem activated measurements of the
ROTEM delta analyser which was not investigated in our study was studied in blood
from healthy and unhealthy horses by two different operators using three different
ROTEM devices Results indicated acceptable reproducibility (Junge et al 2016)
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
27
42 Reference intervals
The number of 125 healthy dogs used in the present study to create reference intervals is
regarded adequate according to the guidelines suggested by the American Society of
Veterinary Clinical Pathology (ASVCP) and the National Committee for Clinical
Laboratory Standards (NCCLS) who recommend to include at least 120 individuals
(Friedrichs et al 2012) To the best of our knowledge reference intervals for the most
relevant ROTEMreg delta parameters obtained in an adequate number of samples of whole
citrated canine blood have not been published so far A previous study was performed on
a smaller number of 45 dogs (Falco et al 2012) The median values for reference
intervals obtained in the present study are partially similar to those calculated in the
mentioned study based on 45 healthy dogs (eg ex- tem CT 610 s450 s [Falco et al
2012own results] CFT 150 s 110 s α angle 620deg680deg MCF 530 m 610 mm ML
180 220 in-tem CT 235 s190 s CFT 136 s116 s α angle 660deg680deg MCF
560 mm590 mm ML 30 180 ) However in comparison to our results the
reference intervals in the mentioned study are slightly wider (eg ex-tem CT 29ndash92
s34ndash83 s [Falco et al 2012own results] CFT 540ndash275 s650ndash220 s α angle 470ndash
790deg540ndash790deg MCF 360ndash730 mm460ndash730 mm ML 000ndash560 800ndash590
in-tem CT 126ndash363 s135ndash283 s CFT 470ndash224 s730ndash251 s aα angle 550ndash
810deg510ndash750deg MCF 500-ndash750 mm480ndash680 mm ML 000ndash400 500ndash270 )
although the previous study used the 5 and 95 quantiles to define limits of the
reference range whereas we calculated the commonly used 25 and 975 quantiles
to capture the central 95 of reference values (Friedrichs et al 2012)
Although it is desirable that each laboratory should specifically determine its own
reference intervals the results obtained in this study may provide an orientation for other
laboratories to interpret ROTEM delta parameters analysed in citrated canine blood In
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
28
order to test transference of the reference ranges to another laboratory a validation
procedure with measurement of 20 samples should be performed (Friedrichs et al 2012)
Although intra-individual control values (subject-based reference values) are regarded
superior to population based reference values (Wiinber et al 2007) the latter have to be
routinely used in clinical patients due to lacking individual controls especially for specific
tests such as ROTEM analyses In addition the calculated reference values cannot be
used for the fully automated device ROTEM sigma because - according to an actual
human study - values of part of the parameters do not correlate well between both
instruments (Gillissen et al 2019)
43 Influence of size age sex and neuter status
In previous studies in humans (Attard et al 2013) and animals (Mischke 1994 Borrelli
et al 2017) age has been described as an influencing factor for different haemostasis
parameters We also found an influence of age on the CT with shorter times in senior
dogs (gt 84 months) compared with young dogs (up to 24 months) which was limited to
measurements activated by the in-tem reagent
This age dependency confirms in principle a previous study on 20 healthy dogs
(Barthelemy et al 2015) which found shorter CTs in elderly dogs and results of a human
study where individuals with an age gt 45 years showed shorter CTs compared with
individuals lt 45 years old (Sucher et al 2011) However in the cited studies this effect
was only seen when ex-tem but not when the in-tem reagent was used
Based on the age distribution of our dogs which were primarily recruited to establish
reference intervals we cannot deliver information regarding very young dogs A human
study focusing on ROTEM reference ranges in children demonstrated significant
differences for nearly all ROTEM parameters with the most striking differences in
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
29
subjects aged 0-3 months (Oswald et al 2010) The limited number of dogs with an age
lt 1 year which did not allow to consider these separately in the present study may also
be one reason for the different results in a feline study (Doumlderlein and Mischke 2015) In
the cited study on healthy cats age showed an influence on CT but contrary to our study
the CT was shorter in young individuals (6-12 months) compared with older ones In
addition this was only the case in measurements using kaolin and in non-activated
measurements When compared to humans and cats uni- or multivariate analyses
considering factors age and breed or size based on do populations including different
breeds have the limitation that they can be biased by the variate life expectancy between
small medium and large dog breeds (Willems et al 2017) Apart from age we found a
significant influence only for the neuter status in bitches reflected by a significant
interaction effect between factors neuter status and sex in the ANOVA analysis
Especially for CFT values (eg activated by ex-tem median values of 121 s [spayed] vs
96 s [intact]) this difference seems to be also clinically relevant with respect to the
interpretation of ROTEM results In contrast although also significantly different
absolute and relative differences of CT values were small (eg activated by ex-tem 46 s
[spayed] vs 43 s [intact]) These differences may not only be considered for the result
interpretation of clinical patients but indicate also the necessity for sex-matched patient
groups in scientific studies
The obviously more active haemostasis system in intact females is probably related to an
influence of sex hormones (specifically estrogens) on the coagulation system
Accordingly ex vivo and in vivo studies demonstrated the activating properties of
endogenous estrogens on the haemostasis system In one ex vivo study addition of
endogenous estrogens (estrone [E1] 17szlig-estradiol [E2] and estriol [E3]) to blood samples
from men resulted in shorter mean values of the reaction time in TEG analyses
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
30
corresponding to CT in ROTEM measurements (452-537 s vs 698 s [control]) and of
the TEG variable kinetics (corresponding to the CFT in ROTEM analyses) (Swanepoel
et al 2017) In a further study subcutaneous injection of E2 into male rats induced a
significant increase in activities of coagulation factors X (by 8 ) and XI (by 10-24 )
however did not show any significant effect on PT aPTT and factor VII activity (Franco-
Murillo and Jaimez 2017)
In the cited studies also the effects of the synthetic estrogen ethinylestradiol [EE] were
studied which interestingly seems to have an inhibiting effect as indicated by prolonged
reaction time and kinetics (TEG) compared with the control in the human ex vivo study
and significantly longer PTs and aPTTs and lower factor VII and X activities in the study
on rats As expected we did not find a significant influence of size on ROTEM parameters
in dogs To the best of the authoracutes knowledge size has not been examined as an
influencing factor on viscoelastic measurements or any other haemostatic variable in
dogs Apart from Greyhounds there is limited literature on breed comparisons of
haemostatic variables (Clemons and Meyers 1984 Mischke 1994 Nielsen et al 2007
Nielsen et al 2010 Boyd et al 2018) indicating very limited breed differences for
example for factor VII (Mischke 1994) However these also do not indicate systematic
influence of size and in addition possible breed-specific effects have to be considered
As already stated one limitation of the study is the limited number of young animals
which does not allow to verify reference values for animals in this stage of life In
addition with respect to the main aim of the study possible influencing factors such as
platelet count and haematocrit were not considered in the statistical analysis Finally the
relatively small number of dogs used for the precision analysis which express the fact
that this was only a secondary aspect of the study may be regarded as a limitation At the
same time these considerations provide space for further investigations
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
31
5 Conclusions
Results of the precision analysis revealed acceptable results and thereby confirm that the
ROTEMreg delta analyser delivers repeatable results to measure overall haemostasis in
canine citrated blood samples The established reference values should offer a reliable
basis for the interpretation of thromboelastometric measurements in whole blood using
the device ROTEMreg delta in dogs with suspected haemostasis disorders Neuter status
in female dogs appeared to be a relevant influencing factor on most important ROTEMreg
parameters and should be considered for result interpretation and with respect to
experimental designs
6 Acknowledgments
Jose Mauricio Pereira received a scholarship from the Universidad Nacional de Costa
Rica (reference code JB-C-1107-2016)
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
32
7 References
1 Attard C van der Straaten T Karlafitis V Monagle P Ignjativic V 2013 Developmental
hemostasis age-specific differences in the levels of hemostatic proteins J Thromb and
Haemost 11 1850ndash1854
2 Bagge A Schoumltt U Kander T 2016 Effects of naturopathic medicines on Multiplate and
ROTEMreg A prospective experimental pilot study in healthy volunteers BMC Complemen
Altern Med 17 (16) 64
3 Bartheacutelemy A Rannou B Forterre M Verwaerde P Bonnet-Garin JM Pouzot-Nevoret
C Goy-Thollot I 2015 Differences between coagulation and cytokine profiles in dogs of
different ages Vet J 205 410ndash412
4 Bauer N Eralp O Moritz A 2009 Establishment of reference intervals for kaolin-activated
thromboelastography in dogs including an assessment of the effects of sex and anticoagulat use
J Vet Diagn Investi 21 641ndash648
5 Bollinger D Seeberger MD Tanaka KA 2012 Principles and practice of
thrromboelastography in clinical coagulation management and transfusio practice Transfus Med
Rev 26 1ndash13
6 Borrelli A Botto A Maurella C Falco S Pagani E Miniscaldo B Tarducci A Bruno
B 2017 Thromboelastometric assesment of hemostasis in newborn Piemontese calves J Vet
Diagn Invest 29 293ndash297
7 Boyd CJ Claus MA Raisis AL Hosqood G Sharp CR SmartL 2018
Hypocoagulability and platelet dysfunction are exacerbated by synthetic colloids in a canine
hemorrhagic shock model Front Vet Sci 5 279
8 Bruno B Maurella C Falco S Tarducci A Zanatta R DrsquoAngelo A Borrelli A 2015a
Thromboelastometric evaluation of hemostasis in dogs infected with Leishmania infantum J Vet
Emerg Crit Care (San Antonio) 25 502ndash511
9 Bruno B Maurella C Falco S Tarducci A Zanatta R Gianella P DrsquoAngelo A Piras L
Di Bella A Borrelli A 2015b Assessment of coagulation utilizing thromboelastometry in dogs
undergoing orthopedic surgery J Vet Emerg Crit Care (San Antonio) 25 358ndash363
10 Clemons RM Meyers KM 1984 Acquisition and aggregation of canine blood platelets Basic
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
33
mechanisms of function and differences because of breed origin Am J Vet Res 45 137ndash144
11 Conversy B Blais MC Dunn M Gara-Boivin C del Castillo JRE 2017 Anticoagulant
activity of oral rivaroxaban in healthy dogs Vet J 223 5ndash11
12 Crochemore T Piza FMT Rodriacuteguez RDR Guerra JCC Ferraz LJR Correcirca TD
2017 A new era of thromboelastometry Einstein (Sao Paulo) 15 380ndash385
13 Crochemore T Correcirca TD Lance MD Solomon C Neto AS Guerra JCC Lellis PS
Bernz LM Nunes N Mancio CM Yokoyama APH Silva E 2018 Thromboelastometry
profile in critically ill patients A single-center retrospective observational study PLoS One 13
e0192965
14 Doumlderlein E Mischke R 2015 Reference intervals for thromboelastometry with the ROTEMreg
delta in cats Res Vet Sci 100 271ndash276
15 Falco S Bruno B Maurella C Bellino C DacuteAngello A Gianella P Tarducci A Zannata
R Borrelli A 2012 In vitro evaluation of canine hemostasis following dilution with
hydroxyethyl starch (13004) via thromboelastometry J Vet Emerg Crit Care (San Antonio)
22 640ndash645
16 Franco-Murillo Y Jaimez R 2017 Inhibitory effect of ethinylestradiol on coagulation factors
in rats Exp Anim 66 107ndash113
17 Friedrichs KR Harr KE Freeman KP Szladovits B Walton RM Barnhart KF Blanco-
Chavez J 2012 ASVCP reference interval guidelines Determination of de novo reference
intervals in veterinary species and other related topics Vet Clin Pathol 41 441ndash453
18 Gillissen A va den Akker T Caram-Deelder C Henriquez DDCA Bloemenkamp
RWM Eikenboom J van der Bom JG de Maat MPM 2019 Comparison of
thromboelastometry by ROTEMreg Delta and ROTEMreg Sigma in women with postpartum
haemorrhage Scand J Clin Lab Invest 79 32ndash38
19 Harttert H 1948 Blutgerinnungstudien mit der Thrombelastographie einem neuen
Untersuchungsverfahren Klin Wochenschr 26 577ndash583
20 Horn P Pesce A 2003 Reference intervals and update Clin Chim Acta 334 5ndash23
21 Hua Yanhun Qiu W Xiao Q Wu Q 2018 Precision (repeatability and reproducibility) of
ocular parameters obtained by the Tomey OA-2000 biometer compared to the IOLMaster in
healthy eyes PLoS One 13 e0193023
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
34
22 Junge H Ringer S Mayer N Schwarzwald C 2016 Assesment of method reliability and
determination of reference intervals for rotational thromboelastometry in horses J Vet Emerg
Crit Care (San Antonio) 26 691ndash703
23 Kol A Borjesson DL 2010 Application of thrombelastographythromboelastometry to
veterinary medicine Vet Clin Pathol 39 405ndash416
24 Marschner CB Wiinberg B Tarnow I Markussen B Kuumlhnel L Bochsen L Kristensen
AT 2018 The influence of inflammation and hematocrit on clot strength in canine
thromboelastographic hypercoagulability J Vet Emerg Crit Care (San Antonio) 28 20ndash30
25 McMichael MA Smith SA 2011 Viscoelastic coagulation testing technology applications
and limitations Vet Clin Pathol 40 140ndash153
26 Mischke R 1994 Activity of coagulation factors II V VII and X in healthy dogs- dependence
on age sex and breed Berl Muumlnch Tieraumlrztl Wochenschr 107 289ndash294
27 Nielsen A Zois N Pedersen H Olse L Tarnow I 2007 Platelet function in dogs bread
differences and effect of acetylsalicylic acid administration Vet Clin Pathol 36 267ndash273
28 Nielsen L Kjelgaard‐Hansen M Jensen A Kristensen A 2010 Breed‐specific variation of
hematologic and biochemical analytes in healthy adult Bernese Mountain dogs Vet Clin Pathol
39 20ndash28
29 Nogami K 2016 The utility of thromboelastography in inherited and acquired bleeding
disorders Br J Haematol 174 503ndash514
30 Oswald E Stalzer B Heitz E Weiss M Schmugge M Strasak A Innerhofer P Haas T
2010 Thromboelastometry (ROTEMreg) in children Age-related reference ranges and correlations
with standard coagulation tests Br J Anaesth 105 827ndash835
31 Sankarankutty A Nascimento B Teodoro da Luz L Rizoli S 2012 TEGreg and ROTEMreg
in trauma similar test but different results World J Emerg Surg 7 (Suppl 1) S3
32 Sigrist NE Schefer RJJ Kutter APN 2018 Characteristics of hyperfibrinolysis in dogs and
cats demonstrated by rotational Thromboelastometry (ROTEM) Vet J 242 67-73
33 Steiner J Teague S Williams D 2003 Development and analytic validation of an enzime-
linked immunosorbent assay for the measurement of canine pancreatic lipase immunoreactivity in
serum Can J Vet Res 68 161ndash168
34 Sucker C Tharra K Lithmate J Scharf RE Zotz RB 2011 Rotation thromboelastography
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
35
(ROTEM) parameters are influenced by age gender and oral contraception Perfusion 26 334ndash
340
35 Swanepoel A Emmerson O Pretorius E 2017 The effect of endogenous and synthetic
estrogens on whole blood clot formation and erythrocyte structure Microsc Microanal 23 599ndash
606
36 Theusinger OM Nuumlrnberg J Asmis LM Seifert B Spahn DR 2010 Rotation
thromboelastometry (ROTEM) stability and reproducibility over time Eur J Cardiothorac Surg
37 677ndash683
37 Toulon P Metge S Hangard M Zwahlen S Piaulenne S Besson V 2017 Impact of
different storage times at room temperature of unspun citrated blood samples on routine
coagulation tests results Results of a bicenter study and review of the literature Int J Lab
Hematol 3 458ndash468
38 Turner JS Kutter APN Sigrist NE 2019 Correlation of rotational thrromboelastometry
(ROTEM) parameters with platelet count and their ability to predict thrombocytopenia in dogs
Res Vet Sci 126 45ndash50
39 Versteeg HH Heemskerk JWM Levi M Reitsma PH 2013 New Fundamentals in
Hemostasis Physiol Rev 93 327ndash358
40 Wiinberg B Jensen AL Kjelgaard-Hansen M Rojkjaer R Johansson PI Gade LP
Gram DX Kristensen AT 2007 Study on biological variation of haemostatic parameters in
clinically healthy dogs Vet J 174 62ndash68
41 Wiinberg B Jensen AL Johansson PL Rozanski E Tranholm M Kristensen AT 2008
Thromboelastographic evaluation of hemostatic function in dogs with disseminated intravascular
coagulation J Vet Intern Med 22 357ndash365
42 Wikkelsoslash A Wetterslev J Moslashller AM Afshari A 2016 Thromboelastography (TEG) or
thromboelastometry (ROTEMreg) to monitor haemostatic treatment versus usual care in adults or
children with bleeding Cochrane Database Syst Rev Art
httpsdoiorg10100214651858CD007871pub3 No CD007871
43 Willems A Paepe D Marynissen S Smets P Van de Maele I Picavet P Duchateau L
Daminet S 2017 Results of screening of apparently healthy senior and geriatric dogs J Vet
Intern Med 31 81ndash92
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
36
44 Woodman J Wagg CR Boysen SR Leguillete R Mizen K Roy MF 2015 Evaluation
of coagulation via thromboelastography in healthy horses administered dexamethasone Can Vet
J 56 1271ndash1274
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
37
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of
standard heparin prophylaxis and correlation to haemostatic changes
Pereira JM Hewicker-Trautwein M Rohn K von Depka Prondzinski M
Mischke R
Contributions of the authors
Pereira JM performed (1) the ultrasound evaluations and blood sampling of the
patients under study (2) haematology analysis (3) injection of the anticoagulation
prophylaxis (partially) to the patients under study (4) analysis of samples (collaboration)
at Werlhof Institute Medical Care Center (5) analysis of some haemostasis test
(collaboration) (6) statistical analysis (partially) (7) data documentation (8) and wrote
the manuscript
Hewicker-Trautwein M performed the electron microscopy analysis
Rohn K performed the statistical analysis
von Depka Prondzinski M performed some haemostasis test (thrombin generation
assay and endogenous thrombin potential)
Mischke R performed the coagulation and haemostasis test was involved in designing
and supervision of the study analysis and discussion of the data and its statistical analysis
and revision edition and correction of the manuscript
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
38
Central venous catheter induced thrombosis in dogs ndash efficacy of standard heparin
prophylaxis and correlation to haemostatic changes
Joseacute Mauricio Pereira1 Marion Hewicker-Trautwein2 Karl Rohn3 Mario von Depka
Prondzinski4 Reinhard Mischke1
1Small Animal Clinic University of Veterinary Medicine Hannover Foundation
Buumlnteweg 9 D-30559 Hannover Germany
2Institute for Pathology University of Veterinary Medicine Hannover Foundation
Buumlnteweg 17 D-30559 Hannover Germany
3Institute of Biometry Epidemiology and Information Processing University of
Veterinary Medicine Hannover Foundation Buumlnteweg 2 30559 Hannover Germany
4Werlhof Institute Schillerstrasse 23 30159 Hannover Germany
Abbreviations anti-Xa anti-factor Xa BW body weight CVC central venous catheter
EJV external jugular vein ELMI electron microscopy IV intravenous JV jugular vein
LMWH low molecular weight heparin ML maximum lysis (ROTEM) PIVC
peripheral intravenous catheter ROTEM rotational elastometry SC subcutaneous
TGA thrombin generation assay TID three times a day TT thrombin time UFH
unfractionated heparin
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
39
Corresponding author
E-mail addresses matiscrgmailcom (JM Pereira) MarionHewicker-
Trautweintiho-hannoverde (M Hewicker-Trautwein) KarlRohntiho-hannoverde
(K Rohn) mailwerlhof-institutde (M von Depka Prondzinski)
ReinhardMischketiho-hannoverde (R Mischke)
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
40
ABSTRACT
Central venous catheters (CVC) are of importance for intensive care management of
canine patients Thrombosis of the jugular vein is a severe complication related to the
CVC use However only little is known about the incidence of CVC-associated
thromboses in dogs and the necessity and efficacy of prophylactic antithrombotic
regimens The objectives of the present study were (1) to determine the incidence of
catheter-induced thrombosis in canine intensive care patients and thereby (2) to evaluate
the efficacy of a routine prophylactic heparin treatment and finally (3) to assess whether
initial changes of selected haemostasis parameters are predictive for an increased risk for
thrombosis formation 29 dogs hospitalised in the Small Animal Clinic University of
Veterinary Medicine Hannover Foundation and receiving a central venous catheter
(CVC) in the external jugular vein (EJV) for medical reasons were included in the final
assessment 17 dogs received the standard antithrombotic dosage regimen of
unfractionated heparin (UFH 150 IUKG BW TID sc) and 12 dogs (usually surgical
patients) the reduced dosage (75 IUKG BW TID sc) A colour Doppler ultrasound of
the respective EJV in order to detect thrombus formation and blood collection for
haemostasis tests were performed immediately before the installation procedure (day 0)
and on days 1 3 5 7 and so on after the CVC insertion Haemostasis tests included
prothrombin time activated partial thromboplastin time thrombin time antithrombin
activity D-dimer concentration thrombin generation and rotational elastometry as well
as the heparin activity using a chromogenic anti-factor Xa test Finally electron
microscopy (ELMI) of sections of the removed CVC was performed In 8 dogs
sonographic examination revealed thrombus formation in the lumen of EJV and in 1927
CVCs assessed using ELMI thrombi on the external CVC surface were detected without
significant correlation between both methods Plasmatic heparin activities showed great
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
41
variability but no significant differences were found between dogs with or without CVC-
associated thrombus formation From the initially performed haemostasis tests high
fibrinogen concentrations and low maximum lysis values as measured by the ROTEM
delta device (ex-tem reagent) were associated with sonographically detectable thrombi
The results of the present study indicate that CVC-associated thromboses occur (eg 28
[829] of the patients as detected by ultrasound) although severe thromboses (high
grades) are relatively rare in intensive care canine patients receiving a routine
anticoagulant treatment A high fibrinogen concentration and low maximum lysis
(ROTEM ex-tem) may be useful indicators for an increased thrombotic risk in individual
patients which may require special anticoagulatory treatment
Keywords CVC complication venal occlusion antithrombotic treatment ultrasound
electron microscopy rotational elastometry
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
42
1 Introduction
Central venous catheters (CVCs) are very important tools for care of patients in critical
care units allowing parenteral nutrition support therapy administration of high volumes
of intravenous fluids safe drugs administration that cannot be given through peripheral
veins plasmapheresis haemodialysis blood transfusions as well as periodical blood
sampling (Geerts 2014 Vose et al 2019) CVC related thrombosis is one of the most
severe complications (apart from infections and pneumothorax) of CVCs in humans
(Geerts 2014 Comerlato et al 2017 Evans and Ratchford 2018) leading to patientrsquos
distress CVC dysfunction venous stenosis and in some cases to the development of
thromboembolism (Geerts 2014)
In the human literature there are various studies dealing with epidemiology with reference
to different anticoagulatory regimens including placebo-controlled studies (Lee and
Kamphuisen 2012) In humans CVC related thromboses represent 70ndash80 of all upper
extremity thrombosis cases and approximately 10 of thromboembolism cases in
humans (Kucher 2011) The incidence of CVC related thrombosis reported in humans
based on 25 studies ranged widely from 2 to 67 (median = 30 ) (van Rooden et al
2005 review article) This wide range of calculated incidences of CVC related
thromboses in humans reflects the use of different CVC types different locations of entry
site of the CVC (jugular femoral subclavian axillary cephalic vein) different thrombus
detection technique (venography or ultrasonography) unequal durations of follow up
varying diseases of patients (eg haematological oncological) and presence of
predisposing factors (eg previous venous thrombosis history presence of cancer) (van
Rooden et al 2005 Geerts 2014 Liu et al 2015)
Part of the authors empirically observed CVC-associated thrombosis of the external
jugular vein (EJV) as a relevant complication in their canine intensive care patients which
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
43
awakened their interest in that topic To the best of the authoracutes knowledge studies about
extra luminal CVC related thromboses in dogs are rare in the available veterinary
literature One study evaluating the complications associated with 100 CVCs placed in
the jugular vein of dogs and cats reported only 2 cases of thrombosis (confirmed by
palpation or ultrasound) but obviously did not assess the appearance of subclinical CVC
related thromboses (Adamantos et al 2010) Thereby the incidence of CVC-induced
thromboses in dogs is unknown and systematic studies regarding an effective
anticoagulatory management are lacking
In this context it is of interest whether a possible development of thrombosis or an
increased risk (despite the prevention by a standard application of unfractionated heparin
[UFH]) can be predicted by means of changes of haemostasis related parameters during
the initial examination In one study on 168 human patients without indication of
anticoagulation prophylaxis for example elevated leukocyte counts high plasminogen
activator inhibitor and high coagulation factor VIII levels (all measured on blood samples
taken after CVC placement) were associated with an increased incidence of symptomatic
CVC-related thrombosis diagnosed by ultrasonography (Boersma et al 2016)
Therefore aims of the present study were (1) to determine the incidence of catheter-
induced thrombosis in canine intensive care patients (as detected by ultrasonography and
electron microscopy ELMI) and thereby (2) to evaluate the efficacy of a standard
antithrombotic regimen with unfractionated heparin (UFH) routinely used in the Small
Animal Clinic of the University of Veterinary Medicine Hannover and (3) to assess
whether selected haemostasis test results are predictive for an increased risk for
thrombosis formation
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
44
2 Material and methods
21 Study design
Canine in-patients of the Small Animal Clinic University of Veterinary Medicine
Hannover receiving a CVC in the EJV for medical reasons from March 2017 to
December 2019 were included Anticoagulatory treatment was performed according to
the standard protocol of the clinic Colour Doppler ultrasonographic controls of the EJV
and blood samplings (for heparin activity basic haematology and haemostasis tests) were
done immediately before the catheter was placed (day 0) and on days 1 3 5 etc (2 hours
after the first daily UFH injection) until the CVC was removed Only patients were
included which could be examined at least until day 3 Haemostasis tests included
prothrombin time activated partial thromboplastin time thrombin time fibrinogen
concentration antithrombin activity fibrin monomers D-dimers thrombin generation
assay (TGA) and rotational elastometry as well as the heparin activity using a
chromogenic anti-factor Xa test Finally ELMI of sections of the CVC after its removal
was performed
22 Animals
Originally 32 patients hospitalised in the Small Animal Clinic and receiving a CVC in the
EJV for medical reasons entered the study Three of them were subsequently excluded
because they did not reach the defined minimum study period of 3 days Two were
euthanised and one referred back to the referring vet before day 3 22 dogs weighing more
than 15 kg were included so that the withdrawal of about 10 ml of blood on several days
was not a significant burden for the patient (lt 1 of the total blood volume) Five dogs
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
45
with a body weight of less than 15 kg but at least 8 kg were included with special
consideration of the amount of blood taken (no more than 6 ml on individual days)
All animals received standard anticoagulatory treatment with UFH 17 dogs with internal
diseases received 150 IUkg BW TID subcutaneously and 12 dogs (post surgery) received
a reduced dosage of 75 IUkg BW TID subcutaneously
The 29 dogs enclosed in the study included 4 mixed breed dogs and the following
pedigree dogs 5 Labrador Retrievers 3 German Shepherds 2 Australian Shepherds and
one from each of the following breeds Akita Inu Asian Shepherd Bavarian Mountain
Hound Beagle Belgian Shepherd Briard Canaan dog Dalmatian Golden Retriever
Havanese dog Hovawart Rhodesian Ridgeback Schnauzer Shar Pei and Wolfspitz
These dogs were aged between 2 and 164 months (median 77 months) and had body
weights ranging from 81 and 63 kg (median 27 kg) 9 were castrated males 8 intact
males 7 spayed females and 5 intact females
The diseases of the 29 patients requiring a CVC included 3 from each of the following
diseases hepatopathy and esophagus dilatation 2 from each of the following diseases
bacterial cystitis diabetes mellitus and 1 from each of the following diseases abdominal
wall perforation aspiration pneumonia cystitis intestinal round cells sarcoma carcinoma
of the nasal cavity chronic renal insufficiency cystic duct rupture heartworm disease
ivermectine intoxication lymphoma multifocal alveolar histiocytosis pancreatitis post-
surgical bleeding stomach dilatation stomach foreign body tetanus thoracic
pyogranulomatous inflammation thrombocytopenia and trigeminal nerve paresis
Animals were treated in accordance with the German Animal Welfare Law The
experimental design was approved by the Animal Welfare officer of the University of
Veterinary Medicine and reported to the responsible authority (Lower Saxony State
Office for Consumer Protection and Food Safety reference number 17A101)
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
46
23 Positioning and handling of the central venous catheter
Two types of single lumen radiopaque polyurethane CVC (different on length and
diameter) were used during the study The CVC type I (n = 2529) was Cavafixreg Certoreg
Splittocanreg CVC 32 cm length 11 x 17 mm diameter16 G (BBraun Melsungen AG)
the CVC type II used (n = 429) was Certofixreg Mono S 215 15 cm length 08 x 14 mm
diameter18G (BBraun Melsungen AG) Each CVC was inserted into the EJV using a
modified Seldinger technique as follows the skin of the neck was shaved and disinfected
with antiseptic soap alcohol and iodine Local anesthesia (Lidocaine HCl 2) was
administrated subcutaneously on the catheter insertion site (a middle point in the lateral
neck over the jugular sulcus) A small skin incision was performed in the insertion site
and one peripheral plastic cannula with a metallic needle inside (14 G for CVC type I
and 20 G for CVC type II) was inserted in the EJV in cranio-caudal orientation (Hundley
et al 2018) Once the EJV was reached the needle was removed and a flexible wire
guide was inserted through the peripheral cannula following an external landmark
(4thintercostal space) (Reminga et al 2018) The peripheral cannula was removed and
the respective CVC was inserted slowly and with gentle pressure into the EJV guided
with the flexible wire and following the same external landmark to reach the right
position After the insertion of the CVC the flexible wire guide was removed The correct
positioning of the CVC was checked with a latero-lateral thorax x-ray ensuring that the
CVC tip was placed at the junction of the cranial cava vein with the right atrium Each
CVC was fixed to the neck skin (adjacent to the insertion point) with a monofilament
non-absorbable suture (gauge 3-0) Finally a bandage elaborated with Povidone-Iodine
gauze (Betaisodonareg) padded synthetic band (Artiflexreg Natur) and elastic band
(Elastomullreg) was used to protect the CVC output (Claude et al 2010) and changed
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
47
daily Several times day the catheter was flushed with isotonic sodium chloride
containing 1 IUml UFH
24 Heparin preparation and administration
Heparin-sodium-25000-ratiopharm (injection solution containing 5000 IUml sodium
heparin [mucosa]) was used SC injection was performed over the lateral thorax wall
using 1 mL syringes with fine graduation marks
25 Ultrasonography
Colour Doppler ultrasonographic examinations were performed in lateral position using
a LO-GIQ E9 ultrasound device (GE Healthcare Chicago Illinois USA) with a 15
Megahertz linear ultrasound probe The development of a thrombus or fibrin debris in the
EJV lumen around the CVC or inside it was assessed at each control Once the
ultrasound was finished a new protective bandage was placed around the neck of the dog
According to the percentage of lumen occlusion of the EJV (evaluated in cross sectional
views during the ultrasound scanning) the thrombi were classified in four different grades
(Fig 1)
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
48
of the residual vein lumen C ndash catheter T ndash thrombus
Fig 1 Definition of different grades of central venous catheter induced thrombosis of the
jugular vein as detected using colour Doppler ultrasound
26 Blood collection
At each time point approx 6ndash10 ml of blood (according to the body weight) was collected
This included approx 1 ml of EDTA blood for hematological analysis and approx 5ndash9
ml of citrated blood (tubes containing one part of 011 molL [38 ] sodium citrate to
nine parts of blood) for the measurement of hemostasis tests in citrated blood (ROTEM)
or citrated plasma Blood samples were collected by puncture of peripheral veins with
disposable cannulas (09 x 40 mm [20G] or 10 x 40mm [21G]) directly into sample tubes
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
49
or from the CVC using the three-syringe-technique (Villalta-Garciacutea et al 2015) in
situations when the peripheral venipuncture was not indicated due to phlebitis or irritation
andor inflammation of the surrounding skin Briefly 2ndash3 ml of blood were taken in a
sterile disposable syringe and immediately afterwards the blood sample was taken in a
new disposable syringe and transferred into the test tubes containing the respective
anticoagulants finally the first 2-3 ml taken were re-infused to the dog through the CVC
followed by flushing with a solution of sodium chlorideUFH (02) Immediately after
blood collection each sample tube was swayed gently until blood and anticoagulant had
been mixed thoroughly To prepare citrated platelet poor plasma citrated blood was
centrifuged at 16000 g for 10 min at room temperature using a microcentrifuge The
plasma was transferred into plastic tubes and the centrifugation procedure was repeated
This platelet poor plasma was then frozen in aliquots at -70 degC until used for analysis
Directly before analysis the plastic tubes were thawed at 37 degC in a water bath
27 Laboratory tests
Haematological analyses were made automatically with Adviareg 2120i (Siemens
Healthcare GmbH Germany)
Prothrombin time activated partial thromboplastin time and thrombin time were
measured with the autoanalyser Amax Destiny Plus (Tcoag Deutschland GmbH
Germany) and commercial reagents (Thromborel S Siemens Healthcare Diagnostics
Products GmbH Marburg Germany CK Prestreg and Test Thrombin Reagent
Diagnostica Stago SAS Asniegraveres sur Seine Cedex France) Whereas the last two tests
were measured according to the manufacturer`s instructions a modified test optimised
for canine plasma was used to measure prothrombin time (25 microl citrated plasma 120 with
imidazole buffer + 25 microl of a human fibrinogen solution 2 gl prewarmed for 120 s at 37
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
50
oC addition of 25 microl of activating reagent Thromborel S) and the results converted into a
percentage activity based on a calibration curve prepared with canine pooled plasma The
fibrinogen concentration was measured with the method according to Clauss using a test
kit from Stago Diagnostica SAS D-dimer concentration was measured with a
quantitative commercial human latex turbidimetric method in the autoanalyser Cobas
c311 using a completely assembled kit and human standards (Roche Diagnostics
Mannheim Germany) Antithrombin activity was also measured with a completely
assembled chromogenic test kit in the autoanalyser Cobas c311 and calibrated with a
canine pool plasma (n = 100)
The thrombin generation assay (Technothrombinreg TGA (Technoclone GbmH Vienna
Austria) determines thrombin generation based on monitoring of the fluorescence
generated by cleavage of a fluorogenic substrate by thrombin over time after activation
of the coagulation cascade by different concentrations of tissue factor and negatively
charged phospholipid in platelet poor plasma From the fluorescence development over
time the concentration of thrombin in the sample can be calculated using a thrombin
calibration curve The increase in thrombin concentration with time then allows to
calculate generation of thrombin in the sample and to plot thrombin values over time for
the whole coagulation process
The rotational thromboelastometry analysis with ROTEM delta device (Werfen Munich
Germany) was started approx 15 minutes after the blood collection To perform the
analysis and according to the automated ROTEM software 20 microl of recalcification reagent
(200 mmoll calcium chloride solution star-tem) and 20 microl of the respective activating
reagent (ex-tem in-tem) were pipetted into and carefully mixed in a pre-warmed cuvette
using the automated pipette provided in the device immediately afterwards 300 microl of
citrated whole blood (previously gently revolved) were added and mixed with the
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
51
reagents previously deposited Finally the cuvette was carefully placed and fixed to an
oscillating pin (oscillation movements on left and right through an angle of 475deg) in the
ROTEM delta device The interaction between blood cells and subsequently the bond
between fibrin and platelets promoted by the biomechanical changes in pH electrolytes
and temperature start the clot formation with its subsequent retraction and lysis The
magnitude of the resistance produced by the clot over the oscillating pin is quantitatively
represented by different parameters measured during the analysis and also qualitatively
in a graphic illustration (Bagge et al 2016 Crochemore et al 2017)
A chromogenic substrate test (Coatest Heparin Chromogenix-Instrumentation
Laboratory Haemochrom Diagnostica Essen Germany) was used for measurements of
UFH in the auto-analyser Cobas c311 The test application was created based on
manufacturers` instructions Different dilutions of the same batch of the commercial
sodium UFH preparation used in the experiment with normal canine pool plasma
(prepared from identical aliquots of 10 healthy adult dogs) served as standards for the
calibration of the measurement
28 Electron microscopy
Selected areas on the last third of the CVC (5ndash10 cm according with the CVC) with
macroscopically detectable thrombi were analysed and some of the CVCs were cut in
two different places The cut samples were preserved in a fixative medium composed of
1 glutaraldehyde and 4 formalin Once the sample was fixed it was prepared
according to the osmium thiocarbohydrazide procedure (OTOTO-procedure) (Malick et
al 1976) The fixed CVCs were dehydrated in seven series as follow first three series of
120 minutes each one on 30 50 and 70 ethanol respectively followed by two series
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
52
of 120 min each one on 90 ethanol and then two series of 24 hours each one on 100
ethanol After the dehydration process the samples were placed two times during 24
hours each one on isoamyl acetate and then placed on in a critical point drier Finally the
samples were coated with gold in a splutter-coater with 25 kV acceleration voltage in an
argon atmosphere with a current of 20 mA for one minute The samples were examined
in an EVO 15 scanning electron microscope (ZEISS International Carl Zeiss Jena
Germany) and photographed with magnifications of 600x and 6000x avoiding mechanical
artifacts (Toskala et al 1995) Thrombi on the external wall of the CVC were analysed
and measured using the software ImageJ (National Institutes of Health and the Laboratory
for Optical and Computational Instrumentation University of Wisconsin USA)
According to their extension (surface size) and thickness (related to the CVC wall
thickness in a cross sectional view) thrombi on the external wall and additionally within
the lumen of the CVC were each classified in four grades (Fig 2 Fig 1S [suppl
material]) In case of two cuts the highest degree of thrombosis detected at one of the two
sites was used for the final assessment
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
53
surface sizepercent of CVC wall thickness
Fig 2 Definition and illustration of different grades of central venous catheter induced
(extraluminal) thrombosis as detected using electron microscopy
Fig 1S (suppl material) Definition and illustration of different grades of intraluminal
thrombosis in central venous catheters as detected using electron microscopy
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
54
29 Statistical methods
Comparison between thrombus graduation either based on sonographic and ELMI
findings respectively was performed using the McNemar test with calculation of the
concordance index (κ) Standard normal distribution was assessed using the
Kolomogorov-Smirnov test
Due to non-normal distribution of part of data sets heparin activities and results of
haemostasis assays are reported using non-parametric variables (median minimumndash
maximum box and whisker plots) Heparin activities and results of haemostasis tests
between dogs with or without thrombosis were compared using Mann-Whitney-U test
3 Results
31 Sonographic detection of thrombus formation
Thrombus formation was sonographically detected in 829 patients during the study
period Based on examination on the final day in half of these eight patients (n = 4) the
thrombus formation was scored grade 1 in 3 dogs grade 2 and only in one grade 3 In all
of these 8 dogs this was the maximum grade during the study period One dog with fibrin
precipitates was excluded from further analysis because it could not be clearly assigned
to one of the groups In 78 cases thrombi were attached mainly to the venous wall
whereas only in one case was attached to the CVC external wall Seven of these 8 dogs
which developed thrombi received the standard heparin dosage (150 IUkg TID 41
[717] of dogs receiving this dosage) and only one the reduced heparin dosage (75 IUkg
TID 8 [112] of dogs receiving this dosage)
The time point of first detection of thrombus formation varied between days 3 and 11
(median day 8) In addition detritus (presumably composed mainly of fibrin) was
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
55
detectable in the lumen of the EJV in one further dog and within the lumen of the CVC
in further 5 dogs
32 Thrombus formation by electron microscopy
27 from 29 CVC catheter tips were analysed by electron microscopy 827 samples were
without detectable thrombus (grade 0) and 1927 with detectable thrombi 72 showed
thrombus formation of grade 1 427 of grade 2 and 827 of grade 3 There was no
concordance between thrombus graduation based on sonographic and the ELMI
graduation respectively The concordance index (McNemar test) was negative (κ = -
00718) indicating that the degree of agreement is less than by chance
Of the in total 19 dogs with detectable thrombus formation by electron microscopy 11
dogs received 150 IUkg TID (in total 16 dogs of these 27 dogs received that heparin
dosage) and 8 received 75 IUkg TID (in total 11 of these 27 dogs received that heparin
dosage 73 811) and Of the 12 patients with grade 2 and 3 thrombus formation 5 dogs
received 75 IUkg TID and 7 dogs 150 IUkg
The supplementary analysis of intraluminal thrombus formation revealed no detectable
thrombi in 7 cases and thrombus formation of grade 1 (lt 25 occlusion) in 11 cases of
grade 2 (25ndash50 ) in 3 cases and of grade 3 (gt 50 ) in 6 cases
33 Heparin activities and relationship to thrombus formation
Heparin activities varied remarkably in both dosage groups (Fig 3 ab) There was no
significant difference of heparin activities between blood samples collected via
venipuncture or from the CVC (Table 1S supplementary material) In addition there was
no significant difference between heparin activities in dogs with or without
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
56
sonographically or electron microscopically detectable thrombosis (Tables 1 and 2)
Table 1 refers to sonographically detectable thromboses and is based on median heparin
activities calculated from the measurements on day 1 and following days per patient
firstly for the total number of patients and secondly for the patients receiving 150 IUkg
TID In addition in the eight dogs heparin activities on the day of first thrombus detection
(023 IUml 019ndash036 IUml) were not significantly different from the median values
(Table 1 P = 05214 Wilcoxon test) Table 2 compares heparin activities (median values
of all days and results on the final day) between dogs with and without electron
microscopically detectable CVC-associated thrombi
Table 1S (Supplementary material) Heparin activities (IUml median values
minimumndashmaximum) measured in 27 dogs receiving 75 or 150 IUkg unfractionated
(standard) heparin TID subcutaneously 2 hours after the first injection on the day
Comparison of samples collected via puncture of the peripheral veins and those collected
from the central venous catheter (CVC) using the three-syringe-technique
Peripheral vein
puncture CVC
Mann-Whitney-U
test
Total number
(75 and 150
IUkg TID)
019
(00ndash108)
(n = 65)
023
(000ndash105)
(n = 65)
P = 03033
75 IUkg TID
010
(000ndash108)
(n = 25)
012
(000ndash023)
(n = 21)
P = 06907
150 IUkg
TID
030
(000ndash096)
(n = 40)
036
(000ndash105)
(n = 44)
P = 01223
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
57
Fig 3ab Heparin activities (box- and whisker plot arithmetic means) in dogs receiving
unfractionated heparin at a dosage of 75 IUkg BW tid (a) or 150 IUkg BW tid (b) two
hours after the first daily heparin administration
day 1 represents the first day after initiation of heparin treatment
only days were considered with at least results from 5 patients
Table 1 Heparin activities (IUml median values minimumndashmaximum) in 28 dogs
receiving 75 or 150 IUkg unfractionated (standard) heparin TID subcutaneously with
(grades 1ndash3) or without (grade 0) sonographically detectable thrombus Blood collections
were performed 2 hours after the first daily injection
Grade 0
(sonography)
Grades 1ndash3
(sonography)
Mann-Whitney U
test
Total number
(75 and 150 IUkg
TID)
020 (000ndash072)
(n = 20)
027 (018ndash059)
(n = 8)
P = 03212
150 IUkg TID 042 (000ndash072)
(n = 10)
031 (018ndash059)
(n = 7)
P = 04593
one dog with fibrin precipitates in the lumen of the jugular vein but without defined thrombus formation
was not considered because it could not be clearly assigned to one of the two groups
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
58
Table 2 Heparin activities (IUml median values minimumndashmaximum) in 27 dogs
receiving 75 or 150 IUkg unfractionated (standard) heparin TID subcutaneously with
(score 1ndash3) or without (score 0) detectable thrombus via electron microscopy
Grade 0
(electron
microscop)
(n = 8)
Grades 1ndash3
(electron
microscop)
(n = 19)
Mann-Whitney U
test
Median value of
different days 028 (005ndash060)
022 (000ndash072)
P = 05770
Final day 020 (001ndash045)
015 (000ndash105)
P = 06706
34 Relationship between haemostasis tests and thrombus formation
From the initially performed haemostasis tests high fibrinogen concentrations and low
maximum lysis values as measured by the ROTEM delta device (using the ex-tem
reagent) were associated with sonographically detectable thrombi (Tab 3) Additional
analyses based on the median values of different days for individual patients revealed that
low maximum lysis values as measured by the ROTEM delta device (using the in-tem
reagent) were associated with sonographically detectable thrombi (maximum lysis score
0 850 [100ndash175 ] score 1ndash3 375 [100ndash900 ] median [minimumndash
maximum] P = 00166) but no other test Comparison of the results of haemostasis tests
on the final day between dogs with and without sonographically detectable thromboses
did not show any significant difference (P gt 005)
Comparison of results of haemostasis tests on the final day in dogs with detectable
thrombosis via ELMI and dogs without significant thrombus formation showed several
significant differences including lower fibrinogen concentrations and longer clotting
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
59
times (ex-tem reagent) and clot formation times (ex-tem and in-tem reagents) using the
ROTEM delta analyser in general expressing a less coagulable status (Tab 4) If values
of dogs graded 0 and 1 (n =15) are compared to those of grades 2 and 3 (n = 12) the last-
mentioned had longer clotting times (ROTEM delta in-tem reagent) as well as longer clot
formation times and lower alpha angles (ROTEM delta in-tem reagent) (P lt 005 results
not shown)
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
60
Table 3 Comparison of initial results of haemostasis parameters (median values
minimumndashmaximum in dogs with (grades 1ndash3) and without (grade 0) sonographically
detectable central venous catheter-induced thromboses
Parameter Unit
Sonographic
thrombus grade
P-value
(Mann-Whitney
U test)
0
(n = 20)
123
(n = 8)
Haematocrit 355 (228ndash641)
382 (245ndash499)
06111
Platelet count x103microl 292 (420ndash483)
218 (860ndash458)
02320
Prothrombin time 944 (359ndash169)
896 (471ndash136)
05758
Activated partial thromboplastin
time s 128
(955ndash249) 140
(117ndash164) 02322
Thrombin time s 129 (121ndash178)
141 (650ndash160)
02028
Fibrinogen gl 448 (153ndash770)
747 (153ndash116)
00235
Antithrombin activity 639 (289ndash107)
670 (464ndash746)
05249
D-dimers microgml 006 (000ndash148)
015 (003ndash372)
03997
Thrombin generation assay nmol 130 (000ndash365)
912 (190ndash277)
08787
Clotting time (ROTEM ex-tem) s 420 (290ndash930)
430 (170ndash159)
07989
Clot formation time (ROTEM ex-
tem) s 665
(310ndash143) 470
(240ndash800) 00633
Alpha angle (ROTEM ex-tem) degrees 795 (620ndash840)
815 (750ndash860)
01667
Max clot firmness (ROTEM ex-
tem) mm 750
(570ndash860) 775
(700ndash830) 01753
Max clot elasticity (ROTEM ex-
tem) - 299
(131ndash619) 347
(235ndash484) 01544
Maximum lysis (ROTEM ex-tem) 135 (300ndash290)
400 (000ndash800)
00037
Clotting time (ROTEM in-tem) s 208 (840ndash810)
249 (720ndash371)
05085
Clot formation time (ROTEM in-
tem) s 835
(370ndash634) 745
(390ndash235) 08988
Alpha angle (ROTEM in-tem) degrees 745 (250ndash820)
750 (500ndash820)
09797
Max clot firmness (ROTEM in-
tem) mm 700
(540ndash810) 740
(610ndash770) 03587
Max clot elasticity (ROTEM in-
tem) - 235
(118ndash424) 287
(156ndash332) 04764
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
61
Maximum lysis (ROTEM in-tem) 850 (000ndash210)
350 (000ndash220)
01327
Table 4 Comparison of results of haemostasis parameters (median values minimumndash
maximum) on the last day in dogs with (grades 1ndash3) and without (grade 0) central venous
catheter-associated thrombus formation as detected by electron microscopy
Parameter Unit
Thrombus grade
(ELMI)
P-value
(Mann Whitney
U test)
0
(n = 8)
123
(n = 19)
Haematocrit 322 (241ndash454)
330 (216ndash510)
05953
Platelet count 103microl 446 (321ndash592)
268 (101ndash675)
00558
Prothrombin time 145 (104ndash264)
120 (561ndash148)
00462
Activated partial thromboplastin
time s 123
(109ndash140) 136
(109ndash188) 00665
Thrombin time s 129 (121ndash178)
141 (650ndash160)
02028
Fibrinogen gl 465 (275ndash866)
347 (189ndash992)
00491
Antithrombin activity 793 (448ndash981)
692 (398ndash889)
04572
D-dimers microgml 018 (000ndash173)
007 (000ndash118)
01920
Thrombin generation assay nmol 501 (000ndash160)
93 (000ndash287)
09562
Clotting time (ROTEM ex-tem) s 360
(270ndash410) 400
(310ndash620) 00238
Clot formation time (ROTEM ex-
tem) s 395
(120ndash740) 650
(240ndash174) 00275
Alpha angle (ROTEM ex-tem) degrees 835 (760ndash880)
790 (580ndash850)
00484
Max clot firmness (ROTEM ex-
tem) mm 780
(700ndash850) 720
(550ndash860) 00586
Max clot elasticity (ROTEM ex-
tem) - 346
(239ndash555) 254
(121ndash637) 00495
Maximum lysis (ROTEM ex-tem) 105 (200ndash190)
110 (000ndash380)
03796
Clotting time (ROTEM in-tem) s 178 (100ndash222)
201 (139ndash554)
00525
Clot formation time (ROTEM in-
tem) s 435
(330ndash111) 790
(350ndash882) 00209
Alpha angle (ROTEM in-tem) degrees 815 (710ndash830)
740 (240ndash830)
00191
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
62
Max clot firmness (ROTEM in-
tem) mm 785
(660ndash810) 690
(270ndash840) 00586
Max clot elasticity (ROTEM in-
tem) - 362
(192ndash434) 218
(370ndash507) 00559
Maximum lysis (ROTEM in-tem) 950 (200ndash190)
800 (100ndash180)
06890
4 Discussion
The main result of our study is that under the study conditions part of the dogs developed
CVC-associated thromboses However only a very low part of these thromboses were
obviously of a clinically significant degree regarding the amount of occlusion of the EJV
and none of the dogs had a symptomatic thrombosis whose definition includes presence
of clinical signs such as localised swelling pain and redness (Boersma et al 2008 Lee
and Kamphuisen 2012) In the present study we particularly refer to the extraluminal
thromboses with regard to the assessment of the antithrombotic management
One major problem of the statistical analysis and result presentation was that the two
methods used to define the occurrence and degree of thrombus formation (ultrasound and
ELMI) revealed discrepant results Therefore we performed a statistical analysis
independently regarding thrombus formation with either of the two methods It is well
known that ELMI which has already been used to detect CVC induced thromboses in
humans (Lucas et al 2017) and experimental animals (Xiang et al 1998 Xiang et al
2001) is a very sensitive diagnostic technique which allows the ex vivo diagnosis of very
small or fibrin rich thrombi that perhaps cannot be detected by ultrasonography or
venography during the in vivo evaluation of patients (Xiang et al 2001) A major reason
for the discrepant results between the two methods used to detect CVC associated
thromboses is that different CVC segments are evaluated Using ultrasonography it was
only possible to evaluate the EJV in the neck area of the patient and thereby only its
proximal third In contrast ELMI assessed mainly the CVC tip and a human study
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
63
revealed that even different positions of the catheter tip are associated with various risks
for development of CVC associated thrombosis (Ballard et al 2016) One additional point
to consider which also may contribute to the discrepancy is that it is possible that thrombi
developed on the CVC surface can be detached during the CVC removal from the vein
and thereby before its analysis by ELMI (Xiang et al 2001)
The contrast venography technique is used as gold standard to confirm thrombosis in
human medicine However this diagnostic technique is normally used only in the case of
a strong clinical suspicion of venous thrombosis that cannot be detected by
ultrasonography (Laurenson et al 2010 Langston and Eatroff 2018) Our study protocol
with privately owned clinic patients did not allow to use this method In a study performed
in 44 human patients requiring a CVC (Koumlksoy et al 1995) a sensitivity of 94 and a
specificity of 96 were determined for the colour Doppler ultrasound diagnosis when it
was compared with the diagnosis performed with the contrast venography technique and
that makes the colour Doppler ultrasound an accurate diagnosis method to detect CVC
related thrombosis even in humans (Koumlksoy et al 1995 van Rooden et al 2003)
The incidences of CVC related thrombosis detected by colour Doppler ultrasound of the
internal jugular vein reported in 3 human studies were 2 30 and 56 (Harter et al
2002 Lordick et al 2003 Wu et al 1999) and although with a wide range the median
value of these rates (30 ) is very well suited to the incidence of 28 (829) which we
calculated for our canine study based on sonographic findings The wide variation of
incidences of CVC related thrombosis in different human studies can in general be
explained by differences regarding the type and entry site of the CVC (jugular femoral
subclavian axillary cephalic vein) the time period with the CVC in place the thrombus
detection technique (venography or ultrasonography comment in the cited 3 studies
consistently the last-mentioned method was used) and criteria for diagnosis of
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
64
thrombosis the duration of follow up diseases and presence of predisposing factors in
these patients (eg previous venous thrombosis history presence of cancer) and finally
prophylactic treatment with anticoagulants (van Rooden et al 2005 Geerts 2014 Liu et
al 2015)
Different pathomechanisms contribute to the development of CVC induced thromboses
During the CVC insertion the puncture of the skin on the CVC insertion site and the
rupture of the blood vessels integrity activate the coagulation cascade and the CVC acts
as a matrix for adherence and accumulation of fibrin and cellular components (Smith et
al 2012) The attachment of proteins and blood cells to the internal andor external wall
of the CVC can lead to a partial or complete occlusion of the catheter lumen and
sometimes to a partial or complete obstruction of the blood flow andor
thromboembolism (Langston and Eatroff 2018) There is a strong interrelationship
between thrombus and biofilm formation (Smith et al 2012) Prevention of thrombus
formation has thereby also relevance regarding the increased risk for microbial
colonisation of the catheter (Boersma et al 2008) leading at least in humans to an
increased number of CVC related systemic infections (Lordick et al 2003)
In this context it has to be considered that CVC produced from polyurethane which are
routinely used in our clinic and thereby in the study are usually associated with a
relatively high thrombosis potential and thereby definitely provide an effective test for
an antithrombotic regimen Silicone catheters have a significantly reduced risk for
thrombotic complications but this advantage has to be purchased by a higher rate of
mechanical failure (eg rupture of the catheter) (Wildgruber et al 2016) Promising
experimental approaches work on surface modifications of the standard catheters For
example one group was able to demonstrate that a coating of the surface of the catheter
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
65
with a zwitterionic polymeric sulfobetaine showed a significant reduction of thrombus
formation in vitro with human blood and in an in vivo canine model (Smith et al 2012)
Due to ethical reasons we did not performed a placebo-controlled study However
interestingly not all human studies report systemic anticoagulation and part of the human
studies was even placebo-controlled (review Lee and Kamphuisen 2012) Obviously
there is no consensus in the available human literature regarding the beneficial use of
antithrombotic prophylaxis to prevent CVC related thrombosis Several studies did not
find a statistically significant difference in the incidence of CVC related thrombosis
between patients receiving the antithrombotic prophylaxis compared with the control
groups without systemic anticoagulation (Verso et al 2005 P = 035 Karthaus et al
2006 P gt 005 Niers et al 2007 P = 049) whereas in other studies a lower incidence
of CVC related thrombosis was found in the group receiving anticoagulants as
prophylaxis compared with the control groups (Abdelfeki et al 2004 150 vs 126
P = 003 De Cicco et al 2009 400 vs 526 P = 005)
Although it is well known that subcutaneously administered UFH has a limited and
variable bioavailability and thereby leads to unpredictable blood activities (Hirsh and
Raschke 2004) the extremely wide range of blood activities after a defined dosage in our
study was relatively unexpected Blood was collected 2 hours after the first UFH
injection and thereby at the time of suspected maximum heparin activity considering the
relatively low UGH dosage (Mischke and Jacobs 2001) The cited study was performed
on healthy experimental dogs Possible additional influencing factors in critical ill
patients include circulatory insufficiency which may reduce and delay absorption from
subcutanenous tissue decreased clearance function of the liver and kidneys as well as
altered blood plasma concentrations of heparin binding plasma proteins such as
fibrinogen and platelet factor 4 and alterations of the heparin binding capacity of
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
66
endothelial cells which altogether are reasons for a great inter- but also intrapatient
variability (Hirsh and Raschke 2004) In individual patients this can lead to heparin
resistance It has also to be considered that antithrombin activities which are necessary
for an adequate heparin effect (Bjoumlrk and Lindahl 1982) were reduced in our patients
Availability of approved blood tests which can predict the risk of development of CVC
associated thrombosis already at the moment of the CVC placement are important
(Boersma et al 2016) One of the most interesting results was that dogs which developed
sonographically detectable thrombi had nearly twofold higher fibrinogen levels Well in
agreement with our results one study performed in anticoagulated human patients
demonstrated that high fibrinogen levels were associated with an increased risk of
perioperative venous thromboembolism (P = 0004) (Kato et al 2016) The cited study
does not exclusively refer to CVC associated thrombosis but 76 (1621) of the venous
thromboses were suggested to be associated with CVCs Fibrinogen measurement a test
which is widely available is therefore a useful and practicable indicator of an increased
risk for CVC induced thromboses and may advice the clinician to treat these patients
especially carefully (eg with low molecular weight heparin) In the study performed by
Boersma et al (2016) in 168 human patients without indication of anticoagulation
prophylaxis elevated leukocyte counts high levels of plasminogen activator inhibitor
and of coagulation factor VIII were associated with an increased incidence of
symptomatic CVC related thrombosis diagnosed by ultrasonography A limitation of that
study is that all measurements were performed on blood samples taken after CVC
placement Interestingly in our study ROTEM results were partly associated with
thrombus formation which reflects the well-known fact that results of viscoelastic
measurements are a good indicator of hypercoagulability and thromboembolic events
(Harahsheh and Ho 2018) It is of further interest and possibly unexpeted that on the final
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
67
day of examination dogs with ELMI signs of thromboses had partly lower coagulation
activities than dogs without thromboses Due to the low number of dogs without
detectable thrombi using ELMI this implausible result has to be interpreted with caution
One possible explanation could be that temporary hypercoagulability in these dogs may
have finally resulted in consumption of haemostasis substrates
Limitations of the present study were as a consequence of the study design based on
clinical patients the relatively low total number of patients inclusion of different UFH
treatment regimens and the lack of a control group
5 Conclusions
The results of the present study indicate that the used UFH regimen was not completely
effective to prevent CVC-associated thromboses which were detectable in 28 [829]
of the patients by ultrasound However severe thromboses (high grades) are relatively
rare in intensive care canine patients receiving a routine anticoagulant treatment Because
heparin plasma activities were not different in dogs with or without CVC-associated
thrombus formation other local and systemic factors must play a significant role as well
A high fibrinogen concentration and low maximum lysis (ROTEM ex-tem) may be
useful indicators for an increased thrombotic risk in individual patients which may
require special anticoagulatory treatment
6 Acknowledgments
Jose Mauricio Pereira received a scholarship from the Universidad Nacional de Costa
Rica (reference code JB-C-1107-2016)
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
68
7 References
1 Abdelkefi A Ben Othman T Kammoun L Chelli M Ben Romdhane N Kriaa A Ladeb S
Torjman L Lakhal A Achour W Ben Hassen A Hsaiumlri M Ladeb F Ben Abdeladhim A
2004 Prevention of central venous line-related thrombosis by continuous infusion of low-dose
unfractionated heparin in patients with haemato-oncological disease A randomized controlled trial
Thromb Haemost 92 654ndash661
2 Adamantos S Brodbelt D Moores A L 2010 Prospective evaluation of complications associated
with jugular venous catheter use in a veterinary hospital J Small Anim Pract 51 254ndash257
3 Bagge A Schoumltt U Kander T 2016 Effects of naturopathic medicines on Multiplate and ROTEM
a prospective experimental pilot study in healthy volunteers BMC Complement Altern Med 16 64
4 Ballard DH Samra NS Gifford KM Roller R Wolfe BM Owings JT 2016 Distance of the
internal central venous catheter tip from the right atrium is positively correlated with central venous
thrombosis Emerg Radiol 23 269ndash273
5 Boersma RS Jie KS Verbon A van Pampus EC Schouten HC 2008 Thrombotic and
infectious complications of central venous catheters in patients with hematological malignancies Ann
Oncol 19 433ndash442
6 Boersma R S Hamulyak K van Oerle R Tuinenburg A Ten Cate-Hoek AJ Schouten HC
2016 Biomarkers for prediction of central venous catheter related-thrombosis in patients with
hematological malignancies Clin Appl Thromb Hemost 22 779ndash784
7 Bjoumlrk I Lindahl U 1982 Mechanism of the anticoagulant action of heparin Mol Cell Biochem
48 161ndash182
8 Claude A Riedesel D Riedesel E 2010 Electrocardiography-guided and retrospective analysis of
central venous catheter placement in the dog Vet Anaesth Analg 37 97ndash105
9 Comerlato PH Rebelatto TF Santiago de Almeida FA Klein LB Boniatti M M Schaan
DB Rados VD 2017 Complications of central venous catheter insertion in a teaching hospital
Rev Assoc Med Bras 63 613ndash620
10 Crochemore T Piza FMT Rodriacutegues RDR Guerra JCC Ferraz LJR Correcirca TD 2017
A new era of thromboelastometry Einstein (Sao Paulo) 15 380ndash385
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
69
11 De Cicco M Matovic M Balestreri L Steffan A Pacenzia R Malafronte M Fantin D
Bertuzzi CA Fabiani F Morassut S Bidoli E Veronesi A 2009 Early and short‐term
acenocumarine or dalteparin for the prevention of central vein catheter‐related thrombosis in cancer
patients a randomized controlled study based on serial venographies Ann Oncol 20 1936ndash1942
12 Evans NS Ratchford EV 2018 Catheter related-venous thrombosis Vasc Med 23 411ndash413
13 Geerts W 2014 Central venous catheterndashrelated thrombosis Hematology Am Soc Hematol Educ
Program 2014 306ndash311
14 Harahsheh Y Ho KM 2018 Use of viscoelastic tests to predict clinical thromboembolic events A
systematic review and meta‐analysis Eur J Haematol 100 113ndash123
15 Harter C Salwender HJ Bach A Egerer G Goldschmidt H Ho AD 2002 Catheter-related
infection and thrombosis of the internal jugular vein in hematologic-oncologic patients undergoing
chemotherapy a prospective comparison of silver-coated and uncoated catheters Cancer 94 245ndash251
16 Hirsh J Raschke R 2004 Heparin and low-molecular-weight heparin The Seventh ACCP
Conference on Antithrombotic and Thrombolytic Therapy Chest 126 (Suppl) 188S-203S
17 Hundley D Brooks A Thomovsky E Jhonson P Freeman L Schafbuch R Heng HG Moore
G 2018 Comparison of ultrasound-guided and landmark-based techniques for central venous
catheterization via the external jugular vein in healthy anesthetized dogs Am J Vet Res 79 628ndash
636
18 Kato F Takeuchi H Matsuda S Kawakubo H Omori T Kitagawa Y 2016 Incidence of and
risk factors for venous thromboembolism during surgical treatment for esophageal cancer a single-
institution study Surg Today 46 445ndash452
19 Karthaus M Kretzschmar A Kroning H Biakhov M Irwin D Marschner N Slabber C
Fountzilas G Garin A Abecasis NG Baronius W Steger GG Sudhoff T Giorgetti C
Reichardt P 2006 Dalteparin for prevention of catheter‐related complications in cancer patients with
central venous catheters final results of a double‐blind placebo‐controlled phase III trial Ann Oncol
17 289ndash296
20 Koumlksoy C Kuzu A Kutlay J Erden I Ozcan H Ergicircn K 1995 The diagnostic value of colour
Doppler ultrasound in central venous catheter related thrombosis Clin Radiol 50 687-689
21 Kucher N 2011 Clinical practice Deep-vein thrombosis of the upper extremities N Engl J Med
364 861ndash869
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
70
22 Langston CE Eatroff AE 2018 Hemodialysis catheter-associated fibrin sheath in a dog J Vet
Emerg Crit Care 28 366ndash371
23 Laurenson MP Hopper K Herrera MA Johnson EG 2010 Concurrent diseases and conditions
in dogs with splenic vein thrombosis J Vet Intern Med 24 1298ndash1304
24 Lee AYY Kamphuisen PW 2012 Epidemiology and prevention of catheter‐related thrombosis in
patients with cancer J Thromb Haemost 10 1491ndash1499
25 Liu G Fu ZQ Zhu P Li SJ 2015 Central venous catheter-related thrombosis in senile male
patients New risk factors and predictors J Huazhong Univ Sci Technolog Med Sci 35 445ndash449
26 Lordick F Hentrich M Decker T Hennig M Pohlmann H Hartenstein R Peschel C 2003
Ultrasound screening for internal jugular vein thrombosis aids the detection of central venous catheter-
related infections in patients with haemato-oncological diseases a prospective observational study Br
J Haematol 120 1073ndash1078
27 Lucas TC Silva EED Souza DOD Santos ARD Lara MO 2017 Microstructural
evaluation by confocal and electron microscopy in thrombi developed in central venous catheters Rev
Esc Enferm USP 51 1ndash9
28 Malick L Wilson R Stetson D 1976 Modified thiocarbo- hydrazide procedure for scanning
electron microscopy routine use for normal pathological and experimental tissue Stain Technol 50
265ndash269
29 Mischke R Jacobs C 2001 The monitoring of heparin administration by screening tests in
experimental dogs Res Vet Sci 70 101ndash108
30 Niers TM Di Nisio M Klerk CP Baarslag HJ Buller HR Biemond BJ 2007 Prevention
of catheter‐related venous thrombosis with nadroparin in patients receiving chemotherapy for
hematologic malignancies a randomized placebo‐controlled study J Thromb Haemost 5 1878ndash
1882
31 Reminga C Silverstein D Drobatz K Clarke D 2018 Evaluation of the placement and
maintenance of central venous jugular catheters in critically ill dogs and cats J Vet Emerg Crit Care
28 232ndash243
32 Smith RS Zhang Z Bouchard M Li J Lapp HS Brotske GR Lucchino DL Weaver D
Roth LA Coury A Biggerstaff J Sukavaneshvar S Langer R Loose C 2012 Vascular
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
71
catheters with a nonleaching poly-sulfobetaine surface modification reduce thrombus formation and
microbial attachment Sci Transl Med 4 153ra132 doi101126scitranslmed3004120
33 Toskala E Nuutinen J Rautiainen M 1995 Scanning electron microscopy findings on human
respiratory cilia in chronic sinusitis and in recurrent respiratory infections J Laryngol Otol 109 509ndash
514
34 Van Rooden CJ Tesselaar ME Osanto S Rosendaal FR Huisman MV 2005 Deep vein
thrombosis associated with central venous catheters - a review J Thromb Haemost 3 2409ndash2419
35 Verso M Agnelli G Bertoglio S Di Somma FC Paoletti F Ageno W Bazzan M Parise P
Quintavalle R Naglieri E Santoro A Imberti D Soraru M Mosca S 2005 Enoxaparin for the
prevention of venous thromboembolism associated with central vein catheter a double‐blind placebo‐
controlled randomized study in cancer patients J Clin Oncol 23 4057ndash4062
36 Villalta-Garciacutea P Loacutepez-Herranz M Mazo-Pascual S Honrubia-Fernaacutendez T Jaacutentildeez-Escalada
L Fernaacutendez-Peacuterez C 2015 Reliability of blood test results in samples obtained using a 2-mL discard
volume from the proximal lumen of a triple-lumen central venous catheter in the critically ill patient
Nurs Crit Care 22 298ndash304
37 Vose J Odunayo A Price JM Daves M Schildt JC Tolbert MK 2019 Comparison of
heparinized saline and 09 sodium chloride for maintaining central venous catheter patency in healthy
dogs PeerJ 7 e7072 httpsdoiorg107717peerj7072
38 Wildgruber M Lueg C Borgmeyer S Karimov I Braun U Kiechle M Meier R Koehler M
Ettl J Berger H 2016 Polyurethane versus silicone catheters for central venous port devices
implanted at the forearm Eur J Cancer 59 113ndash124 doi101016jejca201602011
39 Wu X Studer W Skarvan K Seeberger MD 1999 High incidence of intravenous thrombi after
short-term central venous catheterization of the internal jugular vein J Clin Anesth 11 482ndash485
40 Xiang DZ Verbeken EK Van Lommel AT Stas M De Wever I 1998 Composition and
formation of the sleeve enveloping a central venous catheter J Vasc Surg 28 260ndash271
41 Xiang DZ Verbeken EK Van Lommel AT Stas M De Wever I 2001 Sleeve-related
thrombosis a new form of catheter-related thrombosis Thromb Res 104 7ndash14
Chapter 4 General discussion
72
Chapter 4 General discussion
Incidence of CVC related thrombosis
In our study the incidence of CVC related thrombosis detected by ultrasound in critical
care canine patients was 28 To the best of the authoracutes knowledge similar studies
performed in dogs are rare in the available veterinary literature In one study performed
in 81 dogs and 12 cats (Adamantos et al 2010) an incidence of 2 was found for vein
thrombosis related to CVC This low incidence can be due to a lacking of systematic
ultrasound evaluations of the jugular vein containing the CVC In the mentioned study in
dogs and cats the vein thrombosis was confirmed by palpation or ultrasound presumably
after the presentation of clinical symptoms however in many cases as in our study
thrombus into the jugular vein can be developed without manifestation of clinical
symptoms what makes a routinely ultrasound evaluation of the vein containing the CVC
very important Our results are well aligned with the findings in humans in which a
median incidence of 30 was found for thrombosis related to CVC based on 25 studies
performed in patients requiring a CVC (review article van Rooden et al 2005) In the
mentioned 25 human studies the incidence of CVC related thrombosis varied within a
wide range (2ndash67 ) according to factors related to (1) the CVC (eg material type
entry site [jugular femoral subclavian axillary cephalic vein] time period in place (2)
diagnostic thrombus detection technique (ultrasonography or venography) and criteria
(3) patient collective (diseases and presence of predisposing factors [eg previous venous
thrombosis history presence of cancer] and (4) prophylactic antithrombotic treatment
(Geerts 2014 Liu et al 2015 van Rooden et al 2005)
The incidence of CVC related thrombosis detected by colour Doppler ultrasound in our
study fits also with the median incidence (30 ) found in three studies performed in
humans in which colour Doppler ultrasound evaluations of the jugular vein containing a
Chapter 4 General discussion
73
CVC were performed (Harter et al 2002 Lordick et al 2003 Wu et al 1999) In these
three studies the individual incidence of CVC related thrombosis was 2 (Harter et al
2002) 30 (Lordick et al 2003) and 56 (Wu et al 1999) The high thrombus
incidence reported in the study performed by Wu et al (1999) could be due to the criteria
for diagnosis of thrombosis because in that study both fibrin sleeves and compact
thrombi were considered as cases of thrombosis related to CVC The incidence for
compact thrombi was 25 which is more similar to our results In the study performed
by Harter et al (2002) the CVC related thrombosis incidence (2 ) was very low
compared with the incidence found in our study In that study the jugular veins were
examined just before or less than 24 hours after CVC removal (median period of CVC
use = 1025 days) In our study the ultrasound evaluations were performed before the
CVC placement (day 0) and then on days 1 3 5 etc until the CVC removal (median
period of CVC use = 7 days median interval until thrombus detection = 7 days) In our
study we detected thrombus formation as early as on the 3rd day so it could be possible
that in the cited study possibly only temporarily existing thrombi could have been missed
by ultrasonography in the final stage and thereby for the incidence calculation However
this scenario is not very likely because results of the present study show that
sonographically detectable thrombi during the observation period had in all cases their
maximum grade on the final day
In the study performed by Lordick et al (2003) the CVC related thrombosis incidence
determined for 43 human patients was very similar to our study and in both studies only
compact thrombi detectable by ultrasound were considered for assessment of the
incidence In the mentioned study all the human patients were screened for the presence
of thrombosis every 4 days after CVC insertion with real time B-mode ultrasound
(Lordick et al 2003)
Chapter 4 General discussion
74
In our study 70 of CVCs analysed by ELMI showed thrombi of grade 1ndash3 on the
external CVC wall To the best of the authoracutes knowledge studies about incidence of CVC
related thrombosis on the outside surface of the CVC detected by ELMI cannot be found
in the human and small animal literature In one study performed in humans (Lucas et al
2017) thrombus formation was analysed by ELMI in 78 central venous catheters That
study focussed on microstructural assessment of thrombus formation inside the distal
openings of the CVC which were found in 85 of the evaluated CVCs The percentage
of occurrence of thrombosis in the mentioned study in humans was nearly similar to the
incidence of intraluminal thrombus formation as detected in the presented study by ELMI
(74 ) In general intraluminal thrombi were not specially considered in the present
study because they mainly express the efficacy of the flushing technique rather than the
effectivity of the antithrombotic treatment In one study performed in rats the
microstructure of thrombi developed in the jugular vein containing the CVC was also
evaluated by ELMI (Xiang et al 1998) In the mentioned study pericatheter thrombosis
was detected on the proximal section of the CVC in 100 of rats (n = 12) after 1 and 3
days of catheterisation The analyses of the CVCs placed in rats were performed with the
catheter inside the vein in contrast to our study where the CVC was removed from the
jugular vein for the respective analysis Xiang et al (2001) hypothesised in one study
performed in rabbits and rats in which CVCs were placed in the jugular vein that fibrin
rich thrombi associated to the CVC can be detached during the catheter removal and this
could explain the difference between the thrombosis incidence assessed in our study and
that of the study performed by Xiang et al (1998)
In our study the incidence of CVC related thrombosis detected by ultrasonography in the
lumen of the EJV did not correlate with the incidence of CVC related thrombosis detected
by ELMI on the external wall of the CVC This was mainly due to the fact that the CVC
Chapter 4 General discussion
75
segment evaluated with both methods was different Using ultrasonography it was only
possible to evaluate the EJV in the neck area of the patient and thereby only the proximal
third of the CVC In contrast with the ELMI analysis only the distal section of the CVC
(last third) was evaluated because macroscopically detected thrombi mainly developed
near the CVC tip which was routinely positioned at the junction of the cranial cava vein
with the right atrium In a retrospective review of medical records of 169 human patients
requiring a CVC (Ballard et al 2016) the position of the CVC tip was determined by
radiologic evaluation and it was determined that the catheter tip position can be a
predisposing factor for central venous thrombosis development Thereby it could be of
interest to evaluate in further studies in dogs if a high incidence of thrombosis detected
by ELMI as was shown in our study could be related to the CVC tip position
In our study polyurethane catheters were tested which are widely used and are the
standard CVCs used in the Small Animal Clinic University of Veterinary Medicine
Hannover In vivo and in vitro studies have assessed the thrombogenicity of different
catheter materials (Borow and Crowley 1985 Smith et al 2012) In general terms
polyurethane catheters (central or peripheral) had shown a higher thrombogenicity
compared with coated (eg with hydromer) and silicone catheters Due to this property
of polyurethane catheters they can be supposed as a good test system for antithrombotic
regimens as evaluated in our study On the other hand despite silicone catheters have been
associated with a lower thrombogenicity their mechanical flexibility could be a
disadvantage in small clinical practice due to the increased risk of rupture detected even
in humans (Wildgruber et al 2016)
Chapter 4 General discussion
76
Heparin thromboprophylaxis for CVC related thrombosis
The occurence of CVC related thrombosis detected by colour Doppler ultrasound and
ELMI in our study demonstrates that the UFH in the given dosage was not completely
effective to prevent CVC related thrombosis in canine intensive care patients In human
medicine LMWH is commonly used in prophylactic anticoagulation regimens however
studies performed in human patients requiring a CVC and receiving a prophylactic
anticoagulation (with LMWH or UFH) did not achieve consensus regarding the efficacy
of prophylactic heparin to prevent CVC related thrombosis (Abdelfeki et al 2004
Lordick et al 2003 Wu et al 1999) To the best of the authoracutes knowledge studies
performed in dogs evaluating systemic prophylactic anticoagulation to prevent CVC
related thrombosis cannot be found However according to the evidence found in the
human literature and the results of our study further investigations using comparative
groups with different drugs dosages andor administration routes are required to establish
an effective antithrombotic treatment to prevent CVC related thrombosis
The plasma heparin activities measured in canine patients in our study varied significantly
after a defined subcutaneous dosage The limited and variable bioavailability of SC
administered UFH is well documented in humans (Hirsh and Raschke 2004)
Considerable variations of the circulatory status of the liver- and kidney function and
concentration of heparin-binding proteins in intensive care patients obviously amplify
this phenomenon leading to an extremely high inter-individual and intra-individual
variation of the heparin blood levels in our patients A different application route (IV)
may have reduced the influence of part of these factors and therefore could have led to
more predictable UFH blood levels However continuous infusion requires more
technical equipment and is also associated with a wide range of heparin activities in
canine patients (Scott et al 2009)
Chapter 4 General discussion
77
Prediction of CVC related thrombosis
Of 7 haemostasis tests (including ROTEM with 12 different parameters [6 for each
reagent ex-tem and in-tem]) evaluated as possible predictors of CVC related thrombosis
in our study initial values of high fibrinogen and low maximum lysis (ROTEM ex-tem)
were associated with sonographically detectable thrombus formation Some studies in
humans have established the association of laboratory biomarkers as predictors of risk
thrombosis (Boersma et al 2016 Cheng et al 2013 Kato et al 2016 Liu et al 2015)
One study performed in anticoagulated human patients showed ndash well in agreement with
our results ndash that high fibrinogen levels were associated with an increased risk of
perioperative venous thromboembolism (P = 0004) (Kato et al 2016) The cited study
does not exclusively refer to CVC associated thrombosis but 762 (1621) of the
venous thromboses were suggested to be associated with CVCs Fibrinogen concentration
measurements are normally available in the human and veterinary practice and the
association of this parameter with a high risk of development of CVC related thrombosis
can be useful to choose special antithrombotic treatment in individual critical care patients
to prevent such severe complications
Thrombin generation assay (TGA) abnormalities such as higher velocity and endogenous
thrombin potential (ETP) values measured initially ie before any anticoagulation
therapy have been associated with a higher thromboembolism risk in general (ie without
specific association with CVCs) in one study performed on 105 human patients (Espitia
et al 2017) In contrast in our study the initial TGA assay results did not show an
association with formation of CVC related thrombosis
The ROTEM parameters evaluated in the present study were partially associated with
formation of CVC thrombosis detected by colour Doppler ultrasound This finding
confirms the results obtained in humans in which ROTEM measurements are regarded as
Chapter 4 General discussion
78
a valuable indicator of hypercoagulability (Harahsheh and Ho 2018 Tuumlrk et al 2018)
However in our study individual ROTEM parameters showed partly lower coagulation
activities in dogs with CVC related thrombosis detected by ELMI at the final day of
evaluation Because the number of patients without signs of thrombosis detected by ELMI
was low compared with those who showed signs these implausible results should be
interpreted carefully The usefulness of ROTEM analysis which has also many other
indications demonstrates that is was worth to perform a detailed methodological approval
of the device including the establishment of reference values in the preliminary study
Conclusion
In conclusion the prethrombotic or thrombotic changes detected in our study did not
correlate between ultrasonography and ELMI however both methods show accurately
signs of CVC related thrombosis The UFH in the given dosage was not completely
effective to prevent CVC induced thrombus formation in canine intensive care patients
Plasma heparin activities varied significantly after a defined subcutaneous dosage
However plasma heparin activities were not different between dogs with and without
detectable CVC associated thromboses Initial high fibrinogen values and low maximum
lysis (ROTEM ex-tem) values were associated with sonographically detectable thrombi
and may be useful as indicators for an increased thrombotic risk
Finally our study in canine intensive care patients is to the best of the authoracutes
knowledge the first study in the available veterinary literature performed to evaluate the
efficacy of an antithrombotic treatment with UFH to prevent CVC related thrombosis
Chapter 5 Summary
79
Chapter 5 Summary
Central venous catheter induced thrombosis in dogs ndash occurrence under an
antithrombotic treatment regimen and possible indicators
Joseacute Mauricio Pereira Mora
Central venous catheters (CVC) are of importance for intensive care management of
canine patients Thrombosis of the jugular vein is a severe complication related to the
CVC use However only little is known about the incidence of CVC-associated
thromboses in dogs and the necessity and efficacy of prophylactic antithrombotic
regimens
Therefore the aims of the present study were (1) to determine the incidence of catheter-
induced thrombosis in canine intensive care patients (2) to evaluate the efficacy of a
prophylactic heparin regimen routinely used in the Small Animal Clinic of the University
of Veterinary Medicine Hannover and (3) to assess whether selected haemostasis test
results are predictive for an increased risk for thrombosis formation
The thesis also includes a preliminary methodological study on the use of rotational
elastometry in dogs using the ROTEM delta device In this preliminary study reference
intervals were determined based on 125 healthy dogs for different clot activator reagents
and for measurements without activator Coefficients of variation (CVs) were calculated
based on results of fourfold measurements of 3 normal and 3 abnormal blood samples in
order to assess precision of the method Influence of age groups sex neuter status and
size were also assessed The median CVs for most of the parameters evaluated in the
precision analysis were less than 10 and this confirmed that the ROTEM delta analyser
delivers repeatable results Regarding the influence of age sex neuter status and animal
Chapter 5 Summary
80
size only neuter status in female dogs appeared to be a relevant influencing factor on
most of important ROTEM parameters
The main study of the thesis comprises 29 patients hospitalised at the Small Animal Clinic
requiring a CVC for medical reasons These patients received a prophylactic
anticoagulation treatment with unfractionated heparin (UFH 150 IU [surgical dogs
received 75 IU]kg BW TID subcutaneously) A colour Doppler ultrasound of the
respective external jugular vein (EJV) in order to detect thrombus formation in the lumen
of the EJV and blood collection for haemostasis tests were performed directly before the
installation procedure (day 0) and on days 1 3 5 7 and so on after the CVC insertion
Haemostasis tests included prothrombin time activated partial thromboplastin time
thrombin time antithrombin activity D-dimer concentration thrombin generation and
rotational elastometry as well as the heparin activity using a chromogenic anti-factor Xa
test Finally when the CVC was removed electron microscopy (ELMI) of sections of the
CVC with macroscopically visible thrombi in the last third (5ndash10 cm) of the CVC was
performed 3 different grades were defined for CVC-associated (extraluminal) thrombi
detected by either ultrasound or ELMI respectively
829 patients evaluated by colour Doppler ultrasound during the study developed thrombi
into the lumen of EJV (4 x grade 1 3 x grade 2 and 1 x grade 3) 1927 CVCs assessed
using ELMI showed detectable thrombi on the external CVC surface (7 x grade 1 4 x
grade 2 and 8 x grade 3) There was no significant correlation between thrombus
formation detected by ultrasound or ELMI Thus statistical group comparisons of
patients with or without CVC-associated thrombus formation was performed
independently for both diagnostic procedures The plasmatic heparin activities showed
great variability but no significant differences were found between dogs with or without
CVC-associated thrombus formation regardless whether this was based on sonographic
Chapter 5 Summary
81
or ELMI findings From the initially performed haemostasis tests high fibrinogen
concentrations and low maximum lysis values as measured by the ROTEM delta device
(using the ex-tem reagent) were associated with sonographically detectable thrombi
In conclusion the preliminary methodological study on the ROTEM delta in dogs shows
that the method delivers repeatable results in canine citrated whole blood and the
reference values obtained should offer a reliable basis for the interpretation of test results
of this device in dogs with suspected haemostasis disorders The results of the main study
indicate that CVC-associated thromboses occur (eg 28 [829] of the patients as
detected by ultrasound) although severe thromboses (high grades) are relatively rare in
intensive care canine patients receiving a routine anticoagulant treatment Thereby
prophylactic antithrombotic protocol (unfractionated heparin [UFH]) in the given dosage
was not completely effective to prevent CVC induced thrombus formation in canine
intensive care patients A high fibrinogen concentration and low maximum lysis
(ROTEM ex-tem) may be useful indicators for an increased thrombotic risk in individual
patients which may require special anticoagulatory treatment Further studies are
required to evaluate whether different thromboprophylactic treatment regimens eg with
low molecular weight heparin are more effective in preventing CVC-associated
thromboses in dogs
Chapter 6 Zusammenfassung
82
Chapter 6 Zusammenfassung
Zentralvenenkatheter-induzierte Thrombose bei Hunden - Auftreten unter einem
antithrombotischen Behandlungsschema und moumlgliche Indikatoren
Joseacute Mauricio Pereira Mora
Zentralvenenkatheter (ZVK) sind fuumlr das Intensivmanagement von Hunden von
Bedeutung Die Entstehung einer Thrombose der Vena jugularis ist eine schwerwiegende
Komplikation im Zusammenhang mit der Verwendung eines ZVKs Uumlber die Inzidenz
von ZVK-assoziierten Thrombosen bei Hunden und die Notwendigkeit und Wirksamkeit
prophylaktischer antithrombotischer Protokolle liegen jedoch nur wenige fundierte
Angaben in der Literatur vor
Daher waren die Ziele der vorliegenden Studie (1) die Inzidenz der Katheter-induzierten
Thrombose bei caninen Intensivpatienten zu bestimmen (2) die Wirksamkeit eines
prophylaktischen Heparin-Regimes zu bewerten das routinemaumlszligig in der Klinik fuumlr
Kleintiere der Stiftung Tieraumlrztliche Hochschule Hannover angewendet wird und (3) um
zu beurteilen ob ausgewaumlhlte Haumlmostasetestergebnisse ein erhoumlhtes Risiko fuumlr die
Thrombosebildung vorhersagen koumlnnen
Die PhD-Arbeit enthaumllt zudem eine vorbereitende methodische Studie zur Verwendung
der Rotationselastometrie bei Hunden mit Hilfe des ROTEM delta-Geraumltes In dieser
Vorstudie wurden Referenzintervalle basierend auf 125 gesunden Hunden fuumlr
verschiedene Gerinnungsaktivatorreagenzien und fuumlr Messungen ohne Aktivator
bestimmt Um die Genauigkeit der Methode einzustufen wurden Variationskoeffizienten
(VKs) basierend auf den Ergebnissen von Vierfachmessungen von drei normalen und drei
abnormalen Blutproben berechnet Der Einfluss von Alter Geschlecht Kastration und
Groumlszlige wurde ebenfalls getestet Die Medianwerte der VKs fuumlr die meisten in der
Chapter 6 Zusammenfassung
83
Praumlzisionsanalyse bewerteten Parameter betrugen weniger als 10 und dies bestaumltigte
dass das ROTEM delta-Analysegeraumlt wiederholbare Ergebnisse liefert In Bezug auf den
Einfluss von Alter Geschlecht Kastration und Tiergroumlszlige erwies sich nur eine Kastration
bei Huumlndinnen als relevanter Einflussfaktor fuumlr die meisten wichtigen ROTEM-
Parameter
Die Hauptstudie der Arbeit umfasst 29 Patienten welche zum Zeitpunkt der
Untersuchung stationaumlr in der Kleintierklinik aufgenommen worden waren und aus
medizinischer Indikation einen ZVK benoumltigten Diese Patienten erhielten eine
prophylaktische Antikoagulation mit unfraktioniertem Heparin (UFH 150 IE
[chirurgische Hunde erhielten 75 IE]kg KG 3 x taumlglich subkutan) Bildgebende
Untersuchungen mittels Farbdoppler-Ultraschall der jeweiligen Vena jugularis externa
(VJE) zum Nachweis der Thrombusbildung im Lumen der VJE und Blutentnahmen fuumlr
Haumlmostasetests wurden jeweils direkt vor dem Installationsvorgang (Tag 0) sowie an den
Tagen 1 3 5 und 7 nach Legen des ZVKs durchgefuumlhrt Haumlmostasetests umfassten die
Prothrombinzeit die aktivierte partielle Thromboplastinzeit die Thrombinzeit die
Antithrombinaktivitaumlt die D-Dimer-Konzentration die Thrombingenerierung und die
Rotationselastometrie sowie die Heparinaktivitaumlt unter Verwendung eines chromogenen
Anti-Faktor-Xa-Tests Schlieszliglich wurde nach Entfernung des ZVKs eine
Elektronenmikroskopie (ELMI) von Abschnitten aus dem letzten Drittel (5ndash10 cm) des
ZVKs mit makroskopisch sichtbaren Thromben durchgefuumlhrt Fuumlr ZVK-assoziierte
(extraluminale) Thromben die entweder durch Ultraschall oder ELMI nachgewiesen
wurden wurden drei verschiedene Grade definiert
829 Patienten die waumlhrend der Studie mittels Farbdoppler-Ultraschall untersucht
wurden entwickelten Thromben im Lumen der VJE (4 x Grad 1 3 x Grad 2 und 1 x Grad
3) 1927 ZVKs die unter Verwendung von ELMI bewertet wurden zeigten
Chapter 6 Zusammenfassung
84
nachweisbare Thromben auf der aumluszligeren ZVK-Oberflaumlche (7 x Grad 1 4 x Grad 2 und 8
x Grad 3) Es gab keine signifikante Korrelation zwischen der durch Ultraschall oder
ELMI festgestellten Thrombusbildung Daher wurden statistische Gruppenvergleiche
von Patienten mit oder ohne ZVK-assoziierter Thrombusbildung fuumlr beide diagnostische
Verfahren unabhaumlngig voneinander durchgefuumlhrt Die plasmatischen Heparinaktivitaumlten
zeigten eine groszlige Variabilitaumlt es wurden jedoch keine signifikanten Unterschiede
zwischen Hunden mit oder ohne ZVK-assoziierter Thrombusbildung gefunden
unabhaumlngig davon ob diese auf sonographischen oder ELMI-Befunden beruhte Von den
initial durchgefuumlhrten Haumlmostasetests waren hohe Fibrinogenkonzentrationen und
niedrige maximale Lyse-Werte der Messungen mit ROTEM delta (ex-tem Reagenz) mit
sonographisch nachweisbaren Thromben assoziiert
Zusammenfassend zeigt die vorbereitende methodische Studie zum ROTEM delta bei
Hunden dass die Methode wiederholbare Ergebnisse in mit Zitrat
antikoaguliertemVollblut liefert Die erarbeiteten Referenzwerte sollten eine zuverlaumlssige
Grundlage fuumlr die Interpretation der Testergebnisse dieses Geraumlts bei Hunden mit
Verdacht auf Haumlmostasestoumlrungen bieten Die Ergebnisse der Hauptstudie zeigen dass
ZVK-assoziierte Thrombosen auftreten (z B 28 [829] der Patienten bezogen auf den
Ultraschall-gestuumltzten Nachweis) obwohl schwere Thrombosen (hohe Grade) bei
intensivmedizinisch versorgten Hundepatienten die eine Routineantikoagulation
erhalten relativ selten sind Somit war das prophylaktische antithrombotische Protokoll
(unfraktioniertes Heparin [UFH]) in der angewendeten Dosierung nicht vollstaumlndig
wirksam um die ZVK-induzierte Thrombusbildung bei caninen Intensivpatienten zu
verhindern Eine hohe Fibrinogenkonzentration und eine niedrige maximale Lyse
(ROTEM ex-tem) koumlnnen als Indikatoren eines erhoumlhten Thromboserisikos bei einzelnen
Patienten nuumltzlich sein fuumlr die moumlglicherweise eine spezielle gerinnungshemmende
Chapter 6 Zusammenfassung
85
Behandlung notwendig ist Weitere Studien sind erforderlich um zu untersuchen ob
verschiedene thromboprophylaktische Behandlungsschemata z B mit
niedermolekularem Heparin bezuumlglich der Verhinderung von ZVK-assoziierten
Thrombosen bei Hunden wirksamer sind
Chapter 7 References
86
Chapter 7 References
1 Abdelkefi A Ben Othman T Kammoun L Chelli M Ben Romdhane N Kriaa A Ladeb
S Torjman L Lakhal A Achour W Ben Hassen A Hsaiumlri M Ladeb F Ben Abdeladhim
A 2004 Prevention of central venous line-related thrombosis by continuous infusion of low-dose
unfractionated heparin in patients with haemato-oncological disease A randomized controlled
trial Thromb Haemost 92 654ndash661
2 Adamantos S Brodbelt D Moores A L 2010 Prospective evaluation of complications
associated with jugular venous catheter use in a veterinary hospital J Small Anim Pract 51 254ndash
257
3 Ballard D H Samra N S Gifford K M Roller R Wolfe B M Owings J T 2016
Distance of the internal central venous catheter tip from the right atrium is positively correlated
with central venous thrombosis Emerg Radiol 23 269ndash273
4 Baumann-Kreuziger L Onwuemene O Kolesar E Crowther M Lim W 2015 Systematic
review of anticoagulant treatment of catheter-related thrombosis Thromb Res 136 1103ndash1109
5 Bliss SP Bliss SK Harvey HJ 2002 Use of recombinant tissue‐plasminogen activator in a
dog with chylothorax secondary to catheter‐associated thrombosis of the cranial vena cava J Am
Anim Hosp Assoc 38 431ndash435
6 Boersma RS Hamulyak K van Oerle R Tuinenburg A Ten Cate-Hoek AJ Schouten
HC 2016 Biomarkers for prediction of central venous catheter related-thrombosis in patients
with hematological malignancies Clin Appl Thromb Hemost 22 779ndash784
7 Boersma RS Jie KS Verbon A van Pampus EC Schouten HC 2008 Thrombotic and
infectious complications of central venous catheters in patients with hematological
malignancies Ann Oncol 19 433ndash442
8 Bonnet F Loriferne JF Texier JP Texier M Salvat A Vasile N 1989 Evaluation of
Doppler examination for diagnosis of catheter-related deep vein thrombosis Intensive Care Med
15 238ndash240
9 Borow M Crowley J G 1985 Evaluation of central venous catheter thrombogenicity Acta
Anaesthesiol Scand Suppl 81 59ndash64
10 Cheng Y Cui T Fu P Liu F Zhou L 2013 Dyslipidemia is associated with tunneled-cuffed
catheter-related central venous thrombosis in hemodialysis patients a retrospective multicenter
Chapter 7 References
87
study Artif Organs 37 155ndash161
11 Comerlato PH Rebelatto TF Santiago de Almeida FA Klein LB Boniatti MM
Schaan BD Rados DV 2017 Complications of central venous catheter insertion in a teaching
hospital Rev Assoc Med Bras 63 613ndash620
12 De Cicco M Matovic M Balestreri L Steffan A Pacenzia R Malafronte M Fantin
D Bertuzzi CA Fabiani F Morassut S Bidoli E Veronesi A 2009 Early and short‐term
acenocumarine or dalteparin for the prevention of central vein catheter‐related thrombosis in
cancer patients a randomized controlled study based on serial venographies Ann
Oncol 20 1936ndash1942
13 Espitia O Fouassier M Hardouin JB Pistorius MA Agard C Planchon B Trossaert M
Pottier P 2017 Thrombin generation assay in hospitalized nonsurgical patients A new tool to
assess venous thromboembolism risk Clin Appl Thromb Hemost 23 45ndash51
14 Evans NS Ratchford EV 2018 Catheter related-venous thrombosis Vasc Med 23 411ndash413
15 Geerts W 2014 Central Venous Catheter-related thrombosis Hematology Am Soc Hematol
Educ Program 2014 306ndash311
16 Harahsheh Y Ho KM 2018 Use of viscoelastic tests to predict clinical thromboembolic
events A systematic review and meta‐analysis Eur J Haematol 100 113ndash123
17 Harter C Salwender HJ Bach A Egerer G Goldschmidt H Ho AD 2002 Catheter-
related infection and thrombosis of the internal jugular vein in hematologic-oncologic patients
undergoing chemotherapy a prospective comparison of silver-coated and uncoated
catheters Cancer 94 245ndash251
18 Hirsh J Raschke R 2004 Heparin and low-molecular-weight heparin The Seventh ACCP
Conference on Antithrombotic and Thrombolytic Therapy Chest 126 (Suppl) 188Sndash203S
19 Karthaus M Kretzschmar A Kroning H Biakhov M Irwin D Marschner N Slabber
C Fountzilas G Garin A Abecasis NG Baronius W Steger GG Sudhoff T Giorgetti
C Reichardt P 2006 Dalteparin for prevention of catheter‐related complications in cancer
patients with central venous catheters final results of a double‐blind placebo‐controlled phase III
trial Ann Oncol 17 289ndash296
20 Kato F Takeuchi H Matsuda S Kawakubo H Omori T Kitagawa Y 2016 Incidence of
and risk factors for venous thromboembolism during surgical treatment for esophageal cancer a
Chapter 7 References
88
single-institution study Surg Today 46 445ndash452
21 Kohler TR Kirkman TR 1998 Central venous catheter failure is induced by injury and can
be prevented by stabilizing the catheter tip J Vasc Surg 28 59ndash66
22 Koumlksoy C Kuzu A Kutlay J Erden I Ozcan H Ergicircn K 1995 The diagnostic value of
colour Doppler ultrasound in central venous catheter related thrombosis Clin Radiol 50 687ndash
689
23 Kucher N 2011 Clinical practice Deep-vein thrombosis of the upper extremities N Engl J
Med 364 861ndash869
24 Langston CE Eatroff AE 2018 Hemodialysis catheter-associated fibrin sheath in a dog J
Vet Emerg Crit Care 28 366ndash371
25 Langston CE Eatroff AE Poeppel K 2014 Use of tissue plasminogen activator in catheters
used for extracorporeal renal replacement therapy J Vet Intern Med 28 270ndash276
26 Laurenson MP Hopper K Herrera MA Johnson EG 2010 Concurrent diseases and
conditions in dogs with splenic vein thrombosis J Vet Intern Med 24 1298ndash1304
27 Lee AYY Kamphuisen PW 2012 Epidemiology and prevention of catheter‐related
thrombosis in patients with cancer J Thromb Haemost 10 1491ndash1499
28 Liu G Fu ZQ Zhu P Li SJ 2015 Central venous catheter-related thrombosis in senile male
patients New risk factors and predictors J Huazhong Univ Sci Technolog Med Sci 35 445ndash
449
29 Lordick F Hentrich M Decker T Hennig M Pohlmann H Hartenstein R Peschel C
2003 Ultrasound screening for internal jugular vein thrombosis aids the detection of central
venous catheter-related infections in patients with haemato-oncological diseases a prospective
observational study Br J Haematol 120 1073ndash1078
30 Lucas TC Silva EED Souza DOD Santos ARD Lara MO 2017 Microstructural
evaluation by confocal and electron microscopy in thrombi developed in central venous catheters
Rev Esc Enferm USP 51 1ndash9
31 Mismetti P Mille D Laporte S Charlet V Buchmuller‐Cordier A Jacquin JP Fournel
P Dutrey‐Dupagne C Decousus H 2003 Low‐molecular‐weight heparin (nadroparin) and
very low doses of warfarin in the prevention of upper extremity thrombosis in cancer patients with
indwelling long‐term central venous catheters a pilot randomized trial Haematologica 88 67ndash
Chapter 7 References
89
73
32 Niers TM Di Nisio M Klerk CP Baarslag HJ Buller HR Biemond BJ 2007
Prevention of catheter‐related venous thrombosis with nadroparin in patients receiving
chemotherapy for hematologic malignancies a randomized placebo‐controlled study J Thromb
Haemost 5 1878ndash1882
33 Scott K C Hansen B D DeFrancesco T C (2009) Coagulation effects of low molecular
weight heparin compared with heparin in dogs considered to be at risk for clinically significant
venous thrombosis J Vet Emerg Crit Care 19 74ndash80
34 Smith RS Zhang Z Bouchard M Li J Lapp HS Brotske GR Lucchino DL Weaver
D Roth LA Coury A Biggerstaff J Sukavaneshvar S Langer R Loose C 2012
Vascular catheters with a nonleaching poly-sulfobetaine surface modification reduce thrombus
formation and microbial attachment Sci Transl Med 4 153
35 Tuumlrk S M Cansu D Uuml Teke H Uuml Kaşifoğlu T Meltem Akay O Bilgin M amp Korkmaz
C 2018 Can we predict thrombotic tendency in rheumatoid arthritis A thromboelastographic
analysis (with ROTEM) Clin Rheumatol 37 2341ndash2349
36 van Rooden CJ Rosendaal FR Barge RM van Oostayen JA van der Meer FJM
Meinders AE Huisman MV 2003 Central venous catheter related thrombosis in haematology
patients and prediction of risk by screening with Doppler-ultrasound Br J Haematol 123 507ndash
512
37 van Rooden CJ Tesselaar ME Osanto S Rosendaal FR Huisman MV 2005 Deep vein
thrombosis associated with central venous catheters - a review J Thromb Haemost 3 2409ndash
2419
38 Verso M Agnelli G Bertoglio S Di Somma FC Paoletti F Ageno W Bazzan M Parise
P Quintavalle R Naglieri E Santoro A Imberti D Soraru M Mosca S 2005 Enoxaparin
for the prevention of venous thromboembolism associated with central vein catheter a double‐
blind placebo‐controlled randomized study in cancer patients J Clin Oncol 23 4057ndash4062
39 Vose J Odunayo A Price JM Daves M Schildt JC Tolbert MK 2019 Comparison of
heparinized saline and 09 sodium chloride for maintaining central venous catheter patency in
healthy dogs PeerJ 7e7072 httpdoi107717peerj7072
40 Wang LH Wei F Jia L Zhi Lu Wang B Dong HY Yu HY Sun GJ YangJ Li B
Chapter 7 References
90
Meng J Zhang RN Bi XQ Chen HY Jiang AL 2015 Fibrin sheath formation and
intimal thickening after catheter placement in dog model role of hemodynamic wall shear stress J
Vasc Access 16 275ndash284
41 Wildgruber M Lueg C Borgmeyer S Karimov I Braun U Kiechle M Meier R Koehler
M Ettl J Berger H 2016 Polyurethane versus silicone catheters for central venous port devices
implanted at the forearm Eur J Cancer 59 113ndash124
42 Whitesell RT Steenburg SD 2014 Imaging findings of acute intravascular thrombus on non-
enhanced computed tomography Emerg Radiol 21 271ndash277
43 Wu X Studer W Skarvan K Seeberger MD 1999 High incidence of intravenous thrombi
after short-term central venous catheterization of the internal jugular vein J Clin Anesth 11
482ndash485
44 Xiang DZ Verbeken EK Van Lommel AT Stas M De Wever I 1998 Composition and
formation of the sleeve enveloping a central venous catheter J Vasc Surg 28 260ndash271
45 Xiang DZ Verbeken EK Van Lommel AT Stas M De Wever I 2001 Sleeve-related
thrombosis a new form of catheter-related thrombosis Thromb Res 104 7ndash14
91
Acknowledgements
First I thank God for every day in my life for His guide and because He was is
and will be always with me and that certainty gives me strength to take a step
forward
My sincere thanks to Prof Dr Reinhard Mischke for the opportunity to perform
my PhD under his supervision for his support and guidance for all that I learned
with him and for his friendship
I also thank to Prof Dr Manfred Kietzmann and PD Dr Mario von Depka
Prondzinski for being part of my supervision group for their time and beneficial
advices and discussion during my PhD
My gratitude to Prof Hewicker-Trautwein and Mrs Rohn for their valuable work
and availability to help me
I thank the Werlhof Institute Medical Care Center Hannover for the valuable
cooperation especially to Mrs Ober-Cassebaum Anne and Franziska for all their
help
I thank all the staff of the Small Animal Clinic of the University of Veterinary
Medicine Hannover (TiHo) My special gratitude to the internal medicine and
critical care surgery neurology ultrasound and anesthesia departments and to
the TiHo students all their cooperation made possible my research
I thank the haematology clinical chemistry and haemostasis laboratory
department of the Small Animal Clinic TiHo especially to Dirk because he was
always available for me thanks for his guide and friendship all his explanations
in the laboratory were of great value but also the conversations about different
topics of the life were of big value for me
92
I thank the Hannover Graduate School for Veterinary Pathobiology
Neuroinfectology and translational Medicine (HGNI) for the well-organized
PhD program and especially thanks to Prof Dr Beatrice Grummer Dr Tina
Selle and Mrs Tania Czeslik for his guide and help during my studies
My sincere thanks to Mrs Maritta Ledwoch for her guidance at the International
office of the TiHo ldquoI remember you since my externship in Germanyrdquo
I also thank the Student Union Hannover for all the help with the accommodation
for me and my family
I want to thank the Universidad Nacional de Costa Rica and therefore to Costa
Rica (my loved country) for the financial support to perform my PhD studies
but also for my professional formation as a veterinarian Special thanks to the
Veterinary Medicine School from Universidad Nacional de Costa Rica To all the
academic and administrative staff for their support and for believing in me
Sincerely thanks to Dr Mauricio Jimenez Soto my mentor my adviser but most
important my friend Thanks for believing in me and for always being by my side
ldquoAgradezco profundamente a mis papaacutes ldquodon Tito (Papillo) y dontildea Eida (Mami)rdquo
Todo lo bueno que hay en mi proviene de Dios y de ellos Gracias a mis hermanos
(as) cuntildeados (as) sobrinos y a toda la familia relacionada a ellos Siempre han
sido un gran apoyo y los amordquo
ldquoGracias a dontildea Alba Fran y David Gracias por toda la ayuda y por estar siempre
pendientesrdquo
I would like to thank all my friends that during a short part or during my whole
life have been with me For all the good time shared really thanks
To Willy thanks ldquogordordquo
93
I sincerely thank to Adriano and Johanna for their help and hospitality Thanks
Adriano for your guidance and support during my beginning at the TiHo for make
me feel in Costa Rica with your friendship for the tourist walks and for the bike
training I missed the coffee times
I want to thank Ina the first person to give me a smile at the start of my PhD
Thanks for your hugs and friendship
I also thank Franz for his help and for being a very nice partner Thanks for help
me always when I requested you
ldquoFinalmente quiero expresar mi gratitud y amor a Karo Faacutetima y Abigaiacutel A Karo
por tu sacrificio y soporte por las palabras de aliento por estar a mi lado en las
buenas pero en especial en las malas A Faacute por tu sacrificio por tus sonrisas por
tus abrazos y besos por bailar con migo siempre por ser mi modelo e inspiracioacuten
A Abi por ser una nueva razoacuten in mi vida para seguir adelante por todo lo que
deseo compartir con tigo por mostrarme junto con Faacutetima la felicidad en una
sonrisardquo
List of abbreviations
i
List of abbreviations
CV coefficient of variation
CVC central venous catheter
EJV external jugular vein
ETP endogenous thrombin potential
HDL high-density lipoprotein
IV intravenous
LDL low-density lipoprotein
LMWH low molecular weight heparin
ROTEM rotational elastometry
SC subcutaneous
TGA thrombin generation assay
UFH unfractionated heparin
Chapters 2 and 3 have their own abbreviations included
List of figures and tables
ii
List of figures and tables
Chapter 2
Table 1 Coefficients of variation () for rotational thromboelastometry using the
ROTEMreg delta analyser in normal blood samples 20
Table 2 Coefficients of variation () for rotational thromboelastometry using the
ROTEMreg delta analyser in canine blood samples with haemostatic alterations 20
Table 3 Reference values (median reference interval based on 25 - and 975 -
quantiles) for thromboelastometric measurements with the ROTEMreg delta analyser in
dogs (n=125 95 confidence intervals in brackets) 23
Table 4 Median values (minimum and maximum in brackets) for different ROTEMreg
variables using ex-tem reagent stratified by sex and neuter status 24
Table 5 Median values (minimum and maximum in brackets) for different ROTEMreg
variables using the in-tem reagent stratified by sex and neutering status 25
Chapter 3
Fig 1 Definition of different grades of central venous catheter induced thrombosis of the
jugular vein as detected using colour Doppler ultrasound 48
Fig 2 Definition and illustration of different grades of central venous catheter induced
(extraluminal) thrombosis as detected using electron microscopy 53
Fig 1S (suppl material) Definition and illustration of different grades of intraluminal
thrombosis in central venous catheters as detected using electron microscopy 53
Table 1S (Supplementary material) Heparin activities (IUml median values
minimumndashmaximum) measured in 27 dogs receiving 75 or 150 IUkg unfractionated
(standard) heparin TID subcutaneously 2 hours after the first injection on the day
Comparison of samples collected via puncture of the peripheral veins and those collected
from the central venous catheter (CVC) using the three-syringe-technique 56
List of figures and tables
iii
Fig 3ab Heparin activities (box- and whisker plot arithmetic means) in dogs receiving
unfractionated heparin at a dosage of 75 IUkg BW tid (a) or 150 IUkg BW tid (b) two
hours after the first daily heparin administration 57
Table 1 Heparin activities (IUml median values minimumndashmaximum) in 28 dogs
receiving 75 or 150 IUkg unfractionated (standard) heparin TID subcutaneously with
(grades 1ndash3) or without (grade 0) sonographically detectable thrombus Blood collections
were performed 2 hours after the first daily injection 57
Table 2 Heparin activities (IUml median values minimumndashmaximum) in 27 dogs
receiving 75 or 150 IUkg unfractionated (standard) heparin TID subcutaneously with
(score 1ndash3) or without (score 0) detectable thrombus via electron microscopy 58
Table 3 Comparison of initial results of haemostasis parameters (median values
minimumndashmaximum in dogs with (grades 1ndash3) and without (grade 0) sonographically
detectable central venous catheter-induced thromboses 60
Table 4 Comparison of results of haemostasis parameters (median values minimumndash
maximum) on the last day in dogs with (grades 1ndash3) and without (grade 0) central venous
catheter-associated thrombus formation as detected by electron microscopy 61
Chapter 1 Introduction
1
Chapter 1 Introduction
Central venous catheters (CVC) are mainly used at critical care units in human and small
animal patients requiring infusion of large amounts of fluids or blood parenteral nutrition
support routine drug therapy haemodialysis chemotherapy plasmapheresis or
periodical blood sampling (Geerts 2014 Vose et al 2019) CVC related thrombosis is
one of the most severe complications (apart from infections and pneumothorax) of CVCs
in humans (Comerlato et al 2017 Evans and Ratchford 2018 Geerts 2014) leading to
patientrsquos distress CVC dysfunction venous stenosis and in some cases to the
development of thromboembolism (Geerts 2014) In the human literature there are
various studies dealing with epidemiologic and prophylactic aspects which are described
below in detail In contrast to the best of the authoracutes knowledge studies about extra
luminal CVC related thrombosis in dogs are rare in the available veterinary literature
Studies are mainly dealing with development of intraluminal CVC thrombi occurring
frequently in dogs and cats (Langston et al 2014) and leading to CVC malfunction (Vose
et al 2019)
Epidemiology of CVC related thrombosis
CVC related thrombosis represents 70ndash80 of all upper extremity thrombosis cases and
approximately 10 of thromboembolism cases in humans (Kucher 2011) The incidence
of CVC related thrombosis reported in humans based in 25 studies ranged widely from
2 to 67 (median = 30 ) (van Rooden et al 2005) The incidence of CVC related
thrombosis detected by colour Doppler ultrasound of the internal jugular vein reported in
3 studies performed in humans was 2 (Harter et al 2002) 30 (Lordick et al 2003)
and 56 (Wu et al 1999)
Chapter 1 Introduction
2
The incidence of CVC related thrombosis in humans can vary between studies according
to the CVC type used entry site of the CVC (jugular femoral subclavian axillary
cephalic vein) and time period with the CVC in place the diagnostic thrombus detection
technique (venography or ultrasonography) and criteria for diagnosis duration of follow
up diseases and presence of predisposing factors (eg previous venous thrombosis
history presence of cancer) and finally prophylactic anticoagulation treatment (Geerts
2014 Liu et al 2015 van Rooden et al 2005)
To the best of the authoracutes knowledge data about the incidence of CVC related
thrombosis detected by colour Doppler ultrasound into the lumen of the jugular vein in
dogs are rare in the available veterinary literature In one study performed in 81 dogs and
12 cats (Adamantos et al 2010) the incidence of vein thrombosis detected by palpation
or ultrasound was 2 and the incidence of intraluminal CVC thrombosis (presumed by
the inability to draw blood back) was 15 Additionally regarding to intraluminal CVC
thrombosis an incidence of 8 was reported in one case control study performed in 24
dogs receiving CVC flushing with heparinised solution (Vose et al 2019) Data about
incidence in other animal species was also found In another case-control study performed
on 37 pigs thrombosis in the caval vein related to CVC placed in the external jugular
vein (EJV) was evaluated by histological analysis however no ultrasound detection was
performed (Kohler and Kirkman 1998) In this study in pigs 94 of patients with CVC
developed thrombi in the vena cava based on sections at the site of the end of the catheter
tip and 05 and 1 cm proximal of the first section (Kohler and Kirkman 1998)
Pathogenesis of CVC related thrombosis
Two types of CVC related thrombosis are distinguished in the human medicine literature
(Geerts 2014 van Rooden et al 2005) First symptomatic thrombosis is defined as
Chapter 1 Introduction
3
thrombosis detected by diagnostic images (ultrasonography venography) when
symptoms and signs (eg localised swelling pain redness) are present (Lee and
Kamphuisen 2012) Second subclinical thrombosis is defined as thrombosis
demonstrated by the previously mentioned diagnostic images without overt symptoms or
signs (Boersma et al 2008) Additionally the CVC related thrombi can be classified as
occlusive thrombus into the CVC lumen peri-catheter related fibrin sheaths and mural
thrombi (Geerts 2014)
During the CVC insertion the puncture of the skin on the CVC insertion site and the
rupture of the blood vessels integrity activate the coagulation cascade and the CVC acts
as a matrix for adherence and accumulation of fibrin and cellular components (Smith et
al 2012) The attachment of proteins and blood cells to the internal andor external wall
of the CVC can lead to (1) (partial or complete) occlusion of the catheter lumen and (2)
sometimes if a thrombus formation starts into the lumen of the vein (attached to the vein
wall or to the CVC) a partial or complete obstruction of the blood flow andor embolism
can develop (Langston and Eatroff 2018) Additionally a study performed in humans
(Lordick et al 2003) demonstrated a relationship between CVC related thrombosis and
microbial colonisation of the catheter leading to CVC related systemic infections This
relationship was explained among others by the ease of microorganisms like
Staphylococcus aureus and Staphylococcus epidermidis to adhere to CVC related
thrombin sheaths (Boersma et al 2008)
Diagnosis of CVC related thrombosis
Venous thrombosis is commonly diagnosed in human and small animal medicine
(especially in dogs) with vascular ultrasonography with or without use of the colour
Doppler technique which is normally used to evaluate the blood flow through the blood
Chapter 1 Introduction
4
vessels (Evans and Ratchford 2018 Laurenson et al 2010) According to the literature
in humans cited below the CVC related thrombosis detected by colour Doppler
ultrasound is present if any of the following aspects are observed (1) well defined
thrombus visualised in the lumen of the vein (2) impossibility to perform compression
of the vein by slight pressure with the ultrasound probe andor (3) no detectable blood
flow signal by colour Doppler ultrasound (Bonnet et al 1989 Koumlksoy et al 1995)
Similarly in dogs the CVC related thrombosis is usually diagnosed by colour Doppler
ultrasonography through the detection of the thrombus within the lumen of the vein
andor the obstruction (partial or complete) of the normal blood flow (Bliss et al 2002)
Especially in humans the contrast venography technique is used as gold standard to
confirm thrombosis For this purpose a contrast solution is injected through a peripheral
vein to assess its passage through the bloodstream by x-ray However this diagnostic
technique is normally used only in the case of a strong clinical suspicion of venous
thrombosis that cannot be detected by ultrasonography (Laurenson et al 2010 Langston
and Eatroff 2018) In a study performed in 44 human patients requiring a CVC (Koumlksoy
et al 1995) a sensitivity of 94 and a specificity of 96 were determined for the colour
Doppler ultrasound diagnosis when it was compared with the diagnosis performed with
the contrast venography technique That results make the colour Doppler ultrasound an
accurate diagnosis method to detect CVC related thrombosis in humans (Koumlksoy et al
1995 van Rooden et al 2003)
Advanced diagnostic image techniques like computed tomography or magnetic resonance
tomography are also accurate to perform the diagnosis of CVC related thrombosis in
humans (Whitesell and Steenburg 2014) In dogs multidetector computed tomography
venography was used in one study for the diagnosis of CVC related thrombosis allowing
the identification of catheter related sheaths around the CVC external wall (Wang at al
Chapter 1 Introduction
5
2015) However the high costs of these advanced diagnostic imaging procedures and the
requirement to perform at least sedation or general anesthesia are disadvantages when
compared with ultrasonography
Finally electron microscopy (ELMI) is a diagnostic technique that has been used in
humans (Lucas et al 2017) and experimental animals (Xiang et al 1998 Xiang et al
2001) mainly to perform a microstructural evaluation of thrombi developed into the
lumen of the CVC or in the vein containing the CVC ELMI allows the ex vivo diagnosis
of very small thrombi or sleeve related thrombosis that perhaps cannot be detected by
ultrasonography or venography during the evaluation of patients with CVCs (Xiang et al
2001) However one point to consider is that it is possible that compact thrombi
developed on the CVC can be detached during the CVC removal from the vein and
thereby before its analysis by ELMI (Xiang et al 2001)
Heparin thromboprophylaxis for CVC related thrombosis
Application of CVCs in human medicine is commonly accompanied by prophylactic
anticoagulation in order to decrease the risk of development of partial or complete vein
occlusion andor thromboembolism and thereby to improve the patientrsquos comfort
(Baumann-Kreuziger et al 2015) Apart from vitamin K antagonists (Mismetti et al
2003) unfractionated heparin (UFH) (Abdelfeki et al 2004) and low molecular weight
heparin (LMWH) (Karthaus et al 2006) are anticoagulants that have been used on
studies in humans administrated by intravenous (IV) infusion or by subcutaneous (SC)
injection to prevent thrombi development into the lumen of the veins containing the CVC
(review article Baumann-Kreuziger et al 2015) Different heparin dosages have been
used as thromboprophylactic anticoagulation in humans with CVC (review article Lee
and Kamphuisen 2012) For example in five different case-control studies performed in
Chapter 1 Introduction
6
human haemato-oncological patients UFH at a daily dosage of 100 IUkg given by IV
infusion (Abdelfeki et al 2004) and LMWH at SC daily dosages of 40 mg enoxaparin
(Verso et al 2005) 5000 IU AXa dalteparin (Karthaus et al 2006 De Cicco et al 2009)
and 2850 IU AXa nadroparin (Niers et al 2007) were used Only in part of these studies
it is stated that systemic heparin was combined with flushing of the CVC using
heparinised saline solution to reduce the risk of catheter occlusion (Karthaus et al 2006)
In 3 of the 5 previously mentioned studies no statistically significant difference was found
in the incidence of CVC related thrombosis between patients receiving the
anticoagulation prophylaxis compared with the control groups without systemic
anticoagulation (Verso et al 2005 p = 035 Karthaus et al 2006 p gt 005 Niers et al
2007 p = 049) In contrast in the residual two studies a lower incidence of CVC related
thrombosis was found in the group receiving an anticoagulation prophylaxis compared
with the control groups (Abdelfeki et al 2004 150 vs 126 p = 003 De Cicco et
al 2009 400 vs 526 p = 005)
Obviously there is no consensus in the available human literature regarding the beneficial
use of anticoagulation prophylaxis to prevent CVC related thrombosis and this may
reflect great variations for example according to the study design period trial drug
administration route (Lee and Kamphuisen 2012) and the moment of the initial
anticoagulant dosage administration (Decicco et al 2009)
To the best of the authoracutes knowledge studies about thromboprophylaxis for extraluminal
CVC related thrombosis in dogs cannot be found in the available veterinary literature
However similarly to humans prophylactic anticoagulation with heparinised saline
(administrated through CVC flushing) to prevent CVC occlusion by intraluminal thrombi
was used in one case-control study performed in 24 dogs (Vose et al 2019) In the cited
study 12 dogs received dosages of 3 ml of heparinised saline solution (10 IU UFHml) to
Chapter 1 Introduction
7
flush the catheter every 6 hours during 72 hours whereas 12 control dogs received
flushing with the same volume of non-heparinised saline solution administered at the
same frequency and no statistically significant difference was found
Prediction of CVC related thrombosis
Studies regarding the relationship between laboratory biomarkers and CVC related
thrombosis are available in the human literature (Boersma et al 2016 Cheng et al 2013
Liu et al 2015) however to the best of the authoracutes knowledge literature about laboratory
tests used as predictors for CVC related thrombosis in small animals cannot be found at
all Availability of approved blood tests which can predict the risk of thrombosis
development associated to CVC already at the moment of the CVC placement are
important in order to identify an increased risk of CVC related thrombosis (Boersma et
al 2016) In the study performed by Boersma et al (2016) in 168 human patients without
indication of anticoagulation prophylaxis elevated leukocyte counts high levels of
plasminogen activator inhibitor and of coagulation factor VIII (all measured on blood
samples taken after CVC placement) were associated with an increased incidence of
symptomatic CVC-related thrombosis diagnosed by ultrasonography In another study
(Liu et al 2015) high blood levels of lactate dehydrogenase and low concentrations of
HDL and albumin were associated with the incidence for CVC related thrombosis
diagnosed by ultrasound in 324 senile patients which was in total 617 Additionally
dyslipidemia (high triglycerides low HDL and high LDLHDL ratio) was associated
with CVC related thrombosis (diagnosed by ultrasound and computer tomography
angiography) in human patients undergoing haemodialysis (Cheng et al 2013) Studies
about predictive tests for CVC related thrombosis in dogs were not found in the available
literature to the best of the authoracutes knowledge
Chapter 1 Introduction
8
Aims of the study
The aims of the preliminary methodological study of the present scientific project were
(1) to define reference intervals for viscoelastic analyses of canine haemostasis using the
ROTEMreg delta analyser and (2) to determine the precision (repeatability) of this
method And the aims of the main study of the present scientific project were (1) to
determine the incidence of catheter-induced thrombosis in canine intensive care patients
(2) to evaluate the efficacy of a prophylactic heparin regimen routinely used in the Small
Animal Clinic of the University of Veterinary Medicine Hannover and (3) to assess
whether selected haemostasis test results are predictive for an increased risk for
thrombosis formation
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
9
Chapter 2 Reference intervals for rotational thromboelastometry measurements
using the ROTEMreg delta device in dogs
Pereira JM Rohn K Mischke R
Contributions of the authors
Pereira JM performed (1) the patients data and blood samples collection (2)
haematology and ROTEMreg analysis (3) generated the reference intervals and
coefficients of variation included in the study and (4) wrote the manuscript
Rohn K performed the statistical analysis
Mischke R performed the clinical chemistry and coagulation tests and was involved in
designing and supervision of the study analysis and discussion of the data and its
statistical analysis and revision edition and correction of the manuscript
Original source Pereira J M Rohn K amp Mischke R 2020 Reference intervals
for rotational thromboelastometry measurements using the ROTEMreg delta device
in dogs Res Vet Sci 130 26ndash32 httpsdoiorg101016jrvsc202001019
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
10
Reference intervals for rotational thromboelastometry measurements using the
ROTEMreg delta device in dogs
Pereira JMa Rohn Kb Mischke Ra
aSmall Animal Clinic University of Veterinary Medicine Hannover Buumlnteweg 9 D-
30559 Hannover Germany
bInstitute of Biometry Epidemiology and Information Processing University of
Veterinary Medicine Hannover Buumlnteweg 2 30559 Hannover Germany
Corresponding author
E-mail ReinhardMischketiho-hannoverde
Original source Pereira J M Rohn K amp Mischke R 2020 Reference intervals
for rotational thromboelastometry measurements using the ROTEMreg delta device
in dogs Res Vet Sci 130 26ndash32 httpsdoiorg101016jrvsc202001019
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
11
ABSTRACT
Aims of the present study were to define reference intervals for viscoelastic analyses of
canine haemostasis using the ROTEMreg delta analyser and as a secondary aspect to
determine the precision (repeatability) of this method Blood samples from 125 clinically
healthy dogs were included Measurements were performed with commercially available
activating reagents (ex-tem in-tem and kaolin solution) as well as without activation
Additional fourfold measurements were done in 3 of the normal blood samples and in 3
samples with haemostatic alterations to evaluate the precision of the method
Coefficients of Variation (CVs) for most of the ROTEM variables were lt 10 Clot
formation time and maximum clot elasticity showed a wide inter-individual variation in
comparison with alpha angle and maximum clot firmness A multivariate analysis on
various ROTEM parameters revealed particularly a significant influence of neuter status
and a significant interrelationship between the factors sex and neuter status for
measurements with different activating reagents These results reflected the fact that
significant differences occurred only between intact and neutered females but not in
males No or only occasionally significant differences were found between groups of sex
age and size
In conclusion CVs demonstrated that the method delivers repeatable results in canine
citrated whole blood Established reference intervals should deliver valuable orientation
for the evaluation of viscoelastic properties of clotting whole blood in dogs using the
ROTEM delta analyser Neuter status in females appeared to be the most relevant
influencing factor and should be considered for the interpretation of ROTEM delta test
results
Keywords Viscoelastic measurements reference values canine age sex neuter status
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
12
1 Introduction
Heamostasis is a physiologic and dynamic process in which plasma soluble coagulation
factors blood cells and blood vessels are involved Clot formation and possible
subsequent clot lysis as well as vascular tissue repair aim to prevent haemorrhages on the
one hand and thromboses on the other hand (Kol and Borjesson 2010 Versteeg et al
2013) Conventional assays of coagulation such as prothrombin time (PT) activated
partial thromboplastin time (aPTT) and thrombin time TT have limitations especially
cellular elements are not incorporated into the test and thereby these tests reflect only
limitedly in vivo functionality of haemostasis (McMichael and Smith 2011 Crochemore
et al 2018)
The rotational thromboelastometry (ROTEMreg) currently termed as thromboelastometry
(TEM) and thromboelastography (TEG) are methods for the rapid evaluation of the
overall haemostasis capacity through the measurement of viscoelastic properties of
clotting whole blood (Kol and Borjesson 2010 Theusinger et al 2010 Sankarankutty
et al 2012 Versteeg et al 2013) In TEG the cup with the blood sample is rotating
whereas the torsion wire is fixed In ROTEMTEM the cup is fixed whereas the pin is
rotating Changes in torque ie resistance to rotation by the fibrin bridge created between
pin and cup in the clotting blood are detected electromechanically in TEG and optically
in ROTEM (Bollinger et al 2012) and displayed as a graph that indicates the time for the
formation of blood clot and its strength (Bagge et al 2016) The resulting graph reflects
the initiation and propagation of the coagulation system fibrin-platelet interactions and
clot lysis
TEG was first described in 1948 by Hellmut Harten (Hartert 1948) as an in vitro method
to evaluate the clot formation process while resembling the in vivo blood flow The
development of the actually used instrument types TEG and ROTEMregTEM were based
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
13
on that original principle Viscoelastic measurements have been widely used since the
1980s in human medicine to evaluate haemostasis and hypercoagulable states in surgical
patients (eg cardiac and hepatic surgeries) and in general in critical care units (Kol and
Borjesson 2010 Nogami 2016 Wikkelso et al 2016 Crochemore et al 2017) In
veterinary medicine viscoelastic measurements using TEG and ROTEMTEM have been
validated for use in dogs cats and horses and used to study haemostasis in different
diseases (eg infectious diseases liver diseases) and medical conditions (eg
orthopaedic surgery hyperfibrinolysis) and also to investigate the effect of
anticoagulants and other drugs (eg corticosteroids) on haemostasis (Kol and Borjesson
2010 Bruno et al 2015a 2015b Woodman et al 2015 Conversy et al 2017 Sigrist et
al 2018 Turner et al 2019) The methodology seems to be especially suitable to detect
hypercoagulability (Wiinberg et al 2008 Kol and Borjesson 2010 Marschner et al
2018)
In the available veterinary literature information about reference values for TEG were
found using kaolin as activation reagent (Bauer et al 2009) Falco et al (2012)
determined reference values for the ROTEM analyser in dogs using ex-tem in-tem and
fib-tem reagents for selected ROTEM parameters in a study on forty five healthy dogs
However data related to reference intervals obtained in an adequate number of samples
(at least 120) according to the American Society for Veterinary Clinical Pathology
(ASVCP) (Friedrichs et al 2012) and the National Committee for Clinical Laboratory
Standards (NCCLS) (Horn and Pesce 2003) could not be found in the available literature
The aim of this study is to determine reference values for the ROTEMreg delta device in
dogs using citrated whole blood with different clot activator reagents (ex-tem in-tem
kaolin) and without activator
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
14
2 Materials and methods
21 Study design
Reference intervals Single thromboelastometric measurements were performed in 125
clinically healthy dogs using the ROTEMreg analyser and different activating reagents (ex-
tem in-tem and kaolin) as well as without activation Reference values were calculated
and influences of age groups (young adult and senior) sex neuter status and size
(groups small medium and large) were assessed
Precision analysis Additional fourfold measurements using ex-tem or in-tem activating
reagents respectively were performed on 3 of the normal and also in 3 abnormal blood
samples each in order to assess within-run imprecision for these measurements
22 Animals
The clinically healthy dogs with a body weight of at least 5 kg were recruited by launching
a call in the University of Veterinary Medicine Hannover network for students and staff
members Interested dog owners received a free health check of their animals (clinical
examination and blood tests specified below) in combination with the blood collection
In addition individual privately owned patient dogs of the Small Animal Clinic with
undisturbed general health were also included if blood was collected due to another
indication (eg general health check pre-anaesthetic examination test for hereditary
diseases etc) Before taking the blood samples all dogs were fasted for at least 12 h with
free access to water 125 animals for which the examination of the health status (clinical
examination routine haematology and clinical chemistry basis coagulation profile) did
not reveal any deviations from the reference were included in the study whereas 3 of the
128 initially recruited animals had to be excluded due to laboratory abnormalities
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
15
Haematological and clinical chemistry profiles performed in order to confirm the health
status included white and red cell count haematocrit platelet counts and reticulocytes
plasma activities of alanine aminotransferase glutamate dehydrogenase alkaline
phosphatase and pseudocholinesterase as well as concentrations of total bilirubin urea
creatinine glucose total protein albumin cholesterol and ionised calcium A basis
coagulation profile including PT PTT and TT was also performed to exclude significant
abnormalities within the blood coagulation system
The 125 healthy dogs enclosed in the establishment of reference values included 48 mixed
breed dogs and the following pedigree dogs 13 Labrador Retrievers 6 Australian
Shepherds 4 Havanese dogs 3 from each of the following breeds American
Staffordshire Terriers and Cocker Spaniels 2 from each of the following breeds Beagles
Border Collies Dalmatians Elo dogs (breed not recognised by Feacutedeacuteration cynologique
international [FCI]) German Longhaired Pointers German Shorthaired Pointers Giant
Schnauzers and Golden Retrievers 1 from each of the following breeds Belgian
Malinois Bolonka Zwetna Briard Brussels Griffon American English Coonhound
Italian Mastiff Flat Coated Retriever French Bulldog German Shepherd Great Dane
Greater Swiss Mountain Dog Giant Spitz Goldendoodle Hovawart Hungarian Vizsla
Italian Bloodhound Lycanis Wolfdog Miniature American Shepherd Miniature
Schnauzer Old German Shepherd Podenco Poodle Pyrenean Shepherd Rhodesian
Ridgeback Rottweiler Schafpudel (breed not recognised by FCI) Small
Munsterlander Spanish Mastiff Tibet Terrier Weimaraner West Highland White
Terrier and Wirehaired Pointer
These dogs were aged between 6 and 154 months (median = 410 months) and had body
weights ranging from 50 to 707 kg (median = 226 kg) 30 were castrated males 33
intact males 41 spayed females and 21 intact females According to the height at withers
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
16
the dogs were divided into 3 different size groups small (le40 cm n = 22) medium (41ndash
60 cm n = 77) and large (gt60 cm n = 26) Dogs were also divided in three different age
groups young (le 24 months n = 34) adult (25ndash84 months n = 59) and senior dogs (gt84
months n = 32)
The three blood samples with haemostatic abnormalities were taken from two dogs with
complex haemostatic disorders (Disseminated Intravascular Coagulation [DIC])
including reduced platelet count and abnormal PT and APTT and one dog with
thrombocytopenia
Animals were treated in accordance with the German Animal Welfare Law The
experimental design was approved by the Animal Welfare officer of the University of
Veterinary Medicine Hannover Foundation and by the ethics committee of the
responsible authority (Lower Saxony State Office for Consumer Protection and Food
Safety reference number 17A101)
23 Blood sampling
The venipuncture area was shaved and then disinfected with alcohol The saphenous or
cephalic vein was visualised with slight pressure and punctured with disposable cannulas
(diameter 09 or 10 mm) The continuously running blood was directly collected from
the needle into the tubes containing the respective anticoagulants From each dog
approximately 10 ml of blood were collected including approximately 8 ml of citrated
blood for the measurement of haemostasis tests (ROTEM PT aPTT TT) in tubes
containing sodium citrate 313 (Eifelfangoreg GmbH amp Co KG Bad Neuenahr-
Ahrweiler Germany) in a ratio of 1 volume9 volumes of blood In addition
approximately 1 ml of EDTA blood for haematological analysis and approximately 1 ml
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
17
of lithium heparin blood for clinical chemical measurements were collected into
commercial 13 ml tubes (Sarstedt AG amp Co Nuumlmbrecht Germany)
Citrated blood was initially kept at room temperature (20-25 C) in order to avoid errors
within the preanalytical phase (Toulon et al 2017) Two ml of citrated blood were used
for the ROTEMreg analysis the residual citrate blood was centrifuged for 10 min at 3800
rpm and 5 degC then the plasma was separated by careful pipetting and it was centrifuged
a second time under the same conditions to get platelet poor plasma which was stored in
aliquots of 500 microl and 250 microl at -80 degC for coagulation tests
24 Rotational elastometry
Viscoelastic measurements using the ROTEMreg delta for the establishment of reference
intervals were performed approximately 15 min after the blood collection using activating
reagents (liquid reagents) provided by the manufacturer of the instrument (r-ex-temreg in-
temreg) and a kaolin solution (5 g1 as part of the PTT reagent CK Prest [Diagnostica
Stago SAS Asniegraveres sur Seine Cedex France]) In addition non-activated analyses
were performed using isotonic NaCl solution instead of the activating reagent in the fourth
channel of the device Fourfold measurements for precision analysis were performed
simultaneously in the four channels of the device in separate runs
According to the standard test protocol provided by the manufacturer of the ROTEMreg
delta (Werfen GmbHInstrumentation Laboratory Munich Germany) 20 μl of the
reagent for recalcification (star-temreg20) and 20 μl of the activating reagent were pipetted
into a cuvette using the automated pipette provided with the device Immediately
thereafter 300 μl of citrated whole blood (prewarmed to 37 degC for five minutes in the
heating device of the instrument and gently rocked to resuspend possibly sedimented
cells) were added to the test preparation in the cuvette using the automated pipette and
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
18
then the cuvette was laced carefully in the ROTEMreg device whereby the rotating pin
entered into the test approach
Different parameters are provided automatically by the ROTEMreg delta device and the
following variables (described according to the Pentapharm GmbH ROTEMreg delta
Manual [2009]) were used in the study
Clotting time (CT) the time (in s) from the beginning of the test (by adding the clot
activator) until the formation of fibrin starts (defined as the moment when the curve
achieves an amplitude of 2 mm) This Parameter facilitates the decision to replace clotting
factors when it is required
Clot formation time (CFT) describes the next phase of clotting by the formation of a
stable clot through the activation of thrombocytes and fibrin formation It is defined as
the time (in s) between the 2 mm amplitude and 20 mm amplitude of the curve
Alpha angle (α angle) is the angle (in degrees) between the middle axis and the tangent
to the clotting curve through the 2 mm amplitude point
Amplitude after ldquoXXrdquo minutes (eg A30 after 30 min) is the amplitude (in mm) at a
certain time after the CT has finished
Maximum clot firmness (MCF) corresponds to the maximum amplitude (in mm) reached
before the clot is dissolved by fibrinolysis This parameter measures the firmness of the
clot and its quality Like CFT and α angle it facilitates the decision for substitution therapy
with platelet concentrate andor fibrinogen especially in humans
Maximum clot elasticity (MCE) is a parameter calculated from MCF and may be superior
at high amplitudes as compared to the MCF
Lysis index at 30 45 and 60 min (LI30 LI45 LI60) represents the fibrinolysis (in
percentage) 30 45 and 60 min after the CT It may facilitate the decision for or against a
therapy with anti-fibrinolytic drugs
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
19
Maximum lysis (ML) describes the degree of fibrinolysis relative to the MCF achieved
during the measurement (percent loss of clot firmness)
25 Statistical methods
Original data obtained from the ROTEMreg delta device software were transferred into the
Microsoftreg Excelreg formal (Microsoft Office Professional Plus 2013) The Statistical
analyses were performed using the SAS Enterprise Guide Version 71 (SAS Institute Inc
Cary NC USA)
Coefficients of Variation (CVs) for selected relevant parameters of the ROTEM delta
analysis using ex-tem and in-tem regents were calculated for the fourfold measurements
on 3 normal and 3 abnormal blood samples in order to assess within-run imprecision CVs
for the results of each sample were calculated by dividing the standard deviation of the
data set by its arithmetic mean Finally the median of the CVs for each group of 3 samples
was calculated The reference values were calculated according to recommendations of
the American College of Veterinary Clinical Pathology (ACVCP) based on non-
parametric methods (25 and 975 quantiles) supplemented by 95 confidence
intervals (Friedrichs et al 2012)
Data were analysed for Standard normal distribution by Kolmogorov Smirnov test and
assessment of residuals of the analysis of variance (ANOVA) Because these tests
indicated almost normally distributed values for the studied ROTEM variables data were
analysed by a fourway ANOVA stratified by the activating reagent (ex-tem in-tem
kaolin or without activation) to test the influence of size age sex and neuter status on
several parameters and taking all interactions into account Post hoc t-tests were
performed when indicated The significance level was set to P lt 05
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
20
3 Results
31 Precision of the method
For all the parameters evaluated in the study the CVs for the fourfold measurement in
healthy dogs were less than 10 (Table 1) In the case of dogs with haemostatic
abnormalities CVs of the vast majority of ROTEM variables were also less than 10
except the variables CFT and ML irrespective whether ex-tem or in-tem reagents were
used (Table 2)
Table 1 Coefficients of variation () for rotational thromboelastometry using the
ROTEMreg delta analyser in normal blood samples
Activator CT (s) CFT (s) α (deg) A30 (mm) MCF (mm) MCE(mm) LI30 () LI45 () LI60 () ML ()
Ex-temreg 875 629 262 213 179 634 000 020 037 534
In-temreg 245 596 166 117 135 299 000 000 046 216
Abbreviations CT ndash clotting time CFT ndash clot formation time α ndash alpha angle A30 ndash
amplitude after 30 min MCF ndash maximum clot firmness MCE ndash maximum clot elasticity
LI 30 LI 45 LI 60 ndash Lysis index at 30 45 and 60 min ML ndash maximum lysis
Median values of the measurements of 3 samples
Table 2 Coefficients of variation () for rotational thromboelastometry using the
ROTEMreg delta analyser in canine blood samples with haemostatic alterations
Activator CT (s) CFT (s) α (deg) A30 (mm) MCF (mm) MCE(mm) LI30 () LI45 () LI60 () ML ()
Ex-temreg 668 1478 393 218 268 582 0 018 000 848
In-temreg 228 799 461 219 251 371 0 0 017 283
Median values of the measurements of 3 samples from patients with DIC (n = 2)
thrombocytopenia (n = 1) abbreviations please see legend to Table 1
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
21
32 Reference intervals
Activation by ex-tem reagent (tissue factor) resulted in faster CTs when compared to
activation via the intrinsic pathway using ellagic acid (in-tem) or kaolin reagents whereas
the CFT was much less affected by the activating reagent (Table 3) Especially CFT and
MCE showed a wide inter-individual variation in comparison for example with α angle
and MCF regardless of the used activating reagent (Table 3)
33 Influence of age sex neuter status and size
ANOVA did not reveal statistically significant differences in ROTEM delta parameters
between dog groups of variable size or sex (P gt 05) Age had influence only on CT and
only when the in-tem reagent was used (P = 0169 ANOVA) but not when using the ex-
tem reagent (P = 7105) Post hoc comparisons using t-test of the in-tem results between
age groups revealed shorter CTs in the group of senior dogs when compared with young
dogs (P = 0194) but not between adult dogs and these two groups ANOVA showed
particularly influences of the neuter status and a significant interrelationship between the
factors sex and neuter status on different ROTEM delta parameters using ex-tem and in-
tem respectively as activator (Tables 4 and 5) ANOVA revealed also a significant
influence of neuter status on different ROTEM parameters when kaolin was used as
activating reagent (CFT α angle MCF MCE) whereas non-activated measurements
were not subject to any significant influence of the assessed factors (results not shown)
The interrelationship between sex and neuter status mentioned above reflected the fact
that significant differences between neutered and not neutered dogs were only found in
females The statistical analysis using the t-test to compare intact vs neutered dogs
stratified by sex showed statistically significantly (P lt 05) longer CTs and CFTs and
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
22
lower α angle A30 MCF and MCE values in spayed when compared to intact females
(Tables 4 and 5)
Chapter 2 Reference intervals for rotational thromboelastometry measurements using the ROTEMreg delta device in dogs
23
Table 3 Reference values (median reference interval based on 25 - and 975 - quantiles) for thromboelastometric measurements with the ROTEMreg delta analyser in
dogs (n=125 95 confidence intervals in brackets) Abbreviations please see legend to Table 1
Activator
Statistical
para-meter
CT
(s)
CFT
(s)
Α
(deg)
A30
(mm)
MCF
(mm)
MCE
(mm)
LI30
()
LI45
()
LI60
()
ML
()
Ex-temreg
Median 450
[430ndash470]
110
[105ndash115]
680
[670ndash690]
600
[580ndash610]
610
[600ndash620]
154
[147ndash164]
100
[960ndash100]
100
[100ndash100]
970
[960ndash980]
220
[210ndash240]
25 340
[270ndash350]
650
[490ndash710]
540
[420ndash560]
460
[410ndash480]
460
[420-490]
870
[720ndash950]
100
[100-100]
870
[800ndash960]
700
[640ndash820]
800
[100ndash110]
975 830
[790ndash990]
220
[201ndash315]
790
[770ndash800]
770
[690ndash770]
730
[700ndash770]
264
[230-334]
100
[100ndash100]
100
[100ndash100]
100
[990ndash100]
590
[530ndash680]
In-tem
Median 190
[187ndash201]
116
[111ndash125]
680
[670ndash690]
570
[560ndash580]
590
[580ndash600]
143
[135ndash149]
100
[100ndash100]
100
[100ndash100]
980
[980ndash980]
180
[170ndash190]
25 135
[112ndash142]
730
[590ndash800]
510
[460ndash570]
450
[410ndash470]
480
[410ndash500]
920
[710ndash101]
100
[950ndash100]
990
[820ndash990]
930
[740ndash930]
500
[100ndash700]
975 283
[268ndash291]
251
[198ndash227]
750
[750ndash780]
660
[660ndash690]
680
[660ndash690]
210
[195ndash227]
100
[100ndash100]
100
[100ndash100]
100
[100ndash100]
270
[260ndash380]
Activator
Statistical
para-meter
CT
(s)
CFT
(s)
Α
(deg)
A30
(mm)
MCF
(mm)
MCE
(mm)
LI30
()
LI45
()
LI60
()
ML
()
Kaolin
Median 162
[154ndash173]
870
[820ndash910]
740
[720ndash750]
500
[470ndash530]
550
[530ndash560]
121
[115ndash127]
960
[940ndash980]
840
[790ndash880]
710
[640ndash770]
500
[450ndash540]
25
109
[990ndash115]
470
[440ndash580]
560
[490ndash600]
110
[700ndash230]
390
[380ndash400]
640
[600ndash680]
270
[140ndash480]
110
[800ndash260]
800
[400ndash170]
200
[160ndash240]
975 246
[230ndash337]
214
[185ndash290]
810
[790ndash810]
640
[630ndash690]
650
[650ndash690]
187
[182ndash223]
100
[100ndash100]
990
[980ndash100]
940
[930ndash990]
970
[930ndash100]
Without activation
Median 607
[495ndash662]
221
[194ndash265]
510
[460ndash550]
520
[500ndash530]
540
[520ndash550]
115
[109ndash123]
100
[100ndash100]
100
[100ndash100]
980
[980ndash990]
170
[160ndash190]
25 203
[123ndash215]
860
[550ndash930]
190
[120ndash230]
350
[230ndash370]
370
[260ndash400]
600
[350ndash670]
100
[950ndash100]
980
[890ndash990]
930
[840ndash950]
400
[000ndash500]
975 1356
[1305ndash1721]
811
[688ndash1333]
730
[710ndash790]
660
[640ndash720]
660
[640ndash730]
197
[179ndash265]
100
[100ndash100]
100
[100ndash100]
100
[100ndash100]
290
[280ndash320]
Chapter 2 Reference intervals for rotational thromboelastometry measurements using the ROTEMreg delta device in dogs
24
Table 4 Median values (minimum and maximum in brackets) for different ROTEMreg variables using ex-tem reagent stratified by sex and neuter status
Group
CT
(s)
CFT
(s)
Α
(deg)
A30
(mm)
MCF
(mm)
MCE
(mm)
LI30
()
LI45
()
LI60
()
ML
()
Female intact 430
(320ndash530)
960
(590ndash198)
710
(570ndash800)
620
(480ndash730)
640
(510ndash740)
179
(103ndash282)
100
(100ndash100)
100
(980ndash100)
960
(800ndash100)
230
(100ndash640)
Female spayed 460
(270ndash830)
121
(650ndash224)
660
(520ndash770)
580
(430ndash690)
590
(440ndash700)
146
(790ndash230)
100
(960ndash100)
100
(800ndash100)
970
(690ndash100)
240
(100ndash590)
p-value (t-test)
Female
intact vs spayed
00025 00063 00049 00023 00016 00015 na na na na
Male intact 450
(320ndash990)
105
(650ndash315)
690
(420ndash790)
590
(420ndash700)
610
(440ndash710)
154
(770ndash244)
100
(100ndash100)
100
(870ndash100)
970
(650ndash100)
220
(700ndash680)
Male castrated 460
(340ndash930)
111
(490ndash255)
685
(480ndash800)
590
(410ndash770)
600
(420ndash770)
152
(720ndash334)
100
(100ndash100)
100
(830ndash100)
980
(640ndash100)
205
(500ndash590)
p-value (t-test)
Male
Intact vs castrated
08326 05106 04756 01799 01568 02163 na na na na
p-values (ANOVA)
Factor Sex
05428 04847 06612 02206 01887 01630 08489 07893 07689 02758
Factor neuter status 01872 00167 00173 00039 00037 00028 07141 04737 09356 05948
Interrelationship
sexneuter status 00383 00968 00782 00488 00472 00319 04943 08611 05422 00160
na ndash not analysed all significant P-values (lt 005) are highlighted in bold print
Chapter 2 Reference intervals for rotational thromboelastometry measurements using the ROTEMreg delta device in dogs
25
Table 5 Median values (minimum and maximum in brackets) for different ROTEMreg variables using the in-tem reagent stratified by sex and neutering status
Group
CT
(s)
CFT
(s)
Α
(deg)
A30
(mm)
MCF
(mm)
MCE
(mm)
LI30
()
LI45
()
LI60
()
ML
()
Female intact 186
(126ndash238)
940
(590ndash164)
720
(620ndash780)
610
(490ndash690)
620
(530ndash690)
166
(113ndash227)
100
(100ndash100)
100
(990ndash100)
980
(930ndash100)
170
(100ndash300)
Female spayed 201
(149ndash291)
1220
(820ndash262)
670
(500ndash740)
550
(450ndash660)
570
(480ndash680)
133
(910ndash214)
100
(950ndash100)
100
(820ndash100)
980
(740ndash100)
190
(500ndash380)
p-value (t-test)
Female
intact vs spayed
na na lt0001 00009 00005 00005 na na na na
Male intact 199
(117ndash283)
122
(730ndash277)
680
(460ndash750)
570
(430ndash660)
590
(450ndash660)
146
(810ndash192)
100
(100ndash100)
100
(990ndash100)
980
(930ndash100)
180
(600ndash270)
Male castrated 181
(112ndash288)
123
(820ndash222)
670
(540ndash740)
565
(410ndash630)
585
(410ndash640)
138
(710ndash180)
100
(100ndash100)
100
(970ndash100)
980
(890ndash100)
180
(800ndash260)
p-value (t-test)
Male
intact vs castrated
na na 08232 01401 01162 00849 na na na na
p-values (ANOVA)
Factor Sex
02105 03317 03370 00877 01258 00510 09564 07832 08117 03638
Factor neuter status 04289 00660 00619 00107 00088 00054 07152 09795 07056 08299
Interrelationship
sexneuter status
04310 00550 00408 00707 01733 00996 05240 05982 08047 04578
na ndash not analysed all significant P-values (lt 005) are highlighted in bold print
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
26
4 Discussion
41 Coefficients of variation
In accordance with the literature CVs below 10 indicate adequate repeatability in intra-
assays evaluations (Steiner et al 2003 Hua et al 2018) According to our results
obtained from the fourfold measurements in citrated whole blood ROTEM delta
measurements on healthy dogs and dogs with haemostatic alterations fulfil these
requirements with only a few exceptions concerning CFT and ML values in abnormal
samples
Our results regarding repeatability are similar to those obtained for several TEM
parameters in previous studies performed in animals In one study the imprecision of the
ROTEM analyser was evaluated by duplicate measurements in 10 blood samples of
healthy dogs and as result of that evaluation a good precision (CVs below 10 ) was
calculated for CT CFT MCF and α angle but in contrary to our results a low
repeatability was found for ML (CVs of 233 [ex-tem] and 285 [in-tem] own values
534 and 216 ) (Falco et al 2012)
In a previous feline study using ex-tem and in-tem as activating reagents for the ROTEM
delta repeatability was assessed similarly to the present study based on fourfold
measurements of blood samples of two healthy individuals (Doumlderlein and Mischke
2015) The results also revealed CVs below 10 for most of the parameters evaluated
The partially low repeatability of ML values may reflect the vulnerability of this variable
which is not only influenced by the complex clot formation process but also by its
subsequent lysis Reproducibility of ex-tem or in-tem activated measurements of the
ROTEM delta analyser which was not investigated in our study was studied in blood
from healthy and unhealthy horses by two different operators using three different
ROTEM devices Results indicated acceptable reproducibility (Junge et al 2016)
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
27
42 Reference intervals
The number of 125 healthy dogs used in the present study to create reference intervals is
regarded adequate according to the guidelines suggested by the American Society of
Veterinary Clinical Pathology (ASVCP) and the National Committee for Clinical
Laboratory Standards (NCCLS) who recommend to include at least 120 individuals
(Friedrichs et al 2012) To the best of our knowledge reference intervals for the most
relevant ROTEMreg delta parameters obtained in an adequate number of samples of whole
citrated canine blood have not been published so far A previous study was performed on
a smaller number of 45 dogs (Falco et al 2012) The median values for reference
intervals obtained in the present study are partially similar to those calculated in the
mentioned study based on 45 healthy dogs (eg ex- tem CT 610 s450 s [Falco et al
2012own results] CFT 150 s 110 s α angle 620deg680deg MCF 530 m 610 mm ML
180 220 in-tem CT 235 s190 s CFT 136 s116 s α angle 660deg680deg MCF
560 mm590 mm ML 30 180 ) However in comparison to our results the
reference intervals in the mentioned study are slightly wider (eg ex-tem CT 29ndash92
s34ndash83 s [Falco et al 2012own results] CFT 540ndash275 s650ndash220 s α angle 470ndash
790deg540ndash790deg MCF 360ndash730 mm460ndash730 mm ML 000ndash560 800ndash590
in-tem CT 126ndash363 s135ndash283 s CFT 470ndash224 s730ndash251 s aα angle 550ndash
810deg510ndash750deg MCF 500-ndash750 mm480ndash680 mm ML 000ndash400 500ndash270 )
although the previous study used the 5 and 95 quantiles to define limits of the
reference range whereas we calculated the commonly used 25 and 975 quantiles
to capture the central 95 of reference values (Friedrichs et al 2012)
Although it is desirable that each laboratory should specifically determine its own
reference intervals the results obtained in this study may provide an orientation for other
laboratories to interpret ROTEM delta parameters analysed in citrated canine blood In
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
28
order to test transference of the reference ranges to another laboratory a validation
procedure with measurement of 20 samples should be performed (Friedrichs et al 2012)
Although intra-individual control values (subject-based reference values) are regarded
superior to population based reference values (Wiinber et al 2007) the latter have to be
routinely used in clinical patients due to lacking individual controls especially for specific
tests such as ROTEM analyses In addition the calculated reference values cannot be
used for the fully automated device ROTEM sigma because - according to an actual
human study - values of part of the parameters do not correlate well between both
instruments (Gillissen et al 2019)
43 Influence of size age sex and neuter status
In previous studies in humans (Attard et al 2013) and animals (Mischke 1994 Borrelli
et al 2017) age has been described as an influencing factor for different haemostasis
parameters We also found an influence of age on the CT with shorter times in senior
dogs (gt 84 months) compared with young dogs (up to 24 months) which was limited to
measurements activated by the in-tem reagent
This age dependency confirms in principle a previous study on 20 healthy dogs
(Barthelemy et al 2015) which found shorter CTs in elderly dogs and results of a human
study where individuals with an age gt 45 years showed shorter CTs compared with
individuals lt 45 years old (Sucher et al 2011) However in the cited studies this effect
was only seen when ex-tem but not when the in-tem reagent was used
Based on the age distribution of our dogs which were primarily recruited to establish
reference intervals we cannot deliver information regarding very young dogs A human
study focusing on ROTEM reference ranges in children demonstrated significant
differences for nearly all ROTEM parameters with the most striking differences in
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
29
subjects aged 0-3 months (Oswald et al 2010) The limited number of dogs with an age
lt 1 year which did not allow to consider these separately in the present study may also
be one reason for the different results in a feline study (Doumlderlein and Mischke 2015) In
the cited study on healthy cats age showed an influence on CT but contrary to our study
the CT was shorter in young individuals (6-12 months) compared with older ones In
addition this was only the case in measurements using kaolin and in non-activated
measurements When compared to humans and cats uni- or multivariate analyses
considering factors age and breed or size based on do populations including different
breeds have the limitation that they can be biased by the variate life expectancy between
small medium and large dog breeds (Willems et al 2017) Apart from age we found a
significant influence only for the neuter status in bitches reflected by a significant
interaction effect between factors neuter status and sex in the ANOVA analysis
Especially for CFT values (eg activated by ex-tem median values of 121 s [spayed] vs
96 s [intact]) this difference seems to be also clinically relevant with respect to the
interpretation of ROTEM results In contrast although also significantly different
absolute and relative differences of CT values were small (eg activated by ex-tem 46 s
[spayed] vs 43 s [intact]) These differences may not only be considered for the result
interpretation of clinical patients but indicate also the necessity for sex-matched patient
groups in scientific studies
The obviously more active haemostasis system in intact females is probably related to an
influence of sex hormones (specifically estrogens) on the coagulation system
Accordingly ex vivo and in vivo studies demonstrated the activating properties of
endogenous estrogens on the haemostasis system In one ex vivo study addition of
endogenous estrogens (estrone [E1] 17szlig-estradiol [E2] and estriol [E3]) to blood samples
from men resulted in shorter mean values of the reaction time in TEG analyses
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
30
corresponding to CT in ROTEM measurements (452-537 s vs 698 s [control]) and of
the TEG variable kinetics (corresponding to the CFT in ROTEM analyses) (Swanepoel
et al 2017) In a further study subcutaneous injection of E2 into male rats induced a
significant increase in activities of coagulation factors X (by 8 ) and XI (by 10-24 )
however did not show any significant effect on PT aPTT and factor VII activity (Franco-
Murillo and Jaimez 2017)
In the cited studies also the effects of the synthetic estrogen ethinylestradiol [EE] were
studied which interestingly seems to have an inhibiting effect as indicated by prolonged
reaction time and kinetics (TEG) compared with the control in the human ex vivo study
and significantly longer PTs and aPTTs and lower factor VII and X activities in the study
on rats As expected we did not find a significant influence of size on ROTEM parameters
in dogs To the best of the authoracutes knowledge size has not been examined as an
influencing factor on viscoelastic measurements or any other haemostatic variable in
dogs Apart from Greyhounds there is limited literature on breed comparisons of
haemostatic variables (Clemons and Meyers 1984 Mischke 1994 Nielsen et al 2007
Nielsen et al 2010 Boyd et al 2018) indicating very limited breed differences for
example for factor VII (Mischke 1994) However these also do not indicate systematic
influence of size and in addition possible breed-specific effects have to be considered
As already stated one limitation of the study is the limited number of young animals
which does not allow to verify reference values for animals in this stage of life In
addition with respect to the main aim of the study possible influencing factors such as
platelet count and haematocrit were not considered in the statistical analysis Finally the
relatively small number of dogs used for the precision analysis which express the fact
that this was only a secondary aspect of the study may be regarded as a limitation At the
same time these considerations provide space for further investigations
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
31
5 Conclusions
Results of the precision analysis revealed acceptable results and thereby confirm that the
ROTEMreg delta analyser delivers repeatable results to measure overall haemostasis in
canine citrated blood samples The established reference values should offer a reliable
basis for the interpretation of thromboelastometric measurements in whole blood using
the device ROTEMreg delta in dogs with suspected haemostasis disorders Neuter status
in female dogs appeared to be a relevant influencing factor on most important ROTEMreg
parameters and should be considered for result interpretation and with respect to
experimental designs
6 Acknowledgments
Jose Mauricio Pereira received a scholarship from the Universidad Nacional de Costa
Rica (reference code JB-C-1107-2016)
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
32
7 References
1 Attard C van der Straaten T Karlafitis V Monagle P Ignjativic V 2013 Developmental
hemostasis age-specific differences in the levels of hemostatic proteins J Thromb and
Haemost 11 1850ndash1854
2 Bagge A Schoumltt U Kander T 2016 Effects of naturopathic medicines on Multiplate and
ROTEMreg A prospective experimental pilot study in healthy volunteers BMC Complemen
Altern Med 17 (16) 64
3 Bartheacutelemy A Rannou B Forterre M Verwaerde P Bonnet-Garin JM Pouzot-Nevoret
C Goy-Thollot I 2015 Differences between coagulation and cytokine profiles in dogs of
different ages Vet J 205 410ndash412
4 Bauer N Eralp O Moritz A 2009 Establishment of reference intervals for kaolin-activated
thromboelastography in dogs including an assessment of the effects of sex and anticoagulat use
J Vet Diagn Investi 21 641ndash648
5 Bollinger D Seeberger MD Tanaka KA 2012 Principles and practice of
thrromboelastography in clinical coagulation management and transfusio practice Transfus Med
Rev 26 1ndash13
6 Borrelli A Botto A Maurella C Falco S Pagani E Miniscaldo B Tarducci A Bruno
B 2017 Thromboelastometric assesment of hemostasis in newborn Piemontese calves J Vet
Diagn Invest 29 293ndash297
7 Boyd CJ Claus MA Raisis AL Hosqood G Sharp CR SmartL 2018
Hypocoagulability and platelet dysfunction are exacerbated by synthetic colloids in a canine
hemorrhagic shock model Front Vet Sci 5 279
8 Bruno B Maurella C Falco S Tarducci A Zanatta R DrsquoAngelo A Borrelli A 2015a
Thromboelastometric evaluation of hemostasis in dogs infected with Leishmania infantum J Vet
Emerg Crit Care (San Antonio) 25 502ndash511
9 Bruno B Maurella C Falco S Tarducci A Zanatta R Gianella P DrsquoAngelo A Piras L
Di Bella A Borrelli A 2015b Assessment of coagulation utilizing thromboelastometry in dogs
undergoing orthopedic surgery J Vet Emerg Crit Care (San Antonio) 25 358ndash363
10 Clemons RM Meyers KM 1984 Acquisition and aggregation of canine blood platelets Basic
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
33
mechanisms of function and differences because of breed origin Am J Vet Res 45 137ndash144
11 Conversy B Blais MC Dunn M Gara-Boivin C del Castillo JRE 2017 Anticoagulant
activity of oral rivaroxaban in healthy dogs Vet J 223 5ndash11
12 Crochemore T Piza FMT Rodriacuteguez RDR Guerra JCC Ferraz LJR Correcirca TD
2017 A new era of thromboelastometry Einstein (Sao Paulo) 15 380ndash385
13 Crochemore T Correcirca TD Lance MD Solomon C Neto AS Guerra JCC Lellis PS
Bernz LM Nunes N Mancio CM Yokoyama APH Silva E 2018 Thromboelastometry
profile in critically ill patients A single-center retrospective observational study PLoS One 13
e0192965
14 Doumlderlein E Mischke R 2015 Reference intervals for thromboelastometry with the ROTEMreg
delta in cats Res Vet Sci 100 271ndash276
15 Falco S Bruno B Maurella C Bellino C DacuteAngello A Gianella P Tarducci A Zannata
R Borrelli A 2012 In vitro evaluation of canine hemostasis following dilution with
hydroxyethyl starch (13004) via thromboelastometry J Vet Emerg Crit Care (San Antonio)
22 640ndash645
16 Franco-Murillo Y Jaimez R 2017 Inhibitory effect of ethinylestradiol on coagulation factors
in rats Exp Anim 66 107ndash113
17 Friedrichs KR Harr KE Freeman KP Szladovits B Walton RM Barnhart KF Blanco-
Chavez J 2012 ASVCP reference interval guidelines Determination of de novo reference
intervals in veterinary species and other related topics Vet Clin Pathol 41 441ndash453
18 Gillissen A va den Akker T Caram-Deelder C Henriquez DDCA Bloemenkamp
RWM Eikenboom J van der Bom JG de Maat MPM 2019 Comparison of
thromboelastometry by ROTEMreg Delta and ROTEMreg Sigma in women with postpartum
haemorrhage Scand J Clin Lab Invest 79 32ndash38
19 Harttert H 1948 Blutgerinnungstudien mit der Thrombelastographie einem neuen
Untersuchungsverfahren Klin Wochenschr 26 577ndash583
20 Horn P Pesce A 2003 Reference intervals and update Clin Chim Acta 334 5ndash23
21 Hua Yanhun Qiu W Xiao Q Wu Q 2018 Precision (repeatability and reproducibility) of
ocular parameters obtained by the Tomey OA-2000 biometer compared to the IOLMaster in
healthy eyes PLoS One 13 e0193023
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
34
22 Junge H Ringer S Mayer N Schwarzwald C 2016 Assesment of method reliability and
determination of reference intervals for rotational thromboelastometry in horses J Vet Emerg
Crit Care (San Antonio) 26 691ndash703
23 Kol A Borjesson DL 2010 Application of thrombelastographythromboelastometry to
veterinary medicine Vet Clin Pathol 39 405ndash416
24 Marschner CB Wiinberg B Tarnow I Markussen B Kuumlhnel L Bochsen L Kristensen
AT 2018 The influence of inflammation and hematocrit on clot strength in canine
thromboelastographic hypercoagulability J Vet Emerg Crit Care (San Antonio) 28 20ndash30
25 McMichael MA Smith SA 2011 Viscoelastic coagulation testing technology applications
and limitations Vet Clin Pathol 40 140ndash153
26 Mischke R 1994 Activity of coagulation factors II V VII and X in healthy dogs- dependence
on age sex and breed Berl Muumlnch Tieraumlrztl Wochenschr 107 289ndash294
27 Nielsen A Zois N Pedersen H Olse L Tarnow I 2007 Platelet function in dogs bread
differences and effect of acetylsalicylic acid administration Vet Clin Pathol 36 267ndash273
28 Nielsen L Kjelgaard‐Hansen M Jensen A Kristensen A 2010 Breed‐specific variation of
hematologic and biochemical analytes in healthy adult Bernese Mountain dogs Vet Clin Pathol
39 20ndash28
29 Nogami K 2016 The utility of thromboelastography in inherited and acquired bleeding
disorders Br J Haematol 174 503ndash514
30 Oswald E Stalzer B Heitz E Weiss M Schmugge M Strasak A Innerhofer P Haas T
2010 Thromboelastometry (ROTEMreg) in children Age-related reference ranges and correlations
with standard coagulation tests Br J Anaesth 105 827ndash835
31 Sankarankutty A Nascimento B Teodoro da Luz L Rizoli S 2012 TEGreg and ROTEMreg
in trauma similar test but different results World J Emerg Surg 7 (Suppl 1) S3
32 Sigrist NE Schefer RJJ Kutter APN 2018 Characteristics of hyperfibrinolysis in dogs and
cats demonstrated by rotational Thromboelastometry (ROTEM) Vet J 242 67-73
33 Steiner J Teague S Williams D 2003 Development and analytic validation of an enzime-
linked immunosorbent assay for the measurement of canine pancreatic lipase immunoreactivity in
serum Can J Vet Res 68 161ndash168
34 Sucker C Tharra K Lithmate J Scharf RE Zotz RB 2011 Rotation thromboelastography
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
35
(ROTEM) parameters are influenced by age gender and oral contraception Perfusion 26 334ndash
340
35 Swanepoel A Emmerson O Pretorius E 2017 The effect of endogenous and synthetic
estrogens on whole blood clot formation and erythrocyte structure Microsc Microanal 23 599ndash
606
36 Theusinger OM Nuumlrnberg J Asmis LM Seifert B Spahn DR 2010 Rotation
thromboelastometry (ROTEM) stability and reproducibility over time Eur J Cardiothorac Surg
37 677ndash683
37 Toulon P Metge S Hangard M Zwahlen S Piaulenne S Besson V 2017 Impact of
different storage times at room temperature of unspun citrated blood samples on routine
coagulation tests results Results of a bicenter study and review of the literature Int J Lab
Hematol 3 458ndash468
38 Turner JS Kutter APN Sigrist NE 2019 Correlation of rotational thrromboelastometry
(ROTEM) parameters with platelet count and their ability to predict thrombocytopenia in dogs
Res Vet Sci 126 45ndash50
39 Versteeg HH Heemskerk JWM Levi M Reitsma PH 2013 New Fundamentals in
Hemostasis Physiol Rev 93 327ndash358
40 Wiinberg B Jensen AL Kjelgaard-Hansen M Rojkjaer R Johansson PI Gade LP
Gram DX Kristensen AT 2007 Study on biological variation of haemostatic parameters in
clinically healthy dogs Vet J 174 62ndash68
41 Wiinberg B Jensen AL Johansson PL Rozanski E Tranholm M Kristensen AT 2008
Thromboelastographic evaluation of hemostatic function in dogs with disseminated intravascular
coagulation J Vet Intern Med 22 357ndash365
42 Wikkelsoslash A Wetterslev J Moslashller AM Afshari A 2016 Thromboelastography (TEG) or
thromboelastometry (ROTEMreg) to monitor haemostatic treatment versus usual care in adults or
children with bleeding Cochrane Database Syst Rev Art
httpsdoiorg10100214651858CD007871pub3 No CD007871
43 Willems A Paepe D Marynissen S Smets P Van de Maele I Picavet P Duchateau L
Daminet S 2017 Results of screening of apparently healthy senior and geriatric dogs J Vet
Intern Med 31 81ndash92
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
36
44 Woodman J Wagg CR Boysen SR Leguillete R Mizen K Roy MF 2015 Evaluation
of coagulation via thromboelastography in healthy horses administered dexamethasone Can Vet
J 56 1271ndash1274
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
37
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of
standard heparin prophylaxis and correlation to haemostatic changes
Pereira JM Hewicker-Trautwein M Rohn K von Depka Prondzinski M
Mischke R
Contributions of the authors
Pereira JM performed (1) the ultrasound evaluations and blood sampling of the
patients under study (2) haematology analysis (3) injection of the anticoagulation
prophylaxis (partially) to the patients under study (4) analysis of samples (collaboration)
at Werlhof Institute Medical Care Center (5) analysis of some haemostasis test
(collaboration) (6) statistical analysis (partially) (7) data documentation (8) and wrote
the manuscript
Hewicker-Trautwein M performed the electron microscopy analysis
Rohn K performed the statistical analysis
von Depka Prondzinski M performed some haemostasis test (thrombin generation
assay and endogenous thrombin potential)
Mischke R performed the coagulation and haemostasis test was involved in designing
and supervision of the study analysis and discussion of the data and its statistical analysis
and revision edition and correction of the manuscript
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
38
Central venous catheter induced thrombosis in dogs ndash efficacy of standard heparin
prophylaxis and correlation to haemostatic changes
Joseacute Mauricio Pereira1 Marion Hewicker-Trautwein2 Karl Rohn3 Mario von Depka
Prondzinski4 Reinhard Mischke1
1Small Animal Clinic University of Veterinary Medicine Hannover Foundation
Buumlnteweg 9 D-30559 Hannover Germany
2Institute for Pathology University of Veterinary Medicine Hannover Foundation
Buumlnteweg 17 D-30559 Hannover Germany
3Institute of Biometry Epidemiology and Information Processing University of
Veterinary Medicine Hannover Foundation Buumlnteweg 2 30559 Hannover Germany
4Werlhof Institute Schillerstrasse 23 30159 Hannover Germany
Abbreviations anti-Xa anti-factor Xa BW body weight CVC central venous catheter
EJV external jugular vein ELMI electron microscopy IV intravenous JV jugular vein
LMWH low molecular weight heparin ML maximum lysis (ROTEM) PIVC
peripheral intravenous catheter ROTEM rotational elastometry SC subcutaneous
TGA thrombin generation assay TID three times a day TT thrombin time UFH
unfractionated heparin
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
39
Corresponding author
E-mail addresses matiscrgmailcom (JM Pereira) MarionHewicker-
Trautweintiho-hannoverde (M Hewicker-Trautwein) KarlRohntiho-hannoverde
(K Rohn) mailwerlhof-institutde (M von Depka Prondzinski)
ReinhardMischketiho-hannoverde (R Mischke)
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
40
ABSTRACT
Central venous catheters (CVC) are of importance for intensive care management of
canine patients Thrombosis of the jugular vein is a severe complication related to the
CVC use However only little is known about the incidence of CVC-associated
thromboses in dogs and the necessity and efficacy of prophylactic antithrombotic
regimens The objectives of the present study were (1) to determine the incidence of
catheter-induced thrombosis in canine intensive care patients and thereby (2) to evaluate
the efficacy of a routine prophylactic heparin treatment and finally (3) to assess whether
initial changes of selected haemostasis parameters are predictive for an increased risk for
thrombosis formation 29 dogs hospitalised in the Small Animal Clinic University of
Veterinary Medicine Hannover Foundation and receiving a central venous catheter
(CVC) in the external jugular vein (EJV) for medical reasons were included in the final
assessment 17 dogs received the standard antithrombotic dosage regimen of
unfractionated heparin (UFH 150 IUKG BW TID sc) and 12 dogs (usually surgical
patients) the reduced dosage (75 IUKG BW TID sc) A colour Doppler ultrasound of
the respective EJV in order to detect thrombus formation and blood collection for
haemostasis tests were performed immediately before the installation procedure (day 0)
and on days 1 3 5 7 and so on after the CVC insertion Haemostasis tests included
prothrombin time activated partial thromboplastin time thrombin time antithrombin
activity D-dimer concentration thrombin generation and rotational elastometry as well
as the heparin activity using a chromogenic anti-factor Xa test Finally electron
microscopy (ELMI) of sections of the removed CVC was performed In 8 dogs
sonographic examination revealed thrombus formation in the lumen of EJV and in 1927
CVCs assessed using ELMI thrombi on the external CVC surface were detected without
significant correlation between both methods Plasmatic heparin activities showed great
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
41
variability but no significant differences were found between dogs with or without CVC-
associated thrombus formation From the initially performed haemostasis tests high
fibrinogen concentrations and low maximum lysis values as measured by the ROTEM
delta device (ex-tem reagent) were associated with sonographically detectable thrombi
The results of the present study indicate that CVC-associated thromboses occur (eg 28
[829] of the patients as detected by ultrasound) although severe thromboses (high
grades) are relatively rare in intensive care canine patients receiving a routine
anticoagulant treatment A high fibrinogen concentration and low maximum lysis
(ROTEM ex-tem) may be useful indicators for an increased thrombotic risk in individual
patients which may require special anticoagulatory treatment
Keywords CVC complication venal occlusion antithrombotic treatment ultrasound
electron microscopy rotational elastometry
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
42
1 Introduction
Central venous catheters (CVCs) are very important tools for care of patients in critical
care units allowing parenteral nutrition support therapy administration of high volumes
of intravenous fluids safe drugs administration that cannot be given through peripheral
veins plasmapheresis haemodialysis blood transfusions as well as periodical blood
sampling (Geerts 2014 Vose et al 2019) CVC related thrombosis is one of the most
severe complications (apart from infections and pneumothorax) of CVCs in humans
(Geerts 2014 Comerlato et al 2017 Evans and Ratchford 2018) leading to patientrsquos
distress CVC dysfunction venous stenosis and in some cases to the development of
thromboembolism (Geerts 2014)
In the human literature there are various studies dealing with epidemiology with reference
to different anticoagulatory regimens including placebo-controlled studies (Lee and
Kamphuisen 2012) In humans CVC related thromboses represent 70ndash80 of all upper
extremity thrombosis cases and approximately 10 of thromboembolism cases in
humans (Kucher 2011) The incidence of CVC related thrombosis reported in humans
based on 25 studies ranged widely from 2 to 67 (median = 30 ) (van Rooden et al
2005 review article) This wide range of calculated incidences of CVC related
thromboses in humans reflects the use of different CVC types different locations of entry
site of the CVC (jugular femoral subclavian axillary cephalic vein) different thrombus
detection technique (venography or ultrasonography) unequal durations of follow up
varying diseases of patients (eg haematological oncological) and presence of
predisposing factors (eg previous venous thrombosis history presence of cancer) (van
Rooden et al 2005 Geerts 2014 Liu et al 2015)
Part of the authors empirically observed CVC-associated thrombosis of the external
jugular vein (EJV) as a relevant complication in their canine intensive care patients which
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
43
awakened their interest in that topic To the best of the authoracutes knowledge studies about
extra luminal CVC related thromboses in dogs are rare in the available veterinary
literature One study evaluating the complications associated with 100 CVCs placed in
the jugular vein of dogs and cats reported only 2 cases of thrombosis (confirmed by
palpation or ultrasound) but obviously did not assess the appearance of subclinical CVC
related thromboses (Adamantos et al 2010) Thereby the incidence of CVC-induced
thromboses in dogs is unknown and systematic studies regarding an effective
anticoagulatory management are lacking
In this context it is of interest whether a possible development of thrombosis or an
increased risk (despite the prevention by a standard application of unfractionated heparin
[UFH]) can be predicted by means of changes of haemostasis related parameters during
the initial examination In one study on 168 human patients without indication of
anticoagulation prophylaxis for example elevated leukocyte counts high plasminogen
activator inhibitor and high coagulation factor VIII levels (all measured on blood samples
taken after CVC placement) were associated with an increased incidence of symptomatic
CVC-related thrombosis diagnosed by ultrasonography (Boersma et al 2016)
Therefore aims of the present study were (1) to determine the incidence of catheter-
induced thrombosis in canine intensive care patients (as detected by ultrasonography and
electron microscopy ELMI) and thereby (2) to evaluate the efficacy of a standard
antithrombotic regimen with unfractionated heparin (UFH) routinely used in the Small
Animal Clinic of the University of Veterinary Medicine Hannover and (3) to assess
whether selected haemostasis test results are predictive for an increased risk for
thrombosis formation
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
44
2 Material and methods
21 Study design
Canine in-patients of the Small Animal Clinic University of Veterinary Medicine
Hannover receiving a CVC in the EJV for medical reasons from March 2017 to
December 2019 were included Anticoagulatory treatment was performed according to
the standard protocol of the clinic Colour Doppler ultrasonographic controls of the EJV
and blood samplings (for heparin activity basic haematology and haemostasis tests) were
done immediately before the catheter was placed (day 0) and on days 1 3 5 etc (2 hours
after the first daily UFH injection) until the CVC was removed Only patients were
included which could be examined at least until day 3 Haemostasis tests included
prothrombin time activated partial thromboplastin time thrombin time fibrinogen
concentration antithrombin activity fibrin monomers D-dimers thrombin generation
assay (TGA) and rotational elastometry as well as the heparin activity using a
chromogenic anti-factor Xa test Finally ELMI of sections of the CVC after its removal
was performed
22 Animals
Originally 32 patients hospitalised in the Small Animal Clinic and receiving a CVC in the
EJV for medical reasons entered the study Three of them were subsequently excluded
because they did not reach the defined minimum study period of 3 days Two were
euthanised and one referred back to the referring vet before day 3 22 dogs weighing more
than 15 kg were included so that the withdrawal of about 10 ml of blood on several days
was not a significant burden for the patient (lt 1 of the total blood volume) Five dogs
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
45
with a body weight of less than 15 kg but at least 8 kg were included with special
consideration of the amount of blood taken (no more than 6 ml on individual days)
All animals received standard anticoagulatory treatment with UFH 17 dogs with internal
diseases received 150 IUkg BW TID subcutaneously and 12 dogs (post surgery) received
a reduced dosage of 75 IUkg BW TID subcutaneously
The 29 dogs enclosed in the study included 4 mixed breed dogs and the following
pedigree dogs 5 Labrador Retrievers 3 German Shepherds 2 Australian Shepherds and
one from each of the following breeds Akita Inu Asian Shepherd Bavarian Mountain
Hound Beagle Belgian Shepherd Briard Canaan dog Dalmatian Golden Retriever
Havanese dog Hovawart Rhodesian Ridgeback Schnauzer Shar Pei and Wolfspitz
These dogs were aged between 2 and 164 months (median 77 months) and had body
weights ranging from 81 and 63 kg (median 27 kg) 9 were castrated males 8 intact
males 7 spayed females and 5 intact females
The diseases of the 29 patients requiring a CVC included 3 from each of the following
diseases hepatopathy and esophagus dilatation 2 from each of the following diseases
bacterial cystitis diabetes mellitus and 1 from each of the following diseases abdominal
wall perforation aspiration pneumonia cystitis intestinal round cells sarcoma carcinoma
of the nasal cavity chronic renal insufficiency cystic duct rupture heartworm disease
ivermectine intoxication lymphoma multifocal alveolar histiocytosis pancreatitis post-
surgical bleeding stomach dilatation stomach foreign body tetanus thoracic
pyogranulomatous inflammation thrombocytopenia and trigeminal nerve paresis
Animals were treated in accordance with the German Animal Welfare Law The
experimental design was approved by the Animal Welfare officer of the University of
Veterinary Medicine and reported to the responsible authority (Lower Saxony State
Office for Consumer Protection and Food Safety reference number 17A101)
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
46
23 Positioning and handling of the central venous catheter
Two types of single lumen radiopaque polyurethane CVC (different on length and
diameter) were used during the study The CVC type I (n = 2529) was Cavafixreg Certoreg
Splittocanreg CVC 32 cm length 11 x 17 mm diameter16 G (BBraun Melsungen AG)
the CVC type II used (n = 429) was Certofixreg Mono S 215 15 cm length 08 x 14 mm
diameter18G (BBraun Melsungen AG) Each CVC was inserted into the EJV using a
modified Seldinger technique as follows the skin of the neck was shaved and disinfected
with antiseptic soap alcohol and iodine Local anesthesia (Lidocaine HCl 2) was
administrated subcutaneously on the catheter insertion site (a middle point in the lateral
neck over the jugular sulcus) A small skin incision was performed in the insertion site
and one peripheral plastic cannula with a metallic needle inside (14 G for CVC type I
and 20 G for CVC type II) was inserted in the EJV in cranio-caudal orientation (Hundley
et al 2018) Once the EJV was reached the needle was removed and a flexible wire
guide was inserted through the peripheral cannula following an external landmark
(4thintercostal space) (Reminga et al 2018) The peripheral cannula was removed and
the respective CVC was inserted slowly and with gentle pressure into the EJV guided
with the flexible wire and following the same external landmark to reach the right
position After the insertion of the CVC the flexible wire guide was removed The correct
positioning of the CVC was checked with a latero-lateral thorax x-ray ensuring that the
CVC tip was placed at the junction of the cranial cava vein with the right atrium Each
CVC was fixed to the neck skin (adjacent to the insertion point) with a monofilament
non-absorbable suture (gauge 3-0) Finally a bandage elaborated with Povidone-Iodine
gauze (Betaisodonareg) padded synthetic band (Artiflexreg Natur) and elastic band
(Elastomullreg) was used to protect the CVC output (Claude et al 2010) and changed
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
47
daily Several times day the catheter was flushed with isotonic sodium chloride
containing 1 IUml UFH
24 Heparin preparation and administration
Heparin-sodium-25000-ratiopharm (injection solution containing 5000 IUml sodium
heparin [mucosa]) was used SC injection was performed over the lateral thorax wall
using 1 mL syringes with fine graduation marks
25 Ultrasonography
Colour Doppler ultrasonographic examinations were performed in lateral position using
a LO-GIQ E9 ultrasound device (GE Healthcare Chicago Illinois USA) with a 15
Megahertz linear ultrasound probe The development of a thrombus or fibrin debris in the
EJV lumen around the CVC or inside it was assessed at each control Once the
ultrasound was finished a new protective bandage was placed around the neck of the dog
According to the percentage of lumen occlusion of the EJV (evaluated in cross sectional
views during the ultrasound scanning) the thrombi were classified in four different grades
(Fig 1)
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
48
of the residual vein lumen C ndash catheter T ndash thrombus
Fig 1 Definition of different grades of central venous catheter induced thrombosis of the
jugular vein as detected using colour Doppler ultrasound
26 Blood collection
At each time point approx 6ndash10 ml of blood (according to the body weight) was collected
This included approx 1 ml of EDTA blood for hematological analysis and approx 5ndash9
ml of citrated blood (tubes containing one part of 011 molL [38 ] sodium citrate to
nine parts of blood) for the measurement of hemostasis tests in citrated blood (ROTEM)
or citrated plasma Blood samples were collected by puncture of peripheral veins with
disposable cannulas (09 x 40 mm [20G] or 10 x 40mm [21G]) directly into sample tubes
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
49
or from the CVC using the three-syringe-technique (Villalta-Garciacutea et al 2015) in
situations when the peripheral venipuncture was not indicated due to phlebitis or irritation
andor inflammation of the surrounding skin Briefly 2ndash3 ml of blood were taken in a
sterile disposable syringe and immediately afterwards the blood sample was taken in a
new disposable syringe and transferred into the test tubes containing the respective
anticoagulants finally the first 2-3 ml taken were re-infused to the dog through the CVC
followed by flushing with a solution of sodium chlorideUFH (02) Immediately after
blood collection each sample tube was swayed gently until blood and anticoagulant had
been mixed thoroughly To prepare citrated platelet poor plasma citrated blood was
centrifuged at 16000 g for 10 min at room temperature using a microcentrifuge The
plasma was transferred into plastic tubes and the centrifugation procedure was repeated
This platelet poor plasma was then frozen in aliquots at -70 degC until used for analysis
Directly before analysis the plastic tubes were thawed at 37 degC in a water bath
27 Laboratory tests
Haematological analyses were made automatically with Adviareg 2120i (Siemens
Healthcare GmbH Germany)
Prothrombin time activated partial thromboplastin time and thrombin time were
measured with the autoanalyser Amax Destiny Plus (Tcoag Deutschland GmbH
Germany) and commercial reagents (Thromborel S Siemens Healthcare Diagnostics
Products GmbH Marburg Germany CK Prestreg and Test Thrombin Reagent
Diagnostica Stago SAS Asniegraveres sur Seine Cedex France) Whereas the last two tests
were measured according to the manufacturer`s instructions a modified test optimised
for canine plasma was used to measure prothrombin time (25 microl citrated plasma 120 with
imidazole buffer + 25 microl of a human fibrinogen solution 2 gl prewarmed for 120 s at 37
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
50
oC addition of 25 microl of activating reagent Thromborel S) and the results converted into a
percentage activity based on a calibration curve prepared with canine pooled plasma The
fibrinogen concentration was measured with the method according to Clauss using a test
kit from Stago Diagnostica SAS D-dimer concentration was measured with a
quantitative commercial human latex turbidimetric method in the autoanalyser Cobas
c311 using a completely assembled kit and human standards (Roche Diagnostics
Mannheim Germany) Antithrombin activity was also measured with a completely
assembled chromogenic test kit in the autoanalyser Cobas c311 and calibrated with a
canine pool plasma (n = 100)
The thrombin generation assay (Technothrombinreg TGA (Technoclone GbmH Vienna
Austria) determines thrombin generation based on monitoring of the fluorescence
generated by cleavage of a fluorogenic substrate by thrombin over time after activation
of the coagulation cascade by different concentrations of tissue factor and negatively
charged phospholipid in platelet poor plasma From the fluorescence development over
time the concentration of thrombin in the sample can be calculated using a thrombin
calibration curve The increase in thrombin concentration with time then allows to
calculate generation of thrombin in the sample and to plot thrombin values over time for
the whole coagulation process
The rotational thromboelastometry analysis with ROTEM delta device (Werfen Munich
Germany) was started approx 15 minutes after the blood collection To perform the
analysis and according to the automated ROTEM software 20 microl of recalcification reagent
(200 mmoll calcium chloride solution star-tem) and 20 microl of the respective activating
reagent (ex-tem in-tem) were pipetted into and carefully mixed in a pre-warmed cuvette
using the automated pipette provided in the device immediately afterwards 300 microl of
citrated whole blood (previously gently revolved) were added and mixed with the
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
51
reagents previously deposited Finally the cuvette was carefully placed and fixed to an
oscillating pin (oscillation movements on left and right through an angle of 475deg) in the
ROTEM delta device The interaction between blood cells and subsequently the bond
between fibrin and platelets promoted by the biomechanical changes in pH electrolytes
and temperature start the clot formation with its subsequent retraction and lysis The
magnitude of the resistance produced by the clot over the oscillating pin is quantitatively
represented by different parameters measured during the analysis and also qualitatively
in a graphic illustration (Bagge et al 2016 Crochemore et al 2017)
A chromogenic substrate test (Coatest Heparin Chromogenix-Instrumentation
Laboratory Haemochrom Diagnostica Essen Germany) was used for measurements of
UFH in the auto-analyser Cobas c311 The test application was created based on
manufacturers` instructions Different dilutions of the same batch of the commercial
sodium UFH preparation used in the experiment with normal canine pool plasma
(prepared from identical aliquots of 10 healthy adult dogs) served as standards for the
calibration of the measurement
28 Electron microscopy
Selected areas on the last third of the CVC (5ndash10 cm according with the CVC) with
macroscopically detectable thrombi were analysed and some of the CVCs were cut in
two different places The cut samples were preserved in a fixative medium composed of
1 glutaraldehyde and 4 formalin Once the sample was fixed it was prepared
according to the osmium thiocarbohydrazide procedure (OTOTO-procedure) (Malick et
al 1976) The fixed CVCs were dehydrated in seven series as follow first three series of
120 minutes each one on 30 50 and 70 ethanol respectively followed by two series
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
52
of 120 min each one on 90 ethanol and then two series of 24 hours each one on 100
ethanol After the dehydration process the samples were placed two times during 24
hours each one on isoamyl acetate and then placed on in a critical point drier Finally the
samples were coated with gold in a splutter-coater with 25 kV acceleration voltage in an
argon atmosphere with a current of 20 mA for one minute The samples were examined
in an EVO 15 scanning electron microscope (ZEISS International Carl Zeiss Jena
Germany) and photographed with magnifications of 600x and 6000x avoiding mechanical
artifacts (Toskala et al 1995) Thrombi on the external wall of the CVC were analysed
and measured using the software ImageJ (National Institutes of Health and the Laboratory
for Optical and Computational Instrumentation University of Wisconsin USA)
According to their extension (surface size) and thickness (related to the CVC wall
thickness in a cross sectional view) thrombi on the external wall and additionally within
the lumen of the CVC were each classified in four grades (Fig 2 Fig 1S [suppl
material]) In case of two cuts the highest degree of thrombosis detected at one of the two
sites was used for the final assessment
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
53
surface sizepercent of CVC wall thickness
Fig 2 Definition and illustration of different grades of central venous catheter induced
(extraluminal) thrombosis as detected using electron microscopy
Fig 1S (suppl material) Definition and illustration of different grades of intraluminal
thrombosis in central venous catheters as detected using electron microscopy
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
54
29 Statistical methods
Comparison between thrombus graduation either based on sonographic and ELMI
findings respectively was performed using the McNemar test with calculation of the
concordance index (κ) Standard normal distribution was assessed using the
Kolomogorov-Smirnov test
Due to non-normal distribution of part of data sets heparin activities and results of
haemostasis assays are reported using non-parametric variables (median minimumndash
maximum box and whisker plots) Heparin activities and results of haemostasis tests
between dogs with or without thrombosis were compared using Mann-Whitney-U test
3 Results
31 Sonographic detection of thrombus formation
Thrombus formation was sonographically detected in 829 patients during the study
period Based on examination on the final day in half of these eight patients (n = 4) the
thrombus formation was scored grade 1 in 3 dogs grade 2 and only in one grade 3 In all
of these 8 dogs this was the maximum grade during the study period One dog with fibrin
precipitates was excluded from further analysis because it could not be clearly assigned
to one of the groups In 78 cases thrombi were attached mainly to the venous wall
whereas only in one case was attached to the CVC external wall Seven of these 8 dogs
which developed thrombi received the standard heparin dosage (150 IUkg TID 41
[717] of dogs receiving this dosage) and only one the reduced heparin dosage (75 IUkg
TID 8 [112] of dogs receiving this dosage)
The time point of first detection of thrombus formation varied between days 3 and 11
(median day 8) In addition detritus (presumably composed mainly of fibrin) was
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
55
detectable in the lumen of the EJV in one further dog and within the lumen of the CVC
in further 5 dogs
32 Thrombus formation by electron microscopy
27 from 29 CVC catheter tips were analysed by electron microscopy 827 samples were
without detectable thrombus (grade 0) and 1927 with detectable thrombi 72 showed
thrombus formation of grade 1 427 of grade 2 and 827 of grade 3 There was no
concordance between thrombus graduation based on sonographic and the ELMI
graduation respectively The concordance index (McNemar test) was negative (κ = -
00718) indicating that the degree of agreement is less than by chance
Of the in total 19 dogs with detectable thrombus formation by electron microscopy 11
dogs received 150 IUkg TID (in total 16 dogs of these 27 dogs received that heparin
dosage) and 8 received 75 IUkg TID (in total 11 of these 27 dogs received that heparin
dosage 73 811) and Of the 12 patients with grade 2 and 3 thrombus formation 5 dogs
received 75 IUkg TID and 7 dogs 150 IUkg
The supplementary analysis of intraluminal thrombus formation revealed no detectable
thrombi in 7 cases and thrombus formation of grade 1 (lt 25 occlusion) in 11 cases of
grade 2 (25ndash50 ) in 3 cases and of grade 3 (gt 50 ) in 6 cases
33 Heparin activities and relationship to thrombus formation
Heparin activities varied remarkably in both dosage groups (Fig 3 ab) There was no
significant difference of heparin activities between blood samples collected via
venipuncture or from the CVC (Table 1S supplementary material) In addition there was
no significant difference between heparin activities in dogs with or without
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
56
sonographically or electron microscopically detectable thrombosis (Tables 1 and 2)
Table 1 refers to sonographically detectable thromboses and is based on median heparin
activities calculated from the measurements on day 1 and following days per patient
firstly for the total number of patients and secondly for the patients receiving 150 IUkg
TID In addition in the eight dogs heparin activities on the day of first thrombus detection
(023 IUml 019ndash036 IUml) were not significantly different from the median values
(Table 1 P = 05214 Wilcoxon test) Table 2 compares heparin activities (median values
of all days and results on the final day) between dogs with and without electron
microscopically detectable CVC-associated thrombi
Table 1S (Supplementary material) Heparin activities (IUml median values
minimumndashmaximum) measured in 27 dogs receiving 75 or 150 IUkg unfractionated
(standard) heparin TID subcutaneously 2 hours after the first injection on the day
Comparison of samples collected via puncture of the peripheral veins and those collected
from the central venous catheter (CVC) using the three-syringe-technique
Peripheral vein
puncture CVC
Mann-Whitney-U
test
Total number
(75 and 150
IUkg TID)
019
(00ndash108)
(n = 65)
023
(000ndash105)
(n = 65)
P = 03033
75 IUkg TID
010
(000ndash108)
(n = 25)
012
(000ndash023)
(n = 21)
P = 06907
150 IUkg
TID
030
(000ndash096)
(n = 40)
036
(000ndash105)
(n = 44)
P = 01223
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
57
Fig 3ab Heparin activities (box- and whisker plot arithmetic means) in dogs receiving
unfractionated heparin at a dosage of 75 IUkg BW tid (a) or 150 IUkg BW tid (b) two
hours after the first daily heparin administration
day 1 represents the first day after initiation of heparin treatment
only days were considered with at least results from 5 patients
Table 1 Heparin activities (IUml median values minimumndashmaximum) in 28 dogs
receiving 75 or 150 IUkg unfractionated (standard) heparin TID subcutaneously with
(grades 1ndash3) or without (grade 0) sonographically detectable thrombus Blood collections
were performed 2 hours after the first daily injection
Grade 0
(sonography)
Grades 1ndash3
(sonography)
Mann-Whitney U
test
Total number
(75 and 150 IUkg
TID)
020 (000ndash072)
(n = 20)
027 (018ndash059)
(n = 8)
P = 03212
150 IUkg TID 042 (000ndash072)
(n = 10)
031 (018ndash059)
(n = 7)
P = 04593
one dog with fibrin precipitates in the lumen of the jugular vein but without defined thrombus formation
was not considered because it could not be clearly assigned to one of the two groups
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
58
Table 2 Heparin activities (IUml median values minimumndashmaximum) in 27 dogs
receiving 75 or 150 IUkg unfractionated (standard) heparin TID subcutaneously with
(score 1ndash3) or without (score 0) detectable thrombus via electron microscopy
Grade 0
(electron
microscop)
(n = 8)
Grades 1ndash3
(electron
microscop)
(n = 19)
Mann-Whitney U
test
Median value of
different days 028 (005ndash060)
022 (000ndash072)
P = 05770
Final day 020 (001ndash045)
015 (000ndash105)
P = 06706
34 Relationship between haemostasis tests and thrombus formation
From the initially performed haemostasis tests high fibrinogen concentrations and low
maximum lysis values as measured by the ROTEM delta device (using the ex-tem
reagent) were associated with sonographically detectable thrombi (Tab 3) Additional
analyses based on the median values of different days for individual patients revealed that
low maximum lysis values as measured by the ROTEM delta device (using the in-tem
reagent) were associated with sonographically detectable thrombi (maximum lysis score
0 850 [100ndash175 ] score 1ndash3 375 [100ndash900 ] median [minimumndash
maximum] P = 00166) but no other test Comparison of the results of haemostasis tests
on the final day between dogs with and without sonographically detectable thromboses
did not show any significant difference (P gt 005)
Comparison of results of haemostasis tests on the final day in dogs with detectable
thrombosis via ELMI and dogs without significant thrombus formation showed several
significant differences including lower fibrinogen concentrations and longer clotting
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
59
times (ex-tem reagent) and clot formation times (ex-tem and in-tem reagents) using the
ROTEM delta analyser in general expressing a less coagulable status (Tab 4) If values
of dogs graded 0 and 1 (n =15) are compared to those of grades 2 and 3 (n = 12) the last-
mentioned had longer clotting times (ROTEM delta in-tem reagent) as well as longer clot
formation times and lower alpha angles (ROTEM delta in-tem reagent) (P lt 005 results
not shown)
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
60
Table 3 Comparison of initial results of haemostasis parameters (median values
minimumndashmaximum in dogs with (grades 1ndash3) and without (grade 0) sonographically
detectable central venous catheter-induced thromboses
Parameter Unit
Sonographic
thrombus grade
P-value
(Mann-Whitney
U test)
0
(n = 20)
123
(n = 8)
Haematocrit 355 (228ndash641)
382 (245ndash499)
06111
Platelet count x103microl 292 (420ndash483)
218 (860ndash458)
02320
Prothrombin time 944 (359ndash169)
896 (471ndash136)
05758
Activated partial thromboplastin
time s 128
(955ndash249) 140
(117ndash164) 02322
Thrombin time s 129 (121ndash178)
141 (650ndash160)
02028
Fibrinogen gl 448 (153ndash770)
747 (153ndash116)
00235
Antithrombin activity 639 (289ndash107)
670 (464ndash746)
05249
D-dimers microgml 006 (000ndash148)
015 (003ndash372)
03997
Thrombin generation assay nmol 130 (000ndash365)
912 (190ndash277)
08787
Clotting time (ROTEM ex-tem) s 420 (290ndash930)
430 (170ndash159)
07989
Clot formation time (ROTEM ex-
tem) s 665
(310ndash143) 470
(240ndash800) 00633
Alpha angle (ROTEM ex-tem) degrees 795 (620ndash840)
815 (750ndash860)
01667
Max clot firmness (ROTEM ex-
tem) mm 750
(570ndash860) 775
(700ndash830) 01753
Max clot elasticity (ROTEM ex-
tem) - 299
(131ndash619) 347
(235ndash484) 01544
Maximum lysis (ROTEM ex-tem) 135 (300ndash290)
400 (000ndash800)
00037
Clotting time (ROTEM in-tem) s 208 (840ndash810)
249 (720ndash371)
05085
Clot formation time (ROTEM in-
tem) s 835
(370ndash634) 745
(390ndash235) 08988
Alpha angle (ROTEM in-tem) degrees 745 (250ndash820)
750 (500ndash820)
09797
Max clot firmness (ROTEM in-
tem) mm 700
(540ndash810) 740
(610ndash770) 03587
Max clot elasticity (ROTEM in-
tem) - 235
(118ndash424) 287
(156ndash332) 04764
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
61
Maximum lysis (ROTEM in-tem) 850 (000ndash210)
350 (000ndash220)
01327
Table 4 Comparison of results of haemostasis parameters (median values minimumndash
maximum) on the last day in dogs with (grades 1ndash3) and without (grade 0) central venous
catheter-associated thrombus formation as detected by electron microscopy
Parameter Unit
Thrombus grade
(ELMI)
P-value
(Mann Whitney
U test)
0
(n = 8)
123
(n = 19)
Haematocrit 322 (241ndash454)
330 (216ndash510)
05953
Platelet count 103microl 446 (321ndash592)
268 (101ndash675)
00558
Prothrombin time 145 (104ndash264)
120 (561ndash148)
00462
Activated partial thromboplastin
time s 123
(109ndash140) 136
(109ndash188) 00665
Thrombin time s 129 (121ndash178)
141 (650ndash160)
02028
Fibrinogen gl 465 (275ndash866)
347 (189ndash992)
00491
Antithrombin activity 793 (448ndash981)
692 (398ndash889)
04572
D-dimers microgml 018 (000ndash173)
007 (000ndash118)
01920
Thrombin generation assay nmol 501 (000ndash160)
93 (000ndash287)
09562
Clotting time (ROTEM ex-tem) s 360
(270ndash410) 400
(310ndash620) 00238
Clot formation time (ROTEM ex-
tem) s 395
(120ndash740) 650
(240ndash174) 00275
Alpha angle (ROTEM ex-tem) degrees 835 (760ndash880)
790 (580ndash850)
00484
Max clot firmness (ROTEM ex-
tem) mm 780
(700ndash850) 720
(550ndash860) 00586
Max clot elasticity (ROTEM ex-
tem) - 346
(239ndash555) 254
(121ndash637) 00495
Maximum lysis (ROTEM ex-tem) 105 (200ndash190)
110 (000ndash380)
03796
Clotting time (ROTEM in-tem) s 178 (100ndash222)
201 (139ndash554)
00525
Clot formation time (ROTEM in-
tem) s 435
(330ndash111) 790
(350ndash882) 00209
Alpha angle (ROTEM in-tem) degrees 815 (710ndash830)
740 (240ndash830)
00191
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
62
Max clot firmness (ROTEM in-
tem) mm 785
(660ndash810) 690
(270ndash840) 00586
Max clot elasticity (ROTEM in-
tem) - 362
(192ndash434) 218
(370ndash507) 00559
Maximum lysis (ROTEM in-tem) 950 (200ndash190)
800 (100ndash180)
06890
4 Discussion
The main result of our study is that under the study conditions part of the dogs developed
CVC-associated thromboses However only a very low part of these thromboses were
obviously of a clinically significant degree regarding the amount of occlusion of the EJV
and none of the dogs had a symptomatic thrombosis whose definition includes presence
of clinical signs such as localised swelling pain and redness (Boersma et al 2008 Lee
and Kamphuisen 2012) In the present study we particularly refer to the extraluminal
thromboses with regard to the assessment of the antithrombotic management
One major problem of the statistical analysis and result presentation was that the two
methods used to define the occurrence and degree of thrombus formation (ultrasound and
ELMI) revealed discrepant results Therefore we performed a statistical analysis
independently regarding thrombus formation with either of the two methods It is well
known that ELMI which has already been used to detect CVC induced thromboses in
humans (Lucas et al 2017) and experimental animals (Xiang et al 1998 Xiang et al
2001) is a very sensitive diagnostic technique which allows the ex vivo diagnosis of very
small or fibrin rich thrombi that perhaps cannot be detected by ultrasonography or
venography during the in vivo evaluation of patients (Xiang et al 2001) A major reason
for the discrepant results between the two methods used to detect CVC associated
thromboses is that different CVC segments are evaluated Using ultrasonography it was
only possible to evaluate the EJV in the neck area of the patient and thereby only its
proximal third In contrast ELMI assessed mainly the CVC tip and a human study
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
63
revealed that even different positions of the catheter tip are associated with various risks
for development of CVC associated thrombosis (Ballard et al 2016) One additional point
to consider which also may contribute to the discrepancy is that it is possible that thrombi
developed on the CVC surface can be detached during the CVC removal from the vein
and thereby before its analysis by ELMI (Xiang et al 2001)
The contrast venography technique is used as gold standard to confirm thrombosis in
human medicine However this diagnostic technique is normally used only in the case of
a strong clinical suspicion of venous thrombosis that cannot be detected by
ultrasonography (Laurenson et al 2010 Langston and Eatroff 2018) Our study protocol
with privately owned clinic patients did not allow to use this method In a study performed
in 44 human patients requiring a CVC (Koumlksoy et al 1995) a sensitivity of 94 and a
specificity of 96 were determined for the colour Doppler ultrasound diagnosis when it
was compared with the diagnosis performed with the contrast venography technique and
that makes the colour Doppler ultrasound an accurate diagnosis method to detect CVC
related thrombosis even in humans (Koumlksoy et al 1995 van Rooden et al 2003)
The incidences of CVC related thrombosis detected by colour Doppler ultrasound of the
internal jugular vein reported in 3 human studies were 2 30 and 56 (Harter et al
2002 Lordick et al 2003 Wu et al 1999) and although with a wide range the median
value of these rates (30 ) is very well suited to the incidence of 28 (829) which we
calculated for our canine study based on sonographic findings The wide variation of
incidences of CVC related thrombosis in different human studies can in general be
explained by differences regarding the type and entry site of the CVC (jugular femoral
subclavian axillary cephalic vein) the time period with the CVC in place the thrombus
detection technique (venography or ultrasonography comment in the cited 3 studies
consistently the last-mentioned method was used) and criteria for diagnosis of
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
64
thrombosis the duration of follow up diseases and presence of predisposing factors in
these patients (eg previous venous thrombosis history presence of cancer) and finally
prophylactic treatment with anticoagulants (van Rooden et al 2005 Geerts 2014 Liu et
al 2015)
Different pathomechanisms contribute to the development of CVC induced thromboses
During the CVC insertion the puncture of the skin on the CVC insertion site and the
rupture of the blood vessels integrity activate the coagulation cascade and the CVC acts
as a matrix for adherence and accumulation of fibrin and cellular components (Smith et
al 2012) The attachment of proteins and blood cells to the internal andor external wall
of the CVC can lead to a partial or complete occlusion of the catheter lumen and
sometimes to a partial or complete obstruction of the blood flow andor
thromboembolism (Langston and Eatroff 2018) There is a strong interrelationship
between thrombus and biofilm formation (Smith et al 2012) Prevention of thrombus
formation has thereby also relevance regarding the increased risk for microbial
colonisation of the catheter (Boersma et al 2008) leading at least in humans to an
increased number of CVC related systemic infections (Lordick et al 2003)
In this context it has to be considered that CVC produced from polyurethane which are
routinely used in our clinic and thereby in the study are usually associated with a
relatively high thrombosis potential and thereby definitely provide an effective test for
an antithrombotic regimen Silicone catheters have a significantly reduced risk for
thrombotic complications but this advantage has to be purchased by a higher rate of
mechanical failure (eg rupture of the catheter) (Wildgruber et al 2016) Promising
experimental approaches work on surface modifications of the standard catheters For
example one group was able to demonstrate that a coating of the surface of the catheter
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
65
with a zwitterionic polymeric sulfobetaine showed a significant reduction of thrombus
formation in vitro with human blood and in an in vivo canine model (Smith et al 2012)
Due to ethical reasons we did not performed a placebo-controlled study However
interestingly not all human studies report systemic anticoagulation and part of the human
studies was even placebo-controlled (review Lee and Kamphuisen 2012) Obviously
there is no consensus in the available human literature regarding the beneficial use of
antithrombotic prophylaxis to prevent CVC related thrombosis Several studies did not
find a statistically significant difference in the incidence of CVC related thrombosis
between patients receiving the antithrombotic prophylaxis compared with the control
groups without systemic anticoagulation (Verso et al 2005 P = 035 Karthaus et al
2006 P gt 005 Niers et al 2007 P = 049) whereas in other studies a lower incidence
of CVC related thrombosis was found in the group receiving anticoagulants as
prophylaxis compared with the control groups (Abdelfeki et al 2004 150 vs 126
P = 003 De Cicco et al 2009 400 vs 526 P = 005)
Although it is well known that subcutaneously administered UFH has a limited and
variable bioavailability and thereby leads to unpredictable blood activities (Hirsh and
Raschke 2004) the extremely wide range of blood activities after a defined dosage in our
study was relatively unexpected Blood was collected 2 hours after the first UFH
injection and thereby at the time of suspected maximum heparin activity considering the
relatively low UGH dosage (Mischke and Jacobs 2001) The cited study was performed
on healthy experimental dogs Possible additional influencing factors in critical ill
patients include circulatory insufficiency which may reduce and delay absorption from
subcutanenous tissue decreased clearance function of the liver and kidneys as well as
altered blood plasma concentrations of heparin binding plasma proteins such as
fibrinogen and platelet factor 4 and alterations of the heparin binding capacity of
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
66
endothelial cells which altogether are reasons for a great inter- but also intrapatient
variability (Hirsh and Raschke 2004) In individual patients this can lead to heparin
resistance It has also to be considered that antithrombin activities which are necessary
for an adequate heparin effect (Bjoumlrk and Lindahl 1982) were reduced in our patients
Availability of approved blood tests which can predict the risk of development of CVC
associated thrombosis already at the moment of the CVC placement are important
(Boersma et al 2016) One of the most interesting results was that dogs which developed
sonographically detectable thrombi had nearly twofold higher fibrinogen levels Well in
agreement with our results one study performed in anticoagulated human patients
demonstrated that high fibrinogen levels were associated with an increased risk of
perioperative venous thromboembolism (P = 0004) (Kato et al 2016) The cited study
does not exclusively refer to CVC associated thrombosis but 76 (1621) of the venous
thromboses were suggested to be associated with CVCs Fibrinogen measurement a test
which is widely available is therefore a useful and practicable indicator of an increased
risk for CVC induced thromboses and may advice the clinician to treat these patients
especially carefully (eg with low molecular weight heparin) In the study performed by
Boersma et al (2016) in 168 human patients without indication of anticoagulation
prophylaxis elevated leukocyte counts high levels of plasminogen activator inhibitor
and of coagulation factor VIII were associated with an increased incidence of
symptomatic CVC related thrombosis diagnosed by ultrasonography A limitation of that
study is that all measurements were performed on blood samples taken after CVC
placement Interestingly in our study ROTEM results were partly associated with
thrombus formation which reflects the well-known fact that results of viscoelastic
measurements are a good indicator of hypercoagulability and thromboembolic events
(Harahsheh and Ho 2018) It is of further interest and possibly unexpeted that on the final
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
67
day of examination dogs with ELMI signs of thromboses had partly lower coagulation
activities than dogs without thromboses Due to the low number of dogs without
detectable thrombi using ELMI this implausible result has to be interpreted with caution
One possible explanation could be that temporary hypercoagulability in these dogs may
have finally resulted in consumption of haemostasis substrates
Limitations of the present study were as a consequence of the study design based on
clinical patients the relatively low total number of patients inclusion of different UFH
treatment regimens and the lack of a control group
5 Conclusions
The results of the present study indicate that the used UFH regimen was not completely
effective to prevent CVC-associated thromboses which were detectable in 28 [829]
of the patients by ultrasound However severe thromboses (high grades) are relatively
rare in intensive care canine patients receiving a routine anticoagulant treatment Because
heparin plasma activities were not different in dogs with or without CVC-associated
thrombus formation other local and systemic factors must play a significant role as well
A high fibrinogen concentration and low maximum lysis (ROTEM ex-tem) may be
useful indicators for an increased thrombotic risk in individual patients which may
require special anticoagulatory treatment
6 Acknowledgments
Jose Mauricio Pereira received a scholarship from the Universidad Nacional de Costa
Rica (reference code JB-C-1107-2016)
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
68
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Torjman L Lakhal A Achour W Ben Hassen A Hsaiumlri M Ladeb F Ben Abdeladhim A
2004 Prevention of central venous line-related thrombosis by continuous infusion of low-dose
unfractionated heparin in patients with haemato-oncological disease A randomized controlled trial
Thromb Haemost 92 654ndash661
2 Adamantos S Brodbelt D Moores A L 2010 Prospective evaluation of complications associated
with jugular venous catheter use in a veterinary hospital J Small Anim Pract 51 254ndash257
3 Bagge A Schoumltt U Kander T 2016 Effects of naturopathic medicines on Multiplate and ROTEM
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4 Ballard DH Samra NS Gifford KM Roller R Wolfe BM Owings JT 2016 Distance of the
internal central venous catheter tip from the right atrium is positively correlated with central venous
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5 Boersma RS Jie KS Verbon A van Pampus EC Schouten HC 2008 Thrombotic and
infectious complications of central venous catheters in patients with hematological malignancies Ann
Oncol 19 433ndash442
6 Boersma R S Hamulyak K van Oerle R Tuinenburg A Ten Cate-Hoek AJ Schouten HC
2016 Biomarkers for prediction of central venous catheter related-thrombosis in patients with
hematological malignancies Clin Appl Thromb Hemost 22 779ndash784
7 Bjoumlrk I Lindahl U 1982 Mechanism of the anticoagulant action of heparin Mol Cell Biochem
48 161ndash182
8 Claude A Riedesel D Riedesel E 2010 Electrocardiography-guided and retrospective analysis of
central venous catheter placement in the dog Vet Anaesth Analg 37 97ndash105
9 Comerlato PH Rebelatto TF Santiago de Almeida FA Klein LB Boniatti M M Schaan
DB Rados VD 2017 Complications of central venous catheter insertion in a teaching hospital
Rev Assoc Med Bras 63 613ndash620
10 Crochemore T Piza FMT Rodriacutegues RDR Guerra JCC Ferraz LJR Correcirca TD 2017
A new era of thromboelastometry Einstein (Sao Paulo) 15 380ndash385
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
69
11 De Cicco M Matovic M Balestreri L Steffan A Pacenzia R Malafronte M Fantin D
Bertuzzi CA Fabiani F Morassut S Bidoli E Veronesi A 2009 Early and short‐term
acenocumarine or dalteparin for the prevention of central vein catheter‐related thrombosis in cancer
patients a randomized controlled study based on serial venographies Ann Oncol 20 1936ndash1942
12 Evans NS Ratchford EV 2018 Catheter related-venous thrombosis Vasc Med 23 411ndash413
13 Geerts W 2014 Central venous catheterndashrelated thrombosis Hematology Am Soc Hematol Educ
Program 2014 306ndash311
14 Harahsheh Y Ho KM 2018 Use of viscoelastic tests to predict clinical thromboembolic events A
systematic review and meta‐analysis Eur J Haematol 100 113ndash123
15 Harter C Salwender HJ Bach A Egerer G Goldschmidt H Ho AD 2002 Catheter-related
infection and thrombosis of the internal jugular vein in hematologic-oncologic patients undergoing
chemotherapy a prospective comparison of silver-coated and uncoated catheters Cancer 94 245ndash251
16 Hirsh J Raschke R 2004 Heparin and low-molecular-weight heparin The Seventh ACCP
Conference on Antithrombotic and Thrombolytic Therapy Chest 126 (Suppl) 188S-203S
17 Hundley D Brooks A Thomovsky E Jhonson P Freeman L Schafbuch R Heng HG Moore
G 2018 Comparison of ultrasound-guided and landmark-based techniques for central venous
catheterization via the external jugular vein in healthy anesthetized dogs Am J Vet Res 79 628ndash
636
18 Kato F Takeuchi H Matsuda S Kawakubo H Omori T Kitagawa Y 2016 Incidence of and
risk factors for venous thromboembolism during surgical treatment for esophageal cancer a single-
institution study Surg Today 46 445ndash452
19 Karthaus M Kretzschmar A Kroning H Biakhov M Irwin D Marschner N Slabber C
Fountzilas G Garin A Abecasis NG Baronius W Steger GG Sudhoff T Giorgetti C
Reichardt P 2006 Dalteparin for prevention of catheter‐related complications in cancer patients with
central venous catheters final results of a double‐blind placebo‐controlled phase III trial Ann Oncol
17 289ndash296
20 Koumlksoy C Kuzu A Kutlay J Erden I Ozcan H Ergicircn K 1995 The diagnostic value of colour
Doppler ultrasound in central venous catheter related thrombosis Clin Radiol 50 687-689
21 Kucher N 2011 Clinical practice Deep-vein thrombosis of the upper extremities N Engl J Med
364 861ndash869
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heparin prophylaxis and correlation to haemostatic changes
70
22 Langston CE Eatroff AE 2018 Hemodialysis catheter-associated fibrin sheath in a dog J Vet
Emerg Crit Care 28 366ndash371
23 Laurenson MP Hopper K Herrera MA Johnson EG 2010 Concurrent diseases and conditions
in dogs with splenic vein thrombosis J Vet Intern Med 24 1298ndash1304
24 Lee AYY Kamphuisen PW 2012 Epidemiology and prevention of catheter‐related thrombosis in
patients with cancer J Thromb Haemost 10 1491ndash1499
25 Liu G Fu ZQ Zhu P Li SJ 2015 Central venous catheter-related thrombosis in senile male
patients New risk factors and predictors J Huazhong Univ Sci Technolog Med Sci 35 445ndash449
26 Lordick F Hentrich M Decker T Hennig M Pohlmann H Hartenstein R Peschel C 2003
Ultrasound screening for internal jugular vein thrombosis aids the detection of central venous catheter-
related infections in patients with haemato-oncological diseases a prospective observational study Br
J Haematol 120 1073ndash1078
27 Lucas TC Silva EED Souza DOD Santos ARD Lara MO 2017 Microstructural
evaluation by confocal and electron microscopy in thrombi developed in central venous catheters Rev
Esc Enferm USP 51 1ndash9
28 Malick L Wilson R Stetson D 1976 Modified thiocarbo- hydrazide procedure for scanning
electron microscopy routine use for normal pathological and experimental tissue Stain Technol 50
265ndash269
29 Mischke R Jacobs C 2001 The monitoring of heparin administration by screening tests in
experimental dogs Res Vet Sci 70 101ndash108
30 Niers TM Di Nisio M Klerk CP Baarslag HJ Buller HR Biemond BJ 2007 Prevention
of catheter‐related venous thrombosis with nadroparin in patients receiving chemotherapy for
hematologic malignancies a randomized placebo‐controlled study J Thromb Haemost 5 1878ndash
1882
31 Reminga C Silverstein D Drobatz K Clarke D 2018 Evaluation of the placement and
maintenance of central venous jugular catheters in critically ill dogs and cats J Vet Emerg Crit Care
28 232ndash243
32 Smith RS Zhang Z Bouchard M Li J Lapp HS Brotske GR Lucchino DL Weaver D
Roth LA Coury A Biggerstaff J Sukavaneshvar S Langer R Loose C 2012 Vascular
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
71
catheters with a nonleaching poly-sulfobetaine surface modification reduce thrombus formation and
microbial attachment Sci Transl Med 4 153ra132 doi101126scitranslmed3004120
33 Toskala E Nuutinen J Rautiainen M 1995 Scanning electron microscopy findings on human
respiratory cilia in chronic sinusitis and in recurrent respiratory infections J Laryngol Otol 109 509ndash
514
34 Van Rooden CJ Tesselaar ME Osanto S Rosendaal FR Huisman MV 2005 Deep vein
thrombosis associated with central venous catheters - a review J Thromb Haemost 3 2409ndash2419
35 Verso M Agnelli G Bertoglio S Di Somma FC Paoletti F Ageno W Bazzan M Parise P
Quintavalle R Naglieri E Santoro A Imberti D Soraru M Mosca S 2005 Enoxaparin for the
prevention of venous thromboembolism associated with central vein catheter a double‐blind placebo‐
controlled randomized study in cancer patients J Clin Oncol 23 4057ndash4062
36 Villalta-Garciacutea P Loacutepez-Herranz M Mazo-Pascual S Honrubia-Fernaacutendez T Jaacutentildeez-Escalada
L Fernaacutendez-Peacuterez C 2015 Reliability of blood test results in samples obtained using a 2-mL discard
volume from the proximal lumen of a triple-lumen central venous catheter in the critically ill patient
Nurs Crit Care 22 298ndash304
37 Vose J Odunayo A Price JM Daves M Schildt JC Tolbert MK 2019 Comparison of
heparinized saline and 09 sodium chloride for maintaining central venous catheter patency in healthy
dogs PeerJ 7 e7072 httpsdoiorg107717peerj7072
38 Wildgruber M Lueg C Borgmeyer S Karimov I Braun U Kiechle M Meier R Koehler M
Ettl J Berger H 2016 Polyurethane versus silicone catheters for central venous port devices
implanted at the forearm Eur J Cancer 59 113ndash124 doi101016jejca201602011
39 Wu X Studer W Skarvan K Seeberger MD 1999 High incidence of intravenous thrombi after
short-term central venous catheterization of the internal jugular vein J Clin Anesth 11 482ndash485
40 Xiang DZ Verbeken EK Van Lommel AT Stas M De Wever I 1998 Composition and
formation of the sleeve enveloping a central venous catheter J Vasc Surg 28 260ndash271
41 Xiang DZ Verbeken EK Van Lommel AT Stas M De Wever I 2001 Sleeve-related
thrombosis a new form of catheter-related thrombosis Thromb Res 104 7ndash14
Chapter 4 General discussion
72
Chapter 4 General discussion
Incidence of CVC related thrombosis
In our study the incidence of CVC related thrombosis detected by ultrasound in critical
care canine patients was 28 To the best of the authoracutes knowledge similar studies
performed in dogs are rare in the available veterinary literature In one study performed
in 81 dogs and 12 cats (Adamantos et al 2010) an incidence of 2 was found for vein
thrombosis related to CVC This low incidence can be due to a lacking of systematic
ultrasound evaluations of the jugular vein containing the CVC In the mentioned study in
dogs and cats the vein thrombosis was confirmed by palpation or ultrasound presumably
after the presentation of clinical symptoms however in many cases as in our study
thrombus into the jugular vein can be developed without manifestation of clinical
symptoms what makes a routinely ultrasound evaluation of the vein containing the CVC
very important Our results are well aligned with the findings in humans in which a
median incidence of 30 was found for thrombosis related to CVC based on 25 studies
performed in patients requiring a CVC (review article van Rooden et al 2005) In the
mentioned 25 human studies the incidence of CVC related thrombosis varied within a
wide range (2ndash67 ) according to factors related to (1) the CVC (eg material type
entry site [jugular femoral subclavian axillary cephalic vein] time period in place (2)
diagnostic thrombus detection technique (ultrasonography or venography) and criteria
(3) patient collective (diseases and presence of predisposing factors [eg previous venous
thrombosis history presence of cancer] and (4) prophylactic antithrombotic treatment
(Geerts 2014 Liu et al 2015 van Rooden et al 2005)
The incidence of CVC related thrombosis detected by colour Doppler ultrasound in our
study fits also with the median incidence (30 ) found in three studies performed in
humans in which colour Doppler ultrasound evaluations of the jugular vein containing a
Chapter 4 General discussion
73
CVC were performed (Harter et al 2002 Lordick et al 2003 Wu et al 1999) In these
three studies the individual incidence of CVC related thrombosis was 2 (Harter et al
2002) 30 (Lordick et al 2003) and 56 (Wu et al 1999) The high thrombus
incidence reported in the study performed by Wu et al (1999) could be due to the criteria
for diagnosis of thrombosis because in that study both fibrin sleeves and compact
thrombi were considered as cases of thrombosis related to CVC The incidence for
compact thrombi was 25 which is more similar to our results In the study performed
by Harter et al (2002) the CVC related thrombosis incidence (2 ) was very low
compared with the incidence found in our study In that study the jugular veins were
examined just before or less than 24 hours after CVC removal (median period of CVC
use = 1025 days) In our study the ultrasound evaluations were performed before the
CVC placement (day 0) and then on days 1 3 5 etc until the CVC removal (median
period of CVC use = 7 days median interval until thrombus detection = 7 days) In our
study we detected thrombus formation as early as on the 3rd day so it could be possible
that in the cited study possibly only temporarily existing thrombi could have been missed
by ultrasonography in the final stage and thereby for the incidence calculation However
this scenario is not very likely because results of the present study show that
sonographically detectable thrombi during the observation period had in all cases their
maximum grade on the final day
In the study performed by Lordick et al (2003) the CVC related thrombosis incidence
determined for 43 human patients was very similar to our study and in both studies only
compact thrombi detectable by ultrasound were considered for assessment of the
incidence In the mentioned study all the human patients were screened for the presence
of thrombosis every 4 days after CVC insertion with real time B-mode ultrasound
(Lordick et al 2003)
Chapter 4 General discussion
74
In our study 70 of CVCs analysed by ELMI showed thrombi of grade 1ndash3 on the
external CVC wall To the best of the authoracutes knowledge studies about incidence of CVC
related thrombosis on the outside surface of the CVC detected by ELMI cannot be found
in the human and small animal literature In one study performed in humans (Lucas et al
2017) thrombus formation was analysed by ELMI in 78 central venous catheters That
study focussed on microstructural assessment of thrombus formation inside the distal
openings of the CVC which were found in 85 of the evaluated CVCs The percentage
of occurrence of thrombosis in the mentioned study in humans was nearly similar to the
incidence of intraluminal thrombus formation as detected in the presented study by ELMI
(74 ) In general intraluminal thrombi were not specially considered in the present
study because they mainly express the efficacy of the flushing technique rather than the
effectivity of the antithrombotic treatment In one study performed in rats the
microstructure of thrombi developed in the jugular vein containing the CVC was also
evaluated by ELMI (Xiang et al 1998) In the mentioned study pericatheter thrombosis
was detected on the proximal section of the CVC in 100 of rats (n = 12) after 1 and 3
days of catheterisation The analyses of the CVCs placed in rats were performed with the
catheter inside the vein in contrast to our study where the CVC was removed from the
jugular vein for the respective analysis Xiang et al (2001) hypothesised in one study
performed in rabbits and rats in which CVCs were placed in the jugular vein that fibrin
rich thrombi associated to the CVC can be detached during the catheter removal and this
could explain the difference between the thrombosis incidence assessed in our study and
that of the study performed by Xiang et al (1998)
In our study the incidence of CVC related thrombosis detected by ultrasonography in the
lumen of the EJV did not correlate with the incidence of CVC related thrombosis detected
by ELMI on the external wall of the CVC This was mainly due to the fact that the CVC
Chapter 4 General discussion
75
segment evaluated with both methods was different Using ultrasonography it was only
possible to evaluate the EJV in the neck area of the patient and thereby only the proximal
third of the CVC In contrast with the ELMI analysis only the distal section of the CVC
(last third) was evaluated because macroscopically detected thrombi mainly developed
near the CVC tip which was routinely positioned at the junction of the cranial cava vein
with the right atrium In a retrospective review of medical records of 169 human patients
requiring a CVC (Ballard et al 2016) the position of the CVC tip was determined by
radiologic evaluation and it was determined that the catheter tip position can be a
predisposing factor for central venous thrombosis development Thereby it could be of
interest to evaluate in further studies in dogs if a high incidence of thrombosis detected
by ELMI as was shown in our study could be related to the CVC tip position
In our study polyurethane catheters were tested which are widely used and are the
standard CVCs used in the Small Animal Clinic University of Veterinary Medicine
Hannover In vivo and in vitro studies have assessed the thrombogenicity of different
catheter materials (Borow and Crowley 1985 Smith et al 2012) In general terms
polyurethane catheters (central or peripheral) had shown a higher thrombogenicity
compared with coated (eg with hydromer) and silicone catheters Due to this property
of polyurethane catheters they can be supposed as a good test system for antithrombotic
regimens as evaluated in our study On the other hand despite silicone catheters have been
associated with a lower thrombogenicity their mechanical flexibility could be a
disadvantage in small clinical practice due to the increased risk of rupture detected even
in humans (Wildgruber et al 2016)
Chapter 4 General discussion
76
Heparin thromboprophylaxis for CVC related thrombosis
The occurence of CVC related thrombosis detected by colour Doppler ultrasound and
ELMI in our study demonstrates that the UFH in the given dosage was not completely
effective to prevent CVC related thrombosis in canine intensive care patients In human
medicine LMWH is commonly used in prophylactic anticoagulation regimens however
studies performed in human patients requiring a CVC and receiving a prophylactic
anticoagulation (with LMWH or UFH) did not achieve consensus regarding the efficacy
of prophylactic heparin to prevent CVC related thrombosis (Abdelfeki et al 2004
Lordick et al 2003 Wu et al 1999) To the best of the authoracutes knowledge studies
performed in dogs evaluating systemic prophylactic anticoagulation to prevent CVC
related thrombosis cannot be found However according to the evidence found in the
human literature and the results of our study further investigations using comparative
groups with different drugs dosages andor administration routes are required to establish
an effective antithrombotic treatment to prevent CVC related thrombosis
The plasma heparin activities measured in canine patients in our study varied significantly
after a defined subcutaneous dosage The limited and variable bioavailability of SC
administered UFH is well documented in humans (Hirsh and Raschke 2004)
Considerable variations of the circulatory status of the liver- and kidney function and
concentration of heparin-binding proteins in intensive care patients obviously amplify
this phenomenon leading to an extremely high inter-individual and intra-individual
variation of the heparin blood levels in our patients A different application route (IV)
may have reduced the influence of part of these factors and therefore could have led to
more predictable UFH blood levels However continuous infusion requires more
technical equipment and is also associated with a wide range of heparin activities in
canine patients (Scott et al 2009)
Chapter 4 General discussion
77
Prediction of CVC related thrombosis
Of 7 haemostasis tests (including ROTEM with 12 different parameters [6 for each
reagent ex-tem and in-tem]) evaluated as possible predictors of CVC related thrombosis
in our study initial values of high fibrinogen and low maximum lysis (ROTEM ex-tem)
were associated with sonographically detectable thrombus formation Some studies in
humans have established the association of laboratory biomarkers as predictors of risk
thrombosis (Boersma et al 2016 Cheng et al 2013 Kato et al 2016 Liu et al 2015)
One study performed in anticoagulated human patients showed ndash well in agreement with
our results ndash that high fibrinogen levels were associated with an increased risk of
perioperative venous thromboembolism (P = 0004) (Kato et al 2016) The cited study
does not exclusively refer to CVC associated thrombosis but 762 (1621) of the
venous thromboses were suggested to be associated with CVCs Fibrinogen concentration
measurements are normally available in the human and veterinary practice and the
association of this parameter with a high risk of development of CVC related thrombosis
can be useful to choose special antithrombotic treatment in individual critical care patients
to prevent such severe complications
Thrombin generation assay (TGA) abnormalities such as higher velocity and endogenous
thrombin potential (ETP) values measured initially ie before any anticoagulation
therapy have been associated with a higher thromboembolism risk in general (ie without
specific association with CVCs) in one study performed on 105 human patients (Espitia
et al 2017) In contrast in our study the initial TGA assay results did not show an
association with formation of CVC related thrombosis
The ROTEM parameters evaluated in the present study were partially associated with
formation of CVC thrombosis detected by colour Doppler ultrasound This finding
confirms the results obtained in humans in which ROTEM measurements are regarded as
Chapter 4 General discussion
78
a valuable indicator of hypercoagulability (Harahsheh and Ho 2018 Tuumlrk et al 2018)
However in our study individual ROTEM parameters showed partly lower coagulation
activities in dogs with CVC related thrombosis detected by ELMI at the final day of
evaluation Because the number of patients without signs of thrombosis detected by ELMI
was low compared with those who showed signs these implausible results should be
interpreted carefully The usefulness of ROTEM analysis which has also many other
indications demonstrates that is was worth to perform a detailed methodological approval
of the device including the establishment of reference values in the preliminary study
Conclusion
In conclusion the prethrombotic or thrombotic changes detected in our study did not
correlate between ultrasonography and ELMI however both methods show accurately
signs of CVC related thrombosis The UFH in the given dosage was not completely
effective to prevent CVC induced thrombus formation in canine intensive care patients
Plasma heparin activities varied significantly after a defined subcutaneous dosage
However plasma heparin activities were not different between dogs with and without
detectable CVC associated thromboses Initial high fibrinogen values and low maximum
lysis (ROTEM ex-tem) values were associated with sonographically detectable thrombi
and may be useful as indicators for an increased thrombotic risk
Finally our study in canine intensive care patients is to the best of the authoracutes
knowledge the first study in the available veterinary literature performed to evaluate the
efficacy of an antithrombotic treatment with UFH to prevent CVC related thrombosis
Chapter 5 Summary
79
Chapter 5 Summary
Central venous catheter induced thrombosis in dogs ndash occurrence under an
antithrombotic treatment regimen and possible indicators
Joseacute Mauricio Pereira Mora
Central venous catheters (CVC) are of importance for intensive care management of
canine patients Thrombosis of the jugular vein is a severe complication related to the
CVC use However only little is known about the incidence of CVC-associated
thromboses in dogs and the necessity and efficacy of prophylactic antithrombotic
regimens
Therefore the aims of the present study were (1) to determine the incidence of catheter-
induced thrombosis in canine intensive care patients (2) to evaluate the efficacy of a
prophylactic heparin regimen routinely used in the Small Animal Clinic of the University
of Veterinary Medicine Hannover and (3) to assess whether selected haemostasis test
results are predictive for an increased risk for thrombosis formation
The thesis also includes a preliminary methodological study on the use of rotational
elastometry in dogs using the ROTEM delta device In this preliminary study reference
intervals were determined based on 125 healthy dogs for different clot activator reagents
and for measurements without activator Coefficients of variation (CVs) were calculated
based on results of fourfold measurements of 3 normal and 3 abnormal blood samples in
order to assess precision of the method Influence of age groups sex neuter status and
size were also assessed The median CVs for most of the parameters evaluated in the
precision analysis were less than 10 and this confirmed that the ROTEM delta analyser
delivers repeatable results Regarding the influence of age sex neuter status and animal
Chapter 5 Summary
80
size only neuter status in female dogs appeared to be a relevant influencing factor on
most of important ROTEM parameters
The main study of the thesis comprises 29 patients hospitalised at the Small Animal Clinic
requiring a CVC for medical reasons These patients received a prophylactic
anticoagulation treatment with unfractionated heparin (UFH 150 IU [surgical dogs
received 75 IU]kg BW TID subcutaneously) A colour Doppler ultrasound of the
respective external jugular vein (EJV) in order to detect thrombus formation in the lumen
of the EJV and blood collection for haemostasis tests were performed directly before the
installation procedure (day 0) and on days 1 3 5 7 and so on after the CVC insertion
Haemostasis tests included prothrombin time activated partial thromboplastin time
thrombin time antithrombin activity D-dimer concentration thrombin generation and
rotational elastometry as well as the heparin activity using a chromogenic anti-factor Xa
test Finally when the CVC was removed electron microscopy (ELMI) of sections of the
CVC with macroscopically visible thrombi in the last third (5ndash10 cm) of the CVC was
performed 3 different grades were defined for CVC-associated (extraluminal) thrombi
detected by either ultrasound or ELMI respectively
829 patients evaluated by colour Doppler ultrasound during the study developed thrombi
into the lumen of EJV (4 x grade 1 3 x grade 2 and 1 x grade 3) 1927 CVCs assessed
using ELMI showed detectable thrombi on the external CVC surface (7 x grade 1 4 x
grade 2 and 8 x grade 3) There was no significant correlation between thrombus
formation detected by ultrasound or ELMI Thus statistical group comparisons of
patients with or without CVC-associated thrombus formation was performed
independently for both diagnostic procedures The plasmatic heparin activities showed
great variability but no significant differences were found between dogs with or without
CVC-associated thrombus formation regardless whether this was based on sonographic
Chapter 5 Summary
81
or ELMI findings From the initially performed haemostasis tests high fibrinogen
concentrations and low maximum lysis values as measured by the ROTEM delta device
(using the ex-tem reagent) were associated with sonographically detectable thrombi
In conclusion the preliminary methodological study on the ROTEM delta in dogs shows
that the method delivers repeatable results in canine citrated whole blood and the
reference values obtained should offer a reliable basis for the interpretation of test results
of this device in dogs with suspected haemostasis disorders The results of the main study
indicate that CVC-associated thromboses occur (eg 28 [829] of the patients as
detected by ultrasound) although severe thromboses (high grades) are relatively rare in
intensive care canine patients receiving a routine anticoagulant treatment Thereby
prophylactic antithrombotic protocol (unfractionated heparin [UFH]) in the given dosage
was not completely effective to prevent CVC induced thrombus formation in canine
intensive care patients A high fibrinogen concentration and low maximum lysis
(ROTEM ex-tem) may be useful indicators for an increased thrombotic risk in individual
patients which may require special anticoagulatory treatment Further studies are
required to evaluate whether different thromboprophylactic treatment regimens eg with
low molecular weight heparin are more effective in preventing CVC-associated
thromboses in dogs
Chapter 6 Zusammenfassung
82
Chapter 6 Zusammenfassung
Zentralvenenkatheter-induzierte Thrombose bei Hunden - Auftreten unter einem
antithrombotischen Behandlungsschema und moumlgliche Indikatoren
Joseacute Mauricio Pereira Mora
Zentralvenenkatheter (ZVK) sind fuumlr das Intensivmanagement von Hunden von
Bedeutung Die Entstehung einer Thrombose der Vena jugularis ist eine schwerwiegende
Komplikation im Zusammenhang mit der Verwendung eines ZVKs Uumlber die Inzidenz
von ZVK-assoziierten Thrombosen bei Hunden und die Notwendigkeit und Wirksamkeit
prophylaktischer antithrombotischer Protokolle liegen jedoch nur wenige fundierte
Angaben in der Literatur vor
Daher waren die Ziele der vorliegenden Studie (1) die Inzidenz der Katheter-induzierten
Thrombose bei caninen Intensivpatienten zu bestimmen (2) die Wirksamkeit eines
prophylaktischen Heparin-Regimes zu bewerten das routinemaumlszligig in der Klinik fuumlr
Kleintiere der Stiftung Tieraumlrztliche Hochschule Hannover angewendet wird und (3) um
zu beurteilen ob ausgewaumlhlte Haumlmostasetestergebnisse ein erhoumlhtes Risiko fuumlr die
Thrombosebildung vorhersagen koumlnnen
Die PhD-Arbeit enthaumllt zudem eine vorbereitende methodische Studie zur Verwendung
der Rotationselastometrie bei Hunden mit Hilfe des ROTEM delta-Geraumltes In dieser
Vorstudie wurden Referenzintervalle basierend auf 125 gesunden Hunden fuumlr
verschiedene Gerinnungsaktivatorreagenzien und fuumlr Messungen ohne Aktivator
bestimmt Um die Genauigkeit der Methode einzustufen wurden Variationskoeffizienten
(VKs) basierend auf den Ergebnissen von Vierfachmessungen von drei normalen und drei
abnormalen Blutproben berechnet Der Einfluss von Alter Geschlecht Kastration und
Groumlszlige wurde ebenfalls getestet Die Medianwerte der VKs fuumlr die meisten in der
Chapter 6 Zusammenfassung
83
Praumlzisionsanalyse bewerteten Parameter betrugen weniger als 10 und dies bestaumltigte
dass das ROTEM delta-Analysegeraumlt wiederholbare Ergebnisse liefert In Bezug auf den
Einfluss von Alter Geschlecht Kastration und Tiergroumlszlige erwies sich nur eine Kastration
bei Huumlndinnen als relevanter Einflussfaktor fuumlr die meisten wichtigen ROTEM-
Parameter
Die Hauptstudie der Arbeit umfasst 29 Patienten welche zum Zeitpunkt der
Untersuchung stationaumlr in der Kleintierklinik aufgenommen worden waren und aus
medizinischer Indikation einen ZVK benoumltigten Diese Patienten erhielten eine
prophylaktische Antikoagulation mit unfraktioniertem Heparin (UFH 150 IE
[chirurgische Hunde erhielten 75 IE]kg KG 3 x taumlglich subkutan) Bildgebende
Untersuchungen mittels Farbdoppler-Ultraschall der jeweiligen Vena jugularis externa
(VJE) zum Nachweis der Thrombusbildung im Lumen der VJE und Blutentnahmen fuumlr
Haumlmostasetests wurden jeweils direkt vor dem Installationsvorgang (Tag 0) sowie an den
Tagen 1 3 5 und 7 nach Legen des ZVKs durchgefuumlhrt Haumlmostasetests umfassten die
Prothrombinzeit die aktivierte partielle Thromboplastinzeit die Thrombinzeit die
Antithrombinaktivitaumlt die D-Dimer-Konzentration die Thrombingenerierung und die
Rotationselastometrie sowie die Heparinaktivitaumlt unter Verwendung eines chromogenen
Anti-Faktor-Xa-Tests Schlieszliglich wurde nach Entfernung des ZVKs eine
Elektronenmikroskopie (ELMI) von Abschnitten aus dem letzten Drittel (5ndash10 cm) des
ZVKs mit makroskopisch sichtbaren Thromben durchgefuumlhrt Fuumlr ZVK-assoziierte
(extraluminale) Thromben die entweder durch Ultraschall oder ELMI nachgewiesen
wurden wurden drei verschiedene Grade definiert
829 Patienten die waumlhrend der Studie mittels Farbdoppler-Ultraschall untersucht
wurden entwickelten Thromben im Lumen der VJE (4 x Grad 1 3 x Grad 2 und 1 x Grad
3) 1927 ZVKs die unter Verwendung von ELMI bewertet wurden zeigten
Chapter 6 Zusammenfassung
84
nachweisbare Thromben auf der aumluszligeren ZVK-Oberflaumlche (7 x Grad 1 4 x Grad 2 und 8
x Grad 3) Es gab keine signifikante Korrelation zwischen der durch Ultraschall oder
ELMI festgestellten Thrombusbildung Daher wurden statistische Gruppenvergleiche
von Patienten mit oder ohne ZVK-assoziierter Thrombusbildung fuumlr beide diagnostische
Verfahren unabhaumlngig voneinander durchgefuumlhrt Die plasmatischen Heparinaktivitaumlten
zeigten eine groszlige Variabilitaumlt es wurden jedoch keine signifikanten Unterschiede
zwischen Hunden mit oder ohne ZVK-assoziierter Thrombusbildung gefunden
unabhaumlngig davon ob diese auf sonographischen oder ELMI-Befunden beruhte Von den
initial durchgefuumlhrten Haumlmostasetests waren hohe Fibrinogenkonzentrationen und
niedrige maximale Lyse-Werte der Messungen mit ROTEM delta (ex-tem Reagenz) mit
sonographisch nachweisbaren Thromben assoziiert
Zusammenfassend zeigt die vorbereitende methodische Studie zum ROTEM delta bei
Hunden dass die Methode wiederholbare Ergebnisse in mit Zitrat
antikoaguliertemVollblut liefert Die erarbeiteten Referenzwerte sollten eine zuverlaumlssige
Grundlage fuumlr die Interpretation der Testergebnisse dieses Geraumlts bei Hunden mit
Verdacht auf Haumlmostasestoumlrungen bieten Die Ergebnisse der Hauptstudie zeigen dass
ZVK-assoziierte Thrombosen auftreten (z B 28 [829] der Patienten bezogen auf den
Ultraschall-gestuumltzten Nachweis) obwohl schwere Thrombosen (hohe Grade) bei
intensivmedizinisch versorgten Hundepatienten die eine Routineantikoagulation
erhalten relativ selten sind Somit war das prophylaktische antithrombotische Protokoll
(unfraktioniertes Heparin [UFH]) in der angewendeten Dosierung nicht vollstaumlndig
wirksam um die ZVK-induzierte Thrombusbildung bei caninen Intensivpatienten zu
verhindern Eine hohe Fibrinogenkonzentration und eine niedrige maximale Lyse
(ROTEM ex-tem) koumlnnen als Indikatoren eines erhoumlhten Thromboserisikos bei einzelnen
Patienten nuumltzlich sein fuumlr die moumlglicherweise eine spezielle gerinnungshemmende
Chapter 6 Zusammenfassung
85
Behandlung notwendig ist Weitere Studien sind erforderlich um zu untersuchen ob
verschiedene thromboprophylaktische Behandlungsschemata z B mit
niedermolekularem Heparin bezuumlglich der Verhinderung von ZVK-assoziierten
Thrombosen bei Hunden wirksamer sind
Chapter 7 References
86
Chapter 7 References
1 Abdelkefi A Ben Othman T Kammoun L Chelli M Ben Romdhane N Kriaa A Ladeb
S Torjman L Lakhal A Achour W Ben Hassen A Hsaiumlri M Ladeb F Ben Abdeladhim
A 2004 Prevention of central venous line-related thrombosis by continuous infusion of low-dose
unfractionated heparin in patients with haemato-oncological disease A randomized controlled
trial Thromb Haemost 92 654ndash661
2 Adamantos S Brodbelt D Moores A L 2010 Prospective evaluation of complications
associated with jugular venous catheter use in a veterinary hospital J Small Anim Pract 51 254ndash
257
3 Ballard D H Samra N S Gifford K M Roller R Wolfe B M Owings J T 2016
Distance of the internal central venous catheter tip from the right atrium is positively correlated
with central venous thrombosis Emerg Radiol 23 269ndash273
4 Baumann-Kreuziger L Onwuemene O Kolesar E Crowther M Lim W 2015 Systematic
review of anticoagulant treatment of catheter-related thrombosis Thromb Res 136 1103ndash1109
5 Bliss SP Bliss SK Harvey HJ 2002 Use of recombinant tissue‐plasminogen activator in a
dog with chylothorax secondary to catheter‐associated thrombosis of the cranial vena cava J Am
Anim Hosp Assoc 38 431ndash435
6 Boersma RS Hamulyak K van Oerle R Tuinenburg A Ten Cate-Hoek AJ Schouten
HC 2016 Biomarkers for prediction of central venous catheter related-thrombosis in patients
with hematological malignancies Clin Appl Thromb Hemost 22 779ndash784
7 Boersma RS Jie KS Verbon A van Pampus EC Schouten HC 2008 Thrombotic and
infectious complications of central venous catheters in patients with hematological
malignancies Ann Oncol 19 433ndash442
8 Bonnet F Loriferne JF Texier JP Texier M Salvat A Vasile N 1989 Evaluation of
Doppler examination for diagnosis of catheter-related deep vein thrombosis Intensive Care Med
15 238ndash240
9 Borow M Crowley J G 1985 Evaluation of central venous catheter thrombogenicity Acta
Anaesthesiol Scand Suppl 81 59ndash64
10 Cheng Y Cui T Fu P Liu F Zhou L 2013 Dyslipidemia is associated with tunneled-cuffed
catheter-related central venous thrombosis in hemodialysis patients a retrospective multicenter
Chapter 7 References
87
study Artif Organs 37 155ndash161
11 Comerlato PH Rebelatto TF Santiago de Almeida FA Klein LB Boniatti MM
Schaan BD Rados DV 2017 Complications of central venous catheter insertion in a teaching
hospital Rev Assoc Med Bras 63 613ndash620
12 De Cicco M Matovic M Balestreri L Steffan A Pacenzia R Malafronte M Fantin
D Bertuzzi CA Fabiani F Morassut S Bidoli E Veronesi A 2009 Early and short‐term
acenocumarine or dalteparin for the prevention of central vein catheter‐related thrombosis in
cancer patients a randomized controlled study based on serial venographies Ann
Oncol 20 1936ndash1942
13 Espitia O Fouassier M Hardouin JB Pistorius MA Agard C Planchon B Trossaert M
Pottier P 2017 Thrombin generation assay in hospitalized nonsurgical patients A new tool to
assess venous thromboembolism risk Clin Appl Thromb Hemost 23 45ndash51
14 Evans NS Ratchford EV 2018 Catheter related-venous thrombosis Vasc Med 23 411ndash413
15 Geerts W 2014 Central Venous Catheter-related thrombosis Hematology Am Soc Hematol
Educ Program 2014 306ndash311
16 Harahsheh Y Ho KM 2018 Use of viscoelastic tests to predict clinical thromboembolic
events A systematic review and meta‐analysis Eur J Haematol 100 113ndash123
17 Harter C Salwender HJ Bach A Egerer G Goldschmidt H Ho AD 2002 Catheter-
related infection and thrombosis of the internal jugular vein in hematologic-oncologic patients
undergoing chemotherapy a prospective comparison of silver-coated and uncoated
catheters Cancer 94 245ndash251
18 Hirsh J Raschke R 2004 Heparin and low-molecular-weight heparin The Seventh ACCP
Conference on Antithrombotic and Thrombolytic Therapy Chest 126 (Suppl) 188Sndash203S
19 Karthaus M Kretzschmar A Kroning H Biakhov M Irwin D Marschner N Slabber
C Fountzilas G Garin A Abecasis NG Baronius W Steger GG Sudhoff T Giorgetti
C Reichardt P 2006 Dalteparin for prevention of catheter‐related complications in cancer
patients with central venous catheters final results of a double‐blind placebo‐controlled phase III
trial Ann Oncol 17 289ndash296
20 Kato F Takeuchi H Matsuda S Kawakubo H Omori T Kitagawa Y 2016 Incidence of
and risk factors for venous thromboembolism during surgical treatment for esophageal cancer a
Chapter 7 References
88
single-institution study Surg Today 46 445ndash452
21 Kohler TR Kirkman TR 1998 Central venous catheter failure is induced by injury and can
be prevented by stabilizing the catheter tip J Vasc Surg 28 59ndash66
22 Koumlksoy C Kuzu A Kutlay J Erden I Ozcan H Ergicircn K 1995 The diagnostic value of
colour Doppler ultrasound in central venous catheter related thrombosis Clin Radiol 50 687ndash
689
23 Kucher N 2011 Clinical practice Deep-vein thrombosis of the upper extremities N Engl J
Med 364 861ndash869
24 Langston CE Eatroff AE 2018 Hemodialysis catheter-associated fibrin sheath in a dog J
Vet Emerg Crit Care 28 366ndash371
25 Langston CE Eatroff AE Poeppel K 2014 Use of tissue plasminogen activator in catheters
used for extracorporeal renal replacement therapy J Vet Intern Med 28 270ndash276
26 Laurenson MP Hopper K Herrera MA Johnson EG 2010 Concurrent diseases and
conditions in dogs with splenic vein thrombosis J Vet Intern Med 24 1298ndash1304
27 Lee AYY Kamphuisen PW 2012 Epidemiology and prevention of catheter‐related
thrombosis in patients with cancer J Thromb Haemost 10 1491ndash1499
28 Liu G Fu ZQ Zhu P Li SJ 2015 Central venous catheter-related thrombosis in senile male
patients New risk factors and predictors J Huazhong Univ Sci Technolog Med Sci 35 445ndash
449
29 Lordick F Hentrich M Decker T Hennig M Pohlmann H Hartenstein R Peschel C
2003 Ultrasound screening for internal jugular vein thrombosis aids the detection of central
venous catheter-related infections in patients with haemato-oncological diseases a prospective
observational study Br J Haematol 120 1073ndash1078
30 Lucas TC Silva EED Souza DOD Santos ARD Lara MO 2017 Microstructural
evaluation by confocal and electron microscopy in thrombi developed in central venous catheters
Rev Esc Enferm USP 51 1ndash9
31 Mismetti P Mille D Laporte S Charlet V Buchmuller‐Cordier A Jacquin JP Fournel
P Dutrey‐Dupagne C Decousus H 2003 Low‐molecular‐weight heparin (nadroparin) and
very low doses of warfarin in the prevention of upper extremity thrombosis in cancer patients with
indwelling long‐term central venous catheters a pilot randomized trial Haematologica 88 67ndash
Chapter 7 References
89
73
32 Niers TM Di Nisio M Klerk CP Baarslag HJ Buller HR Biemond BJ 2007
Prevention of catheter‐related venous thrombosis with nadroparin in patients receiving
chemotherapy for hematologic malignancies a randomized placebo‐controlled study J Thromb
Haemost 5 1878ndash1882
33 Scott K C Hansen B D DeFrancesco T C (2009) Coagulation effects of low molecular
weight heparin compared with heparin in dogs considered to be at risk for clinically significant
venous thrombosis J Vet Emerg Crit Care 19 74ndash80
34 Smith RS Zhang Z Bouchard M Li J Lapp HS Brotske GR Lucchino DL Weaver
D Roth LA Coury A Biggerstaff J Sukavaneshvar S Langer R Loose C 2012
Vascular catheters with a nonleaching poly-sulfobetaine surface modification reduce thrombus
formation and microbial attachment Sci Transl Med 4 153
35 Tuumlrk S M Cansu D Uuml Teke H Uuml Kaşifoğlu T Meltem Akay O Bilgin M amp Korkmaz
C 2018 Can we predict thrombotic tendency in rheumatoid arthritis A thromboelastographic
analysis (with ROTEM) Clin Rheumatol 37 2341ndash2349
36 van Rooden CJ Rosendaal FR Barge RM van Oostayen JA van der Meer FJM
Meinders AE Huisman MV 2003 Central venous catheter related thrombosis in haematology
patients and prediction of risk by screening with Doppler-ultrasound Br J Haematol 123 507ndash
512
37 van Rooden CJ Tesselaar ME Osanto S Rosendaal FR Huisman MV 2005 Deep vein
thrombosis associated with central venous catheters - a review J Thromb Haemost 3 2409ndash
2419
38 Verso M Agnelli G Bertoglio S Di Somma FC Paoletti F Ageno W Bazzan M Parise
P Quintavalle R Naglieri E Santoro A Imberti D Soraru M Mosca S 2005 Enoxaparin
for the prevention of venous thromboembolism associated with central vein catheter a double‐
blind placebo‐controlled randomized study in cancer patients J Clin Oncol 23 4057ndash4062
39 Vose J Odunayo A Price JM Daves M Schildt JC Tolbert MK 2019 Comparison of
heparinized saline and 09 sodium chloride for maintaining central venous catheter patency in
healthy dogs PeerJ 7e7072 httpdoi107717peerj7072
40 Wang LH Wei F Jia L Zhi Lu Wang B Dong HY Yu HY Sun GJ YangJ Li B
Chapter 7 References
90
Meng J Zhang RN Bi XQ Chen HY Jiang AL 2015 Fibrin sheath formation and
intimal thickening after catheter placement in dog model role of hemodynamic wall shear stress J
Vasc Access 16 275ndash284
41 Wildgruber M Lueg C Borgmeyer S Karimov I Braun U Kiechle M Meier R Koehler
M Ettl J Berger H 2016 Polyurethane versus silicone catheters for central venous port devices
implanted at the forearm Eur J Cancer 59 113ndash124
42 Whitesell RT Steenburg SD 2014 Imaging findings of acute intravascular thrombus on non-
enhanced computed tomography Emerg Radiol 21 271ndash277
43 Wu X Studer W Skarvan K Seeberger MD 1999 High incidence of intravenous thrombi
after short-term central venous catheterization of the internal jugular vein J Clin Anesth 11
482ndash485
44 Xiang DZ Verbeken EK Van Lommel AT Stas M De Wever I 1998 Composition and
formation of the sleeve enveloping a central venous catheter J Vasc Surg 28 260ndash271
45 Xiang DZ Verbeken EK Van Lommel AT Stas M De Wever I 2001 Sleeve-related
thrombosis a new form of catheter-related thrombosis Thromb Res 104 7ndash14
91
Acknowledgements
First I thank God for every day in my life for His guide and because He was is
and will be always with me and that certainty gives me strength to take a step
forward
My sincere thanks to Prof Dr Reinhard Mischke for the opportunity to perform
my PhD under his supervision for his support and guidance for all that I learned
with him and for his friendship
I also thank to Prof Dr Manfred Kietzmann and PD Dr Mario von Depka
Prondzinski for being part of my supervision group for their time and beneficial
advices and discussion during my PhD
My gratitude to Prof Hewicker-Trautwein and Mrs Rohn for their valuable work
and availability to help me
I thank the Werlhof Institute Medical Care Center Hannover for the valuable
cooperation especially to Mrs Ober-Cassebaum Anne and Franziska for all their
help
I thank all the staff of the Small Animal Clinic of the University of Veterinary
Medicine Hannover (TiHo) My special gratitude to the internal medicine and
critical care surgery neurology ultrasound and anesthesia departments and to
the TiHo students all their cooperation made possible my research
I thank the haematology clinical chemistry and haemostasis laboratory
department of the Small Animal Clinic TiHo especially to Dirk because he was
always available for me thanks for his guide and friendship all his explanations
in the laboratory were of great value but also the conversations about different
topics of the life were of big value for me
92
I thank the Hannover Graduate School for Veterinary Pathobiology
Neuroinfectology and translational Medicine (HGNI) for the well-organized
PhD program and especially thanks to Prof Dr Beatrice Grummer Dr Tina
Selle and Mrs Tania Czeslik for his guide and help during my studies
My sincere thanks to Mrs Maritta Ledwoch for her guidance at the International
office of the TiHo ldquoI remember you since my externship in Germanyrdquo
I also thank the Student Union Hannover for all the help with the accommodation
for me and my family
I want to thank the Universidad Nacional de Costa Rica and therefore to Costa
Rica (my loved country) for the financial support to perform my PhD studies
but also for my professional formation as a veterinarian Special thanks to the
Veterinary Medicine School from Universidad Nacional de Costa Rica To all the
academic and administrative staff for their support and for believing in me
Sincerely thanks to Dr Mauricio Jimenez Soto my mentor my adviser but most
important my friend Thanks for believing in me and for always being by my side
ldquoAgradezco profundamente a mis papaacutes ldquodon Tito (Papillo) y dontildea Eida (Mami)rdquo
Todo lo bueno que hay en mi proviene de Dios y de ellos Gracias a mis hermanos
(as) cuntildeados (as) sobrinos y a toda la familia relacionada a ellos Siempre han
sido un gran apoyo y los amordquo
ldquoGracias a dontildea Alba Fran y David Gracias por toda la ayuda y por estar siempre
pendientesrdquo
I would like to thank all my friends that during a short part or during my whole
life have been with me For all the good time shared really thanks
To Willy thanks ldquogordordquo
93
I sincerely thank to Adriano and Johanna for their help and hospitality Thanks
Adriano for your guidance and support during my beginning at the TiHo for make
me feel in Costa Rica with your friendship for the tourist walks and for the bike
training I missed the coffee times
I want to thank Ina the first person to give me a smile at the start of my PhD
Thanks for your hugs and friendship
I also thank Franz for his help and for being a very nice partner Thanks for help
me always when I requested you
ldquoFinalmente quiero expresar mi gratitud y amor a Karo Faacutetima y Abigaiacutel A Karo
por tu sacrificio y soporte por las palabras de aliento por estar a mi lado en las
buenas pero en especial en las malas A Faacute por tu sacrificio por tus sonrisas por
tus abrazos y besos por bailar con migo siempre por ser mi modelo e inspiracioacuten
A Abi por ser una nueva razoacuten in mi vida para seguir adelante por todo lo que
deseo compartir con tigo por mostrarme junto con Faacutetima la felicidad en una
sonrisardquo
List of figures and tables
ii
List of figures and tables
Chapter 2
Table 1 Coefficients of variation () for rotational thromboelastometry using the
ROTEMreg delta analyser in normal blood samples 20
Table 2 Coefficients of variation () for rotational thromboelastometry using the
ROTEMreg delta analyser in canine blood samples with haemostatic alterations 20
Table 3 Reference values (median reference interval based on 25 - and 975 -
quantiles) for thromboelastometric measurements with the ROTEMreg delta analyser in
dogs (n=125 95 confidence intervals in brackets) 23
Table 4 Median values (minimum and maximum in brackets) for different ROTEMreg
variables using ex-tem reagent stratified by sex and neuter status 24
Table 5 Median values (minimum and maximum in brackets) for different ROTEMreg
variables using the in-tem reagent stratified by sex and neutering status 25
Chapter 3
Fig 1 Definition of different grades of central venous catheter induced thrombosis of the
jugular vein as detected using colour Doppler ultrasound 48
Fig 2 Definition and illustration of different grades of central venous catheter induced
(extraluminal) thrombosis as detected using electron microscopy 53
Fig 1S (suppl material) Definition and illustration of different grades of intraluminal
thrombosis in central venous catheters as detected using electron microscopy 53
Table 1S (Supplementary material) Heparin activities (IUml median values
minimumndashmaximum) measured in 27 dogs receiving 75 or 150 IUkg unfractionated
(standard) heparin TID subcutaneously 2 hours after the first injection on the day
Comparison of samples collected via puncture of the peripheral veins and those collected
from the central venous catheter (CVC) using the three-syringe-technique 56
List of figures and tables
iii
Fig 3ab Heparin activities (box- and whisker plot arithmetic means) in dogs receiving
unfractionated heparin at a dosage of 75 IUkg BW tid (a) or 150 IUkg BW tid (b) two
hours after the first daily heparin administration 57
Table 1 Heparin activities (IUml median values minimumndashmaximum) in 28 dogs
receiving 75 or 150 IUkg unfractionated (standard) heparin TID subcutaneously with
(grades 1ndash3) or without (grade 0) sonographically detectable thrombus Blood collections
were performed 2 hours after the first daily injection 57
Table 2 Heparin activities (IUml median values minimumndashmaximum) in 27 dogs
receiving 75 or 150 IUkg unfractionated (standard) heparin TID subcutaneously with
(score 1ndash3) or without (score 0) detectable thrombus via electron microscopy 58
Table 3 Comparison of initial results of haemostasis parameters (median values
minimumndashmaximum in dogs with (grades 1ndash3) and without (grade 0) sonographically
detectable central venous catheter-induced thromboses 60
Table 4 Comparison of results of haemostasis parameters (median values minimumndash
maximum) on the last day in dogs with (grades 1ndash3) and without (grade 0) central venous
catheter-associated thrombus formation as detected by electron microscopy 61
Chapter 1 Introduction
1
Chapter 1 Introduction
Central venous catheters (CVC) are mainly used at critical care units in human and small
animal patients requiring infusion of large amounts of fluids or blood parenteral nutrition
support routine drug therapy haemodialysis chemotherapy plasmapheresis or
periodical blood sampling (Geerts 2014 Vose et al 2019) CVC related thrombosis is
one of the most severe complications (apart from infections and pneumothorax) of CVCs
in humans (Comerlato et al 2017 Evans and Ratchford 2018 Geerts 2014) leading to
patientrsquos distress CVC dysfunction venous stenosis and in some cases to the
development of thromboembolism (Geerts 2014) In the human literature there are
various studies dealing with epidemiologic and prophylactic aspects which are described
below in detail In contrast to the best of the authoracutes knowledge studies about extra
luminal CVC related thrombosis in dogs are rare in the available veterinary literature
Studies are mainly dealing with development of intraluminal CVC thrombi occurring
frequently in dogs and cats (Langston et al 2014) and leading to CVC malfunction (Vose
et al 2019)
Epidemiology of CVC related thrombosis
CVC related thrombosis represents 70ndash80 of all upper extremity thrombosis cases and
approximately 10 of thromboembolism cases in humans (Kucher 2011) The incidence
of CVC related thrombosis reported in humans based in 25 studies ranged widely from
2 to 67 (median = 30 ) (van Rooden et al 2005) The incidence of CVC related
thrombosis detected by colour Doppler ultrasound of the internal jugular vein reported in
3 studies performed in humans was 2 (Harter et al 2002) 30 (Lordick et al 2003)
and 56 (Wu et al 1999)
Chapter 1 Introduction
2
The incidence of CVC related thrombosis in humans can vary between studies according
to the CVC type used entry site of the CVC (jugular femoral subclavian axillary
cephalic vein) and time period with the CVC in place the diagnostic thrombus detection
technique (venography or ultrasonography) and criteria for diagnosis duration of follow
up diseases and presence of predisposing factors (eg previous venous thrombosis
history presence of cancer) and finally prophylactic anticoagulation treatment (Geerts
2014 Liu et al 2015 van Rooden et al 2005)
To the best of the authoracutes knowledge data about the incidence of CVC related
thrombosis detected by colour Doppler ultrasound into the lumen of the jugular vein in
dogs are rare in the available veterinary literature In one study performed in 81 dogs and
12 cats (Adamantos et al 2010) the incidence of vein thrombosis detected by palpation
or ultrasound was 2 and the incidence of intraluminal CVC thrombosis (presumed by
the inability to draw blood back) was 15 Additionally regarding to intraluminal CVC
thrombosis an incidence of 8 was reported in one case control study performed in 24
dogs receiving CVC flushing with heparinised solution (Vose et al 2019) Data about
incidence in other animal species was also found In another case-control study performed
on 37 pigs thrombosis in the caval vein related to CVC placed in the external jugular
vein (EJV) was evaluated by histological analysis however no ultrasound detection was
performed (Kohler and Kirkman 1998) In this study in pigs 94 of patients with CVC
developed thrombi in the vena cava based on sections at the site of the end of the catheter
tip and 05 and 1 cm proximal of the first section (Kohler and Kirkman 1998)
Pathogenesis of CVC related thrombosis
Two types of CVC related thrombosis are distinguished in the human medicine literature
(Geerts 2014 van Rooden et al 2005) First symptomatic thrombosis is defined as
Chapter 1 Introduction
3
thrombosis detected by diagnostic images (ultrasonography venography) when
symptoms and signs (eg localised swelling pain redness) are present (Lee and
Kamphuisen 2012) Second subclinical thrombosis is defined as thrombosis
demonstrated by the previously mentioned diagnostic images without overt symptoms or
signs (Boersma et al 2008) Additionally the CVC related thrombi can be classified as
occlusive thrombus into the CVC lumen peri-catheter related fibrin sheaths and mural
thrombi (Geerts 2014)
During the CVC insertion the puncture of the skin on the CVC insertion site and the
rupture of the blood vessels integrity activate the coagulation cascade and the CVC acts
as a matrix for adherence and accumulation of fibrin and cellular components (Smith et
al 2012) The attachment of proteins and blood cells to the internal andor external wall
of the CVC can lead to (1) (partial or complete) occlusion of the catheter lumen and (2)
sometimes if a thrombus formation starts into the lumen of the vein (attached to the vein
wall or to the CVC) a partial or complete obstruction of the blood flow andor embolism
can develop (Langston and Eatroff 2018) Additionally a study performed in humans
(Lordick et al 2003) demonstrated a relationship between CVC related thrombosis and
microbial colonisation of the catheter leading to CVC related systemic infections This
relationship was explained among others by the ease of microorganisms like
Staphylococcus aureus and Staphylococcus epidermidis to adhere to CVC related
thrombin sheaths (Boersma et al 2008)
Diagnosis of CVC related thrombosis
Venous thrombosis is commonly diagnosed in human and small animal medicine
(especially in dogs) with vascular ultrasonography with or without use of the colour
Doppler technique which is normally used to evaluate the blood flow through the blood
Chapter 1 Introduction
4
vessels (Evans and Ratchford 2018 Laurenson et al 2010) According to the literature
in humans cited below the CVC related thrombosis detected by colour Doppler
ultrasound is present if any of the following aspects are observed (1) well defined
thrombus visualised in the lumen of the vein (2) impossibility to perform compression
of the vein by slight pressure with the ultrasound probe andor (3) no detectable blood
flow signal by colour Doppler ultrasound (Bonnet et al 1989 Koumlksoy et al 1995)
Similarly in dogs the CVC related thrombosis is usually diagnosed by colour Doppler
ultrasonography through the detection of the thrombus within the lumen of the vein
andor the obstruction (partial or complete) of the normal blood flow (Bliss et al 2002)
Especially in humans the contrast venography technique is used as gold standard to
confirm thrombosis For this purpose a contrast solution is injected through a peripheral
vein to assess its passage through the bloodstream by x-ray However this diagnostic
technique is normally used only in the case of a strong clinical suspicion of venous
thrombosis that cannot be detected by ultrasonography (Laurenson et al 2010 Langston
and Eatroff 2018) In a study performed in 44 human patients requiring a CVC (Koumlksoy
et al 1995) a sensitivity of 94 and a specificity of 96 were determined for the colour
Doppler ultrasound diagnosis when it was compared with the diagnosis performed with
the contrast venography technique That results make the colour Doppler ultrasound an
accurate diagnosis method to detect CVC related thrombosis in humans (Koumlksoy et al
1995 van Rooden et al 2003)
Advanced diagnostic image techniques like computed tomography or magnetic resonance
tomography are also accurate to perform the diagnosis of CVC related thrombosis in
humans (Whitesell and Steenburg 2014) In dogs multidetector computed tomography
venography was used in one study for the diagnosis of CVC related thrombosis allowing
the identification of catheter related sheaths around the CVC external wall (Wang at al
Chapter 1 Introduction
5
2015) However the high costs of these advanced diagnostic imaging procedures and the
requirement to perform at least sedation or general anesthesia are disadvantages when
compared with ultrasonography
Finally electron microscopy (ELMI) is a diagnostic technique that has been used in
humans (Lucas et al 2017) and experimental animals (Xiang et al 1998 Xiang et al
2001) mainly to perform a microstructural evaluation of thrombi developed into the
lumen of the CVC or in the vein containing the CVC ELMI allows the ex vivo diagnosis
of very small thrombi or sleeve related thrombosis that perhaps cannot be detected by
ultrasonography or venography during the evaluation of patients with CVCs (Xiang et al
2001) However one point to consider is that it is possible that compact thrombi
developed on the CVC can be detached during the CVC removal from the vein and
thereby before its analysis by ELMI (Xiang et al 2001)
Heparin thromboprophylaxis for CVC related thrombosis
Application of CVCs in human medicine is commonly accompanied by prophylactic
anticoagulation in order to decrease the risk of development of partial or complete vein
occlusion andor thromboembolism and thereby to improve the patientrsquos comfort
(Baumann-Kreuziger et al 2015) Apart from vitamin K antagonists (Mismetti et al
2003) unfractionated heparin (UFH) (Abdelfeki et al 2004) and low molecular weight
heparin (LMWH) (Karthaus et al 2006) are anticoagulants that have been used on
studies in humans administrated by intravenous (IV) infusion or by subcutaneous (SC)
injection to prevent thrombi development into the lumen of the veins containing the CVC
(review article Baumann-Kreuziger et al 2015) Different heparin dosages have been
used as thromboprophylactic anticoagulation in humans with CVC (review article Lee
and Kamphuisen 2012) For example in five different case-control studies performed in
Chapter 1 Introduction
6
human haemato-oncological patients UFH at a daily dosage of 100 IUkg given by IV
infusion (Abdelfeki et al 2004) and LMWH at SC daily dosages of 40 mg enoxaparin
(Verso et al 2005) 5000 IU AXa dalteparin (Karthaus et al 2006 De Cicco et al 2009)
and 2850 IU AXa nadroparin (Niers et al 2007) were used Only in part of these studies
it is stated that systemic heparin was combined with flushing of the CVC using
heparinised saline solution to reduce the risk of catheter occlusion (Karthaus et al 2006)
In 3 of the 5 previously mentioned studies no statistically significant difference was found
in the incidence of CVC related thrombosis between patients receiving the
anticoagulation prophylaxis compared with the control groups without systemic
anticoagulation (Verso et al 2005 p = 035 Karthaus et al 2006 p gt 005 Niers et al
2007 p = 049) In contrast in the residual two studies a lower incidence of CVC related
thrombosis was found in the group receiving an anticoagulation prophylaxis compared
with the control groups (Abdelfeki et al 2004 150 vs 126 p = 003 De Cicco et
al 2009 400 vs 526 p = 005)
Obviously there is no consensus in the available human literature regarding the beneficial
use of anticoagulation prophylaxis to prevent CVC related thrombosis and this may
reflect great variations for example according to the study design period trial drug
administration route (Lee and Kamphuisen 2012) and the moment of the initial
anticoagulant dosage administration (Decicco et al 2009)
To the best of the authoracutes knowledge studies about thromboprophylaxis for extraluminal
CVC related thrombosis in dogs cannot be found in the available veterinary literature
However similarly to humans prophylactic anticoagulation with heparinised saline
(administrated through CVC flushing) to prevent CVC occlusion by intraluminal thrombi
was used in one case-control study performed in 24 dogs (Vose et al 2019) In the cited
study 12 dogs received dosages of 3 ml of heparinised saline solution (10 IU UFHml) to
Chapter 1 Introduction
7
flush the catheter every 6 hours during 72 hours whereas 12 control dogs received
flushing with the same volume of non-heparinised saline solution administered at the
same frequency and no statistically significant difference was found
Prediction of CVC related thrombosis
Studies regarding the relationship between laboratory biomarkers and CVC related
thrombosis are available in the human literature (Boersma et al 2016 Cheng et al 2013
Liu et al 2015) however to the best of the authoracutes knowledge literature about laboratory
tests used as predictors for CVC related thrombosis in small animals cannot be found at
all Availability of approved blood tests which can predict the risk of thrombosis
development associated to CVC already at the moment of the CVC placement are
important in order to identify an increased risk of CVC related thrombosis (Boersma et
al 2016) In the study performed by Boersma et al (2016) in 168 human patients without
indication of anticoagulation prophylaxis elevated leukocyte counts high levels of
plasminogen activator inhibitor and of coagulation factor VIII (all measured on blood
samples taken after CVC placement) were associated with an increased incidence of
symptomatic CVC-related thrombosis diagnosed by ultrasonography In another study
(Liu et al 2015) high blood levels of lactate dehydrogenase and low concentrations of
HDL and albumin were associated with the incidence for CVC related thrombosis
diagnosed by ultrasound in 324 senile patients which was in total 617 Additionally
dyslipidemia (high triglycerides low HDL and high LDLHDL ratio) was associated
with CVC related thrombosis (diagnosed by ultrasound and computer tomography
angiography) in human patients undergoing haemodialysis (Cheng et al 2013) Studies
about predictive tests for CVC related thrombosis in dogs were not found in the available
literature to the best of the authoracutes knowledge
Chapter 1 Introduction
8
Aims of the study
The aims of the preliminary methodological study of the present scientific project were
(1) to define reference intervals for viscoelastic analyses of canine haemostasis using the
ROTEMreg delta analyser and (2) to determine the precision (repeatability) of this
method And the aims of the main study of the present scientific project were (1) to
determine the incidence of catheter-induced thrombosis in canine intensive care patients
(2) to evaluate the efficacy of a prophylactic heparin regimen routinely used in the Small
Animal Clinic of the University of Veterinary Medicine Hannover and (3) to assess
whether selected haemostasis test results are predictive for an increased risk for
thrombosis formation
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
9
Chapter 2 Reference intervals for rotational thromboelastometry measurements
using the ROTEMreg delta device in dogs
Pereira JM Rohn K Mischke R
Contributions of the authors
Pereira JM performed (1) the patients data and blood samples collection (2)
haematology and ROTEMreg analysis (3) generated the reference intervals and
coefficients of variation included in the study and (4) wrote the manuscript
Rohn K performed the statistical analysis
Mischke R performed the clinical chemistry and coagulation tests and was involved in
designing and supervision of the study analysis and discussion of the data and its
statistical analysis and revision edition and correction of the manuscript
Original source Pereira J M Rohn K amp Mischke R 2020 Reference intervals
for rotational thromboelastometry measurements using the ROTEMreg delta device
in dogs Res Vet Sci 130 26ndash32 httpsdoiorg101016jrvsc202001019
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
10
Reference intervals for rotational thromboelastometry measurements using the
ROTEMreg delta device in dogs
Pereira JMa Rohn Kb Mischke Ra
aSmall Animal Clinic University of Veterinary Medicine Hannover Buumlnteweg 9 D-
30559 Hannover Germany
bInstitute of Biometry Epidemiology and Information Processing University of
Veterinary Medicine Hannover Buumlnteweg 2 30559 Hannover Germany
Corresponding author
E-mail ReinhardMischketiho-hannoverde
Original source Pereira J M Rohn K amp Mischke R 2020 Reference intervals
for rotational thromboelastometry measurements using the ROTEMreg delta device
in dogs Res Vet Sci 130 26ndash32 httpsdoiorg101016jrvsc202001019
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
11
ABSTRACT
Aims of the present study were to define reference intervals for viscoelastic analyses of
canine haemostasis using the ROTEMreg delta analyser and as a secondary aspect to
determine the precision (repeatability) of this method Blood samples from 125 clinically
healthy dogs were included Measurements were performed with commercially available
activating reagents (ex-tem in-tem and kaolin solution) as well as without activation
Additional fourfold measurements were done in 3 of the normal blood samples and in 3
samples with haemostatic alterations to evaluate the precision of the method
Coefficients of Variation (CVs) for most of the ROTEM variables were lt 10 Clot
formation time and maximum clot elasticity showed a wide inter-individual variation in
comparison with alpha angle and maximum clot firmness A multivariate analysis on
various ROTEM parameters revealed particularly a significant influence of neuter status
and a significant interrelationship between the factors sex and neuter status for
measurements with different activating reagents These results reflected the fact that
significant differences occurred only between intact and neutered females but not in
males No or only occasionally significant differences were found between groups of sex
age and size
In conclusion CVs demonstrated that the method delivers repeatable results in canine
citrated whole blood Established reference intervals should deliver valuable orientation
for the evaluation of viscoelastic properties of clotting whole blood in dogs using the
ROTEM delta analyser Neuter status in females appeared to be the most relevant
influencing factor and should be considered for the interpretation of ROTEM delta test
results
Keywords Viscoelastic measurements reference values canine age sex neuter status
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
12
1 Introduction
Heamostasis is a physiologic and dynamic process in which plasma soluble coagulation
factors blood cells and blood vessels are involved Clot formation and possible
subsequent clot lysis as well as vascular tissue repair aim to prevent haemorrhages on the
one hand and thromboses on the other hand (Kol and Borjesson 2010 Versteeg et al
2013) Conventional assays of coagulation such as prothrombin time (PT) activated
partial thromboplastin time (aPTT) and thrombin time TT have limitations especially
cellular elements are not incorporated into the test and thereby these tests reflect only
limitedly in vivo functionality of haemostasis (McMichael and Smith 2011 Crochemore
et al 2018)
The rotational thromboelastometry (ROTEMreg) currently termed as thromboelastometry
(TEM) and thromboelastography (TEG) are methods for the rapid evaluation of the
overall haemostasis capacity through the measurement of viscoelastic properties of
clotting whole blood (Kol and Borjesson 2010 Theusinger et al 2010 Sankarankutty
et al 2012 Versteeg et al 2013) In TEG the cup with the blood sample is rotating
whereas the torsion wire is fixed In ROTEMTEM the cup is fixed whereas the pin is
rotating Changes in torque ie resistance to rotation by the fibrin bridge created between
pin and cup in the clotting blood are detected electromechanically in TEG and optically
in ROTEM (Bollinger et al 2012) and displayed as a graph that indicates the time for the
formation of blood clot and its strength (Bagge et al 2016) The resulting graph reflects
the initiation and propagation of the coagulation system fibrin-platelet interactions and
clot lysis
TEG was first described in 1948 by Hellmut Harten (Hartert 1948) as an in vitro method
to evaluate the clot formation process while resembling the in vivo blood flow The
development of the actually used instrument types TEG and ROTEMregTEM were based
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
13
on that original principle Viscoelastic measurements have been widely used since the
1980s in human medicine to evaluate haemostasis and hypercoagulable states in surgical
patients (eg cardiac and hepatic surgeries) and in general in critical care units (Kol and
Borjesson 2010 Nogami 2016 Wikkelso et al 2016 Crochemore et al 2017) In
veterinary medicine viscoelastic measurements using TEG and ROTEMTEM have been
validated for use in dogs cats and horses and used to study haemostasis in different
diseases (eg infectious diseases liver diseases) and medical conditions (eg
orthopaedic surgery hyperfibrinolysis) and also to investigate the effect of
anticoagulants and other drugs (eg corticosteroids) on haemostasis (Kol and Borjesson
2010 Bruno et al 2015a 2015b Woodman et al 2015 Conversy et al 2017 Sigrist et
al 2018 Turner et al 2019) The methodology seems to be especially suitable to detect
hypercoagulability (Wiinberg et al 2008 Kol and Borjesson 2010 Marschner et al
2018)
In the available veterinary literature information about reference values for TEG were
found using kaolin as activation reagent (Bauer et al 2009) Falco et al (2012)
determined reference values for the ROTEM analyser in dogs using ex-tem in-tem and
fib-tem reagents for selected ROTEM parameters in a study on forty five healthy dogs
However data related to reference intervals obtained in an adequate number of samples
(at least 120) according to the American Society for Veterinary Clinical Pathology
(ASVCP) (Friedrichs et al 2012) and the National Committee for Clinical Laboratory
Standards (NCCLS) (Horn and Pesce 2003) could not be found in the available literature
The aim of this study is to determine reference values for the ROTEMreg delta device in
dogs using citrated whole blood with different clot activator reagents (ex-tem in-tem
kaolin) and without activator
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
14
2 Materials and methods
21 Study design
Reference intervals Single thromboelastometric measurements were performed in 125
clinically healthy dogs using the ROTEMreg analyser and different activating reagents (ex-
tem in-tem and kaolin) as well as without activation Reference values were calculated
and influences of age groups (young adult and senior) sex neuter status and size
(groups small medium and large) were assessed
Precision analysis Additional fourfold measurements using ex-tem or in-tem activating
reagents respectively were performed on 3 of the normal and also in 3 abnormal blood
samples each in order to assess within-run imprecision for these measurements
22 Animals
The clinically healthy dogs with a body weight of at least 5 kg were recruited by launching
a call in the University of Veterinary Medicine Hannover network for students and staff
members Interested dog owners received a free health check of their animals (clinical
examination and blood tests specified below) in combination with the blood collection
In addition individual privately owned patient dogs of the Small Animal Clinic with
undisturbed general health were also included if blood was collected due to another
indication (eg general health check pre-anaesthetic examination test for hereditary
diseases etc) Before taking the blood samples all dogs were fasted for at least 12 h with
free access to water 125 animals for which the examination of the health status (clinical
examination routine haematology and clinical chemistry basis coagulation profile) did
not reveal any deviations from the reference were included in the study whereas 3 of the
128 initially recruited animals had to be excluded due to laboratory abnormalities
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
15
Haematological and clinical chemistry profiles performed in order to confirm the health
status included white and red cell count haematocrit platelet counts and reticulocytes
plasma activities of alanine aminotransferase glutamate dehydrogenase alkaline
phosphatase and pseudocholinesterase as well as concentrations of total bilirubin urea
creatinine glucose total protein albumin cholesterol and ionised calcium A basis
coagulation profile including PT PTT and TT was also performed to exclude significant
abnormalities within the blood coagulation system
The 125 healthy dogs enclosed in the establishment of reference values included 48 mixed
breed dogs and the following pedigree dogs 13 Labrador Retrievers 6 Australian
Shepherds 4 Havanese dogs 3 from each of the following breeds American
Staffordshire Terriers and Cocker Spaniels 2 from each of the following breeds Beagles
Border Collies Dalmatians Elo dogs (breed not recognised by Feacutedeacuteration cynologique
international [FCI]) German Longhaired Pointers German Shorthaired Pointers Giant
Schnauzers and Golden Retrievers 1 from each of the following breeds Belgian
Malinois Bolonka Zwetna Briard Brussels Griffon American English Coonhound
Italian Mastiff Flat Coated Retriever French Bulldog German Shepherd Great Dane
Greater Swiss Mountain Dog Giant Spitz Goldendoodle Hovawart Hungarian Vizsla
Italian Bloodhound Lycanis Wolfdog Miniature American Shepherd Miniature
Schnauzer Old German Shepherd Podenco Poodle Pyrenean Shepherd Rhodesian
Ridgeback Rottweiler Schafpudel (breed not recognised by FCI) Small
Munsterlander Spanish Mastiff Tibet Terrier Weimaraner West Highland White
Terrier and Wirehaired Pointer
These dogs were aged between 6 and 154 months (median = 410 months) and had body
weights ranging from 50 to 707 kg (median = 226 kg) 30 were castrated males 33
intact males 41 spayed females and 21 intact females According to the height at withers
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
16
the dogs were divided into 3 different size groups small (le40 cm n = 22) medium (41ndash
60 cm n = 77) and large (gt60 cm n = 26) Dogs were also divided in three different age
groups young (le 24 months n = 34) adult (25ndash84 months n = 59) and senior dogs (gt84
months n = 32)
The three blood samples with haemostatic abnormalities were taken from two dogs with
complex haemostatic disorders (Disseminated Intravascular Coagulation [DIC])
including reduced platelet count and abnormal PT and APTT and one dog with
thrombocytopenia
Animals were treated in accordance with the German Animal Welfare Law The
experimental design was approved by the Animal Welfare officer of the University of
Veterinary Medicine Hannover Foundation and by the ethics committee of the
responsible authority (Lower Saxony State Office for Consumer Protection and Food
Safety reference number 17A101)
23 Blood sampling
The venipuncture area was shaved and then disinfected with alcohol The saphenous or
cephalic vein was visualised with slight pressure and punctured with disposable cannulas
(diameter 09 or 10 mm) The continuously running blood was directly collected from
the needle into the tubes containing the respective anticoagulants From each dog
approximately 10 ml of blood were collected including approximately 8 ml of citrated
blood for the measurement of haemostasis tests (ROTEM PT aPTT TT) in tubes
containing sodium citrate 313 (Eifelfangoreg GmbH amp Co KG Bad Neuenahr-
Ahrweiler Germany) in a ratio of 1 volume9 volumes of blood In addition
approximately 1 ml of EDTA blood for haematological analysis and approximately 1 ml
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
17
of lithium heparin blood for clinical chemical measurements were collected into
commercial 13 ml tubes (Sarstedt AG amp Co Nuumlmbrecht Germany)
Citrated blood was initially kept at room temperature (20-25 C) in order to avoid errors
within the preanalytical phase (Toulon et al 2017) Two ml of citrated blood were used
for the ROTEMreg analysis the residual citrate blood was centrifuged for 10 min at 3800
rpm and 5 degC then the plasma was separated by careful pipetting and it was centrifuged
a second time under the same conditions to get platelet poor plasma which was stored in
aliquots of 500 microl and 250 microl at -80 degC for coagulation tests
24 Rotational elastometry
Viscoelastic measurements using the ROTEMreg delta for the establishment of reference
intervals were performed approximately 15 min after the blood collection using activating
reagents (liquid reagents) provided by the manufacturer of the instrument (r-ex-temreg in-
temreg) and a kaolin solution (5 g1 as part of the PTT reagent CK Prest [Diagnostica
Stago SAS Asniegraveres sur Seine Cedex France]) In addition non-activated analyses
were performed using isotonic NaCl solution instead of the activating reagent in the fourth
channel of the device Fourfold measurements for precision analysis were performed
simultaneously in the four channels of the device in separate runs
According to the standard test protocol provided by the manufacturer of the ROTEMreg
delta (Werfen GmbHInstrumentation Laboratory Munich Germany) 20 μl of the
reagent for recalcification (star-temreg20) and 20 μl of the activating reagent were pipetted
into a cuvette using the automated pipette provided with the device Immediately
thereafter 300 μl of citrated whole blood (prewarmed to 37 degC for five minutes in the
heating device of the instrument and gently rocked to resuspend possibly sedimented
cells) were added to the test preparation in the cuvette using the automated pipette and
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
18
then the cuvette was laced carefully in the ROTEMreg device whereby the rotating pin
entered into the test approach
Different parameters are provided automatically by the ROTEMreg delta device and the
following variables (described according to the Pentapharm GmbH ROTEMreg delta
Manual [2009]) were used in the study
Clotting time (CT) the time (in s) from the beginning of the test (by adding the clot
activator) until the formation of fibrin starts (defined as the moment when the curve
achieves an amplitude of 2 mm) This Parameter facilitates the decision to replace clotting
factors when it is required
Clot formation time (CFT) describes the next phase of clotting by the formation of a
stable clot through the activation of thrombocytes and fibrin formation It is defined as
the time (in s) between the 2 mm amplitude and 20 mm amplitude of the curve
Alpha angle (α angle) is the angle (in degrees) between the middle axis and the tangent
to the clotting curve through the 2 mm amplitude point
Amplitude after ldquoXXrdquo minutes (eg A30 after 30 min) is the amplitude (in mm) at a
certain time after the CT has finished
Maximum clot firmness (MCF) corresponds to the maximum amplitude (in mm) reached
before the clot is dissolved by fibrinolysis This parameter measures the firmness of the
clot and its quality Like CFT and α angle it facilitates the decision for substitution therapy
with platelet concentrate andor fibrinogen especially in humans
Maximum clot elasticity (MCE) is a parameter calculated from MCF and may be superior
at high amplitudes as compared to the MCF
Lysis index at 30 45 and 60 min (LI30 LI45 LI60) represents the fibrinolysis (in
percentage) 30 45 and 60 min after the CT It may facilitate the decision for or against a
therapy with anti-fibrinolytic drugs
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
19
Maximum lysis (ML) describes the degree of fibrinolysis relative to the MCF achieved
during the measurement (percent loss of clot firmness)
25 Statistical methods
Original data obtained from the ROTEMreg delta device software were transferred into the
Microsoftreg Excelreg formal (Microsoft Office Professional Plus 2013) The Statistical
analyses were performed using the SAS Enterprise Guide Version 71 (SAS Institute Inc
Cary NC USA)
Coefficients of Variation (CVs) for selected relevant parameters of the ROTEM delta
analysis using ex-tem and in-tem regents were calculated for the fourfold measurements
on 3 normal and 3 abnormal blood samples in order to assess within-run imprecision CVs
for the results of each sample were calculated by dividing the standard deviation of the
data set by its arithmetic mean Finally the median of the CVs for each group of 3 samples
was calculated The reference values were calculated according to recommendations of
the American College of Veterinary Clinical Pathology (ACVCP) based on non-
parametric methods (25 and 975 quantiles) supplemented by 95 confidence
intervals (Friedrichs et al 2012)
Data were analysed for Standard normal distribution by Kolmogorov Smirnov test and
assessment of residuals of the analysis of variance (ANOVA) Because these tests
indicated almost normally distributed values for the studied ROTEM variables data were
analysed by a fourway ANOVA stratified by the activating reagent (ex-tem in-tem
kaolin or without activation) to test the influence of size age sex and neuter status on
several parameters and taking all interactions into account Post hoc t-tests were
performed when indicated The significance level was set to P lt 05
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
20
3 Results
31 Precision of the method
For all the parameters evaluated in the study the CVs for the fourfold measurement in
healthy dogs were less than 10 (Table 1) In the case of dogs with haemostatic
abnormalities CVs of the vast majority of ROTEM variables were also less than 10
except the variables CFT and ML irrespective whether ex-tem or in-tem reagents were
used (Table 2)
Table 1 Coefficients of variation () for rotational thromboelastometry using the
ROTEMreg delta analyser in normal blood samples
Activator CT (s) CFT (s) α (deg) A30 (mm) MCF (mm) MCE(mm) LI30 () LI45 () LI60 () ML ()
Ex-temreg 875 629 262 213 179 634 000 020 037 534
In-temreg 245 596 166 117 135 299 000 000 046 216
Abbreviations CT ndash clotting time CFT ndash clot formation time α ndash alpha angle A30 ndash
amplitude after 30 min MCF ndash maximum clot firmness MCE ndash maximum clot elasticity
LI 30 LI 45 LI 60 ndash Lysis index at 30 45 and 60 min ML ndash maximum lysis
Median values of the measurements of 3 samples
Table 2 Coefficients of variation () for rotational thromboelastometry using the
ROTEMreg delta analyser in canine blood samples with haemostatic alterations
Activator CT (s) CFT (s) α (deg) A30 (mm) MCF (mm) MCE(mm) LI30 () LI45 () LI60 () ML ()
Ex-temreg 668 1478 393 218 268 582 0 018 000 848
In-temreg 228 799 461 219 251 371 0 0 017 283
Median values of the measurements of 3 samples from patients with DIC (n = 2)
thrombocytopenia (n = 1) abbreviations please see legend to Table 1
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
21
32 Reference intervals
Activation by ex-tem reagent (tissue factor) resulted in faster CTs when compared to
activation via the intrinsic pathway using ellagic acid (in-tem) or kaolin reagents whereas
the CFT was much less affected by the activating reagent (Table 3) Especially CFT and
MCE showed a wide inter-individual variation in comparison for example with α angle
and MCF regardless of the used activating reagent (Table 3)
33 Influence of age sex neuter status and size
ANOVA did not reveal statistically significant differences in ROTEM delta parameters
between dog groups of variable size or sex (P gt 05) Age had influence only on CT and
only when the in-tem reagent was used (P = 0169 ANOVA) but not when using the ex-
tem reagent (P = 7105) Post hoc comparisons using t-test of the in-tem results between
age groups revealed shorter CTs in the group of senior dogs when compared with young
dogs (P = 0194) but not between adult dogs and these two groups ANOVA showed
particularly influences of the neuter status and a significant interrelationship between the
factors sex and neuter status on different ROTEM delta parameters using ex-tem and in-
tem respectively as activator (Tables 4 and 5) ANOVA revealed also a significant
influence of neuter status on different ROTEM parameters when kaolin was used as
activating reagent (CFT α angle MCF MCE) whereas non-activated measurements
were not subject to any significant influence of the assessed factors (results not shown)
The interrelationship between sex and neuter status mentioned above reflected the fact
that significant differences between neutered and not neutered dogs were only found in
females The statistical analysis using the t-test to compare intact vs neutered dogs
stratified by sex showed statistically significantly (P lt 05) longer CTs and CFTs and
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
22
lower α angle A30 MCF and MCE values in spayed when compared to intact females
(Tables 4 and 5)
Chapter 2 Reference intervals for rotational thromboelastometry measurements using the ROTEMreg delta device in dogs
23
Table 3 Reference values (median reference interval based on 25 - and 975 - quantiles) for thromboelastometric measurements with the ROTEMreg delta analyser in
dogs (n=125 95 confidence intervals in brackets) Abbreviations please see legend to Table 1
Activator
Statistical
para-meter
CT
(s)
CFT
(s)
Α
(deg)
A30
(mm)
MCF
(mm)
MCE
(mm)
LI30
()
LI45
()
LI60
()
ML
()
Ex-temreg
Median 450
[430ndash470]
110
[105ndash115]
680
[670ndash690]
600
[580ndash610]
610
[600ndash620]
154
[147ndash164]
100
[960ndash100]
100
[100ndash100]
970
[960ndash980]
220
[210ndash240]
25 340
[270ndash350]
650
[490ndash710]
540
[420ndash560]
460
[410ndash480]
460
[420-490]
870
[720ndash950]
100
[100-100]
870
[800ndash960]
700
[640ndash820]
800
[100ndash110]
975 830
[790ndash990]
220
[201ndash315]
790
[770ndash800]
770
[690ndash770]
730
[700ndash770]
264
[230-334]
100
[100ndash100]
100
[100ndash100]
100
[990ndash100]
590
[530ndash680]
In-tem
Median 190
[187ndash201]
116
[111ndash125]
680
[670ndash690]
570
[560ndash580]
590
[580ndash600]
143
[135ndash149]
100
[100ndash100]
100
[100ndash100]
980
[980ndash980]
180
[170ndash190]
25 135
[112ndash142]
730
[590ndash800]
510
[460ndash570]
450
[410ndash470]
480
[410ndash500]
920
[710ndash101]
100
[950ndash100]
990
[820ndash990]
930
[740ndash930]
500
[100ndash700]
975 283
[268ndash291]
251
[198ndash227]
750
[750ndash780]
660
[660ndash690]
680
[660ndash690]
210
[195ndash227]
100
[100ndash100]
100
[100ndash100]
100
[100ndash100]
270
[260ndash380]
Activator
Statistical
para-meter
CT
(s)
CFT
(s)
Α
(deg)
A30
(mm)
MCF
(mm)
MCE
(mm)
LI30
()
LI45
()
LI60
()
ML
()
Kaolin
Median 162
[154ndash173]
870
[820ndash910]
740
[720ndash750]
500
[470ndash530]
550
[530ndash560]
121
[115ndash127]
960
[940ndash980]
840
[790ndash880]
710
[640ndash770]
500
[450ndash540]
25
109
[990ndash115]
470
[440ndash580]
560
[490ndash600]
110
[700ndash230]
390
[380ndash400]
640
[600ndash680]
270
[140ndash480]
110
[800ndash260]
800
[400ndash170]
200
[160ndash240]
975 246
[230ndash337]
214
[185ndash290]
810
[790ndash810]
640
[630ndash690]
650
[650ndash690]
187
[182ndash223]
100
[100ndash100]
990
[980ndash100]
940
[930ndash990]
970
[930ndash100]
Without activation
Median 607
[495ndash662]
221
[194ndash265]
510
[460ndash550]
520
[500ndash530]
540
[520ndash550]
115
[109ndash123]
100
[100ndash100]
100
[100ndash100]
980
[980ndash990]
170
[160ndash190]
25 203
[123ndash215]
860
[550ndash930]
190
[120ndash230]
350
[230ndash370]
370
[260ndash400]
600
[350ndash670]
100
[950ndash100]
980
[890ndash990]
930
[840ndash950]
400
[000ndash500]
975 1356
[1305ndash1721]
811
[688ndash1333]
730
[710ndash790]
660
[640ndash720]
660
[640ndash730]
197
[179ndash265]
100
[100ndash100]
100
[100ndash100]
100
[100ndash100]
290
[280ndash320]
Chapter 2 Reference intervals for rotational thromboelastometry measurements using the ROTEMreg delta device in dogs
24
Table 4 Median values (minimum and maximum in brackets) for different ROTEMreg variables using ex-tem reagent stratified by sex and neuter status
Group
CT
(s)
CFT
(s)
Α
(deg)
A30
(mm)
MCF
(mm)
MCE
(mm)
LI30
()
LI45
()
LI60
()
ML
()
Female intact 430
(320ndash530)
960
(590ndash198)
710
(570ndash800)
620
(480ndash730)
640
(510ndash740)
179
(103ndash282)
100
(100ndash100)
100
(980ndash100)
960
(800ndash100)
230
(100ndash640)
Female spayed 460
(270ndash830)
121
(650ndash224)
660
(520ndash770)
580
(430ndash690)
590
(440ndash700)
146
(790ndash230)
100
(960ndash100)
100
(800ndash100)
970
(690ndash100)
240
(100ndash590)
p-value (t-test)
Female
intact vs spayed
00025 00063 00049 00023 00016 00015 na na na na
Male intact 450
(320ndash990)
105
(650ndash315)
690
(420ndash790)
590
(420ndash700)
610
(440ndash710)
154
(770ndash244)
100
(100ndash100)
100
(870ndash100)
970
(650ndash100)
220
(700ndash680)
Male castrated 460
(340ndash930)
111
(490ndash255)
685
(480ndash800)
590
(410ndash770)
600
(420ndash770)
152
(720ndash334)
100
(100ndash100)
100
(830ndash100)
980
(640ndash100)
205
(500ndash590)
p-value (t-test)
Male
Intact vs castrated
08326 05106 04756 01799 01568 02163 na na na na
p-values (ANOVA)
Factor Sex
05428 04847 06612 02206 01887 01630 08489 07893 07689 02758
Factor neuter status 01872 00167 00173 00039 00037 00028 07141 04737 09356 05948
Interrelationship
sexneuter status 00383 00968 00782 00488 00472 00319 04943 08611 05422 00160
na ndash not analysed all significant P-values (lt 005) are highlighted in bold print
Chapter 2 Reference intervals for rotational thromboelastometry measurements using the ROTEMreg delta device in dogs
25
Table 5 Median values (minimum and maximum in brackets) for different ROTEMreg variables using the in-tem reagent stratified by sex and neutering status
Group
CT
(s)
CFT
(s)
Α
(deg)
A30
(mm)
MCF
(mm)
MCE
(mm)
LI30
()
LI45
()
LI60
()
ML
()
Female intact 186
(126ndash238)
940
(590ndash164)
720
(620ndash780)
610
(490ndash690)
620
(530ndash690)
166
(113ndash227)
100
(100ndash100)
100
(990ndash100)
980
(930ndash100)
170
(100ndash300)
Female spayed 201
(149ndash291)
1220
(820ndash262)
670
(500ndash740)
550
(450ndash660)
570
(480ndash680)
133
(910ndash214)
100
(950ndash100)
100
(820ndash100)
980
(740ndash100)
190
(500ndash380)
p-value (t-test)
Female
intact vs spayed
na na lt0001 00009 00005 00005 na na na na
Male intact 199
(117ndash283)
122
(730ndash277)
680
(460ndash750)
570
(430ndash660)
590
(450ndash660)
146
(810ndash192)
100
(100ndash100)
100
(990ndash100)
980
(930ndash100)
180
(600ndash270)
Male castrated 181
(112ndash288)
123
(820ndash222)
670
(540ndash740)
565
(410ndash630)
585
(410ndash640)
138
(710ndash180)
100
(100ndash100)
100
(970ndash100)
980
(890ndash100)
180
(800ndash260)
p-value (t-test)
Male
intact vs castrated
na na 08232 01401 01162 00849 na na na na
p-values (ANOVA)
Factor Sex
02105 03317 03370 00877 01258 00510 09564 07832 08117 03638
Factor neuter status 04289 00660 00619 00107 00088 00054 07152 09795 07056 08299
Interrelationship
sexneuter status
04310 00550 00408 00707 01733 00996 05240 05982 08047 04578
na ndash not analysed all significant P-values (lt 005) are highlighted in bold print
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
26
4 Discussion
41 Coefficients of variation
In accordance with the literature CVs below 10 indicate adequate repeatability in intra-
assays evaluations (Steiner et al 2003 Hua et al 2018) According to our results
obtained from the fourfold measurements in citrated whole blood ROTEM delta
measurements on healthy dogs and dogs with haemostatic alterations fulfil these
requirements with only a few exceptions concerning CFT and ML values in abnormal
samples
Our results regarding repeatability are similar to those obtained for several TEM
parameters in previous studies performed in animals In one study the imprecision of the
ROTEM analyser was evaluated by duplicate measurements in 10 blood samples of
healthy dogs and as result of that evaluation a good precision (CVs below 10 ) was
calculated for CT CFT MCF and α angle but in contrary to our results a low
repeatability was found for ML (CVs of 233 [ex-tem] and 285 [in-tem] own values
534 and 216 ) (Falco et al 2012)
In a previous feline study using ex-tem and in-tem as activating reagents for the ROTEM
delta repeatability was assessed similarly to the present study based on fourfold
measurements of blood samples of two healthy individuals (Doumlderlein and Mischke
2015) The results also revealed CVs below 10 for most of the parameters evaluated
The partially low repeatability of ML values may reflect the vulnerability of this variable
which is not only influenced by the complex clot formation process but also by its
subsequent lysis Reproducibility of ex-tem or in-tem activated measurements of the
ROTEM delta analyser which was not investigated in our study was studied in blood
from healthy and unhealthy horses by two different operators using three different
ROTEM devices Results indicated acceptable reproducibility (Junge et al 2016)
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
27
42 Reference intervals
The number of 125 healthy dogs used in the present study to create reference intervals is
regarded adequate according to the guidelines suggested by the American Society of
Veterinary Clinical Pathology (ASVCP) and the National Committee for Clinical
Laboratory Standards (NCCLS) who recommend to include at least 120 individuals
(Friedrichs et al 2012) To the best of our knowledge reference intervals for the most
relevant ROTEMreg delta parameters obtained in an adequate number of samples of whole
citrated canine blood have not been published so far A previous study was performed on
a smaller number of 45 dogs (Falco et al 2012) The median values for reference
intervals obtained in the present study are partially similar to those calculated in the
mentioned study based on 45 healthy dogs (eg ex- tem CT 610 s450 s [Falco et al
2012own results] CFT 150 s 110 s α angle 620deg680deg MCF 530 m 610 mm ML
180 220 in-tem CT 235 s190 s CFT 136 s116 s α angle 660deg680deg MCF
560 mm590 mm ML 30 180 ) However in comparison to our results the
reference intervals in the mentioned study are slightly wider (eg ex-tem CT 29ndash92
s34ndash83 s [Falco et al 2012own results] CFT 540ndash275 s650ndash220 s α angle 470ndash
790deg540ndash790deg MCF 360ndash730 mm460ndash730 mm ML 000ndash560 800ndash590
in-tem CT 126ndash363 s135ndash283 s CFT 470ndash224 s730ndash251 s aα angle 550ndash
810deg510ndash750deg MCF 500-ndash750 mm480ndash680 mm ML 000ndash400 500ndash270 )
although the previous study used the 5 and 95 quantiles to define limits of the
reference range whereas we calculated the commonly used 25 and 975 quantiles
to capture the central 95 of reference values (Friedrichs et al 2012)
Although it is desirable that each laboratory should specifically determine its own
reference intervals the results obtained in this study may provide an orientation for other
laboratories to interpret ROTEM delta parameters analysed in citrated canine blood In
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
28
order to test transference of the reference ranges to another laboratory a validation
procedure with measurement of 20 samples should be performed (Friedrichs et al 2012)
Although intra-individual control values (subject-based reference values) are regarded
superior to population based reference values (Wiinber et al 2007) the latter have to be
routinely used in clinical patients due to lacking individual controls especially for specific
tests such as ROTEM analyses In addition the calculated reference values cannot be
used for the fully automated device ROTEM sigma because - according to an actual
human study - values of part of the parameters do not correlate well between both
instruments (Gillissen et al 2019)
43 Influence of size age sex and neuter status
In previous studies in humans (Attard et al 2013) and animals (Mischke 1994 Borrelli
et al 2017) age has been described as an influencing factor for different haemostasis
parameters We also found an influence of age on the CT with shorter times in senior
dogs (gt 84 months) compared with young dogs (up to 24 months) which was limited to
measurements activated by the in-tem reagent
This age dependency confirms in principle a previous study on 20 healthy dogs
(Barthelemy et al 2015) which found shorter CTs in elderly dogs and results of a human
study where individuals with an age gt 45 years showed shorter CTs compared with
individuals lt 45 years old (Sucher et al 2011) However in the cited studies this effect
was only seen when ex-tem but not when the in-tem reagent was used
Based on the age distribution of our dogs which were primarily recruited to establish
reference intervals we cannot deliver information regarding very young dogs A human
study focusing on ROTEM reference ranges in children demonstrated significant
differences for nearly all ROTEM parameters with the most striking differences in
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
29
subjects aged 0-3 months (Oswald et al 2010) The limited number of dogs with an age
lt 1 year which did not allow to consider these separately in the present study may also
be one reason for the different results in a feline study (Doumlderlein and Mischke 2015) In
the cited study on healthy cats age showed an influence on CT but contrary to our study
the CT was shorter in young individuals (6-12 months) compared with older ones In
addition this was only the case in measurements using kaolin and in non-activated
measurements When compared to humans and cats uni- or multivariate analyses
considering factors age and breed or size based on do populations including different
breeds have the limitation that they can be biased by the variate life expectancy between
small medium and large dog breeds (Willems et al 2017) Apart from age we found a
significant influence only for the neuter status in bitches reflected by a significant
interaction effect between factors neuter status and sex in the ANOVA analysis
Especially for CFT values (eg activated by ex-tem median values of 121 s [spayed] vs
96 s [intact]) this difference seems to be also clinically relevant with respect to the
interpretation of ROTEM results In contrast although also significantly different
absolute and relative differences of CT values were small (eg activated by ex-tem 46 s
[spayed] vs 43 s [intact]) These differences may not only be considered for the result
interpretation of clinical patients but indicate also the necessity for sex-matched patient
groups in scientific studies
The obviously more active haemostasis system in intact females is probably related to an
influence of sex hormones (specifically estrogens) on the coagulation system
Accordingly ex vivo and in vivo studies demonstrated the activating properties of
endogenous estrogens on the haemostasis system In one ex vivo study addition of
endogenous estrogens (estrone [E1] 17szlig-estradiol [E2] and estriol [E3]) to blood samples
from men resulted in shorter mean values of the reaction time in TEG analyses
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
30
corresponding to CT in ROTEM measurements (452-537 s vs 698 s [control]) and of
the TEG variable kinetics (corresponding to the CFT in ROTEM analyses) (Swanepoel
et al 2017) In a further study subcutaneous injection of E2 into male rats induced a
significant increase in activities of coagulation factors X (by 8 ) and XI (by 10-24 )
however did not show any significant effect on PT aPTT and factor VII activity (Franco-
Murillo and Jaimez 2017)
In the cited studies also the effects of the synthetic estrogen ethinylestradiol [EE] were
studied which interestingly seems to have an inhibiting effect as indicated by prolonged
reaction time and kinetics (TEG) compared with the control in the human ex vivo study
and significantly longer PTs and aPTTs and lower factor VII and X activities in the study
on rats As expected we did not find a significant influence of size on ROTEM parameters
in dogs To the best of the authoracutes knowledge size has not been examined as an
influencing factor on viscoelastic measurements or any other haemostatic variable in
dogs Apart from Greyhounds there is limited literature on breed comparisons of
haemostatic variables (Clemons and Meyers 1984 Mischke 1994 Nielsen et al 2007
Nielsen et al 2010 Boyd et al 2018) indicating very limited breed differences for
example for factor VII (Mischke 1994) However these also do not indicate systematic
influence of size and in addition possible breed-specific effects have to be considered
As already stated one limitation of the study is the limited number of young animals
which does not allow to verify reference values for animals in this stage of life In
addition with respect to the main aim of the study possible influencing factors such as
platelet count and haematocrit were not considered in the statistical analysis Finally the
relatively small number of dogs used for the precision analysis which express the fact
that this was only a secondary aspect of the study may be regarded as a limitation At the
same time these considerations provide space for further investigations
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
31
5 Conclusions
Results of the precision analysis revealed acceptable results and thereby confirm that the
ROTEMreg delta analyser delivers repeatable results to measure overall haemostasis in
canine citrated blood samples The established reference values should offer a reliable
basis for the interpretation of thromboelastometric measurements in whole blood using
the device ROTEMreg delta in dogs with suspected haemostasis disorders Neuter status
in female dogs appeared to be a relevant influencing factor on most important ROTEMreg
parameters and should be considered for result interpretation and with respect to
experimental designs
6 Acknowledgments
Jose Mauricio Pereira received a scholarship from the Universidad Nacional de Costa
Rica (reference code JB-C-1107-2016)
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
32
7 References
1 Attard C van der Straaten T Karlafitis V Monagle P Ignjativic V 2013 Developmental
hemostasis age-specific differences in the levels of hemostatic proteins J Thromb and
Haemost 11 1850ndash1854
2 Bagge A Schoumltt U Kander T 2016 Effects of naturopathic medicines on Multiplate and
ROTEMreg A prospective experimental pilot study in healthy volunteers BMC Complemen
Altern Med 17 (16) 64
3 Bartheacutelemy A Rannou B Forterre M Verwaerde P Bonnet-Garin JM Pouzot-Nevoret
C Goy-Thollot I 2015 Differences between coagulation and cytokine profiles in dogs of
different ages Vet J 205 410ndash412
4 Bauer N Eralp O Moritz A 2009 Establishment of reference intervals for kaolin-activated
thromboelastography in dogs including an assessment of the effects of sex and anticoagulat use
J Vet Diagn Investi 21 641ndash648
5 Bollinger D Seeberger MD Tanaka KA 2012 Principles and practice of
thrromboelastography in clinical coagulation management and transfusio practice Transfus Med
Rev 26 1ndash13
6 Borrelli A Botto A Maurella C Falco S Pagani E Miniscaldo B Tarducci A Bruno
B 2017 Thromboelastometric assesment of hemostasis in newborn Piemontese calves J Vet
Diagn Invest 29 293ndash297
7 Boyd CJ Claus MA Raisis AL Hosqood G Sharp CR SmartL 2018
Hypocoagulability and platelet dysfunction are exacerbated by synthetic colloids in a canine
hemorrhagic shock model Front Vet Sci 5 279
8 Bruno B Maurella C Falco S Tarducci A Zanatta R DrsquoAngelo A Borrelli A 2015a
Thromboelastometric evaluation of hemostasis in dogs infected with Leishmania infantum J Vet
Emerg Crit Care (San Antonio) 25 502ndash511
9 Bruno B Maurella C Falco S Tarducci A Zanatta R Gianella P DrsquoAngelo A Piras L
Di Bella A Borrelli A 2015b Assessment of coagulation utilizing thromboelastometry in dogs
undergoing orthopedic surgery J Vet Emerg Crit Care (San Antonio) 25 358ndash363
10 Clemons RM Meyers KM 1984 Acquisition and aggregation of canine blood platelets Basic
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
33
mechanisms of function and differences because of breed origin Am J Vet Res 45 137ndash144
11 Conversy B Blais MC Dunn M Gara-Boivin C del Castillo JRE 2017 Anticoagulant
activity of oral rivaroxaban in healthy dogs Vet J 223 5ndash11
12 Crochemore T Piza FMT Rodriacuteguez RDR Guerra JCC Ferraz LJR Correcirca TD
2017 A new era of thromboelastometry Einstein (Sao Paulo) 15 380ndash385
13 Crochemore T Correcirca TD Lance MD Solomon C Neto AS Guerra JCC Lellis PS
Bernz LM Nunes N Mancio CM Yokoyama APH Silva E 2018 Thromboelastometry
profile in critically ill patients A single-center retrospective observational study PLoS One 13
e0192965
14 Doumlderlein E Mischke R 2015 Reference intervals for thromboelastometry with the ROTEMreg
delta in cats Res Vet Sci 100 271ndash276
15 Falco S Bruno B Maurella C Bellino C DacuteAngello A Gianella P Tarducci A Zannata
R Borrelli A 2012 In vitro evaluation of canine hemostasis following dilution with
hydroxyethyl starch (13004) via thromboelastometry J Vet Emerg Crit Care (San Antonio)
22 640ndash645
16 Franco-Murillo Y Jaimez R 2017 Inhibitory effect of ethinylestradiol on coagulation factors
in rats Exp Anim 66 107ndash113
17 Friedrichs KR Harr KE Freeman KP Szladovits B Walton RM Barnhart KF Blanco-
Chavez J 2012 ASVCP reference interval guidelines Determination of de novo reference
intervals in veterinary species and other related topics Vet Clin Pathol 41 441ndash453
18 Gillissen A va den Akker T Caram-Deelder C Henriquez DDCA Bloemenkamp
RWM Eikenboom J van der Bom JG de Maat MPM 2019 Comparison of
thromboelastometry by ROTEMreg Delta and ROTEMreg Sigma in women with postpartum
haemorrhage Scand J Clin Lab Invest 79 32ndash38
19 Harttert H 1948 Blutgerinnungstudien mit der Thrombelastographie einem neuen
Untersuchungsverfahren Klin Wochenschr 26 577ndash583
20 Horn P Pesce A 2003 Reference intervals and update Clin Chim Acta 334 5ndash23
21 Hua Yanhun Qiu W Xiao Q Wu Q 2018 Precision (repeatability and reproducibility) of
ocular parameters obtained by the Tomey OA-2000 biometer compared to the IOLMaster in
healthy eyes PLoS One 13 e0193023
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
34
22 Junge H Ringer S Mayer N Schwarzwald C 2016 Assesment of method reliability and
determination of reference intervals for rotational thromboelastometry in horses J Vet Emerg
Crit Care (San Antonio) 26 691ndash703
23 Kol A Borjesson DL 2010 Application of thrombelastographythromboelastometry to
veterinary medicine Vet Clin Pathol 39 405ndash416
24 Marschner CB Wiinberg B Tarnow I Markussen B Kuumlhnel L Bochsen L Kristensen
AT 2018 The influence of inflammation and hematocrit on clot strength in canine
thromboelastographic hypercoagulability J Vet Emerg Crit Care (San Antonio) 28 20ndash30
25 McMichael MA Smith SA 2011 Viscoelastic coagulation testing technology applications
and limitations Vet Clin Pathol 40 140ndash153
26 Mischke R 1994 Activity of coagulation factors II V VII and X in healthy dogs- dependence
on age sex and breed Berl Muumlnch Tieraumlrztl Wochenschr 107 289ndash294
27 Nielsen A Zois N Pedersen H Olse L Tarnow I 2007 Platelet function in dogs bread
differences and effect of acetylsalicylic acid administration Vet Clin Pathol 36 267ndash273
28 Nielsen L Kjelgaard‐Hansen M Jensen A Kristensen A 2010 Breed‐specific variation of
hematologic and biochemical analytes in healthy adult Bernese Mountain dogs Vet Clin Pathol
39 20ndash28
29 Nogami K 2016 The utility of thromboelastography in inherited and acquired bleeding
disorders Br J Haematol 174 503ndash514
30 Oswald E Stalzer B Heitz E Weiss M Schmugge M Strasak A Innerhofer P Haas T
2010 Thromboelastometry (ROTEMreg) in children Age-related reference ranges and correlations
with standard coagulation tests Br J Anaesth 105 827ndash835
31 Sankarankutty A Nascimento B Teodoro da Luz L Rizoli S 2012 TEGreg and ROTEMreg
in trauma similar test but different results World J Emerg Surg 7 (Suppl 1) S3
32 Sigrist NE Schefer RJJ Kutter APN 2018 Characteristics of hyperfibrinolysis in dogs and
cats demonstrated by rotational Thromboelastometry (ROTEM) Vet J 242 67-73
33 Steiner J Teague S Williams D 2003 Development and analytic validation of an enzime-
linked immunosorbent assay for the measurement of canine pancreatic lipase immunoreactivity in
serum Can J Vet Res 68 161ndash168
34 Sucker C Tharra K Lithmate J Scharf RE Zotz RB 2011 Rotation thromboelastography
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
35
(ROTEM) parameters are influenced by age gender and oral contraception Perfusion 26 334ndash
340
35 Swanepoel A Emmerson O Pretorius E 2017 The effect of endogenous and synthetic
estrogens on whole blood clot formation and erythrocyte structure Microsc Microanal 23 599ndash
606
36 Theusinger OM Nuumlrnberg J Asmis LM Seifert B Spahn DR 2010 Rotation
thromboelastometry (ROTEM) stability and reproducibility over time Eur J Cardiothorac Surg
37 677ndash683
37 Toulon P Metge S Hangard M Zwahlen S Piaulenne S Besson V 2017 Impact of
different storage times at room temperature of unspun citrated blood samples on routine
coagulation tests results Results of a bicenter study and review of the literature Int J Lab
Hematol 3 458ndash468
38 Turner JS Kutter APN Sigrist NE 2019 Correlation of rotational thrromboelastometry
(ROTEM) parameters with platelet count and their ability to predict thrombocytopenia in dogs
Res Vet Sci 126 45ndash50
39 Versteeg HH Heemskerk JWM Levi M Reitsma PH 2013 New Fundamentals in
Hemostasis Physiol Rev 93 327ndash358
40 Wiinberg B Jensen AL Kjelgaard-Hansen M Rojkjaer R Johansson PI Gade LP
Gram DX Kristensen AT 2007 Study on biological variation of haemostatic parameters in
clinically healthy dogs Vet J 174 62ndash68
41 Wiinberg B Jensen AL Johansson PL Rozanski E Tranholm M Kristensen AT 2008
Thromboelastographic evaluation of hemostatic function in dogs with disseminated intravascular
coagulation J Vet Intern Med 22 357ndash365
42 Wikkelsoslash A Wetterslev J Moslashller AM Afshari A 2016 Thromboelastography (TEG) or
thromboelastometry (ROTEMreg) to monitor haemostatic treatment versus usual care in adults or
children with bleeding Cochrane Database Syst Rev Art
httpsdoiorg10100214651858CD007871pub3 No CD007871
43 Willems A Paepe D Marynissen S Smets P Van de Maele I Picavet P Duchateau L
Daminet S 2017 Results of screening of apparently healthy senior and geriatric dogs J Vet
Intern Med 31 81ndash92
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
36
44 Woodman J Wagg CR Boysen SR Leguillete R Mizen K Roy MF 2015 Evaluation
of coagulation via thromboelastography in healthy horses administered dexamethasone Can Vet
J 56 1271ndash1274
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
37
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of
standard heparin prophylaxis and correlation to haemostatic changes
Pereira JM Hewicker-Trautwein M Rohn K von Depka Prondzinski M
Mischke R
Contributions of the authors
Pereira JM performed (1) the ultrasound evaluations and blood sampling of the
patients under study (2) haematology analysis (3) injection of the anticoagulation
prophylaxis (partially) to the patients under study (4) analysis of samples (collaboration)
at Werlhof Institute Medical Care Center (5) analysis of some haemostasis test
(collaboration) (6) statistical analysis (partially) (7) data documentation (8) and wrote
the manuscript
Hewicker-Trautwein M performed the electron microscopy analysis
Rohn K performed the statistical analysis
von Depka Prondzinski M performed some haemostasis test (thrombin generation
assay and endogenous thrombin potential)
Mischke R performed the coagulation and haemostasis test was involved in designing
and supervision of the study analysis and discussion of the data and its statistical analysis
and revision edition and correction of the manuscript
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
38
Central venous catheter induced thrombosis in dogs ndash efficacy of standard heparin
prophylaxis and correlation to haemostatic changes
Joseacute Mauricio Pereira1 Marion Hewicker-Trautwein2 Karl Rohn3 Mario von Depka
Prondzinski4 Reinhard Mischke1
1Small Animal Clinic University of Veterinary Medicine Hannover Foundation
Buumlnteweg 9 D-30559 Hannover Germany
2Institute for Pathology University of Veterinary Medicine Hannover Foundation
Buumlnteweg 17 D-30559 Hannover Germany
3Institute of Biometry Epidemiology and Information Processing University of
Veterinary Medicine Hannover Foundation Buumlnteweg 2 30559 Hannover Germany
4Werlhof Institute Schillerstrasse 23 30159 Hannover Germany
Abbreviations anti-Xa anti-factor Xa BW body weight CVC central venous catheter
EJV external jugular vein ELMI electron microscopy IV intravenous JV jugular vein
LMWH low molecular weight heparin ML maximum lysis (ROTEM) PIVC
peripheral intravenous catheter ROTEM rotational elastometry SC subcutaneous
TGA thrombin generation assay TID three times a day TT thrombin time UFH
unfractionated heparin
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
39
Corresponding author
E-mail addresses matiscrgmailcom (JM Pereira) MarionHewicker-
Trautweintiho-hannoverde (M Hewicker-Trautwein) KarlRohntiho-hannoverde
(K Rohn) mailwerlhof-institutde (M von Depka Prondzinski)
ReinhardMischketiho-hannoverde (R Mischke)
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
40
ABSTRACT
Central venous catheters (CVC) are of importance for intensive care management of
canine patients Thrombosis of the jugular vein is a severe complication related to the
CVC use However only little is known about the incidence of CVC-associated
thromboses in dogs and the necessity and efficacy of prophylactic antithrombotic
regimens The objectives of the present study were (1) to determine the incidence of
catheter-induced thrombosis in canine intensive care patients and thereby (2) to evaluate
the efficacy of a routine prophylactic heparin treatment and finally (3) to assess whether
initial changes of selected haemostasis parameters are predictive for an increased risk for
thrombosis formation 29 dogs hospitalised in the Small Animal Clinic University of
Veterinary Medicine Hannover Foundation and receiving a central venous catheter
(CVC) in the external jugular vein (EJV) for medical reasons were included in the final
assessment 17 dogs received the standard antithrombotic dosage regimen of
unfractionated heparin (UFH 150 IUKG BW TID sc) and 12 dogs (usually surgical
patients) the reduced dosage (75 IUKG BW TID sc) A colour Doppler ultrasound of
the respective EJV in order to detect thrombus formation and blood collection for
haemostasis tests were performed immediately before the installation procedure (day 0)
and on days 1 3 5 7 and so on after the CVC insertion Haemostasis tests included
prothrombin time activated partial thromboplastin time thrombin time antithrombin
activity D-dimer concentration thrombin generation and rotational elastometry as well
as the heparin activity using a chromogenic anti-factor Xa test Finally electron
microscopy (ELMI) of sections of the removed CVC was performed In 8 dogs
sonographic examination revealed thrombus formation in the lumen of EJV and in 1927
CVCs assessed using ELMI thrombi on the external CVC surface were detected without
significant correlation between both methods Plasmatic heparin activities showed great
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
41
variability but no significant differences were found between dogs with or without CVC-
associated thrombus formation From the initially performed haemostasis tests high
fibrinogen concentrations and low maximum lysis values as measured by the ROTEM
delta device (ex-tem reagent) were associated with sonographically detectable thrombi
The results of the present study indicate that CVC-associated thromboses occur (eg 28
[829] of the patients as detected by ultrasound) although severe thromboses (high
grades) are relatively rare in intensive care canine patients receiving a routine
anticoagulant treatment A high fibrinogen concentration and low maximum lysis
(ROTEM ex-tem) may be useful indicators for an increased thrombotic risk in individual
patients which may require special anticoagulatory treatment
Keywords CVC complication venal occlusion antithrombotic treatment ultrasound
electron microscopy rotational elastometry
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
42
1 Introduction
Central venous catheters (CVCs) are very important tools for care of patients in critical
care units allowing parenteral nutrition support therapy administration of high volumes
of intravenous fluids safe drugs administration that cannot be given through peripheral
veins plasmapheresis haemodialysis blood transfusions as well as periodical blood
sampling (Geerts 2014 Vose et al 2019) CVC related thrombosis is one of the most
severe complications (apart from infections and pneumothorax) of CVCs in humans
(Geerts 2014 Comerlato et al 2017 Evans and Ratchford 2018) leading to patientrsquos
distress CVC dysfunction venous stenosis and in some cases to the development of
thromboembolism (Geerts 2014)
In the human literature there are various studies dealing with epidemiology with reference
to different anticoagulatory regimens including placebo-controlled studies (Lee and
Kamphuisen 2012) In humans CVC related thromboses represent 70ndash80 of all upper
extremity thrombosis cases and approximately 10 of thromboembolism cases in
humans (Kucher 2011) The incidence of CVC related thrombosis reported in humans
based on 25 studies ranged widely from 2 to 67 (median = 30 ) (van Rooden et al
2005 review article) This wide range of calculated incidences of CVC related
thromboses in humans reflects the use of different CVC types different locations of entry
site of the CVC (jugular femoral subclavian axillary cephalic vein) different thrombus
detection technique (venography or ultrasonography) unequal durations of follow up
varying diseases of patients (eg haematological oncological) and presence of
predisposing factors (eg previous venous thrombosis history presence of cancer) (van
Rooden et al 2005 Geerts 2014 Liu et al 2015)
Part of the authors empirically observed CVC-associated thrombosis of the external
jugular vein (EJV) as a relevant complication in their canine intensive care patients which
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
43
awakened their interest in that topic To the best of the authoracutes knowledge studies about
extra luminal CVC related thromboses in dogs are rare in the available veterinary
literature One study evaluating the complications associated with 100 CVCs placed in
the jugular vein of dogs and cats reported only 2 cases of thrombosis (confirmed by
palpation or ultrasound) but obviously did not assess the appearance of subclinical CVC
related thromboses (Adamantos et al 2010) Thereby the incidence of CVC-induced
thromboses in dogs is unknown and systematic studies regarding an effective
anticoagulatory management are lacking
In this context it is of interest whether a possible development of thrombosis or an
increased risk (despite the prevention by a standard application of unfractionated heparin
[UFH]) can be predicted by means of changes of haemostasis related parameters during
the initial examination In one study on 168 human patients without indication of
anticoagulation prophylaxis for example elevated leukocyte counts high plasminogen
activator inhibitor and high coagulation factor VIII levels (all measured on blood samples
taken after CVC placement) were associated with an increased incidence of symptomatic
CVC-related thrombosis diagnosed by ultrasonography (Boersma et al 2016)
Therefore aims of the present study were (1) to determine the incidence of catheter-
induced thrombosis in canine intensive care patients (as detected by ultrasonography and
electron microscopy ELMI) and thereby (2) to evaluate the efficacy of a standard
antithrombotic regimen with unfractionated heparin (UFH) routinely used in the Small
Animal Clinic of the University of Veterinary Medicine Hannover and (3) to assess
whether selected haemostasis test results are predictive for an increased risk for
thrombosis formation
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
44
2 Material and methods
21 Study design
Canine in-patients of the Small Animal Clinic University of Veterinary Medicine
Hannover receiving a CVC in the EJV for medical reasons from March 2017 to
December 2019 were included Anticoagulatory treatment was performed according to
the standard protocol of the clinic Colour Doppler ultrasonographic controls of the EJV
and blood samplings (for heparin activity basic haematology and haemostasis tests) were
done immediately before the catheter was placed (day 0) and on days 1 3 5 etc (2 hours
after the first daily UFH injection) until the CVC was removed Only patients were
included which could be examined at least until day 3 Haemostasis tests included
prothrombin time activated partial thromboplastin time thrombin time fibrinogen
concentration antithrombin activity fibrin monomers D-dimers thrombin generation
assay (TGA) and rotational elastometry as well as the heparin activity using a
chromogenic anti-factor Xa test Finally ELMI of sections of the CVC after its removal
was performed
22 Animals
Originally 32 patients hospitalised in the Small Animal Clinic and receiving a CVC in the
EJV for medical reasons entered the study Three of them were subsequently excluded
because they did not reach the defined minimum study period of 3 days Two were
euthanised and one referred back to the referring vet before day 3 22 dogs weighing more
than 15 kg were included so that the withdrawal of about 10 ml of blood on several days
was not a significant burden for the patient (lt 1 of the total blood volume) Five dogs
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
45
with a body weight of less than 15 kg but at least 8 kg were included with special
consideration of the amount of blood taken (no more than 6 ml on individual days)
All animals received standard anticoagulatory treatment with UFH 17 dogs with internal
diseases received 150 IUkg BW TID subcutaneously and 12 dogs (post surgery) received
a reduced dosage of 75 IUkg BW TID subcutaneously
The 29 dogs enclosed in the study included 4 mixed breed dogs and the following
pedigree dogs 5 Labrador Retrievers 3 German Shepherds 2 Australian Shepherds and
one from each of the following breeds Akita Inu Asian Shepherd Bavarian Mountain
Hound Beagle Belgian Shepherd Briard Canaan dog Dalmatian Golden Retriever
Havanese dog Hovawart Rhodesian Ridgeback Schnauzer Shar Pei and Wolfspitz
These dogs were aged between 2 and 164 months (median 77 months) and had body
weights ranging from 81 and 63 kg (median 27 kg) 9 were castrated males 8 intact
males 7 spayed females and 5 intact females
The diseases of the 29 patients requiring a CVC included 3 from each of the following
diseases hepatopathy and esophagus dilatation 2 from each of the following diseases
bacterial cystitis diabetes mellitus and 1 from each of the following diseases abdominal
wall perforation aspiration pneumonia cystitis intestinal round cells sarcoma carcinoma
of the nasal cavity chronic renal insufficiency cystic duct rupture heartworm disease
ivermectine intoxication lymphoma multifocal alveolar histiocytosis pancreatitis post-
surgical bleeding stomach dilatation stomach foreign body tetanus thoracic
pyogranulomatous inflammation thrombocytopenia and trigeminal nerve paresis
Animals were treated in accordance with the German Animal Welfare Law The
experimental design was approved by the Animal Welfare officer of the University of
Veterinary Medicine and reported to the responsible authority (Lower Saxony State
Office for Consumer Protection and Food Safety reference number 17A101)
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
46
23 Positioning and handling of the central venous catheter
Two types of single lumen radiopaque polyurethane CVC (different on length and
diameter) were used during the study The CVC type I (n = 2529) was Cavafixreg Certoreg
Splittocanreg CVC 32 cm length 11 x 17 mm diameter16 G (BBraun Melsungen AG)
the CVC type II used (n = 429) was Certofixreg Mono S 215 15 cm length 08 x 14 mm
diameter18G (BBraun Melsungen AG) Each CVC was inserted into the EJV using a
modified Seldinger technique as follows the skin of the neck was shaved and disinfected
with antiseptic soap alcohol and iodine Local anesthesia (Lidocaine HCl 2) was
administrated subcutaneously on the catheter insertion site (a middle point in the lateral
neck over the jugular sulcus) A small skin incision was performed in the insertion site
and one peripheral plastic cannula with a metallic needle inside (14 G for CVC type I
and 20 G for CVC type II) was inserted in the EJV in cranio-caudal orientation (Hundley
et al 2018) Once the EJV was reached the needle was removed and a flexible wire
guide was inserted through the peripheral cannula following an external landmark
(4thintercostal space) (Reminga et al 2018) The peripheral cannula was removed and
the respective CVC was inserted slowly and with gentle pressure into the EJV guided
with the flexible wire and following the same external landmark to reach the right
position After the insertion of the CVC the flexible wire guide was removed The correct
positioning of the CVC was checked with a latero-lateral thorax x-ray ensuring that the
CVC tip was placed at the junction of the cranial cava vein with the right atrium Each
CVC was fixed to the neck skin (adjacent to the insertion point) with a monofilament
non-absorbable suture (gauge 3-0) Finally a bandage elaborated with Povidone-Iodine
gauze (Betaisodonareg) padded synthetic band (Artiflexreg Natur) and elastic band
(Elastomullreg) was used to protect the CVC output (Claude et al 2010) and changed
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
47
daily Several times day the catheter was flushed with isotonic sodium chloride
containing 1 IUml UFH
24 Heparin preparation and administration
Heparin-sodium-25000-ratiopharm (injection solution containing 5000 IUml sodium
heparin [mucosa]) was used SC injection was performed over the lateral thorax wall
using 1 mL syringes with fine graduation marks
25 Ultrasonography
Colour Doppler ultrasonographic examinations were performed in lateral position using
a LO-GIQ E9 ultrasound device (GE Healthcare Chicago Illinois USA) with a 15
Megahertz linear ultrasound probe The development of a thrombus or fibrin debris in the
EJV lumen around the CVC or inside it was assessed at each control Once the
ultrasound was finished a new protective bandage was placed around the neck of the dog
According to the percentage of lumen occlusion of the EJV (evaluated in cross sectional
views during the ultrasound scanning) the thrombi were classified in four different grades
(Fig 1)
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
48
of the residual vein lumen C ndash catheter T ndash thrombus
Fig 1 Definition of different grades of central venous catheter induced thrombosis of the
jugular vein as detected using colour Doppler ultrasound
26 Blood collection
At each time point approx 6ndash10 ml of blood (according to the body weight) was collected
This included approx 1 ml of EDTA blood for hematological analysis and approx 5ndash9
ml of citrated blood (tubes containing one part of 011 molL [38 ] sodium citrate to
nine parts of blood) for the measurement of hemostasis tests in citrated blood (ROTEM)
or citrated plasma Blood samples were collected by puncture of peripheral veins with
disposable cannulas (09 x 40 mm [20G] or 10 x 40mm [21G]) directly into sample tubes
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
49
or from the CVC using the three-syringe-technique (Villalta-Garciacutea et al 2015) in
situations when the peripheral venipuncture was not indicated due to phlebitis or irritation
andor inflammation of the surrounding skin Briefly 2ndash3 ml of blood were taken in a
sterile disposable syringe and immediately afterwards the blood sample was taken in a
new disposable syringe and transferred into the test tubes containing the respective
anticoagulants finally the first 2-3 ml taken were re-infused to the dog through the CVC
followed by flushing with a solution of sodium chlorideUFH (02) Immediately after
blood collection each sample tube was swayed gently until blood and anticoagulant had
been mixed thoroughly To prepare citrated platelet poor plasma citrated blood was
centrifuged at 16000 g for 10 min at room temperature using a microcentrifuge The
plasma was transferred into plastic tubes and the centrifugation procedure was repeated
This platelet poor plasma was then frozen in aliquots at -70 degC until used for analysis
Directly before analysis the plastic tubes were thawed at 37 degC in a water bath
27 Laboratory tests
Haematological analyses were made automatically with Adviareg 2120i (Siemens
Healthcare GmbH Germany)
Prothrombin time activated partial thromboplastin time and thrombin time were
measured with the autoanalyser Amax Destiny Plus (Tcoag Deutschland GmbH
Germany) and commercial reagents (Thromborel S Siemens Healthcare Diagnostics
Products GmbH Marburg Germany CK Prestreg and Test Thrombin Reagent
Diagnostica Stago SAS Asniegraveres sur Seine Cedex France) Whereas the last two tests
were measured according to the manufacturer`s instructions a modified test optimised
for canine plasma was used to measure prothrombin time (25 microl citrated plasma 120 with
imidazole buffer + 25 microl of a human fibrinogen solution 2 gl prewarmed for 120 s at 37
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
50
oC addition of 25 microl of activating reagent Thromborel S) and the results converted into a
percentage activity based on a calibration curve prepared with canine pooled plasma The
fibrinogen concentration was measured with the method according to Clauss using a test
kit from Stago Diagnostica SAS D-dimer concentration was measured with a
quantitative commercial human latex turbidimetric method in the autoanalyser Cobas
c311 using a completely assembled kit and human standards (Roche Diagnostics
Mannheim Germany) Antithrombin activity was also measured with a completely
assembled chromogenic test kit in the autoanalyser Cobas c311 and calibrated with a
canine pool plasma (n = 100)
The thrombin generation assay (Technothrombinreg TGA (Technoclone GbmH Vienna
Austria) determines thrombin generation based on monitoring of the fluorescence
generated by cleavage of a fluorogenic substrate by thrombin over time after activation
of the coagulation cascade by different concentrations of tissue factor and negatively
charged phospholipid in platelet poor plasma From the fluorescence development over
time the concentration of thrombin in the sample can be calculated using a thrombin
calibration curve The increase in thrombin concentration with time then allows to
calculate generation of thrombin in the sample and to plot thrombin values over time for
the whole coagulation process
The rotational thromboelastometry analysis with ROTEM delta device (Werfen Munich
Germany) was started approx 15 minutes after the blood collection To perform the
analysis and according to the automated ROTEM software 20 microl of recalcification reagent
(200 mmoll calcium chloride solution star-tem) and 20 microl of the respective activating
reagent (ex-tem in-tem) were pipetted into and carefully mixed in a pre-warmed cuvette
using the automated pipette provided in the device immediately afterwards 300 microl of
citrated whole blood (previously gently revolved) were added and mixed with the
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
51
reagents previously deposited Finally the cuvette was carefully placed and fixed to an
oscillating pin (oscillation movements on left and right through an angle of 475deg) in the
ROTEM delta device The interaction between blood cells and subsequently the bond
between fibrin and platelets promoted by the biomechanical changes in pH electrolytes
and temperature start the clot formation with its subsequent retraction and lysis The
magnitude of the resistance produced by the clot over the oscillating pin is quantitatively
represented by different parameters measured during the analysis and also qualitatively
in a graphic illustration (Bagge et al 2016 Crochemore et al 2017)
A chromogenic substrate test (Coatest Heparin Chromogenix-Instrumentation
Laboratory Haemochrom Diagnostica Essen Germany) was used for measurements of
UFH in the auto-analyser Cobas c311 The test application was created based on
manufacturers` instructions Different dilutions of the same batch of the commercial
sodium UFH preparation used in the experiment with normal canine pool plasma
(prepared from identical aliquots of 10 healthy adult dogs) served as standards for the
calibration of the measurement
28 Electron microscopy
Selected areas on the last third of the CVC (5ndash10 cm according with the CVC) with
macroscopically detectable thrombi were analysed and some of the CVCs were cut in
two different places The cut samples were preserved in a fixative medium composed of
1 glutaraldehyde and 4 formalin Once the sample was fixed it was prepared
according to the osmium thiocarbohydrazide procedure (OTOTO-procedure) (Malick et
al 1976) The fixed CVCs were dehydrated in seven series as follow first three series of
120 minutes each one on 30 50 and 70 ethanol respectively followed by two series
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
52
of 120 min each one on 90 ethanol and then two series of 24 hours each one on 100
ethanol After the dehydration process the samples were placed two times during 24
hours each one on isoamyl acetate and then placed on in a critical point drier Finally the
samples were coated with gold in a splutter-coater with 25 kV acceleration voltage in an
argon atmosphere with a current of 20 mA for one minute The samples were examined
in an EVO 15 scanning electron microscope (ZEISS International Carl Zeiss Jena
Germany) and photographed with magnifications of 600x and 6000x avoiding mechanical
artifacts (Toskala et al 1995) Thrombi on the external wall of the CVC were analysed
and measured using the software ImageJ (National Institutes of Health and the Laboratory
for Optical and Computational Instrumentation University of Wisconsin USA)
According to their extension (surface size) and thickness (related to the CVC wall
thickness in a cross sectional view) thrombi on the external wall and additionally within
the lumen of the CVC were each classified in four grades (Fig 2 Fig 1S [suppl
material]) In case of two cuts the highest degree of thrombosis detected at one of the two
sites was used for the final assessment
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
53
surface sizepercent of CVC wall thickness
Fig 2 Definition and illustration of different grades of central venous catheter induced
(extraluminal) thrombosis as detected using electron microscopy
Fig 1S (suppl material) Definition and illustration of different grades of intraluminal
thrombosis in central venous catheters as detected using electron microscopy
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
54
29 Statistical methods
Comparison between thrombus graduation either based on sonographic and ELMI
findings respectively was performed using the McNemar test with calculation of the
concordance index (κ) Standard normal distribution was assessed using the
Kolomogorov-Smirnov test
Due to non-normal distribution of part of data sets heparin activities and results of
haemostasis assays are reported using non-parametric variables (median minimumndash
maximum box and whisker plots) Heparin activities and results of haemostasis tests
between dogs with or without thrombosis were compared using Mann-Whitney-U test
3 Results
31 Sonographic detection of thrombus formation
Thrombus formation was sonographically detected in 829 patients during the study
period Based on examination on the final day in half of these eight patients (n = 4) the
thrombus formation was scored grade 1 in 3 dogs grade 2 and only in one grade 3 In all
of these 8 dogs this was the maximum grade during the study period One dog with fibrin
precipitates was excluded from further analysis because it could not be clearly assigned
to one of the groups In 78 cases thrombi were attached mainly to the venous wall
whereas only in one case was attached to the CVC external wall Seven of these 8 dogs
which developed thrombi received the standard heparin dosage (150 IUkg TID 41
[717] of dogs receiving this dosage) and only one the reduced heparin dosage (75 IUkg
TID 8 [112] of dogs receiving this dosage)
The time point of first detection of thrombus formation varied between days 3 and 11
(median day 8) In addition detritus (presumably composed mainly of fibrin) was
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
55
detectable in the lumen of the EJV in one further dog and within the lumen of the CVC
in further 5 dogs
32 Thrombus formation by electron microscopy
27 from 29 CVC catheter tips were analysed by electron microscopy 827 samples were
without detectable thrombus (grade 0) and 1927 with detectable thrombi 72 showed
thrombus formation of grade 1 427 of grade 2 and 827 of grade 3 There was no
concordance between thrombus graduation based on sonographic and the ELMI
graduation respectively The concordance index (McNemar test) was negative (κ = -
00718) indicating that the degree of agreement is less than by chance
Of the in total 19 dogs with detectable thrombus formation by electron microscopy 11
dogs received 150 IUkg TID (in total 16 dogs of these 27 dogs received that heparin
dosage) and 8 received 75 IUkg TID (in total 11 of these 27 dogs received that heparin
dosage 73 811) and Of the 12 patients with grade 2 and 3 thrombus formation 5 dogs
received 75 IUkg TID and 7 dogs 150 IUkg
The supplementary analysis of intraluminal thrombus formation revealed no detectable
thrombi in 7 cases and thrombus formation of grade 1 (lt 25 occlusion) in 11 cases of
grade 2 (25ndash50 ) in 3 cases and of grade 3 (gt 50 ) in 6 cases
33 Heparin activities and relationship to thrombus formation
Heparin activities varied remarkably in both dosage groups (Fig 3 ab) There was no
significant difference of heparin activities between blood samples collected via
venipuncture or from the CVC (Table 1S supplementary material) In addition there was
no significant difference between heparin activities in dogs with or without
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
56
sonographically or electron microscopically detectable thrombosis (Tables 1 and 2)
Table 1 refers to sonographically detectable thromboses and is based on median heparin
activities calculated from the measurements on day 1 and following days per patient
firstly for the total number of patients and secondly for the patients receiving 150 IUkg
TID In addition in the eight dogs heparin activities on the day of first thrombus detection
(023 IUml 019ndash036 IUml) were not significantly different from the median values
(Table 1 P = 05214 Wilcoxon test) Table 2 compares heparin activities (median values
of all days and results on the final day) between dogs with and without electron
microscopically detectable CVC-associated thrombi
Table 1S (Supplementary material) Heparin activities (IUml median values
minimumndashmaximum) measured in 27 dogs receiving 75 or 150 IUkg unfractionated
(standard) heparin TID subcutaneously 2 hours after the first injection on the day
Comparison of samples collected via puncture of the peripheral veins and those collected
from the central venous catheter (CVC) using the three-syringe-technique
Peripheral vein
puncture CVC
Mann-Whitney-U
test
Total number
(75 and 150
IUkg TID)
019
(00ndash108)
(n = 65)
023
(000ndash105)
(n = 65)
P = 03033
75 IUkg TID
010
(000ndash108)
(n = 25)
012
(000ndash023)
(n = 21)
P = 06907
150 IUkg
TID
030
(000ndash096)
(n = 40)
036
(000ndash105)
(n = 44)
P = 01223
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
57
Fig 3ab Heparin activities (box- and whisker plot arithmetic means) in dogs receiving
unfractionated heparin at a dosage of 75 IUkg BW tid (a) or 150 IUkg BW tid (b) two
hours after the first daily heparin administration
day 1 represents the first day after initiation of heparin treatment
only days were considered with at least results from 5 patients
Table 1 Heparin activities (IUml median values minimumndashmaximum) in 28 dogs
receiving 75 or 150 IUkg unfractionated (standard) heparin TID subcutaneously with
(grades 1ndash3) or without (grade 0) sonographically detectable thrombus Blood collections
were performed 2 hours after the first daily injection
Grade 0
(sonography)
Grades 1ndash3
(sonography)
Mann-Whitney U
test
Total number
(75 and 150 IUkg
TID)
020 (000ndash072)
(n = 20)
027 (018ndash059)
(n = 8)
P = 03212
150 IUkg TID 042 (000ndash072)
(n = 10)
031 (018ndash059)
(n = 7)
P = 04593
one dog with fibrin precipitates in the lumen of the jugular vein but without defined thrombus formation
was not considered because it could not be clearly assigned to one of the two groups
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
58
Table 2 Heparin activities (IUml median values minimumndashmaximum) in 27 dogs
receiving 75 or 150 IUkg unfractionated (standard) heparin TID subcutaneously with
(score 1ndash3) or without (score 0) detectable thrombus via electron microscopy
Grade 0
(electron
microscop)
(n = 8)
Grades 1ndash3
(electron
microscop)
(n = 19)
Mann-Whitney U
test
Median value of
different days 028 (005ndash060)
022 (000ndash072)
P = 05770
Final day 020 (001ndash045)
015 (000ndash105)
P = 06706
34 Relationship between haemostasis tests and thrombus formation
From the initially performed haemostasis tests high fibrinogen concentrations and low
maximum lysis values as measured by the ROTEM delta device (using the ex-tem
reagent) were associated with sonographically detectable thrombi (Tab 3) Additional
analyses based on the median values of different days for individual patients revealed that
low maximum lysis values as measured by the ROTEM delta device (using the in-tem
reagent) were associated with sonographically detectable thrombi (maximum lysis score
0 850 [100ndash175 ] score 1ndash3 375 [100ndash900 ] median [minimumndash
maximum] P = 00166) but no other test Comparison of the results of haemostasis tests
on the final day between dogs with and without sonographically detectable thromboses
did not show any significant difference (P gt 005)
Comparison of results of haemostasis tests on the final day in dogs with detectable
thrombosis via ELMI and dogs without significant thrombus formation showed several
significant differences including lower fibrinogen concentrations and longer clotting
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
59
times (ex-tem reagent) and clot formation times (ex-tem and in-tem reagents) using the
ROTEM delta analyser in general expressing a less coagulable status (Tab 4) If values
of dogs graded 0 and 1 (n =15) are compared to those of grades 2 and 3 (n = 12) the last-
mentioned had longer clotting times (ROTEM delta in-tem reagent) as well as longer clot
formation times and lower alpha angles (ROTEM delta in-tem reagent) (P lt 005 results
not shown)
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
60
Table 3 Comparison of initial results of haemostasis parameters (median values
minimumndashmaximum in dogs with (grades 1ndash3) and without (grade 0) sonographically
detectable central venous catheter-induced thromboses
Parameter Unit
Sonographic
thrombus grade
P-value
(Mann-Whitney
U test)
0
(n = 20)
123
(n = 8)
Haematocrit 355 (228ndash641)
382 (245ndash499)
06111
Platelet count x103microl 292 (420ndash483)
218 (860ndash458)
02320
Prothrombin time 944 (359ndash169)
896 (471ndash136)
05758
Activated partial thromboplastin
time s 128
(955ndash249) 140
(117ndash164) 02322
Thrombin time s 129 (121ndash178)
141 (650ndash160)
02028
Fibrinogen gl 448 (153ndash770)
747 (153ndash116)
00235
Antithrombin activity 639 (289ndash107)
670 (464ndash746)
05249
D-dimers microgml 006 (000ndash148)
015 (003ndash372)
03997
Thrombin generation assay nmol 130 (000ndash365)
912 (190ndash277)
08787
Clotting time (ROTEM ex-tem) s 420 (290ndash930)
430 (170ndash159)
07989
Clot formation time (ROTEM ex-
tem) s 665
(310ndash143) 470
(240ndash800) 00633
Alpha angle (ROTEM ex-tem) degrees 795 (620ndash840)
815 (750ndash860)
01667
Max clot firmness (ROTEM ex-
tem) mm 750
(570ndash860) 775
(700ndash830) 01753
Max clot elasticity (ROTEM ex-
tem) - 299
(131ndash619) 347
(235ndash484) 01544
Maximum lysis (ROTEM ex-tem) 135 (300ndash290)
400 (000ndash800)
00037
Clotting time (ROTEM in-tem) s 208 (840ndash810)
249 (720ndash371)
05085
Clot formation time (ROTEM in-
tem) s 835
(370ndash634) 745
(390ndash235) 08988
Alpha angle (ROTEM in-tem) degrees 745 (250ndash820)
750 (500ndash820)
09797
Max clot firmness (ROTEM in-
tem) mm 700
(540ndash810) 740
(610ndash770) 03587
Max clot elasticity (ROTEM in-
tem) - 235
(118ndash424) 287
(156ndash332) 04764
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
61
Maximum lysis (ROTEM in-tem) 850 (000ndash210)
350 (000ndash220)
01327
Table 4 Comparison of results of haemostasis parameters (median values minimumndash
maximum) on the last day in dogs with (grades 1ndash3) and without (grade 0) central venous
catheter-associated thrombus formation as detected by electron microscopy
Parameter Unit
Thrombus grade
(ELMI)
P-value
(Mann Whitney
U test)
0
(n = 8)
123
(n = 19)
Haematocrit 322 (241ndash454)
330 (216ndash510)
05953
Platelet count 103microl 446 (321ndash592)
268 (101ndash675)
00558
Prothrombin time 145 (104ndash264)
120 (561ndash148)
00462
Activated partial thromboplastin
time s 123
(109ndash140) 136
(109ndash188) 00665
Thrombin time s 129 (121ndash178)
141 (650ndash160)
02028
Fibrinogen gl 465 (275ndash866)
347 (189ndash992)
00491
Antithrombin activity 793 (448ndash981)
692 (398ndash889)
04572
D-dimers microgml 018 (000ndash173)
007 (000ndash118)
01920
Thrombin generation assay nmol 501 (000ndash160)
93 (000ndash287)
09562
Clotting time (ROTEM ex-tem) s 360
(270ndash410) 400
(310ndash620) 00238
Clot formation time (ROTEM ex-
tem) s 395
(120ndash740) 650
(240ndash174) 00275
Alpha angle (ROTEM ex-tem) degrees 835 (760ndash880)
790 (580ndash850)
00484
Max clot firmness (ROTEM ex-
tem) mm 780
(700ndash850) 720
(550ndash860) 00586
Max clot elasticity (ROTEM ex-
tem) - 346
(239ndash555) 254
(121ndash637) 00495
Maximum lysis (ROTEM ex-tem) 105 (200ndash190)
110 (000ndash380)
03796
Clotting time (ROTEM in-tem) s 178 (100ndash222)
201 (139ndash554)
00525
Clot formation time (ROTEM in-
tem) s 435
(330ndash111) 790
(350ndash882) 00209
Alpha angle (ROTEM in-tem) degrees 815 (710ndash830)
740 (240ndash830)
00191
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
62
Max clot firmness (ROTEM in-
tem) mm 785
(660ndash810) 690
(270ndash840) 00586
Max clot elasticity (ROTEM in-
tem) - 362
(192ndash434) 218
(370ndash507) 00559
Maximum lysis (ROTEM in-tem) 950 (200ndash190)
800 (100ndash180)
06890
4 Discussion
The main result of our study is that under the study conditions part of the dogs developed
CVC-associated thromboses However only a very low part of these thromboses were
obviously of a clinically significant degree regarding the amount of occlusion of the EJV
and none of the dogs had a symptomatic thrombosis whose definition includes presence
of clinical signs such as localised swelling pain and redness (Boersma et al 2008 Lee
and Kamphuisen 2012) In the present study we particularly refer to the extraluminal
thromboses with regard to the assessment of the antithrombotic management
One major problem of the statistical analysis and result presentation was that the two
methods used to define the occurrence and degree of thrombus formation (ultrasound and
ELMI) revealed discrepant results Therefore we performed a statistical analysis
independently regarding thrombus formation with either of the two methods It is well
known that ELMI which has already been used to detect CVC induced thromboses in
humans (Lucas et al 2017) and experimental animals (Xiang et al 1998 Xiang et al
2001) is a very sensitive diagnostic technique which allows the ex vivo diagnosis of very
small or fibrin rich thrombi that perhaps cannot be detected by ultrasonography or
venography during the in vivo evaluation of patients (Xiang et al 2001) A major reason
for the discrepant results between the two methods used to detect CVC associated
thromboses is that different CVC segments are evaluated Using ultrasonography it was
only possible to evaluate the EJV in the neck area of the patient and thereby only its
proximal third In contrast ELMI assessed mainly the CVC tip and a human study
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
63
revealed that even different positions of the catheter tip are associated with various risks
for development of CVC associated thrombosis (Ballard et al 2016) One additional point
to consider which also may contribute to the discrepancy is that it is possible that thrombi
developed on the CVC surface can be detached during the CVC removal from the vein
and thereby before its analysis by ELMI (Xiang et al 2001)
The contrast venography technique is used as gold standard to confirm thrombosis in
human medicine However this diagnostic technique is normally used only in the case of
a strong clinical suspicion of venous thrombosis that cannot be detected by
ultrasonography (Laurenson et al 2010 Langston and Eatroff 2018) Our study protocol
with privately owned clinic patients did not allow to use this method In a study performed
in 44 human patients requiring a CVC (Koumlksoy et al 1995) a sensitivity of 94 and a
specificity of 96 were determined for the colour Doppler ultrasound diagnosis when it
was compared with the diagnosis performed with the contrast venography technique and
that makes the colour Doppler ultrasound an accurate diagnosis method to detect CVC
related thrombosis even in humans (Koumlksoy et al 1995 van Rooden et al 2003)
The incidences of CVC related thrombosis detected by colour Doppler ultrasound of the
internal jugular vein reported in 3 human studies were 2 30 and 56 (Harter et al
2002 Lordick et al 2003 Wu et al 1999) and although with a wide range the median
value of these rates (30 ) is very well suited to the incidence of 28 (829) which we
calculated for our canine study based on sonographic findings The wide variation of
incidences of CVC related thrombosis in different human studies can in general be
explained by differences regarding the type and entry site of the CVC (jugular femoral
subclavian axillary cephalic vein) the time period with the CVC in place the thrombus
detection technique (venography or ultrasonography comment in the cited 3 studies
consistently the last-mentioned method was used) and criteria for diagnosis of
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
64
thrombosis the duration of follow up diseases and presence of predisposing factors in
these patients (eg previous venous thrombosis history presence of cancer) and finally
prophylactic treatment with anticoagulants (van Rooden et al 2005 Geerts 2014 Liu et
al 2015)
Different pathomechanisms contribute to the development of CVC induced thromboses
During the CVC insertion the puncture of the skin on the CVC insertion site and the
rupture of the blood vessels integrity activate the coagulation cascade and the CVC acts
as a matrix for adherence and accumulation of fibrin and cellular components (Smith et
al 2012) The attachment of proteins and blood cells to the internal andor external wall
of the CVC can lead to a partial or complete occlusion of the catheter lumen and
sometimes to a partial or complete obstruction of the blood flow andor
thromboembolism (Langston and Eatroff 2018) There is a strong interrelationship
between thrombus and biofilm formation (Smith et al 2012) Prevention of thrombus
formation has thereby also relevance regarding the increased risk for microbial
colonisation of the catheter (Boersma et al 2008) leading at least in humans to an
increased number of CVC related systemic infections (Lordick et al 2003)
In this context it has to be considered that CVC produced from polyurethane which are
routinely used in our clinic and thereby in the study are usually associated with a
relatively high thrombosis potential and thereby definitely provide an effective test for
an antithrombotic regimen Silicone catheters have a significantly reduced risk for
thrombotic complications but this advantage has to be purchased by a higher rate of
mechanical failure (eg rupture of the catheter) (Wildgruber et al 2016) Promising
experimental approaches work on surface modifications of the standard catheters For
example one group was able to demonstrate that a coating of the surface of the catheter
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
65
with a zwitterionic polymeric sulfobetaine showed a significant reduction of thrombus
formation in vitro with human blood and in an in vivo canine model (Smith et al 2012)
Due to ethical reasons we did not performed a placebo-controlled study However
interestingly not all human studies report systemic anticoagulation and part of the human
studies was even placebo-controlled (review Lee and Kamphuisen 2012) Obviously
there is no consensus in the available human literature regarding the beneficial use of
antithrombotic prophylaxis to prevent CVC related thrombosis Several studies did not
find a statistically significant difference in the incidence of CVC related thrombosis
between patients receiving the antithrombotic prophylaxis compared with the control
groups without systemic anticoagulation (Verso et al 2005 P = 035 Karthaus et al
2006 P gt 005 Niers et al 2007 P = 049) whereas in other studies a lower incidence
of CVC related thrombosis was found in the group receiving anticoagulants as
prophylaxis compared with the control groups (Abdelfeki et al 2004 150 vs 126
P = 003 De Cicco et al 2009 400 vs 526 P = 005)
Although it is well known that subcutaneously administered UFH has a limited and
variable bioavailability and thereby leads to unpredictable blood activities (Hirsh and
Raschke 2004) the extremely wide range of blood activities after a defined dosage in our
study was relatively unexpected Blood was collected 2 hours after the first UFH
injection and thereby at the time of suspected maximum heparin activity considering the
relatively low UGH dosage (Mischke and Jacobs 2001) The cited study was performed
on healthy experimental dogs Possible additional influencing factors in critical ill
patients include circulatory insufficiency which may reduce and delay absorption from
subcutanenous tissue decreased clearance function of the liver and kidneys as well as
altered blood plasma concentrations of heparin binding plasma proteins such as
fibrinogen and platelet factor 4 and alterations of the heparin binding capacity of
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
66
endothelial cells which altogether are reasons for a great inter- but also intrapatient
variability (Hirsh and Raschke 2004) In individual patients this can lead to heparin
resistance It has also to be considered that antithrombin activities which are necessary
for an adequate heparin effect (Bjoumlrk and Lindahl 1982) were reduced in our patients
Availability of approved blood tests which can predict the risk of development of CVC
associated thrombosis already at the moment of the CVC placement are important
(Boersma et al 2016) One of the most interesting results was that dogs which developed
sonographically detectable thrombi had nearly twofold higher fibrinogen levels Well in
agreement with our results one study performed in anticoagulated human patients
demonstrated that high fibrinogen levels were associated with an increased risk of
perioperative venous thromboembolism (P = 0004) (Kato et al 2016) The cited study
does not exclusively refer to CVC associated thrombosis but 76 (1621) of the venous
thromboses were suggested to be associated with CVCs Fibrinogen measurement a test
which is widely available is therefore a useful and practicable indicator of an increased
risk for CVC induced thromboses and may advice the clinician to treat these patients
especially carefully (eg with low molecular weight heparin) In the study performed by
Boersma et al (2016) in 168 human patients without indication of anticoagulation
prophylaxis elevated leukocyte counts high levels of plasminogen activator inhibitor
and of coagulation factor VIII were associated with an increased incidence of
symptomatic CVC related thrombosis diagnosed by ultrasonography A limitation of that
study is that all measurements were performed on blood samples taken after CVC
placement Interestingly in our study ROTEM results were partly associated with
thrombus formation which reflects the well-known fact that results of viscoelastic
measurements are a good indicator of hypercoagulability and thromboembolic events
(Harahsheh and Ho 2018) It is of further interest and possibly unexpeted that on the final
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
67
day of examination dogs with ELMI signs of thromboses had partly lower coagulation
activities than dogs without thromboses Due to the low number of dogs without
detectable thrombi using ELMI this implausible result has to be interpreted with caution
One possible explanation could be that temporary hypercoagulability in these dogs may
have finally resulted in consumption of haemostasis substrates
Limitations of the present study were as a consequence of the study design based on
clinical patients the relatively low total number of patients inclusion of different UFH
treatment regimens and the lack of a control group
5 Conclusions
The results of the present study indicate that the used UFH regimen was not completely
effective to prevent CVC-associated thromboses which were detectable in 28 [829]
of the patients by ultrasound However severe thromboses (high grades) are relatively
rare in intensive care canine patients receiving a routine anticoagulant treatment Because
heparin plasma activities were not different in dogs with or without CVC-associated
thrombus formation other local and systemic factors must play a significant role as well
A high fibrinogen concentration and low maximum lysis (ROTEM ex-tem) may be
useful indicators for an increased thrombotic risk in individual patients which may
require special anticoagulatory treatment
6 Acknowledgments
Jose Mauricio Pereira received a scholarship from the Universidad Nacional de Costa
Rica (reference code JB-C-1107-2016)
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
68
7 References
1 Abdelkefi A Ben Othman T Kammoun L Chelli M Ben Romdhane N Kriaa A Ladeb S
Torjman L Lakhal A Achour W Ben Hassen A Hsaiumlri M Ladeb F Ben Abdeladhim A
2004 Prevention of central venous line-related thrombosis by continuous infusion of low-dose
unfractionated heparin in patients with haemato-oncological disease A randomized controlled trial
Thromb Haemost 92 654ndash661
2 Adamantos S Brodbelt D Moores A L 2010 Prospective evaluation of complications associated
with jugular venous catheter use in a veterinary hospital J Small Anim Pract 51 254ndash257
3 Bagge A Schoumltt U Kander T 2016 Effects of naturopathic medicines on Multiplate and ROTEM
a prospective experimental pilot study in healthy volunteers BMC Complement Altern Med 16 64
4 Ballard DH Samra NS Gifford KM Roller R Wolfe BM Owings JT 2016 Distance of the
internal central venous catheter tip from the right atrium is positively correlated with central venous
thrombosis Emerg Radiol 23 269ndash273
5 Boersma RS Jie KS Verbon A van Pampus EC Schouten HC 2008 Thrombotic and
infectious complications of central venous catheters in patients with hematological malignancies Ann
Oncol 19 433ndash442
6 Boersma R S Hamulyak K van Oerle R Tuinenburg A Ten Cate-Hoek AJ Schouten HC
2016 Biomarkers for prediction of central venous catheter related-thrombosis in patients with
hematological malignancies Clin Appl Thromb Hemost 22 779ndash784
7 Bjoumlrk I Lindahl U 1982 Mechanism of the anticoagulant action of heparin Mol Cell Biochem
48 161ndash182
8 Claude A Riedesel D Riedesel E 2010 Electrocardiography-guided and retrospective analysis of
central venous catheter placement in the dog Vet Anaesth Analg 37 97ndash105
9 Comerlato PH Rebelatto TF Santiago de Almeida FA Klein LB Boniatti M M Schaan
DB Rados VD 2017 Complications of central venous catheter insertion in a teaching hospital
Rev Assoc Med Bras 63 613ndash620
10 Crochemore T Piza FMT Rodriacutegues RDR Guerra JCC Ferraz LJR Correcirca TD 2017
A new era of thromboelastometry Einstein (Sao Paulo) 15 380ndash385
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
69
11 De Cicco M Matovic M Balestreri L Steffan A Pacenzia R Malafronte M Fantin D
Bertuzzi CA Fabiani F Morassut S Bidoli E Veronesi A 2009 Early and short‐term
acenocumarine or dalteparin for the prevention of central vein catheter‐related thrombosis in cancer
patients a randomized controlled study based on serial venographies Ann Oncol 20 1936ndash1942
12 Evans NS Ratchford EV 2018 Catheter related-venous thrombosis Vasc Med 23 411ndash413
13 Geerts W 2014 Central venous catheterndashrelated thrombosis Hematology Am Soc Hematol Educ
Program 2014 306ndash311
14 Harahsheh Y Ho KM 2018 Use of viscoelastic tests to predict clinical thromboembolic events A
systematic review and meta‐analysis Eur J Haematol 100 113ndash123
15 Harter C Salwender HJ Bach A Egerer G Goldschmidt H Ho AD 2002 Catheter-related
infection and thrombosis of the internal jugular vein in hematologic-oncologic patients undergoing
chemotherapy a prospective comparison of silver-coated and uncoated catheters Cancer 94 245ndash251
16 Hirsh J Raschke R 2004 Heparin and low-molecular-weight heparin The Seventh ACCP
Conference on Antithrombotic and Thrombolytic Therapy Chest 126 (Suppl) 188S-203S
17 Hundley D Brooks A Thomovsky E Jhonson P Freeman L Schafbuch R Heng HG Moore
G 2018 Comparison of ultrasound-guided and landmark-based techniques for central venous
catheterization via the external jugular vein in healthy anesthetized dogs Am J Vet Res 79 628ndash
636
18 Kato F Takeuchi H Matsuda S Kawakubo H Omori T Kitagawa Y 2016 Incidence of and
risk factors for venous thromboembolism during surgical treatment for esophageal cancer a single-
institution study Surg Today 46 445ndash452
19 Karthaus M Kretzschmar A Kroning H Biakhov M Irwin D Marschner N Slabber C
Fountzilas G Garin A Abecasis NG Baronius W Steger GG Sudhoff T Giorgetti C
Reichardt P 2006 Dalteparin for prevention of catheter‐related complications in cancer patients with
central venous catheters final results of a double‐blind placebo‐controlled phase III trial Ann Oncol
17 289ndash296
20 Koumlksoy C Kuzu A Kutlay J Erden I Ozcan H Ergicircn K 1995 The diagnostic value of colour
Doppler ultrasound in central venous catheter related thrombosis Clin Radiol 50 687-689
21 Kucher N 2011 Clinical practice Deep-vein thrombosis of the upper extremities N Engl J Med
364 861ndash869
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
70
22 Langston CE Eatroff AE 2018 Hemodialysis catheter-associated fibrin sheath in a dog J Vet
Emerg Crit Care 28 366ndash371
23 Laurenson MP Hopper K Herrera MA Johnson EG 2010 Concurrent diseases and conditions
in dogs with splenic vein thrombosis J Vet Intern Med 24 1298ndash1304
24 Lee AYY Kamphuisen PW 2012 Epidemiology and prevention of catheter‐related thrombosis in
patients with cancer J Thromb Haemost 10 1491ndash1499
25 Liu G Fu ZQ Zhu P Li SJ 2015 Central venous catheter-related thrombosis in senile male
patients New risk factors and predictors J Huazhong Univ Sci Technolog Med Sci 35 445ndash449
26 Lordick F Hentrich M Decker T Hennig M Pohlmann H Hartenstein R Peschel C 2003
Ultrasound screening for internal jugular vein thrombosis aids the detection of central venous catheter-
related infections in patients with haemato-oncological diseases a prospective observational study Br
J Haematol 120 1073ndash1078
27 Lucas TC Silva EED Souza DOD Santos ARD Lara MO 2017 Microstructural
evaluation by confocal and electron microscopy in thrombi developed in central venous catheters Rev
Esc Enferm USP 51 1ndash9
28 Malick L Wilson R Stetson D 1976 Modified thiocarbo- hydrazide procedure for scanning
electron microscopy routine use for normal pathological and experimental tissue Stain Technol 50
265ndash269
29 Mischke R Jacobs C 2001 The monitoring of heparin administration by screening tests in
experimental dogs Res Vet Sci 70 101ndash108
30 Niers TM Di Nisio M Klerk CP Baarslag HJ Buller HR Biemond BJ 2007 Prevention
of catheter‐related venous thrombosis with nadroparin in patients receiving chemotherapy for
hematologic malignancies a randomized placebo‐controlled study J Thromb Haemost 5 1878ndash
1882
31 Reminga C Silverstein D Drobatz K Clarke D 2018 Evaluation of the placement and
maintenance of central venous jugular catheters in critically ill dogs and cats J Vet Emerg Crit Care
28 232ndash243
32 Smith RS Zhang Z Bouchard M Li J Lapp HS Brotske GR Lucchino DL Weaver D
Roth LA Coury A Biggerstaff J Sukavaneshvar S Langer R Loose C 2012 Vascular
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
71
catheters with a nonleaching poly-sulfobetaine surface modification reduce thrombus formation and
microbial attachment Sci Transl Med 4 153ra132 doi101126scitranslmed3004120
33 Toskala E Nuutinen J Rautiainen M 1995 Scanning electron microscopy findings on human
respiratory cilia in chronic sinusitis and in recurrent respiratory infections J Laryngol Otol 109 509ndash
514
34 Van Rooden CJ Tesselaar ME Osanto S Rosendaal FR Huisman MV 2005 Deep vein
thrombosis associated with central venous catheters - a review J Thromb Haemost 3 2409ndash2419
35 Verso M Agnelli G Bertoglio S Di Somma FC Paoletti F Ageno W Bazzan M Parise P
Quintavalle R Naglieri E Santoro A Imberti D Soraru M Mosca S 2005 Enoxaparin for the
prevention of venous thromboembolism associated with central vein catheter a double‐blind placebo‐
controlled randomized study in cancer patients J Clin Oncol 23 4057ndash4062
36 Villalta-Garciacutea P Loacutepez-Herranz M Mazo-Pascual S Honrubia-Fernaacutendez T Jaacutentildeez-Escalada
L Fernaacutendez-Peacuterez C 2015 Reliability of blood test results in samples obtained using a 2-mL discard
volume from the proximal lumen of a triple-lumen central venous catheter in the critically ill patient
Nurs Crit Care 22 298ndash304
37 Vose J Odunayo A Price JM Daves M Schildt JC Tolbert MK 2019 Comparison of
heparinized saline and 09 sodium chloride for maintaining central venous catheter patency in healthy
dogs PeerJ 7 e7072 httpsdoiorg107717peerj7072
38 Wildgruber M Lueg C Borgmeyer S Karimov I Braun U Kiechle M Meier R Koehler M
Ettl J Berger H 2016 Polyurethane versus silicone catheters for central venous port devices
implanted at the forearm Eur J Cancer 59 113ndash124 doi101016jejca201602011
39 Wu X Studer W Skarvan K Seeberger MD 1999 High incidence of intravenous thrombi after
short-term central venous catheterization of the internal jugular vein J Clin Anesth 11 482ndash485
40 Xiang DZ Verbeken EK Van Lommel AT Stas M De Wever I 1998 Composition and
formation of the sleeve enveloping a central venous catheter J Vasc Surg 28 260ndash271
41 Xiang DZ Verbeken EK Van Lommel AT Stas M De Wever I 2001 Sleeve-related
thrombosis a new form of catheter-related thrombosis Thromb Res 104 7ndash14
Chapter 4 General discussion
72
Chapter 4 General discussion
Incidence of CVC related thrombosis
In our study the incidence of CVC related thrombosis detected by ultrasound in critical
care canine patients was 28 To the best of the authoracutes knowledge similar studies
performed in dogs are rare in the available veterinary literature In one study performed
in 81 dogs and 12 cats (Adamantos et al 2010) an incidence of 2 was found for vein
thrombosis related to CVC This low incidence can be due to a lacking of systematic
ultrasound evaluations of the jugular vein containing the CVC In the mentioned study in
dogs and cats the vein thrombosis was confirmed by palpation or ultrasound presumably
after the presentation of clinical symptoms however in many cases as in our study
thrombus into the jugular vein can be developed without manifestation of clinical
symptoms what makes a routinely ultrasound evaluation of the vein containing the CVC
very important Our results are well aligned with the findings in humans in which a
median incidence of 30 was found for thrombosis related to CVC based on 25 studies
performed in patients requiring a CVC (review article van Rooden et al 2005) In the
mentioned 25 human studies the incidence of CVC related thrombosis varied within a
wide range (2ndash67 ) according to factors related to (1) the CVC (eg material type
entry site [jugular femoral subclavian axillary cephalic vein] time period in place (2)
diagnostic thrombus detection technique (ultrasonography or venography) and criteria
(3) patient collective (diseases and presence of predisposing factors [eg previous venous
thrombosis history presence of cancer] and (4) prophylactic antithrombotic treatment
(Geerts 2014 Liu et al 2015 van Rooden et al 2005)
The incidence of CVC related thrombosis detected by colour Doppler ultrasound in our
study fits also with the median incidence (30 ) found in three studies performed in
humans in which colour Doppler ultrasound evaluations of the jugular vein containing a
Chapter 4 General discussion
73
CVC were performed (Harter et al 2002 Lordick et al 2003 Wu et al 1999) In these
three studies the individual incidence of CVC related thrombosis was 2 (Harter et al
2002) 30 (Lordick et al 2003) and 56 (Wu et al 1999) The high thrombus
incidence reported in the study performed by Wu et al (1999) could be due to the criteria
for diagnosis of thrombosis because in that study both fibrin sleeves and compact
thrombi were considered as cases of thrombosis related to CVC The incidence for
compact thrombi was 25 which is more similar to our results In the study performed
by Harter et al (2002) the CVC related thrombosis incidence (2 ) was very low
compared with the incidence found in our study In that study the jugular veins were
examined just before or less than 24 hours after CVC removal (median period of CVC
use = 1025 days) In our study the ultrasound evaluations were performed before the
CVC placement (day 0) and then on days 1 3 5 etc until the CVC removal (median
period of CVC use = 7 days median interval until thrombus detection = 7 days) In our
study we detected thrombus formation as early as on the 3rd day so it could be possible
that in the cited study possibly only temporarily existing thrombi could have been missed
by ultrasonography in the final stage and thereby for the incidence calculation However
this scenario is not very likely because results of the present study show that
sonographically detectable thrombi during the observation period had in all cases their
maximum grade on the final day
In the study performed by Lordick et al (2003) the CVC related thrombosis incidence
determined for 43 human patients was very similar to our study and in both studies only
compact thrombi detectable by ultrasound were considered for assessment of the
incidence In the mentioned study all the human patients were screened for the presence
of thrombosis every 4 days after CVC insertion with real time B-mode ultrasound
(Lordick et al 2003)
Chapter 4 General discussion
74
In our study 70 of CVCs analysed by ELMI showed thrombi of grade 1ndash3 on the
external CVC wall To the best of the authoracutes knowledge studies about incidence of CVC
related thrombosis on the outside surface of the CVC detected by ELMI cannot be found
in the human and small animal literature In one study performed in humans (Lucas et al
2017) thrombus formation was analysed by ELMI in 78 central venous catheters That
study focussed on microstructural assessment of thrombus formation inside the distal
openings of the CVC which were found in 85 of the evaluated CVCs The percentage
of occurrence of thrombosis in the mentioned study in humans was nearly similar to the
incidence of intraluminal thrombus formation as detected in the presented study by ELMI
(74 ) In general intraluminal thrombi were not specially considered in the present
study because they mainly express the efficacy of the flushing technique rather than the
effectivity of the antithrombotic treatment In one study performed in rats the
microstructure of thrombi developed in the jugular vein containing the CVC was also
evaluated by ELMI (Xiang et al 1998) In the mentioned study pericatheter thrombosis
was detected on the proximal section of the CVC in 100 of rats (n = 12) after 1 and 3
days of catheterisation The analyses of the CVCs placed in rats were performed with the
catheter inside the vein in contrast to our study where the CVC was removed from the
jugular vein for the respective analysis Xiang et al (2001) hypothesised in one study
performed in rabbits and rats in which CVCs were placed in the jugular vein that fibrin
rich thrombi associated to the CVC can be detached during the catheter removal and this
could explain the difference between the thrombosis incidence assessed in our study and
that of the study performed by Xiang et al (1998)
In our study the incidence of CVC related thrombosis detected by ultrasonography in the
lumen of the EJV did not correlate with the incidence of CVC related thrombosis detected
by ELMI on the external wall of the CVC This was mainly due to the fact that the CVC
Chapter 4 General discussion
75
segment evaluated with both methods was different Using ultrasonography it was only
possible to evaluate the EJV in the neck area of the patient and thereby only the proximal
third of the CVC In contrast with the ELMI analysis only the distal section of the CVC
(last third) was evaluated because macroscopically detected thrombi mainly developed
near the CVC tip which was routinely positioned at the junction of the cranial cava vein
with the right atrium In a retrospective review of medical records of 169 human patients
requiring a CVC (Ballard et al 2016) the position of the CVC tip was determined by
radiologic evaluation and it was determined that the catheter tip position can be a
predisposing factor for central venous thrombosis development Thereby it could be of
interest to evaluate in further studies in dogs if a high incidence of thrombosis detected
by ELMI as was shown in our study could be related to the CVC tip position
In our study polyurethane catheters were tested which are widely used and are the
standard CVCs used in the Small Animal Clinic University of Veterinary Medicine
Hannover In vivo and in vitro studies have assessed the thrombogenicity of different
catheter materials (Borow and Crowley 1985 Smith et al 2012) In general terms
polyurethane catheters (central or peripheral) had shown a higher thrombogenicity
compared with coated (eg with hydromer) and silicone catheters Due to this property
of polyurethane catheters they can be supposed as a good test system for antithrombotic
regimens as evaluated in our study On the other hand despite silicone catheters have been
associated with a lower thrombogenicity their mechanical flexibility could be a
disadvantage in small clinical practice due to the increased risk of rupture detected even
in humans (Wildgruber et al 2016)
Chapter 4 General discussion
76
Heparin thromboprophylaxis for CVC related thrombosis
The occurence of CVC related thrombosis detected by colour Doppler ultrasound and
ELMI in our study demonstrates that the UFH in the given dosage was not completely
effective to prevent CVC related thrombosis in canine intensive care patients In human
medicine LMWH is commonly used in prophylactic anticoagulation regimens however
studies performed in human patients requiring a CVC and receiving a prophylactic
anticoagulation (with LMWH or UFH) did not achieve consensus regarding the efficacy
of prophylactic heparin to prevent CVC related thrombosis (Abdelfeki et al 2004
Lordick et al 2003 Wu et al 1999) To the best of the authoracutes knowledge studies
performed in dogs evaluating systemic prophylactic anticoagulation to prevent CVC
related thrombosis cannot be found However according to the evidence found in the
human literature and the results of our study further investigations using comparative
groups with different drugs dosages andor administration routes are required to establish
an effective antithrombotic treatment to prevent CVC related thrombosis
The plasma heparin activities measured in canine patients in our study varied significantly
after a defined subcutaneous dosage The limited and variable bioavailability of SC
administered UFH is well documented in humans (Hirsh and Raschke 2004)
Considerable variations of the circulatory status of the liver- and kidney function and
concentration of heparin-binding proteins in intensive care patients obviously amplify
this phenomenon leading to an extremely high inter-individual and intra-individual
variation of the heparin blood levels in our patients A different application route (IV)
may have reduced the influence of part of these factors and therefore could have led to
more predictable UFH blood levels However continuous infusion requires more
technical equipment and is also associated with a wide range of heparin activities in
canine patients (Scott et al 2009)
Chapter 4 General discussion
77
Prediction of CVC related thrombosis
Of 7 haemostasis tests (including ROTEM with 12 different parameters [6 for each
reagent ex-tem and in-tem]) evaluated as possible predictors of CVC related thrombosis
in our study initial values of high fibrinogen and low maximum lysis (ROTEM ex-tem)
were associated with sonographically detectable thrombus formation Some studies in
humans have established the association of laboratory biomarkers as predictors of risk
thrombosis (Boersma et al 2016 Cheng et al 2013 Kato et al 2016 Liu et al 2015)
One study performed in anticoagulated human patients showed ndash well in agreement with
our results ndash that high fibrinogen levels were associated with an increased risk of
perioperative venous thromboembolism (P = 0004) (Kato et al 2016) The cited study
does not exclusively refer to CVC associated thrombosis but 762 (1621) of the
venous thromboses were suggested to be associated with CVCs Fibrinogen concentration
measurements are normally available in the human and veterinary practice and the
association of this parameter with a high risk of development of CVC related thrombosis
can be useful to choose special antithrombotic treatment in individual critical care patients
to prevent such severe complications
Thrombin generation assay (TGA) abnormalities such as higher velocity and endogenous
thrombin potential (ETP) values measured initially ie before any anticoagulation
therapy have been associated with a higher thromboembolism risk in general (ie without
specific association with CVCs) in one study performed on 105 human patients (Espitia
et al 2017) In contrast in our study the initial TGA assay results did not show an
association with formation of CVC related thrombosis
The ROTEM parameters evaluated in the present study were partially associated with
formation of CVC thrombosis detected by colour Doppler ultrasound This finding
confirms the results obtained in humans in which ROTEM measurements are regarded as
Chapter 4 General discussion
78
a valuable indicator of hypercoagulability (Harahsheh and Ho 2018 Tuumlrk et al 2018)
However in our study individual ROTEM parameters showed partly lower coagulation
activities in dogs with CVC related thrombosis detected by ELMI at the final day of
evaluation Because the number of patients without signs of thrombosis detected by ELMI
was low compared with those who showed signs these implausible results should be
interpreted carefully The usefulness of ROTEM analysis which has also many other
indications demonstrates that is was worth to perform a detailed methodological approval
of the device including the establishment of reference values in the preliminary study
Conclusion
In conclusion the prethrombotic or thrombotic changes detected in our study did not
correlate between ultrasonography and ELMI however both methods show accurately
signs of CVC related thrombosis The UFH in the given dosage was not completely
effective to prevent CVC induced thrombus formation in canine intensive care patients
Plasma heparin activities varied significantly after a defined subcutaneous dosage
However plasma heparin activities were not different between dogs with and without
detectable CVC associated thromboses Initial high fibrinogen values and low maximum
lysis (ROTEM ex-tem) values were associated with sonographically detectable thrombi
and may be useful as indicators for an increased thrombotic risk
Finally our study in canine intensive care patients is to the best of the authoracutes
knowledge the first study in the available veterinary literature performed to evaluate the
efficacy of an antithrombotic treatment with UFH to prevent CVC related thrombosis
Chapter 5 Summary
79
Chapter 5 Summary
Central venous catheter induced thrombosis in dogs ndash occurrence under an
antithrombotic treatment regimen and possible indicators
Joseacute Mauricio Pereira Mora
Central venous catheters (CVC) are of importance for intensive care management of
canine patients Thrombosis of the jugular vein is a severe complication related to the
CVC use However only little is known about the incidence of CVC-associated
thromboses in dogs and the necessity and efficacy of prophylactic antithrombotic
regimens
Therefore the aims of the present study were (1) to determine the incidence of catheter-
induced thrombosis in canine intensive care patients (2) to evaluate the efficacy of a
prophylactic heparin regimen routinely used in the Small Animal Clinic of the University
of Veterinary Medicine Hannover and (3) to assess whether selected haemostasis test
results are predictive for an increased risk for thrombosis formation
The thesis also includes a preliminary methodological study on the use of rotational
elastometry in dogs using the ROTEM delta device In this preliminary study reference
intervals were determined based on 125 healthy dogs for different clot activator reagents
and for measurements without activator Coefficients of variation (CVs) were calculated
based on results of fourfold measurements of 3 normal and 3 abnormal blood samples in
order to assess precision of the method Influence of age groups sex neuter status and
size were also assessed The median CVs for most of the parameters evaluated in the
precision analysis were less than 10 and this confirmed that the ROTEM delta analyser
delivers repeatable results Regarding the influence of age sex neuter status and animal
Chapter 5 Summary
80
size only neuter status in female dogs appeared to be a relevant influencing factor on
most of important ROTEM parameters
The main study of the thesis comprises 29 patients hospitalised at the Small Animal Clinic
requiring a CVC for medical reasons These patients received a prophylactic
anticoagulation treatment with unfractionated heparin (UFH 150 IU [surgical dogs
received 75 IU]kg BW TID subcutaneously) A colour Doppler ultrasound of the
respective external jugular vein (EJV) in order to detect thrombus formation in the lumen
of the EJV and blood collection for haemostasis tests were performed directly before the
installation procedure (day 0) and on days 1 3 5 7 and so on after the CVC insertion
Haemostasis tests included prothrombin time activated partial thromboplastin time
thrombin time antithrombin activity D-dimer concentration thrombin generation and
rotational elastometry as well as the heparin activity using a chromogenic anti-factor Xa
test Finally when the CVC was removed electron microscopy (ELMI) of sections of the
CVC with macroscopically visible thrombi in the last third (5ndash10 cm) of the CVC was
performed 3 different grades were defined for CVC-associated (extraluminal) thrombi
detected by either ultrasound or ELMI respectively
829 patients evaluated by colour Doppler ultrasound during the study developed thrombi
into the lumen of EJV (4 x grade 1 3 x grade 2 and 1 x grade 3) 1927 CVCs assessed
using ELMI showed detectable thrombi on the external CVC surface (7 x grade 1 4 x
grade 2 and 8 x grade 3) There was no significant correlation between thrombus
formation detected by ultrasound or ELMI Thus statistical group comparisons of
patients with or without CVC-associated thrombus formation was performed
independently for both diagnostic procedures The plasmatic heparin activities showed
great variability but no significant differences were found between dogs with or without
CVC-associated thrombus formation regardless whether this was based on sonographic
Chapter 5 Summary
81
or ELMI findings From the initially performed haemostasis tests high fibrinogen
concentrations and low maximum lysis values as measured by the ROTEM delta device
(using the ex-tem reagent) were associated with sonographically detectable thrombi
In conclusion the preliminary methodological study on the ROTEM delta in dogs shows
that the method delivers repeatable results in canine citrated whole blood and the
reference values obtained should offer a reliable basis for the interpretation of test results
of this device in dogs with suspected haemostasis disorders The results of the main study
indicate that CVC-associated thromboses occur (eg 28 [829] of the patients as
detected by ultrasound) although severe thromboses (high grades) are relatively rare in
intensive care canine patients receiving a routine anticoagulant treatment Thereby
prophylactic antithrombotic protocol (unfractionated heparin [UFH]) in the given dosage
was not completely effective to prevent CVC induced thrombus formation in canine
intensive care patients A high fibrinogen concentration and low maximum lysis
(ROTEM ex-tem) may be useful indicators for an increased thrombotic risk in individual
patients which may require special anticoagulatory treatment Further studies are
required to evaluate whether different thromboprophylactic treatment regimens eg with
low molecular weight heparin are more effective in preventing CVC-associated
thromboses in dogs
Chapter 6 Zusammenfassung
82
Chapter 6 Zusammenfassung
Zentralvenenkatheter-induzierte Thrombose bei Hunden - Auftreten unter einem
antithrombotischen Behandlungsschema und moumlgliche Indikatoren
Joseacute Mauricio Pereira Mora
Zentralvenenkatheter (ZVK) sind fuumlr das Intensivmanagement von Hunden von
Bedeutung Die Entstehung einer Thrombose der Vena jugularis ist eine schwerwiegende
Komplikation im Zusammenhang mit der Verwendung eines ZVKs Uumlber die Inzidenz
von ZVK-assoziierten Thrombosen bei Hunden und die Notwendigkeit und Wirksamkeit
prophylaktischer antithrombotischer Protokolle liegen jedoch nur wenige fundierte
Angaben in der Literatur vor
Daher waren die Ziele der vorliegenden Studie (1) die Inzidenz der Katheter-induzierten
Thrombose bei caninen Intensivpatienten zu bestimmen (2) die Wirksamkeit eines
prophylaktischen Heparin-Regimes zu bewerten das routinemaumlszligig in der Klinik fuumlr
Kleintiere der Stiftung Tieraumlrztliche Hochschule Hannover angewendet wird und (3) um
zu beurteilen ob ausgewaumlhlte Haumlmostasetestergebnisse ein erhoumlhtes Risiko fuumlr die
Thrombosebildung vorhersagen koumlnnen
Die PhD-Arbeit enthaumllt zudem eine vorbereitende methodische Studie zur Verwendung
der Rotationselastometrie bei Hunden mit Hilfe des ROTEM delta-Geraumltes In dieser
Vorstudie wurden Referenzintervalle basierend auf 125 gesunden Hunden fuumlr
verschiedene Gerinnungsaktivatorreagenzien und fuumlr Messungen ohne Aktivator
bestimmt Um die Genauigkeit der Methode einzustufen wurden Variationskoeffizienten
(VKs) basierend auf den Ergebnissen von Vierfachmessungen von drei normalen und drei
abnormalen Blutproben berechnet Der Einfluss von Alter Geschlecht Kastration und
Groumlszlige wurde ebenfalls getestet Die Medianwerte der VKs fuumlr die meisten in der
Chapter 6 Zusammenfassung
83
Praumlzisionsanalyse bewerteten Parameter betrugen weniger als 10 und dies bestaumltigte
dass das ROTEM delta-Analysegeraumlt wiederholbare Ergebnisse liefert In Bezug auf den
Einfluss von Alter Geschlecht Kastration und Tiergroumlszlige erwies sich nur eine Kastration
bei Huumlndinnen als relevanter Einflussfaktor fuumlr die meisten wichtigen ROTEM-
Parameter
Die Hauptstudie der Arbeit umfasst 29 Patienten welche zum Zeitpunkt der
Untersuchung stationaumlr in der Kleintierklinik aufgenommen worden waren und aus
medizinischer Indikation einen ZVK benoumltigten Diese Patienten erhielten eine
prophylaktische Antikoagulation mit unfraktioniertem Heparin (UFH 150 IE
[chirurgische Hunde erhielten 75 IE]kg KG 3 x taumlglich subkutan) Bildgebende
Untersuchungen mittels Farbdoppler-Ultraschall der jeweiligen Vena jugularis externa
(VJE) zum Nachweis der Thrombusbildung im Lumen der VJE und Blutentnahmen fuumlr
Haumlmostasetests wurden jeweils direkt vor dem Installationsvorgang (Tag 0) sowie an den
Tagen 1 3 5 und 7 nach Legen des ZVKs durchgefuumlhrt Haumlmostasetests umfassten die
Prothrombinzeit die aktivierte partielle Thromboplastinzeit die Thrombinzeit die
Antithrombinaktivitaumlt die D-Dimer-Konzentration die Thrombingenerierung und die
Rotationselastometrie sowie die Heparinaktivitaumlt unter Verwendung eines chromogenen
Anti-Faktor-Xa-Tests Schlieszliglich wurde nach Entfernung des ZVKs eine
Elektronenmikroskopie (ELMI) von Abschnitten aus dem letzten Drittel (5ndash10 cm) des
ZVKs mit makroskopisch sichtbaren Thromben durchgefuumlhrt Fuumlr ZVK-assoziierte
(extraluminale) Thromben die entweder durch Ultraschall oder ELMI nachgewiesen
wurden wurden drei verschiedene Grade definiert
829 Patienten die waumlhrend der Studie mittels Farbdoppler-Ultraschall untersucht
wurden entwickelten Thromben im Lumen der VJE (4 x Grad 1 3 x Grad 2 und 1 x Grad
3) 1927 ZVKs die unter Verwendung von ELMI bewertet wurden zeigten
Chapter 6 Zusammenfassung
84
nachweisbare Thromben auf der aumluszligeren ZVK-Oberflaumlche (7 x Grad 1 4 x Grad 2 und 8
x Grad 3) Es gab keine signifikante Korrelation zwischen der durch Ultraschall oder
ELMI festgestellten Thrombusbildung Daher wurden statistische Gruppenvergleiche
von Patienten mit oder ohne ZVK-assoziierter Thrombusbildung fuumlr beide diagnostische
Verfahren unabhaumlngig voneinander durchgefuumlhrt Die plasmatischen Heparinaktivitaumlten
zeigten eine groszlige Variabilitaumlt es wurden jedoch keine signifikanten Unterschiede
zwischen Hunden mit oder ohne ZVK-assoziierter Thrombusbildung gefunden
unabhaumlngig davon ob diese auf sonographischen oder ELMI-Befunden beruhte Von den
initial durchgefuumlhrten Haumlmostasetests waren hohe Fibrinogenkonzentrationen und
niedrige maximale Lyse-Werte der Messungen mit ROTEM delta (ex-tem Reagenz) mit
sonographisch nachweisbaren Thromben assoziiert
Zusammenfassend zeigt die vorbereitende methodische Studie zum ROTEM delta bei
Hunden dass die Methode wiederholbare Ergebnisse in mit Zitrat
antikoaguliertemVollblut liefert Die erarbeiteten Referenzwerte sollten eine zuverlaumlssige
Grundlage fuumlr die Interpretation der Testergebnisse dieses Geraumlts bei Hunden mit
Verdacht auf Haumlmostasestoumlrungen bieten Die Ergebnisse der Hauptstudie zeigen dass
ZVK-assoziierte Thrombosen auftreten (z B 28 [829] der Patienten bezogen auf den
Ultraschall-gestuumltzten Nachweis) obwohl schwere Thrombosen (hohe Grade) bei
intensivmedizinisch versorgten Hundepatienten die eine Routineantikoagulation
erhalten relativ selten sind Somit war das prophylaktische antithrombotische Protokoll
(unfraktioniertes Heparin [UFH]) in der angewendeten Dosierung nicht vollstaumlndig
wirksam um die ZVK-induzierte Thrombusbildung bei caninen Intensivpatienten zu
verhindern Eine hohe Fibrinogenkonzentration und eine niedrige maximale Lyse
(ROTEM ex-tem) koumlnnen als Indikatoren eines erhoumlhten Thromboserisikos bei einzelnen
Patienten nuumltzlich sein fuumlr die moumlglicherweise eine spezielle gerinnungshemmende
Chapter 6 Zusammenfassung
85
Behandlung notwendig ist Weitere Studien sind erforderlich um zu untersuchen ob
verschiedene thromboprophylaktische Behandlungsschemata z B mit
niedermolekularem Heparin bezuumlglich der Verhinderung von ZVK-assoziierten
Thrombosen bei Hunden wirksamer sind
Chapter 7 References
86
Chapter 7 References
1 Abdelkefi A Ben Othman T Kammoun L Chelli M Ben Romdhane N Kriaa A Ladeb
S Torjman L Lakhal A Achour W Ben Hassen A Hsaiumlri M Ladeb F Ben Abdeladhim
A 2004 Prevention of central venous line-related thrombosis by continuous infusion of low-dose
unfractionated heparin in patients with haemato-oncological disease A randomized controlled
trial Thromb Haemost 92 654ndash661
2 Adamantos S Brodbelt D Moores A L 2010 Prospective evaluation of complications
associated with jugular venous catheter use in a veterinary hospital J Small Anim Pract 51 254ndash
257
3 Ballard D H Samra N S Gifford K M Roller R Wolfe B M Owings J T 2016
Distance of the internal central venous catheter tip from the right atrium is positively correlated
with central venous thrombosis Emerg Radiol 23 269ndash273
4 Baumann-Kreuziger L Onwuemene O Kolesar E Crowther M Lim W 2015 Systematic
review of anticoagulant treatment of catheter-related thrombosis Thromb Res 136 1103ndash1109
5 Bliss SP Bliss SK Harvey HJ 2002 Use of recombinant tissue‐plasminogen activator in a
dog with chylothorax secondary to catheter‐associated thrombosis of the cranial vena cava J Am
Anim Hosp Assoc 38 431ndash435
6 Boersma RS Hamulyak K van Oerle R Tuinenburg A Ten Cate-Hoek AJ Schouten
HC 2016 Biomarkers for prediction of central venous catheter related-thrombosis in patients
with hematological malignancies Clin Appl Thromb Hemost 22 779ndash784
7 Boersma RS Jie KS Verbon A van Pampus EC Schouten HC 2008 Thrombotic and
infectious complications of central venous catheters in patients with hematological
malignancies Ann Oncol 19 433ndash442
8 Bonnet F Loriferne JF Texier JP Texier M Salvat A Vasile N 1989 Evaluation of
Doppler examination for diagnosis of catheter-related deep vein thrombosis Intensive Care Med
15 238ndash240
9 Borow M Crowley J G 1985 Evaluation of central venous catheter thrombogenicity Acta
Anaesthesiol Scand Suppl 81 59ndash64
10 Cheng Y Cui T Fu P Liu F Zhou L 2013 Dyslipidemia is associated with tunneled-cuffed
catheter-related central venous thrombosis in hemodialysis patients a retrospective multicenter
Chapter 7 References
87
study Artif Organs 37 155ndash161
11 Comerlato PH Rebelatto TF Santiago de Almeida FA Klein LB Boniatti MM
Schaan BD Rados DV 2017 Complications of central venous catheter insertion in a teaching
hospital Rev Assoc Med Bras 63 613ndash620
12 De Cicco M Matovic M Balestreri L Steffan A Pacenzia R Malafronte M Fantin
D Bertuzzi CA Fabiani F Morassut S Bidoli E Veronesi A 2009 Early and short‐term
acenocumarine or dalteparin for the prevention of central vein catheter‐related thrombosis in
cancer patients a randomized controlled study based on serial venographies Ann
Oncol 20 1936ndash1942
13 Espitia O Fouassier M Hardouin JB Pistorius MA Agard C Planchon B Trossaert M
Pottier P 2017 Thrombin generation assay in hospitalized nonsurgical patients A new tool to
assess venous thromboembolism risk Clin Appl Thromb Hemost 23 45ndash51
14 Evans NS Ratchford EV 2018 Catheter related-venous thrombosis Vasc Med 23 411ndash413
15 Geerts W 2014 Central Venous Catheter-related thrombosis Hematology Am Soc Hematol
Educ Program 2014 306ndash311
16 Harahsheh Y Ho KM 2018 Use of viscoelastic tests to predict clinical thromboembolic
events A systematic review and meta‐analysis Eur J Haematol 100 113ndash123
17 Harter C Salwender HJ Bach A Egerer G Goldschmidt H Ho AD 2002 Catheter-
related infection and thrombosis of the internal jugular vein in hematologic-oncologic patients
undergoing chemotherapy a prospective comparison of silver-coated and uncoated
catheters Cancer 94 245ndash251
18 Hirsh J Raschke R 2004 Heparin and low-molecular-weight heparin The Seventh ACCP
Conference on Antithrombotic and Thrombolytic Therapy Chest 126 (Suppl) 188Sndash203S
19 Karthaus M Kretzschmar A Kroning H Biakhov M Irwin D Marschner N Slabber
C Fountzilas G Garin A Abecasis NG Baronius W Steger GG Sudhoff T Giorgetti
C Reichardt P 2006 Dalteparin for prevention of catheter‐related complications in cancer
patients with central venous catheters final results of a double‐blind placebo‐controlled phase III
trial Ann Oncol 17 289ndash296
20 Kato F Takeuchi H Matsuda S Kawakubo H Omori T Kitagawa Y 2016 Incidence of
and risk factors for venous thromboembolism during surgical treatment for esophageal cancer a
Chapter 7 References
88
single-institution study Surg Today 46 445ndash452
21 Kohler TR Kirkman TR 1998 Central venous catheter failure is induced by injury and can
be prevented by stabilizing the catheter tip J Vasc Surg 28 59ndash66
22 Koumlksoy C Kuzu A Kutlay J Erden I Ozcan H Ergicircn K 1995 The diagnostic value of
colour Doppler ultrasound in central venous catheter related thrombosis Clin Radiol 50 687ndash
689
23 Kucher N 2011 Clinical practice Deep-vein thrombosis of the upper extremities N Engl J
Med 364 861ndash869
24 Langston CE Eatroff AE 2018 Hemodialysis catheter-associated fibrin sheath in a dog J
Vet Emerg Crit Care 28 366ndash371
25 Langston CE Eatroff AE Poeppel K 2014 Use of tissue plasminogen activator in catheters
used for extracorporeal renal replacement therapy J Vet Intern Med 28 270ndash276
26 Laurenson MP Hopper K Herrera MA Johnson EG 2010 Concurrent diseases and
conditions in dogs with splenic vein thrombosis J Vet Intern Med 24 1298ndash1304
27 Lee AYY Kamphuisen PW 2012 Epidemiology and prevention of catheter‐related
thrombosis in patients with cancer J Thromb Haemost 10 1491ndash1499
28 Liu G Fu ZQ Zhu P Li SJ 2015 Central venous catheter-related thrombosis in senile male
patients New risk factors and predictors J Huazhong Univ Sci Technolog Med Sci 35 445ndash
449
29 Lordick F Hentrich M Decker T Hennig M Pohlmann H Hartenstein R Peschel C
2003 Ultrasound screening for internal jugular vein thrombosis aids the detection of central
venous catheter-related infections in patients with haemato-oncological diseases a prospective
observational study Br J Haematol 120 1073ndash1078
30 Lucas TC Silva EED Souza DOD Santos ARD Lara MO 2017 Microstructural
evaluation by confocal and electron microscopy in thrombi developed in central venous catheters
Rev Esc Enferm USP 51 1ndash9
31 Mismetti P Mille D Laporte S Charlet V Buchmuller‐Cordier A Jacquin JP Fournel
P Dutrey‐Dupagne C Decousus H 2003 Low‐molecular‐weight heparin (nadroparin) and
very low doses of warfarin in the prevention of upper extremity thrombosis in cancer patients with
indwelling long‐term central venous catheters a pilot randomized trial Haematologica 88 67ndash
Chapter 7 References
89
73
32 Niers TM Di Nisio M Klerk CP Baarslag HJ Buller HR Biemond BJ 2007
Prevention of catheter‐related venous thrombosis with nadroparin in patients receiving
chemotherapy for hematologic malignancies a randomized placebo‐controlled study J Thromb
Haemost 5 1878ndash1882
33 Scott K C Hansen B D DeFrancesco T C (2009) Coagulation effects of low molecular
weight heparin compared with heparin in dogs considered to be at risk for clinically significant
venous thrombosis J Vet Emerg Crit Care 19 74ndash80
34 Smith RS Zhang Z Bouchard M Li J Lapp HS Brotske GR Lucchino DL Weaver
D Roth LA Coury A Biggerstaff J Sukavaneshvar S Langer R Loose C 2012
Vascular catheters with a nonleaching poly-sulfobetaine surface modification reduce thrombus
formation and microbial attachment Sci Transl Med 4 153
35 Tuumlrk S M Cansu D Uuml Teke H Uuml Kaşifoğlu T Meltem Akay O Bilgin M amp Korkmaz
C 2018 Can we predict thrombotic tendency in rheumatoid arthritis A thromboelastographic
analysis (with ROTEM) Clin Rheumatol 37 2341ndash2349
36 van Rooden CJ Rosendaal FR Barge RM van Oostayen JA van der Meer FJM
Meinders AE Huisman MV 2003 Central venous catheter related thrombosis in haematology
patients and prediction of risk by screening with Doppler-ultrasound Br J Haematol 123 507ndash
512
37 van Rooden CJ Tesselaar ME Osanto S Rosendaal FR Huisman MV 2005 Deep vein
thrombosis associated with central venous catheters - a review J Thromb Haemost 3 2409ndash
2419
38 Verso M Agnelli G Bertoglio S Di Somma FC Paoletti F Ageno W Bazzan M Parise
P Quintavalle R Naglieri E Santoro A Imberti D Soraru M Mosca S 2005 Enoxaparin
for the prevention of venous thromboembolism associated with central vein catheter a double‐
blind placebo‐controlled randomized study in cancer patients J Clin Oncol 23 4057ndash4062
39 Vose J Odunayo A Price JM Daves M Schildt JC Tolbert MK 2019 Comparison of
heparinized saline and 09 sodium chloride for maintaining central venous catheter patency in
healthy dogs PeerJ 7e7072 httpdoi107717peerj7072
40 Wang LH Wei F Jia L Zhi Lu Wang B Dong HY Yu HY Sun GJ YangJ Li B
Chapter 7 References
90
Meng J Zhang RN Bi XQ Chen HY Jiang AL 2015 Fibrin sheath formation and
intimal thickening after catheter placement in dog model role of hemodynamic wall shear stress J
Vasc Access 16 275ndash284
41 Wildgruber M Lueg C Borgmeyer S Karimov I Braun U Kiechle M Meier R Koehler
M Ettl J Berger H 2016 Polyurethane versus silicone catheters for central venous port devices
implanted at the forearm Eur J Cancer 59 113ndash124
42 Whitesell RT Steenburg SD 2014 Imaging findings of acute intravascular thrombus on non-
enhanced computed tomography Emerg Radiol 21 271ndash277
43 Wu X Studer W Skarvan K Seeberger MD 1999 High incidence of intravenous thrombi
after short-term central venous catheterization of the internal jugular vein J Clin Anesth 11
482ndash485
44 Xiang DZ Verbeken EK Van Lommel AT Stas M De Wever I 1998 Composition and
formation of the sleeve enveloping a central venous catheter J Vasc Surg 28 260ndash271
45 Xiang DZ Verbeken EK Van Lommel AT Stas M De Wever I 2001 Sleeve-related
thrombosis a new form of catheter-related thrombosis Thromb Res 104 7ndash14
91
Acknowledgements
First I thank God for every day in my life for His guide and because He was is
and will be always with me and that certainty gives me strength to take a step
forward
My sincere thanks to Prof Dr Reinhard Mischke for the opportunity to perform
my PhD under his supervision for his support and guidance for all that I learned
with him and for his friendship
I also thank to Prof Dr Manfred Kietzmann and PD Dr Mario von Depka
Prondzinski for being part of my supervision group for their time and beneficial
advices and discussion during my PhD
My gratitude to Prof Hewicker-Trautwein and Mrs Rohn for their valuable work
and availability to help me
I thank the Werlhof Institute Medical Care Center Hannover for the valuable
cooperation especially to Mrs Ober-Cassebaum Anne and Franziska for all their
help
I thank all the staff of the Small Animal Clinic of the University of Veterinary
Medicine Hannover (TiHo) My special gratitude to the internal medicine and
critical care surgery neurology ultrasound and anesthesia departments and to
the TiHo students all their cooperation made possible my research
I thank the haematology clinical chemistry and haemostasis laboratory
department of the Small Animal Clinic TiHo especially to Dirk because he was
always available for me thanks for his guide and friendship all his explanations
in the laboratory were of great value but also the conversations about different
topics of the life were of big value for me
92
I thank the Hannover Graduate School for Veterinary Pathobiology
Neuroinfectology and translational Medicine (HGNI) for the well-organized
PhD program and especially thanks to Prof Dr Beatrice Grummer Dr Tina
Selle and Mrs Tania Czeslik for his guide and help during my studies
My sincere thanks to Mrs Maritta Ledwoch for her guidance at the International
office of the TiHo ldquoI remember you since my externship in Germanyrdquo
I also thank the Student Union Hannover for all the help with the accommodation
for me and my family
I want to thank the Universidad Nacional de Costa Rica and therefore to Costa
Rica (my loved country) for the financial support to perform my PhD studies
but also for my professional formation as a veterinarian Special thanks to the
Veterinary Medicine School from Universidad Nacional de Costa Rica To all the
academic and administrative staff for their support and for believing in me
Sincerely thanks to Dr Mauricio Jimenez Soto my mentor my adviser but most
important my friend Thanks for believing in me and for always being by my side
ldquoAgradezco profundamente a mis papaacutes ldquodon Tito (Papillo) y dontildea Eida (Mami)rdquo
Todo lo bueno que hay en mi proviene de Dios y de ellos Gracias a mis hermanos
(as) cuntildeados (as) sobrinos y a toda la familia relacionada a ellos Siempre han
sido un gran apoyo y los amordquo
ldquoGracias a dontildea Alba Fran y David Gracias por toda la ayuda y por estar siempre
pendientesrdquo
I would like to thank all my friends that during a short part or during my whole
life have been with me For all the good time shared really thanks
To Willy thanks ldquogordordquo
93
I sincerely thank to Adriano and Johanna for their help and hospitality Thanks
Adriano for your guidance and support during my beginning at the TiHo for make
me feel in Costa Rica with your friendship for the tourist walks and for the bike
training I missed the coffee times
I want to thank Ina the first person to give me a smile at the start of my PhD
Thanks for your hugs and friendship
I also thank Franz for his help and for being a very nice partner Thanks for help
me always when I requested you
ldquoFinalmente quiero expresar mi gratitud y amor a Karo Faacutetima y Abigaiacutel A Karo
por tu sacrificio y soporte por las palabras de aliento por estar a mi lado en las
buenas pero en especial en las malas A Faacute por tu sacrificio por tus sonrisas por
tus abrazos y besos por bailar con migo siempre por ser mi modelo e inspiracioacuten
A Abi por ser una nueva razoacuten in mi vida para seguir adelante por todo lo que
deseo compartir con tigo por mostrarme junto con Faacutetima la felicidad en una
sonrisardquo
List of figures and tables
iii
Fig 3ab Heparin activities (box- and whisker plot arithmetic means) in dogs receiving
unfractionated heparin at a dosage of 75 IUkg BW tid (a) or 150 IUkg BW tid (b) two
hours after the first daily heparin administration 57
Table 1 Heparin activities (IUml median values minimumndashmaximum) in 28 dogs
receiving 75 or 150 IUkg unfractionated (standard) heparin TID subcutaneously with
(grades 1ndash3) or without (grade 0) sonographically detectable thrombus Blood collections
were performed 2 hours after the first daily injection 57
Table 2 Heparin activities (IUml median values minimumndashmaximum) in 27 dogs
receiving 75 or 150 IUkg unfractionated (standard) heparin TID subcutaneously with
(score 1ndash3) or without (score 0) detectable thrombus via electron microscopy 58
Table 3 Comparison of initial results of haemostasis parameters (median values
minimumndashmaximum in dogs with (grades 1ndash3) and without (grade 0) sonographically
detectable central venous catheter-induced thromboses 60
Table 4 Comparison of results of haemostasis parameters (median values minimumndash
maximum) on the last day in dogs with (grades 1ndash3) and without (grade 0) central venous
catheter-associated thrombus formation as detected by electron microscopy 61
Chapter 1 Introduction
1
Chapter 1 Introduction
Central venous catheters (CVC) are mainly used at critical care units in human and small
animal patients requiring infusion of large amounts of fluids or blood parenteral nutrition
support routine drug therapy haemodialysis chemotherapy plasmapheresis or
periodical blood sampling (Geerts 2014 Vose et al 2019) CVC related thrombosis is
one of the most severe complications (apart from infections and pneumothorax) of CVCs
in humans (Comerlato et al 2017 Evans and Ratchford 2018 Geerts 2014) leading to
patientrsquos distress CVC dysfunction venous stenosis and in some cases to the
development of thromboembolism (Geerts 2014) In the human literature there are
various studies dealing with epidemiologic and prophylactic aspects which are described
below in detail In contrast to the best of the authoracutes knowledge studies about extra
luminal CVC related thrombosis in dogs are rare in the available veterinary literature
Studies are mainly dealing with development of intraluminal CVC thrombi occurring
frequently in dogs and cats (Langston et al 2014) and leading to CVC malfunction (Vose
et al 2019)
Epidemiology of CVC related thrombosis
CVC related thrombosis represents 70ndash80 of all upper extremity thrombosis cases and
approximately 10 of thromboembolism cases in humans (Kucher 2011) The incidence
of CVC related thrombosis reported in humans based in 25 studies ranged widely from
2 to 67 (median = 30 ) (van Rooden et al 2005) The incidence of CVC related
thrombosis detected by colour Doppler ultrasound of the internal jugular vein reported in
3 studies performed in humans was 2 (Harter et al 2002) 30 (Lordick et al 2003)
and 56 (Wu et al 1999)
Chapter 1 Introduction
2
The incidence of CVC related thrombosis in humans can vary between studies according
to the CVC type used entry site of the CVC (jugular femoral subclavian axillary
cephalic vein) and time period with the CVC in place the diagnostic thrombus detection
technique (venography or ultrasonography) and criteria for diagnosis duration of follow
up diseases and presence of predisposing factors (eg previous venous thrombosis
history presence of cancer) and finally prophylactic anticoagulation treatment (Geerts
2014 Liu et al 2015 van Rooden et al 2005)
To the best of the authoracutes knowledge data about the incidence of CVC related
thrombosis detected by colour Doppler ultrasound into the lumen of the jugular vein in
dogs are rare in the available veterinary literature In one study performed in 81 dogs and
12 cats (Adamantos et al 2010) the incidence of vein thrombosis detected by palpation
or ultrasound was 2 and the incidence of intraluminal CVC thrombosis (presumed by
the inability to draw blood back) was 15 Additionally regarding to intraluminal CVC
thrombosis an incidence of 8 was reported in one case control study performed in 24
dogs receiving CVC flushing with heparinised solution (Vose et al 2019) Data about
incidence in other animal species was also found In another case-control study performed
on 37 pigs thrombosis in the caval vein related to CVC placed in the external jugular
vein (EJV) was evaluated by histological analysis however no ultrasound detection was
performed (Kohler and Kirkman 1998) In this study in pigs 94 of patients with CVC
developed thrombi in the vena cava based on sections at the site of the end of the catheter
tip and 05 and 1 cm proximal of the first section (Kohler and Kirkman 1998)
Pathogenesis of CVC related thrombosis
Two types of CVC related thrombosis are distinguished in the human medicine literature
(Geerts 2014 van Rooden et al 2005) First symptomatic thrombosis is defined as
Chapter 1 Introduction
3
thrombosis detected by diagnostic images (ultrasonography venography) when
symptoms and signs (eg localised swelling pain redness) are present (Lee and
Kamphuisen 2012) Second subclinical thrombosis is defined as thrombosis
demonstrated by the previously mentioned diagnostic images without overt symptoms or
signs (Boersma et al 2008) Additionally the CVC related thrombi can be classified as
occlusive thrombus into the CVC lumen peri-catheter related fibrin sheaths and mural
thrombi (Geerts 2014)
During the CVC insertion the puncture of the skin on the CVC insertion site and the
rupture of the blood vessels integrity activate the coagulation cascade and the CVC acts
as a matrix for adherence and accumulation of fibrin and cellular components (Smith et
al 2012) The attachment of proteins and blood cells to the internal andor external wall
of the CVC can lead to (1) (partial or complete) occlusion of the catheter lumen and (2)
sometimes if a thrombus formation starts into the lumen of the vein (attached to the vein
wall or to the CVC) a partial or complete obstruction of the blood flow andor embolism
can develop (Langston and Eatroff 2018) Additionally a study performed in humans
(Lordick et al 2003) demonstrated a relationship between CVC related thrombosis and
microbial colonisation of the catheter leading to CVC related systemic infections This
relationship was explained among others by the ease of microorganisms like
Staphylococcus aureus and Staphylococcus epidermidis to adhere to CVC related
thrombin sheaths (Boersma et al 2008)
Diagnosis of CVC related thrombosis
Venous thrombosis is commonly diagnosed in human and small animal medicine
(especially in dogs) with vascular ultrasonography with or without use of the colour
Doppler technique which is normally used to evaluate the blood flow through the blood
Chapter 1 Introduction
4
vessels (Evans and Ratchford 2018 Laurenson et al 2010) According to the literature
in humans cited below the CVC related thrombosis detected by colour Doppler
ultrasound is present if any of the following aspects are observed (1) well defined
thrombus visualised in the lumen of the vein (2) impossibility to perform compression
of the vein by slight pressure with the ultrasound probe andor (3) no detectable blood
flow signal by colour Doppler ultrasound (Bonnet et al 1989 Koumlksoy et al 1995)
Similarly in dogs the CVC related thrombosis is usually diagnosed by colour Doppler
ultrasonography through the detection of the thrombus within the lumen of the vein
andor the obstruction (partial or complete) of the normal blood flow (Bliss et al 2002)
Especially in humans the contrast venography technique is used as gold standard to
confirm thrombosis For this purpose a contrast solution is injected through a peripheral
vein to assess its passage through the bloodstream by x-ray However this diagnostic
technique is normally used only in the case of a strong clinical suspicion of venous
thrombosis that cannot be detected by ultrasonography (Laurenson et al 2010 Langston
and Eatroff 2018) In a study performed in 44 human patients requiring a CVC (Koumlksoy
et al 1995) a sensitivity of 94 and a specificity of 96 were determined for the colour
Doppler ultrasound diagnosis when it was compared with the diagnosis performed with
the contrast venography technique That results make the colour Doppler ultrasound an
accurate diagnosis method to detect CVC related thrombosis in humans (Koumlksoy et al
1995 van Rooden et al 2003)
Advanced diagnostic image techniques like computed tomography or magnetic resonance
tomography are also accurate to perform the diagnosis of CVC related thrombosis in
humans (Whitesell and Steenburg 2014) In dogs multidetector computed tomography
venography was used in one study for the diagnosis of CVC related thrombosis allowing
the identification of catheter related sheaths around the CVC external wall (Wang at al
Chapter 1 Introduction
5
2015) However the high costs of these advanced diagnostic imaging procedures and the
requirement to perform at least sedation or general anesthesia are disadvantages when
compared with ultrasonography
Finally electron microscopy (ELMI) is a diagnostic technique that has been used in
humans (Lucas et al 2017) and experimental animals (Xiang et al 1998 Xiang et al
2001) mainly to perform a microstructural evaluation of thrombi developed into the
lumen of the CVC or in the vein containing the CVC ELMI allows the ex vivo diagnosis
of very small thrombi or sleeve related thrombosis that perhaps cannot be detected by
ultrasonography or venography during the evaluation of patients with CVCs (Xiang et al
2001) However one point to consider is that it is possible that compact thrombi
developed on the CVC can be detached during the CVC removal from the vein and
thereby before its analysis by ELMI (Xiang et al 2001)
Heparin thromboprophylaxis for CVC related thrombosis
Application of CVCs in human medicine is commonly accompanied by prophylactic
anticoagulation in order to decrease the risk of development of partial or complete vein
occlusion andor thromboembolism and thereby to improve the patientrsquos comfort
(Baumann-Kreuziger et al 2015) Apart from vitamin K antagonists (Mismetti et al
2003) unfractionated heparin (UFH) (Abdelfeki et al 2004) and low molecular weight
heparin (LMWH) (Karthaus et al 2006) are anticoagulants that have been used on
studies in humans administrated by intravenous (IV) infusion or by subcutaneous (SC)
injection to prevent thrombi development into the lumen of the veins containing the CVC
(review article Baumann-Kreuziger et al 2015) Different heparin dosages have been
used as thromboprophylactic anticoagulation in humans with CVC (review article Lee
and Kamphuisen 2012) For example in five different case-control studies performed in
Chapter 1 Introduction
6
human haemato-oncological patients UFH at a daily dosage of 100 IUkg given by IV
infusion (Abdelfeki et al 2004) and LMWH at SC daily dosages of 40 mg enoxaparin
(Verso et al 2005) 5000 IU AXa dalteparin (Karthaus et al 2006 De Cicco et al 2009)
and 2850 IU AXa nadroparin (Niers et al 2007) were used Only in part of these studies
it is stated that systemic heparin was combined with flushing of the CVC using
heparinised saline solution to reduce the risk of catheter occlusion (Karthaus et al 2006)
In 3 of the 5 previously mentioned studies no statistically significant difference was found
in the incidence of CVC related thrombosis between patients receiving the
anticoagulation prophylaxis compared with the control groups without systemic
anticoagulation (Verso et al 2005 p = 035 Karthaus et al 2006 p gt 005 Niers et al
2007 p = 049) In contrast in the residual two studies a lower incidence of CVC related
thrombosis was found in the group receiving an anticoagulation prophylaxis compared
with the control groups (Abdelfeki et al 2004 150 vs 126 p = 003 De Cicco et
al 2009 400 vs 526 p = 005)
Obviously there is no consensus in the available human literature regarding the beneficial
use of anticoagulation prophylaxis to prevent CVC related thrombosis and this may
reflect great variations for example according to the study design period trial drug
administration route (Lee and Kamphuisen 2012) and the moment of the initial
anticoagulant dosage administration (Decicco et al 2009)
To the best of the authoracutes knowledge studies about thromboprophylaxis for extraluminal
CVC related thrombosis in dogs cannot be found in the available veterinary literature
However similarly to humans prophylactic anticoagulation with heparinised saline
(administrated through CVC flushing) to prevent CVC occlusion by intraluminal thrombi
was used in one case-control study performed in 24 dogs (Vose et al 2019) In the cited
study 12 dogs received dosages of 3 ml of heparinised saline solution (10 IU UFHml) to
Chapter 1 Introduction
7
flush the catheter every 6 hours during 72 hours whereas 12 control dogs received
flushing with the same volume of non-heparinised saline solution administered at the
same frequency and no statistically significant difference was found
Prediction of CVC related thrombosis
Studies regarding the relationship between laboratory biomarkers and CVC related
thrombosis are available in the human literature (Boersma et al 2016 Cheng et al 2013
Liu et al 2015) however to the best of the authoracutes knowledge literature about laboratory
tests used as predictors for CVC related thrombosis in small animals cannot be found at
all Availability of approved blood tests which can predict the risk of thrombosis
development associated to CVC already at the moment of the CVC placement are
important in order to identify an increased risk of CVC related thrombosis (Boersma et
al 2016) In the study performed by Boersma et al (2016) in 168 human patients without
indication of anticoagulation prophylaxis elevated leukocyte counts high levels of
plasminogen activator inhibitor and of coagulation factor VIII (all measured on blood
samples taken after CVC placement) were associated with an increased incidence of
symptomatic CVC-related thrombosis diagnosed by ultrasonography In another study
(Liu et al 2015) high blood levels of lactate dehydrogenase and low concentrations of
HDL and albumin were associated with the incidence for CVC related thrombosis
diagnosed by ultrasound in 324 senile patients which was in total 617 Additionally
dyslipidemia (high triglycerides low HDL and high LDLHDL ratio) was associated
with CVC related thrombosis (diagnosed by ultrasound and computer tomography
angiography) in human patients undergoing haemodialysis (Cheng et al 2013) Studies
about predictive tests for CVC related thrombosis in dogs were not found in the available
literature to the best of the authoracutes knowledge
Chapter 1 Introduction
8
Aims of the study
The aims of the preliminary methodological study of the present scientific project were
(1) to define reference intervals for viscoelastic analyses of canine haemostasis using the
ROTEMreg delta analyser and (2) to determine the precision (repeatability) of this
method And the aims of the main study of the present scientific project were (1) to
determine the incidence of catheter-induced thrombosis in canine intensive care patients
(2) to evaluate the efficacy of a prophylactic heparin regimen routinely used in the Small
Animal Clinic of the University of Veterinary Medicine Hannover and (3) to assess
whether selected haemostasis test results are predictive for an increased risk for
thrombosis formation
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
9
Chapter 2 Reference intervals for rotational thromboelastometry measurements
using the ROTEMreg delta device in dogs
Pereira JM Rohn K Mischke R
Contributions of the authors
Pereira JM performed (1) the patients data and blood samples collection (2)
haematology and ROTEMreg analysis (3) generated the reference intervals and
coefficients of variation included in the study and (4) wrote the manuscript
Rohn K performed the statistical analysis
Mischke R performed the clinical chemistry and coagulation tests and was involved in
designing and supervision of the study analysis and discussion of the data and its
statistical analysis and revision edition and correction of the manuscript
Original source Pereira J M Rohn K amp Mischke R 2020 Reference intervals
for rotational thromboelastometry measurements using the ROTEMreg delta device
in dogs Res Vet Sci 130 26ndash32 httpsdoiorg101016jrvsc202001019
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
10
Reference intervals for rotational thromboelastometry measurements using the
ROTEMreg delta device in dogs
Pereira JMa Rohn Kb Mischke Ra
aSmall Animal Clinic University of Veterinary Medicine Hannover Buumlnteweg 9 D-
30559 Hannover Germany
bInstitute of Biometry Epidemiology and Information Processing University of
Veterinary Medicine Hannover Buumlnteweg 2 30559 Hannover Germany
Corresponding author
E-mail ReinhardMischketiho-hannoverde
Original source Pereira J M Rohn K amp Mischke R 2020 Reference intervals
for rotational thromboelastometry measurements using the ROTEMreg delta device
in dogs Res Vet Sci 130 26ndash32 httpsdoiorg101016jrvsc202001019
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
11
ABSTRACT
Aims of the present study were to define reference intervals for viscoelastic analyses of
canine haemostasis using the ROTEMreg delta analyser and as a secondary aspect to
determine the precision (repeatability) of this method Blood samples from 125 clinically
healthy dogs were included Measurements were performed with commercially available
activating reagents (ex-tem in-tem and kaolin solution) as well as without activation
Additional fourfold measurements were done in 3 of the normal blood samples and in 3
samples with haemostatic alterations to evaluate the precision of the method
Coefficients of Variation (CVs) for most of the ROTEM variables were lt 10 Clot
formation time and maximum clot elasticity showed a wide inter-individual variation in
comparison with alpha angle and maximum clot firmness A multivariate analysis on
various ROTEM parameters revealed particularly a significant influence of neuter status
and a significant interrelationship between the factors sex and neuter status for
measurements with different activating reagents These results reflected the fact that
significant differences occurred only between intact and neutered females but not in
males No or only occasionally significant differences were found between groups of sex
age and size
In conclusion CVs demonstrated that the method delivers repeatable results in canine
citrated whole blood Established reference intervals should deliver valuable orientation
for the evaluation of viscoelastic properties of clotting whole blood in dogs using the
ROTEM delta analyser Neuter status in females appeared to be the most relevant
influencing factor and should be considered for the interpretation of ROTEM delta test
results
Keywords Viscoelastic measurements reference values canine age sex neuter status
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
12
1 Introduction
Heamostasis is a physiologic and dynamic process in which plasma soluble coagulation
factors blood cells and blood vessels are involved Clot formation and possible
subsequent clot lysis as well as vascular tissue repair aim to prevent haemorrhages on the
one hand and thromboses on the other hand (Kol and Borjesson 2010 Versteeg et al
2013) Conventional assays of coagulation such as prothrombin time (PT) activated
partial thromboplastin time (aPTT) and thrombin time TT have limitations especially
cellular elements are not incorporated into the test and thereby these tests reflect only
limitedly in vivo functionality of haemostasis (McMichael and Smith 2011 Crochemore
et al 2018)
The rotational thromboelastometry (ROTEMreg) currently termed as thromboelastometry
(TEM) and thromboelastography (TEG) are methods for the rapid evaluation of the
overall haemostasis capacity through the measurement of viscoelastic properties of
clotting whole blood (Kol and Borjesson 2010 Theusinger et al 2010 Sankarankutty
et al 2012 Versteeg et al 2013) In TEG the cup with the blood sample is rotating
whereas the torsion wire is fixed In ROTEMTEM the cup is fixed whereas the pin is
rotating Changes in torque ie resistance to rotation by the fibrin bridge created between
pin and cup in the clotting blood are detected electromechanically in TEG and optically
in ROTEM (Bollinger et al 2012) and displayed as a graph that indicates the time for the
formation of blood clot and its strength (Bagge et al 2016) The resulting graph reflects
the initiation and propagation of the coagulation system fibrin-platelet interactions and
clot lysis
TEG was first described in 1948 by Hellmut Harten (Hartert 1948) as an in vitro method
to evaluate the clot formation process while resembling the in vivo blood flow The
development of the actually used instrument types TEG and ROTEMregTEM were based
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
13
on that original principle Viscoelastic measurements have been widely used since the
1980s in human medicine to evaluate haemostasis and hypercoagulable states in surgical
patients (eg cardiac and hepatic surgeries) and in general in critical care units (Kol and
Borjesson 2010 Nogami 2016 Wikkelso et al 2016 Crochemore et al 2017) In
veterinary medicine viscoelastic measurements using TEG and ROTEMTEM have been
validated for use in dogs cats and horses and used to study haemostasis in different
diseases (eg infectious diseases liver diseases) and medical conditions (eg
orthopaedic surgery hyperfibrinolysis) and also to investigate the effect of
anticoagulants and other drugs (eg corticosteroids) on haemostasis (Kol and Borjesson
2010 Bruno et al 2015a 2015b Woodman et al 2015 Conversy et al 2017 Sigrist et
al 2018 Turner et al 2019) The methodology seems to be especially suitable to detect
hypercoagulability (Wiinberg et al 2008 Kol and Borjesson 2010 Marschner et al
2018)
In the available veterinary literature information about reference values for TEG were
found using kaolin as activation reagent (Bauer et al 2009) Falco et al (2012)
determined reference values for the ROTEM analyser in dogs using ex-tem in-tem and
fib-tem reagents for selected ROTEM parameters in a study on forty five healthy dogs
However data related to reference intervals obtained in an adequate number of samples
(at least 120) according to the American Society for Veterinary Clinical Pathology
(ASVCP) (Friedrichs et al 2012) and the National Committee for Clinical Laboratory
Standards (NCCLS) (Horn and Pesce 2003) could not be found in the available literature
The aim of this study is to determine reference values for the ROTEMreg delta device in
dogs using citrated whole blood with different clot activator reagents (ex-tem in-tem
kaolin) and without activator
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
14
2 Materials and methods
21 Study design
Reference intervals Single thromboelastometric measurements were performed in 125
clinically healthy dogs using the ROTEMreg analyser and different activating reagents (ex-
tem in-tem and kaolin) as well as without activation Reference values were calculated
and influences of age groups (young adult and senior) sex neuter status and size
(groups small medium and large) were assessed
Precision analysis Additional fourfold measurements using ex-tem or in-tem activating
reagents respectively were performed on 3 of the normal and also in 3 abnormal blood
samples each in order to assess within-run imprecision for these measurements
22 Animals
The clinically healthy dogs with a body weight of at least 5 kg were recruited by launching
a call in the University of Veterinary Medicine Hannover network for students and staff
members Interested dog owners received a free health check of their animals (clinical
examination and blood tests specified below) in combination with the blood collection
In addition individual privately owned patient dogs of the Small Animal Clinic with
undisturbed general health were also included if blood was collected due to another
indication (eg general health check pre-anaesthetic examination test for hereditary
diseases etc) Before taking the blood samples all dogs were fasted for at least 12 h with
free access to water 125 animals for which the examination of the health status (clinical
examination routine haematology and clinical chemistry basis coagulation profile) did
not reveal any deviations from the reference were included in the study whereas 3 of the
128 initially recruited animals had to be excluded due to laboratory abnormalities
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
15
Haematological and clinical chemistry profiles performed in order to confirm the health
status included white and red cell count haematocrit platelet counts and reticulocytes
plasma activities of alanine aminotransferase glutamate dehydrogenase alkaline
phosphatase and pseudocholinesterase as well as concentrations of total bilirubin urea
creatinine glucose total protein albumin cholesterol and ionised calcium A basis
coagulation profile including PT PTT and TT was also performed to exclude significant
abnormalities within the blood coagulation system
The 125 healthy dogs enclosed in the establishment of reference values included 48 mixed
breed dogs and the following pedigree dogs 13 Labrador Retrievers 6 Australian
Shepherds 4 Havanese dogs 3 from each of the following breeds American
Staffordshire Terriers and Cocker Spaniels 2 from each of the following breeds Beagles
Border Collies Dalmatians Elo dogs (breed not recognised by Feacutedeacuteration cynologique
international [FCI]) German Longhaired Pointers German Shorthaired Pointers Giant
Schnauzers and Golden Retrievers 1 from each of the following breeds Belgian
Malinois Bolonka Zwetna Briard Brussels Griffon American English Coonhound
Italian Mastiff Flat Coated Retriever French Bulldog German Shepherd Great Dane
Greater Swiss Mountain Dog Giant Spitz Goldendoodle Hovawart Hungarian Vizsla
Italian Bloodhound Lycanis Wolfdog Miniature American Shepherd Miniature
Schnauzer Old German Shepherd Podenco Poodle Pyrenean Shepherd Rhodesian
Ridgeback Rottweiler Schafpudel (breed not recognised by FCI) Small
Munsterlander Spanish Mastiff Tibet Terrier Weimaraner West Highland White
Terrier and Wirehaired Pointer
These dogs were aged between 6 and 154 months (median = 410 months) and had body
weights ranging from 50 to 707 kg (median = 226 kg) 30 were castrated males 33
intact males 41 spayed females and 21 intact females According to the height at withers
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
16
the dogs were divided into 3 different size groups small (le40 cm n = 22) medium (41ndash
60 cm n = 77) and large (gt60 cm n = 26) Dogs were also divided in three different age
groups young (le 24 months n = 34) adult (25ndash84 months n = 59) and senior dogs (gt84
months n = 32)
The three blood samples with haemostatic abnormalities were taken from two dogs with
complex haemostatic disorders (Disseminated Intravascular Coagulation [DIC])
including reduced platelet count and abnormal PT and APTT and one dog with
thrombocytopenia
Animals were treated in accordance with the German Animal Welfare Law The
experimental design was approved by the Animal Welfare officer of the University of
Veterinary Medicine Hannover Foundation and by the ethics committee of the
responsible authority (Lower Saxony State Office for Consumer Protection and Food
Safety reference number 17A101)
23 Blood sampling
The venipuncture area was shaved and then disinfected with alcohol The saphenous or
cephalic vein was visualised with slight pressure and punctured with disposable cannulas
(diameter 09 or 10 mm) The continuously running blood was directly collected from
the needle into the tubes containing the respective anticoagulants From each dog
approximately 10 ml of blood were collected including approximately 8 ml of citrated
blood for the measurement of haemostasis tests (ROTEM PT aPTT TT) in tubes
containing sodium citrate 313 (Eifelfangoreg GmbH amp Co KG Bad Neuenahr-
Ahrweiler Germany) in a ratio of 1 volume9 volumes of blood In addition
approximately 1 ml of EDTA blood for haematological analysis and approximately 1 ml
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
17
of lithium heparin blood for clinical chemical measurements were collected into
commercial 13 ml tubes (Sarstedt AG amp Co Nuumlmbrecht Germany)
Citrated blood was initially kept at room temperature (20-25 C) in order to avoid errors
within the preanalytical phase (Toulon et al 2017) Two ml of citrated blood were used
for the ROTEMreg analysis the residual citrate blood was centrifuged for 10 min at 3800
rpm and 5 degC then the plasma was separated by careful pipetting and it was centrifuged
a second time under the same conditions to get platelet poor plasma which was stored in
aliquots of 500 microl and 250 microl at -80 degC for coagulation tests
24 Rotational elastometry
Viscoelastic measurements using the ROTEMreg delta for the establishment of reference
intervals were performed approximately 15 min after the blood collection using activating
reagents (liquid reagents) provided by the manufacturer of the instrument (r-ex-temreg in-
temreg) and a kaolin solution (5 g1 as part of the PTT reagent CK Prest [Diagnostica
Stago SAS Asniegraveres sur Seine Cedex France]) In addition non-activated analyses
were performed using isotonic NaCl solution instead of the activating reagent in the fourth
channel of the device Fourfold measurements for precision analysis were performed
simultaneously in the four channels of the device in separate runs
According to the standard test protocol provided by the manufacturer of the ROTEMreg
delta (Werfen GmbHInstrumentation Laboratory Munich Germany) 20 μl of the
reagent for recalcification (star-temreg20) and 20 μl of the activating reagent were pipetted
into a cuvette using the automated pipette provided with the device Immediately
thereafter 300 μl of citrated whole blood (prewarmed to 37 degC for five minutes in the
heating device of the instrument and gently rocked to resuspend possibly sedimented
cells) were added to the test preparation in the cuvette using the automated pipette and
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
18
then the cuvette was laced carefully in the ROTEMreg device whereby the rotating pin
entered into the test approach
Different parameters are provided automatically by the ROTEMreg delta device and the
following variables (described according to the Pentapharm GmbH ROTEMreg delta
Manual [2009]) were used in the study
Clotting time (CT) the time (in s) from the beginning of the test (by adding the clot
activator) until the formation of fibrin starts (defined as the moment when the curve
achieves an amplitude of 2 mm) This Parameter facilitates the decision to replace clotting
factors when it is required
Clot formation time (CFT) describes the next phase of clotting by the formation of a
stable clot through the activation of thrombocytes and fibrin formation It is defined as
the time (in s) between the 2 mm amplitude and 20 mm amplitude of the curve
Alpha angle (α angle) is the angle (in degrees) between the middle axis and the tangent
to the clotting curve through the 2 mm amplitude point
Amplitude after ldquoXXrdquo minutes (eg A30 after 30 min) is the amplitude (in mm) at a
certain time after the CT has finished
Maximum clot firmness (MCF) corresponds to the maximum amplitude (in mm) reached
before the clot is dissolved by fibrinolysis This parameter measures the firmness of the
clot and its quality Like CFT and α angle it facilitates the decision for substitution therapy
with platelet concentrate andor fibrinogen especially in humans
Maximum clot elasticity (MCE) is a parameter calculated from MCF and may be superior
at high amplitudes as compared to the MCF
Lysis index at 30 45 and 60 min (LI30 LI45 LI60) represents the fibrinolysis (in
percentage) 30 45 and 60 min after the CT It may facilitate the decision for or against a
therapy with anti-fibrinolytic drugs
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
19
Maximum lysis (ML) describes the degree of fibrinolysis relative to the MCF achieved
during the measurement (percent loss of clot firmness)
25 Statistical methods
Original data obtained from the ROTEMreg delta device software were transferred into the
Microsoftreg Excelreg formal (Microsoft Office Professional Plus 2013) The Statistical
analyses were performed using the SAS Enterprise Guide Version 71 (SAS Institute Inc
Cary NC USA)
Coefficients of Variation (CVs) for selected relevant parameters of the ROTEM delta
analysis using ex-tem and in-tem regents were calculated for the fourfold measurements
on 3 normal and 3 abnormal blood samples in order to assess within-run imprecision CVs
for the results of each sample were calculated by dividing the standard deviation of the
data set by its arithmetic mean Finally the median of the CVs for each group of 3 samples
was calculated The reference values were calculated according to recommendations of
the American College of Veterinary Clinical Pathology (ACVCP) based on non-
parametric methods (25 and 975 quantiles) supplemented by 95 confidence
intervals (Friedrichs et al 2012)
Data were analysed for Standard normal distribution by Kolmogorov Smirnov test and
assessment of residuals of the analysis of variance (ANOVA) Because these tests
indicated almost normally distributed values for the studied ROTEM variables data were
analysed by a fourway ANOVA stratified by the activating reagent (ex-tem in-tem
kaolin or without activation) to test the influence of size age sex and neuter status on
several parameters and taking all interactions into account Post hoc t-tests were
performed when indicated The significance level was set to P lt 05
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
20
3 Results
31 Precision of the method
For all the parameters evaluated in the study the CVs for the fourfold measurement in
healthy dogs were less than 10 (Table 1) In the case of dogs with haemostatic
abnormalities CVs of the vast majority of ROTEM variables were also less than 10
except the variables CFT and ML irrespective whether ex-tem or in-tem reagents were
used (Table 2)
Table 1 Coefficients of variation () for rotational thromboelastometry using the
ROTEMreg delta analyser in normal blood samples
Activator CT (s) CFT (s) α (deg) A30 (mm) MCF (mm) MCE(mm) LI30 () LI45 () LI60 () ML ()
Ex-temreg 875 629 262 213 179 634 000 020 037 534
In-temreg 245 596 166 117 135 299 000 000 046 216
Abbreviations CT ndash clotting time CFT ndash clot formation time α ndash alpha angle A30 ndash
amplitude after 30 min MCF ndash maximum clot firmness MCE ndash maximum clot elasticity
LI 30 LI 45 LI 60 ndash Lysis index at 30 45 and 60 min ML ndash maximum lysis
Median values of the measurements of 3 samples
Table 2 Coefficients of variation () for rotational thromboelastometry using the
ROTEMreg delta analyser in canine blood samples with haemostatic alterations
Activator CT (s) CFT (s) α (deg) A30 (mm) MCF (mm) MCE(mm) LI30 () LI45 () LI60 () ML ()
Ex-temreg 668 1478 393 218 268 582 0 018 000 848
In-temreg 228 799 461 219 251 371 0 0 017 283
Median values of the measurements of 3 samples from patients with DIC (n = 2)
thrombocytopenia (n = 1) abbreviations please see legend to Table 1
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
21
32 Reference intervals
Activation by ex-tem reagent (tissue factor) resulted in faster CTs when compared to
activation via the intrinsic pathway using ellagic acid (in-tem) or kaolin reagents whereas
the CFT was much less affected by the activating reagent (Table 3) Especially CFT and
MCE showed a wide inter-individual variation in comparison for example with α angle
and MCF regardless of the used activating reagent (Table 3)
33 Influence of age sex neuter status and size
ANOVA did not reveal statistically significant differences in ROTEM delta parameters
between dog groups of variable size or sex (P gt 05) Age had influence only on CT and
only when the in-tem reagent was used (P = 0169 ANOVA) but not when using the ex-
tem reagent (P = 7105) Post hoc comparisons using t-test of the in-tem results between
age groups revealed shorter CTs in the group of senior dogs when compared with young
dogs (P = 0194) but not between adult dogs and these two groups ANOVA showed
particularly influences of the neuter status and a significant interrelationship between the
factors sex and neuter status on different ROTEM delta parameters using ex-tem and in-
tem respectively as activator (Tables 4 and 5) ANOVA revealed also a significant
influence of neuter status on different ROTEM parameters when kaolin was used as
activating reagent (CFT α angle MCF MCE) whereas non-activated measurements
were not subject to any significant influence of the assessed factors (results not shown)
The interrelationship between sex and neuter status mentioned above reflected the fact
that significant differences between neutered and not neutered dogs were only found in
females The statistical analysis using the t-test to compare intact vs neutered dogs
stratified by sex showed statistically significantly (P lt 05) longer CTs and CFTs and
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
22
lower α angle A30 MCF and MCE values in spayed when compared to intact females
(Tables 4 and 5)
Chapter 2 Reference intervals for rotational thromboelastometry measurements using the ROTEMreg delta device in dogs
23
Table 3 Reference values (median reference interval based on 25 - and 975 - quantiles) for thromboelastometric measurements with the ROTEMreg delta analyser in
dogs (n=125 95 confidence intervals in brackets) Abbreviations please see legend to Table 1
Activator
Statistical
para-meter
CT
(s)
CFT
(s)
Α
(deg)
A30
(mm)
MCF
(mm)
MCE
(mm)
LI30
()
LI45
()
LI60
()
ML
()
Ex-temreg
Median 450
[430ndash470]
110
[105ndash115]
680
[670ndash690]
600
[580ndash610]
610
[600ndash620]
154
[147ndash164]
100
[960ndash100]
100
[100ndash100]
970
[960ndash980]
220
[210ndash240]
25 340
[270ndash350]
650
[490ndash710]
540
[420ndash560]
460
[410ndash480]
460
[420-490]
870
[720ndash950]
100
[100-100]
870
[800ndash960]
700
[640ndash820]
800
[100ndash110]
975 830
[790ndash990]
220
[201ndash315]
790
[770ndash800]
770
[690ndash770]
730
[700ndash770]
264
[230-334]
100
[100ndash100]
100
[100ndash100]
100
[990ndash100]
590
[530ndash680]
In-tem
Median 190
[187ndash201]
116
[111ndash125]
680
[670ndash690]
570
[560ndash580]
590
[580ndash600]
143
[135ndash149]
100
[100ndash100]
100
[100ndash100]
980
[980ndash980]
180
[170ndash190]
25 135
[112ndash142]
730
[590ndash800]
510
[460ndash570]
450
[410ndash470]
480
[410ndash500]
920
[710ndash101]
100
[950ndash100]
990
[820ndash990]
930
[740ndash930]
500
[100ndash700]
975 283
[268ndash291]
251
[198ndash227]
750
[750ndash780]
660
[660ndash690]
680
[660ndash690]
210
[195ndash227]
100
[100ndash100]
100
[100ndash100]
100
[100ndash100]
270
[260ndash380]
Activator
Statistical
para-meter
CT
(s)
CFT
(s)
Α
(deg)
A30
(mm)
MCF
(mm)
MCE
(mm)
LI30
()
LI45
()
LI60
()
ML
()
Kaolin
Median 162
[154ndash173]
870
[820ndash910]
740
[720ndash750]
500
[470ndash530]
550
[530ndash560]
121
[115ndash127]
960
[940ndash980]
840
[790ndash880]
710
[640ndash770]
500
[450ndash540]
25
109
[990ndash115]
470
[440ndash580]
560
[490ndash600]
110
[700ndash230]
390
[380ndash400]
640
[600ndash680]
270
[140ndash480]
110
[800ndash260]
800
[400ndash170]
200
[160ndash240]
975 246
[230ndash337]
214
[185ndash290]
810
[790ndash810]
640
[630ndash690]
650
[650ndash690]
187
[182ndash223]
100
[100ndash100]
990
[980ndash100]
940
[930ndash990]
970
[930ndash100]
Without activation
Median 607
[495ndash662]
221
[194ndash265]
510
[460ndash550]
520
[500ndash530]
540
[520ndash550]
115
[109ndash123]
100
[100ndash100]
100
[100ndash100]
980
[980ndash990]
170
[160ndash190]
25 203
[123ndash215]
860
[550ndash930]
190
[120ndash230]
350
[230ndash370]
370
[260ndash400]
600
[350ndash670]
100
[950ndash100]
980
[890ndash990]
930
[840ndash950]
400
[000ndash500]
975 1356
[1305ndash1721]
811
[688ndash1333]
730
[710ndash790]
660
[640ndash720]
660
[640ndash730]
197
[179ndash265]
100
[100ndash100]
100
[100ndash100]
100
[100ndash100]
290
[280ndash320]
Chapter 2 Reference intervals for rotational thromboelastometry measurements using the ROTEMreg delta device in dogs
24
Table 4 Median values (minimum and maximum in brackets) for different ROTEMreg variables using ex-tem reagent stratified by sex and neuter status
Group
CT
(s)
CFT
(s)
Α
(deg)
A30
(mm)
MCF
(mm)
MCE
(mm)
LI30
()
LI45
()
LI60
()
ML
()
Female intact 430
(320ndash530)
960
(590ndash198)
710
(570ndash800)
620
(480ndash730)
640
(510ndash740)
179
(103ndash282)
100
(100ndash100)
100
(980ndash100)
960
(800ndash100)
230
(100ndash640)
Female spayed 460
(270ndash830)
121
(650ndash224)
660
(520ndash770)
580
(430ndash690)
590
(440ndash700)
146
(790ndash230)
100
(960ndash100)
100
(800ndash100)
970
(690ndash100)
240
(100ndash590)
p-value (t-test)
Female
intact vs spayed
00025 00063 00049 00023 00016 00015 na na na na
Male intact 450
(320ndash990)
105
(650ndash315)
690
(420ndash790)
590
(420ndash700)
610
(440ndash710)
154
(770ndash244)
100
(100ndash100)
100
(870ndash100)
970
(650ndash100)
220
(700ndash680)
Male castrated 460
(340ndash930)
111
(490ndash255)
685
(480ndash800)
590
(410ndash770)
600
(420ndash770)
152
(720ndash334)
100
(100ndash100)
100
(830ndash100)
980
(640ndash100)
205
(500ndash590)
p-value (t-test)
Male
Intact vs castrated
08326 05106 04756 01799 01568 02163 na na na na
p-values (ANOVA)
Factor Sex
05428 04847 06612 02206 01887 01630 08489 07893 07689 02758
Factor neuter status 01872 00167 00173 00039 00037 00028 07141 04737 09356 05948
Interrelationship
sexneuter status 00383 00968 00782 00488 00472 00319 04943 08611 05422 00160
na ndash not analysed all significant P-values (lt 005) are highlighted in bold print
Chapter 2 Reference intervals for rotational thromboelastometry measurements using the ROTEMreg delta device in dogs
25
Table 5 Median values (minimum and maximum in brackets) for different ROTEMreg variables using the in-tem reagent stratified by sex and neutering status
Group
CT
(s)
CFT
(s)
Α
(deg)
A30
(mm)
MCF
(mm)
MCE
(mm)
LI30
()
LI45
()
LI60
()
ML
()
Female intact 186
(126ndash238)
940
(590ndash164)
720
(620ndash780)
610
(490ndash690)
620
(530ndash690)
166
(113ndash227)
100
(100ndash100)
100
(990ndash100)
980
(930ndash100)
170
(100ndash300)
Female spayed 201
(149ndash291)
1220
(820ndash262)
670
(500ndash740)
550
(450ndash660)
570
(480ndash680)
133
(910ndash214)
100
(950ndash100)
100
(820ndash100)
980
(740ndash100)
190
(500ndash380)
p-value (t-test)
Female
intact vs spayed
na na lt0001 00009 00005 00005 na na na na
Male intact 199
(117ndash283)
122
(730ndash277)
680
(460ndash750)
570
(430ndash660)
590
(450ndash660)
146
(810ndash192)
100
(100ndash100)
100
(990ndash100)
980
(930ndash100)
180
(600ndash270)
Male castrated 181
(112ndash288)
123
(820ndash222)
670
(540ndash740)
565
(410ndash630)
585
(410ndash640)
138
(710ndash180)
100
(100ndash100)
100
(970ndash100)
980
(890ndash100)
180
(800ndash260)
p-value (t-test)
Male
intact vs castrated
na na 08232 01401 01162 00849 na na na na
p-values (ANOVA)
Factor Sex
02105 03317 03370 00877 01258 00510 09564 07832 08117 03638
Factor neuter status 04289 00660 00619 00107 00088 00054 07152 09795 07056 08299
Interrelationship
sexneuter status
04310 00550 00408 00707 01733 00996 05240 05982 08047 04578
na ndash not analysed all significant P-values (lt 005) are highlighted in bold print
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
26
4 Discussion
41 Coefficients of variation
In accordance with the literature CVs below 10 indicate adequate repeatability in intra-
assays evaluations (Steiner et al 2003 Hua et al 2018) According to our results
obtained from the fourfold measurements in citrated whole blood ROTEM delta
measurements on healthy dogs and dogs with haemostatic alterations fulfil these
requirements with only a few exceptions concerning CFT and ML values in abnormal
samples
Our results regarding repeatability are similar to those obtained for several TEM
parameters in previous studies performed in animals In one study the imprecision of the
ROTEM analyser was evaluated by duplicate measurements in 10 blood samples of
healthy dogs and as result of that evaluation a good precision (CVs below 10 ) was
calculated for CT CFT MCF and α angle but in contrary to our results a low
repeatability was found for ML (CVs of 233 [ex-tem] and 285 [in-tem] own values
534 and 216 ) (Falco et al 2012)
In a previous feline study using ex-tem and in-tem as activating reagents for the ROTEM
delta repeatability was assessed similarly to the present study based on fourfold
measurements of blood samples of two healthy individuals (Doumlderlein and Mischke
2015) The results also revealed CVs below 10 for most of the parameters evaluated
The partially low repeatability of ML values may reflect the vulnerability of this variable
which is not only influenced by the complex clot formation process but also by its
subsequent lysis Reproducibility of ex-tem or in-tem activated measurements of the
ROTEM delta analyser which was not investigated in our study was studied in blood
from healthy and unhealthy horses by two different operators using three different
ROTEM devices Results indicated acceptable reproducibility (Junge et al 2016)
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
27
42 Reference intervals
The number of 125 healthy dogs used in the present study to create reference intervals is
regarded adequate according to the guidelines suggested by the American Society of
Veterinary Clinical Pathology (ASVCP) and the National Committee for Clinical
Laboratory Standards (NCCLS) who recommend to include at least 120 individuals
(Friedrichs et al 2012) To the best of our knowledge reference intervals for the most
relevant ROTEMreg delta parameters obtained in an adequate number of samples of whole
citrated canine blood have not been published so far A previous study was performed on
a smaller number of 45 dogs (Falco et al 2012) The median values for reference
intervals obtained in the present study are partially similar to those calculated in the
mentioned study based on 45 healthy dogs (eg ex- tem CT 610 s450 s [Falco et al
2012own results] CFT 150 s 110 s α angle 620deg680deg MCF 530 m 610 mm ML
180 220 in-tem CT 235 s190 s CFT 136 s116 s α angle 660deg680deg MCF
560 mm590 mm ML 30 180 ) However in comparison to our results the
reference intervals in the mentioned study are slightly wider (eg ex-tem CT 29ndash92
s34ndash83 s [Falco et al 2012own results] CFT 540ndash275 s650ndash220 s α angle 470ndash
790deg540ndash790deg MCF 360ndash730 mm460ndash730 mm ML 000ndash560 800ndash590
in-tem CT 126ndash363 s135ndash283 s CFT 470ndash224 s730ndash251 s aα angle 550ndash
810deg510ndash750deg MCF 500-ndash750 mm480ndash680 mm ML 000ndash400 500ndash270 )
although the previous study used the 5 and 95 quantiles to define limits of the
reference range whereas we calculated the commonly used 25 and 975 quantiles
to capture the central 95 of reference values (Friedrichs et al 2012)
Although it is desirable that each laboratory should specifically determine its own
reference intervals the results obtained in this study may provide an orientation for other
laboratories to interpret ROTEM delta parameters analysed in citrated canine blood In
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
28
order to test transference of the reference ranges to another laboratory a validation
procedure with measurement of 20 samples should be performed (Friedrichs et al 2012)
Although intra-individual control values (subject-based reference values) are regarded
superior to population based reference values (Wiinber et al 2007) the latter have to be
routinely used in clinical patients due to lacking individual controls especially for specific
tests such as ROTEM analyses In addition the calculated reference values cannot be
used for the fully automated device ROTEM sigma because - according to an actual
human study - values of part of the parameters do not correlate well between both
instruments (Gillissen et al 2019)
43 Influence of size age sex and neuter status
In previous studies in humans (Attard et al 2013) and animals (Mischke 1994 Borrelli
et al 2017) age has been described as an influencing factor for different haemostasis
parameters We also found an influence of age on the CT with shorter times in senior
dogs (gt 84 months) compared with young dogs (up to 24 months) which was limited to
measurements activated by the in-tem reagent
This age dependency confirms in principle a previous study on 20 healthy dogs
(Barthelemy et al 2015) which found shorter CTs in elderly dogs and results of a human
study where individuals with an age gt 45 years showed shorter CTs compared with
individuals lt 45 years old (Sucher et al 2011) However in the cited studies this effect
was only seen when ex-tem but not when the in-tem reagent was used
Based on the age distribution of our dogs which were primarily recruited to establish
reference intervals we cannot deliver information regarding very young dogs A human
study focusing on ROTEM reference ranges in children demonstrated significant
differences for nearly all ROTEM parameters with the most striking differences in
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
29
subjects aged 0-3 months (Oswald et al 2010) The limited number of dogs with an age
lt 1 year which did not allow to consider these separately in the present study may also
be one reason for the different results in a feline study (Doumlderlein and Mischke 2015) In
the cited study on healthy cats age showed an influence on CT but contrary to our study
the CT was shorter in young individuals (6-12 months) compared with older ones In
addition this was only the case in measurements using kaolin and in non-activated
measurements When compared to humans and cats uni- or multivariate analyses
considering factors age and breed or size based on do populations including different
breeds have the limitation that they can be biased by the variate life expectancy between
small medium and large dog breeds (Willems et al 2017) Apart from age we found a
significant influence only for the neuter status in bitches reflected by a significant
interaction effect between factors neuter status and sex in the ANOVA analysis
Especially for CFT values (eg activated by ex-tem median values of 121 s [spayed] vs
96 s [intact]) this difference seems to be also clinically relevant with respect to the
interpretation of ROTEM results In contrast although also significantly different
absolute and relative differences of CT values were small (eg activated by ex-tem 46 s
[spayed] vs 43 s [intact]) These differences may not only be considered for the result
interpretation of clinical patients but indicate also the necessity for sex-matched patient
groups in scientific studies
The obviously more active haemostasis system in intact females is probably related to an
influence of sex hormones (specifically estrogens) on the coagulation system
Accordingly ex vivo and in vivo studies demonstrated the activating properties of
endogenous estrogens on the haemostasis system In one ex vivo study addition of
endogenous estrogens (estrone [E1] 17szlig-estradiol [E2] and estriol [E3]) to blood samples
from men resulted in shorter mean values of the reaction time in TEG analyses
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
30
corresponding to CT in ROTEM measurements (452-537 s vs 698 s [control]) and of
the TEG variable kinetics (corresponding to the CFT in ROTEM analyses) (Swanepoel
et al 2017) In a further study subcutaneous injection of E2 into male rats induced a
significant increase in activities of coagulation factors X (by 8 ) and XI (by 10-24 )
however did not show any significant effect on PT aPTT and factor VII activity (Franco-
Murillo and Jaimez 2017)
In the cited studies also the effects of the synthetic estrogen ethinylestradiol [EE] were
studied which interestingly seems to have an inhibiting effect as indicated by prolonged
reaction time and kinetics (TEG) compared with the control in the human ex vivo study
and significantly longer PTs and aPTTs and lower factor VII and X activities in the study
on rats As expected we did not find a significant influence of size on ROTEM parameters
in dogs To the best of the authoracutes knowledge size has not been examined as an
influencing factor on viscoelastic measurements or any other haemostatic variable in
dogs Apart from Greyhounds there is limited literature on breed comparisons of
haemostatic variables (Clemons and Meyers 1984 Mischke 1994 Nielsen et al 2007
Nielsen et al 2010 Boyd et al 2018) indicating very limited breed differences for
example for factor VII (Mischke 1994) However these also do not indicate systematic
influence of size and in addition possible breed-specific effects have to be considered
As already stated one limitation of the study is the limited number of young animals
which does not allow to verify reference values for animals in this stage of life In
addition with respect to the main aim of the study possible influencing factors such as
platelet count and haematocrit were not considered in the statistical analysis Finally the
relatively small number of dogs used for the precision analysis which express the fact
that this was only a secondary aspect of the study may be regarded as a limitation At the
same time these considerations provide space for further investigations
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
31
5 Conclusions
Results of the precision analysis revealed acceptable results and thereby confirm that the
ROTEMreg delta analyser delivers repeatable results to measure overall haemostasis in
canine citrated blood samples The established reference values should offer a reliable
basis for the interpretation of thromboelastometric measurements in whole blood using
the device ROTEMreg delta in dogs with suspected haemostasis disorders Neuter status
in female dogs appeared to be a relevant influencing factor on most important ROTEMreg
parameters and should be considered for result interpretation and with respect to
experimental designs
6 Acknowledgments
Jose Mauricio Pereira received a scholarship from the Universidad Nacional de Costa
Rica (reference code JB-C-1107-2016)
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
32
7 References
1 Attard C van der Straaten T Karlafitis V Monagle P Ignjativic V 2013 Developmental
hemostasis age-specific differences in the levels of hemostatic proteins J Thromb and
Haemost 11 1850ndash1854
2 Bagge A Schoumltt U Kander T 2016 Effects of naturopathic medicines on Multiplate and
ROTEMreg A prospective experimental pilot study in healthy volunteers BMC Complemen
Altern Med 17 (16) 64
3 Bartheacutelemy A Rannou B Forterre M Verwaerde P Bonnet-Garin JM Pouzot-Nevoret
C Goy-Thollot I 2015 Differences between coagulation and cytokine profiles in dogs of
different ages Vet J 205 410ndash412
4 Bauer N Eralp O Moritz A 2009 Establishment of reference intervals for kaolin-activated
thromboelastography in dogs including an assessment of the effects of sex and anticoagulat use
J Vet Diagn Investi 21 641ndash648
5 Bollinger D Seeberger MD Tanaka KA 2012 Principles and practice of
thrromboelastography in clinical coagulation management and transfusio practice Transfus Med
Rev 26 1ndash13
6 Borrelli A Botto A Maurella C Falco S Pagani E Miniscaldo B Tarducci A Bruno
B 2017 Thromboelastometric assesment of hemostasis in newborn Piemontese calves J Vet
Diagn Invest 29 293ndash297
7 Boyd CJ Claus MA Raisis AL Hosqood G Sharp CR SmartL 2018
Hypocoagulability and platelet dysfunction are exacerbated by synthetic colloids in a canine
hemorrhagic shock model Front Vet Sci 5 279
8 Bruno B Maurella C Falco S Tarducci A Zanatta R DrsquoAngelo A Borrelli A 2015a
Thromboelastometric evaluation of hemostasis in dogs infected with Leishmania infantum J Vet
Emerg Crit Care (San Antonio) 25 502ndash511
9 Bruno B Maurella C Falco S Tarducci A Zanatta R Gianella P DrsquoAngelo A Piras L
Di Bella A Borrelli A 2015b Assessment of coagulation utilizing thromboelastometry in dogs
undergoing orthopedic surgery J Vet Emerg Crit Care (San Antonio) 25 358ndash363
10 Clemons RM Meyers KM 1984 Acquisition and aggregation of canine blood platelets Basic
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
33
mechanisms of function and differences because of breed origin Am J Vet Res 45 137ndash144
11 Conversy B Blais MC Dunn M Gara-Boivin C del Castillo JRE 2017 Anticoagulant
activity of oral rivaroxaban in healthy dogs Vet J 223 5ndash11
12 Crochemore T Piza FMT Rodriacuteguez RDR Guerra JCC Ferraz LJR Correcirca TD
2017 A new era of thromboelastometry Einstein (Sao Paulo) 15 380ndash385
13 Crochemore T Correcirca TD Lance MD Solomon C Neto AS Guerra JCC Lellis PS
Bernz LM Nunes N Mancio CM Yokoyama APH Silva E 2018 Thromboelastometry
profile in critically ill patients A single-center retrospective observational study PLoS One 13
e0192965
14 Doumlderlein E Mischke R 2015 Reference intervals for thromboelastometry with the ROTEMreg
delta in cats Res Vet Sci 100 271ndash276
15 Falco S Bruno B Maurella C Bellino C DacuteAngello A Gianella P Tarducci A Zannata
R Borrelli A 2012 In vitro evaluation of canine hemostasis following dilution with
hydroxyethyl starch (13004) via thromboelastometry J Vet Emerg Crit Care (San Antonio)
22 640ndash645
16 Franco-Murillo Y Jaimez R 2017 Inhibitory effect of ethinylestradiol on coagulation factors
in rats Exp Anim 66 107ndash113
17 Friedrichs KR Harr KE Freeman KP Szladovits B Walton RM Barnhart KF Blanco-
Chavez J 2012 ASVCP reference interval guidelines Determination of de novo reference
intervals in veterinary species and other related topics Vet Clin Pathol 41 441ndash453
18 Gillissen A va den Akker T Caram-Deelder C Henriquez DDCA Bloemenkamp
RWM Eikenboom J van der Bom JG de Maat MPM 2019 Comparison of
thromboelastometry by ROTEMreg Delta and ROTEMreg Sigma in women with postpartum
haemorrhage Scand J Clin Lab Invest 79 32ndash38
19 Harttert H 1948 Blutgerinnungstudien mit der Thrombelastographie einem neuen
Untersuchungsverfahren Klin Wochenschr 26 577ndash583
20 Horn P Pesce A 2003 Reference intervals and update Clin Chim Acta 334 5ndash23
21 Hua Yanhun Qiu W Xiao Q Wu Q 2018 Precision (repeatability and reproducibility) of
ocular parameters obtained by the Tomey OA-2000 biometer compared to the IOLMaster in
healthy eyes PLoS One 13 e0193023
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
34
22 Junge H Ringer S Mayer N Schwarzwald C 2016 Assesment of method reliability and
determination of reference intervals for rotational thromboelastometry in horses J Vet Emerg
Crit Care (San Antonio) 26 691ndash703
23 Kol A Borjesson DL 2010 Application of thrombelastographythromboelastometry to
veterinary medicine Vet Clin Pathol 39 405ndash416
24 Marschner CB Wiinberg B Tarnow I Markussen B Kuumlhnel L Bochsen L Kristensen
AT 2018 The influence of inflammation and hematocrit on clot strength in canine
thromboelastographic hypercoagulability J Vet Emerg Crit Care (San Antonio) 28 20ndash30
25 McMichael MA Smith SA 2011 Viscoelastic coagulation testing technology applications
and limitations Vet Clin Pathol 40 140ndash153
26 Mischke R 1994 Activity of coagulation factors II V VII and X in healthy dogs- dependence
on age sex and breed Berl Muumlnch Tieraumlrztl Wochenschr 107 289ndash294
27 Nielsen A Zois N Pedersen H Olse L Tarnow I 2007 Platelet function in dogs bread
differences and effect of acetylsalicylic acid administration Vet Clin Pathol 36 267ndash273
28 Nielsen L Kjelgaard‐Hansen M Jensen A Kristensen A 2010 Breed‐specific variation of
hematologic and biochemical analytes in healthy adult Bernese Mountain dogs Vet Clin Pathol
39 20ndash28
29 Nogami K 2016 The utility of thromboelastography in inherited and acquired bleeding
disorders Br J Haematol 174 503ndash514
30 Oswald E Stalzer B Heitz E Weiss M Schmugge M Strasak A Innerhofer P Haas T
2010 Thromboelastometry (ROTEMreg) in children Age-related reference ranges and correlations
with standard coagulation tests Br J Anaesth 105 827ndash835
31 Sankarankutty A Nascimento B Teodoro da Luz L Rizoli S 2012 TEGreg and ROTEMreg
in trauma similar test but different results World J Emerg Surg 7 (Suppl 1) S3
32 Sigrist NE Schefer RJJ Kutter APN 2018 Characteristics of hyperfibrinolysis in dogs and
cats demonstrated by rotational Thromboelastometry (ROTEM) Vet J 242 67-73
33 Steiner J Teague S Williams D 2003 Development and analytic validation of an enzime-
linked immunosorbent assay for the measurement of canine pancreatic lipase immunoreactivity in
serum Can J Vet Res 68 161ndash168
34 Sucker C Tharra K Lithmate J Scharf RE Zotz RB 2011 Rotation thromboelastography
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
35
(ROTEM) parameters are influenced by age gender and oral contraception Perfusion 26 334ndash
340
35 Swanepoel A Emmerson O Pretorius E 2017 The effect of endogenous and synthetic
estrogens on whole blood clot formation and erythrocyte structure Microsc Microanal 23 599ndash
606
36 Theusinger OM Nuumlrnberg J Asmis LM Seifert B Spahn DR 2010 Rotation
thromboelastometry (ROTEM) stability and reproducibility over time Eur J Cardiothorac Surg
37 677ndash683
37 Toulon P Metge S Hangard M Zwahlen S Piaulenne S Besson V 2017 Impact of
different storage times at room temperature of unspun citrated blood samples on routine
coagulation tests results Results of a bicenter study and review of the literature Int J Lab
Hematol 3 458ndash468
38 Turner JS Kutter APN Sigrist NE 2019 Correlation of rotational thrromboelastometry
(ROTEM) parameters with platelet count and their ability to predict thrombocytopenia in dogs
Res Vet Sci 126 45ndash50
39 Versteeg HH Heemskerk JWM Levi M Reitsma PH 2013 New Fundamentals in
Hemostasis Physiol Rev 93 327ndash358
40 Wiinberg B Jensen AL Kjelgaard-Hansen M Rojkjaer R Johansson PI Gade LP
Gram DX Kristensen AT 2007 Study on biological variation of haemostatic parameters in
clinically healthy dogs Vet J 174 62ndash68
41 Wiinberg B Jensen AL Johansson PL Rozanski E Tranholm M Kristensen AT 2008
Thromboelastographic evaluation of hemostatic function in dogs with disseminated intravascular
coagulation J Vet Intern Med 22 357ndash365
42 Wikkelsoslash A Wetterslev J Moslashller AM Afshari A 2016 Thromboelastography (TEG) or
thromboelastometry (ROTEMreg) to monitor haemostatic treatment versus usual care in adults or
children with bleeding Cochrane Database Syst Rev Art
httpsdoiorg10100214651858CD007871pub3 No CD007871
43 Willems A Paepe D Marynissen S Smets P Van de Maele I Picavet P Duchateau L
Daminet S 2017 Results of screening of apparently healthy senior and geriatric dogs J Vet
Intern Med 31 81ndash92
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
36
44 Woodman J Wagg CR Boysen SR Leguillete R Mizen K Roy MF 2015 Evaluation
of coagulation via thromboelastography in healthy horses administered dexamethasone Can Vet
J 56 1271ndash1274
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
37
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of
standard heparin prophylaxis and correlation to haemostatic changes
Pereira JM Hewicker-Trautwein M Rohn K von Depka Prondzinski M
Mischke R
Contributions of the authors
Pereira JM performed (1) the ultrasound evaluations and blood sampling of the
patients under study (2) haematology analysis (3) injection of the anticoagulation
prophylaxis (partially) to the patients under study (4) analysis of samples (collaboration)
at Werlhof Institute Medical Care Center (5) analysis of some haemostasis test
(collaboration) (6) statistical analysis (partially) (7) data documentation (8) and wrote
the manuscript
Hewicker-Trautwein M performed the electron microscopy analysis
Rohn K performed the statistical analysis
von Depka Prondzinski M performed some haemostasis test (thrombin generation
assay and endogenous thrombin potential)
Mischke R performed the coagulation and haemostasis test was involved in designing
and supervision of the study analysis and discussion of the data and its statistical analysis
and revision edition and correction of the manuscript
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
38
Central venous catheter induced thrombosis in dogs ndash efficacy of standard heparin
prophylaxis and correlation to haemostatic changes
Joseacute Mauricio Pereira1 Marion Hewicker-Trautwein2 Karl Rohn3 Mario von Depka
Prondzinski4 Reinhard Mischke1
1Small Animal Clinic University of Veterinary Medicine Hannover Foundation
Buumlnteweg 9 D-30559 Hannover Germany
2Institute for Pathology University of Veterinary Medicine Hannover Foundation
Buumlnteweg 17 D-30559 Hannover Germany
3Institute of Biometry Epidemiology and Information Processing University of
Veterinary Medicine Hannover Foundation Buumlnteweg 2 30559 Hannover Germany
4Werlhof Institute Schillerstrasse 23 30159 Hannover Germany
Abbreviations anti-Xa anti-factor Xa BW body weight CVC central venous catheter
EJV external jugular vein ELMI electron microscopy IV intravenous JV jugular vein
LMWH low molecular weight heparin ML maximum lysis (ROTEM) PIVC
peripheral intravenous catheter ROTEM rotational elastometry SC subcutaneous
TGA thrombin generation assay TID three times a day TT thrombin time UFH
unfractionated heparin
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
39
Corresponding author
E-mail addresses matiscrgmailcom (JM Pereira) MarionHewicker-
Trautweintiho-hannoverde (M Hewicker-Trautwein) KarlRohntiho-hannoverde
(K Rohn) mailwerlhof-institutde (M von Depka Prondzinski)
ReinhardMischketiho-hannoverde (R Mischke)
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
40
ABSTRACT
Central venous catheters (CVC) are of importance for intensive care management of
canine patients Thrombosis of the jugular vein is a severe complication related to the
CVC use However only little is known about the incidence of CVC-associated
thromboses in dogs and the necessity and efficacy of prophylactic antithrombotic
regimens The objectives of the present study were (1) to determine the incidence of
catheter-induced thrombosis in canine intensive care patients and thereby (2) to evaluate
the efficacy of a routine prophylactic heparin treatment and finally (3) to assess whether
initial changes of selected haemostasis parameters are predictive for an increased risk for
thrombosis formation 29 dogs hospitalised in the Small Animal Clinic University of
Veterinary Medicine Hannover Foundation and receiving a central venous catheter
(CVC) in the external jugular vein (EJV) for medical reasons were included in the final
assessment 17 dogs received the standard antithrombotic dosage regimen of
unfractionated heparin (UFH 150 IUKG BW TID sc) and 12 dogs (usually surgical
patients) the reduced dosage (75 IUKG BW TID sc) A colour Doppler ultrasound of
the respective EJV in order to detect thrombus formation and blood collection for
haemostasis tests were performed immediately before the installation procedure (day 0)
and on days 1 3 5 7 and so on after the CVC insertion Haemostasis tests included
prothrombin time activated partial thromboplastin time thrombin time antithrombin
activity D-dimer concentration thrombin generation and rotational elastometry as well
as the heparin activity using a chromogenic anti-factor Xa test Finally electron
microscopy (ELMI) of sections of the removed CVC was performed In 8 dogs
sonographic examination revealed thrombus formation in the lumen of EJV and in 1927
CVCs assessed using ELMI thrombi on the external CVC surface were detected without
significant correlation between both methods Plasmatic heparin activities showed great
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
41
variability but no significant differences were found between dogs with or without CVC-
associated thrombus formation From the initially performed haemostasis tests high
fibrinogen concentrations and low maximum lysis values as measured by the ROTEM
delta device (ex-tem reagent) were associated with sonographically detectable thrombi
The results of the present study indicate that CVC-associated thromboses occur (eg 28
[829] of the patients as detected by ultrasound) although severe thromboses (high
grades) are relatively rare in intensive care canine patients receiving a routine
anticoagulant treatment A high fibrinogen concentration and low maximum lysis
(ROTEM ex-tem) may be useful indicators for an increased thrombotic risk in individual
patients which may require special anticoagulatory treatment
Keywords CVC complication venal occlusion antithrombotic treatment ultrasound
electron microscopy rotational elastometry
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
42
1 Introduction
Central venous catheters (CVCs) are very important tools for care of patients in critical
care units allowing parenteral nutrition support therapy administration of high volumes
of intravenous fluids safe drugs administration that cannot be given through peripheral
veins plasmapheresis haemodialysis blood transfusions as well as periodical blood
sampling (Geerts 2014 Vose et al 2019) CVC related thrombosis is one of the most
severe complications (apart from infections and pneumothorax) of CVCs in humans
(Geerts 2014 Comerlato et al 2017 Evans and Ratchford 2018) leading to patientrsquos
distress CVC dysfunction venous stenosis and in some cases to the development of
thromboembolism (Geerts 2014)
In the human literature there are various studies dealing with epidemiology with reference
to different anticoagulatory regimens including placebo-controlled studies (Lee and
Kamphuisen 2012) In humans CVC related thromboses represent 70ndash80 of all upper
extremity thrombosis cases and approximately 10 of thromboembolism cases in
humans (Kucher 2011) The incidence of CVC related thrombosis reported in humans
based on 25 studies ranged widely from 2 to 67 (median = 30 ) (van Rooden et al
2005 review article) This wide range of calculated incidences of CVC related
thromboses in humans reflects the use of different CVC types different locations of entry
site of the CVC (jugular femoral subclavian axillary cephalic vein) different thrombus
detection technique (venography or ultrasonography) unequal durations of follow up
varying diseases of patients (eg haematological oncological) and presence of
predisposing factors (eg previous venous thrombosis history presence of cancer) (van
Rooden et al 2005 Geerts 2014 Liu et al 2015)
Part of the authors empirically observed CVC-associated thrombosis of the external
jugular vein (EJV) as a relevant complication in their canine intensive care patients which
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
43
awakened their interest in that topic To the best of the authoracutes knowledge studies about
extra luminal CVC related thromboses in dogs are rare in the available veterinary
literature One study evaluating the complications associated with 100 CVCs placed in
the jugular vein of dogs and cats reported only 2 cases of thrombosis (confirmed by
palpation or ultrasound) but obviously did not assess the appearance of subclinical CVC
related thromboses (Adamantos et al 2010) Thereby the incidence of CVC-induced
thromboses in dogs is unknown and systematic studies regarding an effective
anticoagulatory management are lacking
In this context it is of interest whether a possible development of thrombosis or an
increased risk (despite the prevention by a standard application of unfractionated heparin
[UFH]) can be predicted by means of changes of haemostasis related parameters during
the initial examination In one study on 168 human patients without indication of
anticoagulation prophylaxis for example elevated leukocyte counts high plasminogen
activator inhibitor and high coagulation factor VIII levels (all measured on blood samples
taken after CVC placement) were associated with an increased incidence of symptomatic
CVC-related thrombosis diagnosed by ultrasonography (Boersma et al 2016)
Therefore aims of the present study were (1) to determine the incidence of catheter-
induced thrombosis in canine intensive care patients (as detected by ultrasonography and
electron microscopy ELMI) and thereby (2) to evaluate the efficacy of a standard
antithrombotic regimen with unfractionated heparin (UFH) routinely used in the Small
Animal Clinic of the University of Veterinary Medicine Hannover and (3) to assess
whether selected haemostasis test results are predictive for an increased risk for
thrombosis formation
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
44
2 Material and methods
21 Study design
Canine in-patients of the Small Animal Clinic University of Veterinary Medicine
Hannover receiving a CVC in the EJV for medical reasons from March 2017 to
December 2019 were included Anticoagulatory treatment was performed according to
the standard protocol of the clinic Colour Doppler ultrasonographic controls of the EJV
and blood samplings (for heparin activity basic haematology and haemostasis tests) were
done immediately before the catheter was placed (day 0) and on days 1 3 5 etc (2 hours
after the first daily UFH injection) until the CVC was removed Only patients were
included which could be examined at least until day 3 Haemostasis tests included
prothrombin time activated partial thromboplastin time thrombin time fibrinogen
concentration antithrombin activity fibrin monomers D-dimers thrombin generation
assay (TGA) and rotational elastometry as well as the heparin activity using a
chromogenic anti-factor Xa test Finally ELMI of sections of the CVC after its removal
was performed
22 Animals
Originally 32 patients hospitalised in the Small Animal Clinic and receiving a CVC in the
EJV for medical reasons entered the study Three of them were subsequently excluded
because they did not reach the defined minimum study period of 3 days Two were
euthanised and one referred back to the referring vet before day 3 22 dogs weighing more
than 15 kg were included so that the withdrawal of about 10 ml of blood on several days
was not a significant burden for the patient (lt 1 of the total blood volume) Five dogs
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
45
with a body weight of less than 15 kg but at least 8 kg were included with special
consideration of the amount of blood taken (no more than 6 ml on individual days)
All animals received standard anticoagulatory treatment with UFH 17 dogs with internal
diseases received 150 IUkg BW TID subcutaneously and 12 dogs (post surgery) received
a reduced dosage of 75 IUkg BW TID subcutaneously
The 29 dogs enclosed in the study included 4 mixed breed dogs and the following
pedigree dogs 5 Labrador Retrievers 3 German Shepherds 2 Australian Shepherds and
one from each of the following breeds Akita Inu Asian Shepherd Bavarian Mountain
Hound Beagle Belgian Shepherd Briard Canaan dog Dalmatian Golden Retriever
Havanese dog Hovawart Rhodesian Ridgeback Schnauzer Shar Pei and Wolfspitz
These dogs were aged between 2 and 164 months (median 77 months) and had body
weights ranging from 81 and 63 kg (median 27 kg) 9 were castrated males 8 intact
males 7 spayed females and 5 intact females
The diseases of the 29 patients requiring a CVC included 3 from each of the following
diseases hepatopathy and esophagus dilatation 2 from each of the following diseases
bacterial cystitis diabetes mellitus and 1 from each of the following diseases abdominal
wall perforation aspiration pneumonia cystitis intestinal round cells sarcoma carcinoma
of the nasal cavity chronic renal insufficiency cystic duct rupture heartworm disease
ivermectine intoxication lymphoma multifocal alveolar histiocytosis pancreatitis post-
surgical bleeding stomach dilatation stomach foreign body tetanus thoracic
pyogranulomatous inflammation thrombocytopenia and trigeminal nerve paresis
Animals were treated in accordance with the German Animal Welfare Law The
experimental design was approved by the Animal Welfare officer of the University of
Veterinary Medicine and reported to the responsible authority (Lower Saxony State
Office for Consumer Protection and Food Safety reference number 17A101)
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
46
23 Positioning and handling of the central venous catheter
Two types of single lumen radiopaque polyurethane CVC (different on length and
diameter) were used during the study The CVC type I (n = 2529) was Cavafixreg Certoreg
Splittocanreg CVC 32 cm length 11 x 17 mm diameter16 G (BBraun Melsungen AG)
the CVC type II used (n = 429) was Certofixreg Mono S 215 15 cm length 08 x 14 mm
diameter18G (BBraun Melsungen AG) Each CVC was inserted into the EJV using a
modified Seldinger technique as follows the skin of the neck was shaved and disinfected
with antiseptic soap alcohol and iodine Local anesthesia (Lidocaine HCl 2) was
administrated subcutaneously on the catheter insertion site (a middle point in the lateral
neck over the jugular sulcus) A small skin incision was performed in the insertion site
and one peripheral plastic cannula with a metallic needle inside (14 G for CVC type I
and 20 G for CVC type II) was inserted in the EJV in cranio-caudal orientation (Hundley
et al 2018) Once the EJV was reached the needle was removed and a flexible wire
guide was inserted through the peripheral cannula following an external landmark
(4thintercostal space) (Reminga et al 2018) The peripheral cannula was removed and
the respective CVC was inserted slowly and with gentle pressure into the EJV guided
with the flexible wire and following the same external landmark to reach the right
position After the insertion of the CVC the flexible wire guide was removed The correct
positioning of the CVC was checked with a latero-lateral thorax x-ray ensuring that the
CVC tip was placed at the junction of the cranial cava vein with the right atrium Each
CVC was fixed to the neck skin (adjacent to the insertion point) with a monofilament
non-absorbable suture (gauge 3-0) Finally a bandage elaborated with Povidone-Iodine
gauze (Betaisodonareg) padded synthetic band (Artiflexreg Natur) and elastic band
(Elastomullreg) was used to protect the CVC output (Claude et al 2010) and changed
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
47
daily Several times day the catheter was flushed with isotonic sodium chloride
containing 1 IUml UFH
24 Heparin preparation and administration
Heparin-sodium-25000-ratiopharm (injection solution containing 5000 IUml sodium
heparin [mucosa]) was used SC injection was performed over the lateral thorax wall
using 1 mL syringes with fine graduation marks
25 Ultrasonography
Colour Doppler ultrasonographic examinations were performed in lateral position using
a LO-GIQ E9 ultrasound device (GE Healthcare Chicago Illinois USA) with a 15
Megahertz linear ultrasound probe The development of a thrombus or fibrin debris in the
EJV lumen around the CVC or inside it was assessed at each control Once the
ultrasound was finished a new protective bandage was placed around the neck of the dog
According to the percentage of lumen occlusion of the EJV (evaluated in cross sectional
views during the ultrasound scanning) the thrombi were classified in four different grades
(Fig 1)
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
48
of the residual vein lumen C ndash catheter T ndash thrombus
Fig 1 Definition of different grades of central venous catheter induced thrombosis of the
jugular vein as detected using colour Doppler ultrasound
26 Blood collection
At each time point approx 6ndash10 ml of blood (according to the body weight) was collected
This included approx 1 ml of EDTA blood for hematological analysis and approx 5ndash9
ml of citrated blood (tubes containing one part of 011 molL [38 ] sodium citrate to
nine parts of blood) for the measurement of hemostasis tests in citrated blood (ROTEM)
or citrated plasma Blood samples were collected by puncture of peripheral veins with
disposable cannulas (09 x 40 mm [20G] or 10 x 40mm [21G]) directly into sample tubes
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
49
or from the CVC using the three-syringe-technique (Villalta-Garciacutea et al 2015) in
situations when the peripheral venipuncture was not indicated due to phlebitis or irritation
andor inflammation of the surrounding skin Briefly 2ndash3 ml of blood were taken in a
sterile disposable syringe and immediately afterwards the blood sample was taken in a
new disposable syringe and transferred into the test tubes containing the respective
anticoagulants finally the first 2-3 ml taken were re-infused to the dog through the CVC
followed by flushing with a solution of sodium chlorideUFH (02) Immediately after
blood collection each sample tube was swayed gently until blood and anticoagulant had
been mixed thoroughly To prepare citrated platelet poor plasma citrated blood was
centrifuged at 16000 g for 10 min at room temperature using a microcentrifuge The
plasma was transferred into plastic tubes and the centrifugation procedure was repeated
This platelet poor plasma was then frozen in aliquots at -70 degC until used for analysis
Directly before analysis the plastic tubes were thawed at 37 degC in a water bath
27 Laboratory tests
Haematological analyses were made automatically with Adviareg 2120i (Siemens
Healthcare GmbH Germany)
Prothrombin time activated partial thromboplastin time and thrombin time were
measured with the autoanalyser Amax Destiny Plus (Tcoag Deutschland GmbH
Germany) and commercial reagents (Thromborel S Siemens Healthcare Diagnostics
Products GmbH Marburg Germany CK Prestreg and Test Thrombin Reagent
Diagnostica Stago SAS Asniegraveres sur Seine Cedex France) Whereas the last two tests
were measured according to the manufacturer`s instructions a modified test optimised
for canine plasma was used to measure prothrombin time (25 microl citrated plasma 120 with
imidazole buffer + 25 microl of a human fibrinogen solution 2 gl prewarmed for 120 s at 37
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
50
oC addition of 25 microl of activating reagent Thromborel S) and the results converted into a
percentage activity based on a calibration curve prepared with canine pooled plasma The
fibrinogen concentration was measured with the method according to Clauss using a test
kit from Stago Diagnostica SAS D-dimer concentration was measured with a
quantitative commercial human latex turbidimetric method in the autoanalyser Cobas
c311 using a completely assembled kit and human standards (Roche Diagnostics
Mannheim Germany) Antithrombin activity was also measured with a completely
assembled chromogenic test kit in the autoanalyser Cobas c311 and calibrated with a
canine pool plasma (n = 100)
The thrombin generation assay (Technothrombinreg TGA (Technoclone GbmH Vienna
Austria) determines thrombin generation based on monitoring of the fluorescence
generated by cleavage of a fluorogenic substrate by thrombin over time after activation
of the coagulation cascade by different concentrations of tissue factor and negatively
charged phospholipid in platelet poor plasma From the fluorescence development over
time the concentration of thrombin in the sample can be calculated using a thrombin
calibration curve The increase in thrombin concentration with time then allows to
calculate generation of thrombin in the sample and to plot thrombin values over time for
the whole coagulation process
The rotational thromboelastometry analysis with ROTEM delta device (Werfen Munich
Germany) was started approx 15 minutes after the blood collection To perform the
analysis and according to the automated ROTEM software 20 microl of recalcification reagent
(200 mmoll calcium chloride solution star-tem) and 20 microl of the respective activating
reagent (ex-tem in-tem) were pipetted into and carefully mixed in a pre-warmed cuvette
using the automated pipette provided in the device immediately afterwards 300 microl of
citrated whole blood (previously gently revolved) were added and mixed with the
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
51
reagents previously deposited Finally the cuvette was carefully placed and fixed to an
oscillating pin (oscillation movements on left and right through an angle of 475deg) in the
ROTEM delta device The interaction between blood cells and subsequently the bond
between fibrin and platelets promoted by the biomechanical changes in pH electrolytes
and temperature start the clot formation with its subsequent retraction and lysis The
magnitude of the resistance produced by the clot over the oscillating pin is quantitatively
represented by different parameters measured during the analysis and also qualitatively
in a graphic illustration (Bagge et al 2016 Crochemore et al 2017)
A chromogenic substrate test (Coatest Heparin Chromogenix-Instrumentation
Laboratory Haemochrom Diagnostica Essen Germany) was used for measurements of
UFH in the auto-analyser Cobas c311 The test application was created based on
manufacturers` instructions Different dilutions of the same batch of the commercial
sodium UFH preparation used in the experiment with normal canine pool plasma
(prepared from identical aliquots of 10 healthy adult dogs) served as standards for the
calibration of the measurement
28 Electron microscopy
Selected areas on the last third of the CVC (5ndash10 cm according with the CVC) with
macroscopically detectable thrombi were analysed and some of the CVCs were cut in
two different places The cut samples were preserved in a fixative medium composed of
1 glutaraldehyde and 4 formalin Once the sample was fixed it was prepared
according to the osmium thiocarbohydrazide procedure (OTOTO-procedure) (Malick et
al 1976) The fixed CVCs were dehydrated in seven series as follow first three series of
120 minutes each one on 30 50 and 70 ethanol respectively followed by two series
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
52
of 120 min each one on 90 ethanol and then two series of 24 hours each one on 100
ethanol After the dehydration process the samples were placed two times during 24
hours each one on isoamyl acetate and then placed on in a critical point drier Finally the
samples were coated with gold in a splutter-coater with 25 kV acceleration voltage in an
argon atmosphere with a current of 20 mA for one minute The samples were examined
in an EVO 15 scanning electron microscope (ZEISS International Carl Zeiss Jena
Germany) and photographed with magnifications of 600x and 6000x avoiding mechanical
artifacts (Toskala et al 1995) Thrombi on the external wall of the CVC were analysed
and measured using the software ImageJ (National Institutes of Health and the Laboratory
for Optical and Computational Instrumentation University of Wisconsin USA)
According to their extension (surface size) and thickness (related to the CVC wall
thickness in a cross sectional view) thrombi on the external wall and additionally within
the lumen of the CVC were each classified in four grades (Fig 2 Fig 1S [suppl
material]) In case of two cuts the highest degree of thrombosis detected at one of the two
sites was used for the final assessment
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
53
surface sizepercent of CVC wall thickness
Fig 2 Definition and illustration of different grades of central venous catheter induced
(extraluminal) thrombosis as detected using electron microscopy
Fig 1S (suppl material) Definition and illustration of different grades of intraluminal
thrombosis in central venous catheters as detected using electron microscopy
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
54
29 Statistical methods
Comparison between thrombus graduation either based on sonographic and ELMI
findings respectively was performed using the McNemar test with calculation of the
concordance index (κ) Standard normal distribution was assessed using the
Kolomogorov-Smirnov test
Due to non-normal distribution of part of data sets heparin activities and results of
haemostasis assays are reported using non-parametric variables (median minimumndash
maximum box and whisker plots) Heparin activities and results of haemostasis tests
between dogs with or without thrombosis were compared using Mann-Whitney-U test
3 Results
31 Sonographic detection of thrombus formation
Thrombus formation was sonographically detected in 829 patients during the study
period Based on examination on the final day in half of these eight patients (n = 4) the
thrombus formation was scored grade 1 in 3 dogs grade 2 and only in one grade 3 In all
of these 8 dogs this was the maximum grade during the study period One dog with fibrin
precipitates was excluded from further analysis because it could not be clearly assigned
to one of the groups In 78 cases thrombi were attached mainly to the venous wall
whereas only in one case was attached to the CVC external wall Seven of these 8 dogs
which developed thrombi received the standard heparin dosage (150 IUkg TID 41
[717] of dogs receiving this dosage) and only one the reduced heparin dosage (75 IUkg
TID 8 [112] of dogs receiving this dosage)
The time point of first detection of thrombus formation varied between days 3 and 11
(median day 8) In addition detritus (presumably composed mainly of fibrin) was
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
55
detectable in the lumen of the EJV in one further dog and within the lumen of the CVC
in further 5 dogs
32 Thrombus formation by electron microscopy
27 from 29 CVC catheter tips were analysed by electron microscopy 827 samples were
without detectable thrombus (grade 0) and 1927 with detectable thrombi 72 showed
thrombus formation of grade 1 427 of grade 2 and 827 of grade 3 There was no
concordance between thrombus graduation based on sonographic and the ELMI
graduation respectively The concordance index (McNemar test) was negative (κ = -
00718) indicating that the degree of agreement is less than by chance
Of the in total 19 dogs with detectable thrombus formation by electron microscopy 11
dogs received 150 IUkg TID (in total 16 dogs of these 27 dogs received that heparin
dosage) and 8 received 75 IUkg TID (in total 11 of these 27 dogs received that heparin
dosage 73 811) and Of the 12 patients with grade 2 and 3 thrombus formation 5 dogs
received 75 IUkg TID and 7 dogs 150 IUkg
The supplementary analysis of intraluminal thrombus formation revealed no detectable
thrombi in 7 cases and thrombus formation of grade 1 (lt 25 occlusion) in 11 cases of
grade 2 (25ndash50 ) in 3 cases and of grade 3 (gt 50 ) in 6 cases
33 Heparin activities and relationship to thrombus formation
Heparin activities varied remarkably in both dosage groups (Fig 3 ab) There was no
significant difference of heparin activities between blood samples collected via
venipuncture or from the CVC (Table 1S supplementary material) In addition there was
no significant difference between heparin activities in dogs with or without
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
56
sonographically or electron microscopically detectable thrombosis (Tables 1 and 2)
Table 1 refers to sonographically detectable thromboses and is based on median heparin
activities calculated from the measurements on day 1 and following days per patient
firstly for the total number of patients and secondly for the patients receiving 150 IUkg
TID In addition in the eight dogs heparin activities on the day of first thrombus detection
(023 IUml 019ndash036 IUml) were not significantly different from the median values
(Table 1 P = 05214 Wilcoxon test) Table 2 compares heparin activities (median values
of all days and results on the final day) between dogs with and without electron
microscopically detectable CVC-associated thrombi
Table 1S (Supplementary material) Heparin activities (IUml median values
minimumndashmaximum) measured in 27 dogs receiving 75 or 150 IUkg unfractionated
(standard) heparin TID subcutaneously 2 hours after the first injection on the day
Comparison of samples collected via puncture of the peripheral veins and those collected
from the central venous catheter (CVC) using the three-syringe-technique
Peripheral vein
puncture CVC
Mann-Whitney-U
test
Total number
(75 and 150
IUkg TID)
019
(00ndash108)
(n = 65)
023
(000ndash105)
(n = 65)
P = 03033
75 IUkg TID
010
(000ndash108)
(n = 25)
012
(000ndash023)
(n = 21)
P = 06907
150 IUkg
TID
030
(000ndash096)
(n = 40)
036
(000ndash105)
(n = 44)
P = 01223
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
57
Fig 3ab Heparin activities (box- and whisker plot arithmetic means) in dogs receiving
unfractionated heparin at a dosage of 75 IUkg BW tid (a) or 150 IUkg BW tid (b) two
hours after the first daily heparin administration
day 1 represents the first day after initiation of heparin treatment
only days were considered with at least results from 5 patients
Table 1 Heparin activities (IUml median values minimumndashmaximum) in 28 dogs
receiving 75 or 150 IUkg unfractionated (standard) heparin TID subcutaneously with
(grades 1ndash3) or without (grade 0) sonographically detectable thrombus Blood collections
were performed 2 hours after the first daily injection
Grade 0
(sonography)
Grades 1ndash3
(sonography)
Mann-Whitney U
test
Total number
(75 and 150 IUkg
TID)
020 (000ndash072)
(n = 20)
027 (018ndash059)
(n = 8)
P = 03212
150 IUkg TID 042 (000ndash072)
(n = 10)
031 (018ndash059)
(n = 7)
P = 04593
one dog with fibrin precipitates in the lumen of the jugular vein but without defined thrombus formation
was not considered because it could not be clearly assigned to one of the two groups
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
58
Table 2 Heparin activities (IUml median values minimumndashmaximum) in 27 dogs
receiving 75 or 150 IUkg unfractionated (standard) heparin TID subcutaneously with
(score 1ndash3) or without (score 0) detectable thrombus via electron microscopy
Grade 0
(electron
microscop)
(n = 8)
Grades 1ndash3
(electron
microscop)
(n = 19)
Mann-Whitney U
test
Median value of
different days 028 (005ndash060)
022 (000ndash072)
P = 05770
Final day 020 (001ndash045)
015 (000ndash105)
P = 06706
34 Relationship between haemostasis tests and thrombus formation
From the initially performed haemostasis tests high fibrinogen concentrations and low
maximum lysis values as measured by the ROTEM delta device (using the ex-tem
reagent) were associated with sonographically detectable thrombi (Tab 3) Additional
analyses based on the median values of different days for individual patients revealed that
low maximum lysis values as measured by the ROTEM delta device (using the in-tem
reagent) were associated with sonographically detectable thrombi (maximum lysis score
0 850 [100ndash175 ] score 1ndash3 375 [100ndash900 ] median [minimumndash
maximum] P = 00166) but no other test Comparison of the results of haemostasis tests
on the final day between dogs with and without sonographically detectable thromboses
did not show any significant difference (P gt 005)
Comparison of results of haemostasis tests on the final day in dogs with detectable
thrombosis via ELMI and dogs without significant thrombus formation showed several
significant differences including lower fibrinogen concentrations and longer clotting
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
59
times (ex-tem reagent) and clot formation times (ex-tem and in-tem reagents) using the
ROTEM delta analyser in general expressing a less coagulable status (Tab 4) If values
of dogs graded 0 and 1 (n =15) are compared to those of grades 2 and 3 (n = 12) the last-
mentioned had longer clotting times (ROTEM delta in-tem reagent) as well as longer clot
formation times and lower alpha angles (ROTEM delta in-tem reagent) (P lt 005 results
not shown)
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
60
Table 3 Comparison of initial results of haemostasis parameters (median values
minimumndashmaximum in dogs with (grades 1ndash3) and without (grade 0) sonographically
detectable central venous catheter-induced thromboses
Parameter Unit
Sonographic
thrombus grade
P-value
(Mann-Whitney
U test)
0
(n = 20)
123
(n = 8)
Haematocrit 355 (228ndash641)
382 (245ndash499)
06111
Platelet count x103microl 292 (420ndash483)
218 (860ndash458)
02320
Prothrombin time 944 (359ndash169)
896 (471ndash136)
05758
Activated partial thromboplastin
time s 128
(955ndash249) 140
(117ndash164) 02322
Thrombin time s 129 (121ndash178)
141 (650ndash160)
02028
Fibrinogen gl 448 (153ndash770)
747 (153ndash116)
00235
Antithrombin activity 639 (289ndash107)
670 (464ndash746)
05249
D-dimers microgml 006 (000ndash148)
015 (003ndash372)
03997
Thrombin generation assay nmol 130 (000ndash365)
912 (190ndash277)
08787
Clotting time (ROTEM ex-tem) s 420 (290ndash930)
430 (170ndash159)
07989
Clot formation time (ROTEM ex-
tem) s 665
(310ndash143) 470
(240ndash800) 00633
Alpha angle (ROTEM ex-tem) degrees 795 (620ndash840)
815 (750ndash860)
01667
Max clot firmness (ROTEM ex-
tem) mm 750
(570ndash860) 775
(700ndash830) 01753
Max clot elasticity (ROTEM ex-
tem) - 299
(131ndash619) 347
(235ndash484) 01544
Maximum lysis (ROTEM ex-tem) 135 (300ndash290)
400 (000ndash800)
00037
Clotting time (ROTEM in-tem) s 208 (840ndash810)
249 (720ndash371)
05085
Clot formation time (ROTEM in-
tem) s 835
(370ndash634) 745
(390ndash235) 08988
Alpha angle (ROTEM in-tem) degrees 745 (250ndash820)
750 (500ndash820)
09797
Max clot firmness (ROTEM in-
tem) mm 700
(540ndash810) 740
(610ndash770) 03587
Max clot elasticity (ROTEM in-
tem) - 235
(118ndash424) 287
(156ndash332) 04764
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
61
Maximum lysis (ROTEM in-tem) 850 (000ndash210)
350 (000ndash220)
01327
Table 4 Comparison of results of haemostasis parameters (median values minimumndash
maximum) on the last day in dogs with (grades 1ndash3) and without (grade 0) central venous
catheter-associated thrombus formation as detected by electron microscopy
Parameter Unit
Thrombus grade
(ELMI)
P-value
(Mann Whitney
U test)
0
(n = 8)
123
(n = 19)
Haematocrit 322 (241ndash454)
330 (216ndash510)
05953
Platelet count 103microl 446 (321ndash592)
268 (101ndash675)
00558
Prothrombin time 145 (104ndash264)
120 (561ndash148)
00462
Activated partial thromboplastin
time s 123
(109ndash140) 136
(109ndash188) 00665
Thrombin time s 129 (121ndash178)
141 (650ndash160)
02028
Fibrinogen gl 465 (275ndash866)
347 (189ndash992)
00491
Antithrombin activity 793 (448ndash981)
692 (398ndash889)
04572
D-dimers microgml 018 (000ndash173)
007 (000ndash118)
01920
Thrombin generation assay nmol 501 (000ndash160)
93 (000ndash287)
09562
Clotting time (ROTEM ex-tem) s 360
(270ndash410) 400
(310ndash620) 00238
Clot formation time (ROTEM ex-
tem) s 395
(120ndash740) 650
(240ndash174) 00275
Alpha angle (ROTEM ex-tem) degrees 835 (760ndash880)
790 (580ndash850)
00484
Max clot firmness (ROTEM ex-
tem) mm 780
(700ndash850) 720
(550ndash860) 00586
Max clot elasticity (ROTEM ex-
tem) - 346
(239ndash555) 254
(121ndash637) 00495
Maximum lysis (ROTEM ex-tem) 105 (200ndash190)
110 (000ndash380)
03796
Clotting time (ROTEM in-tem) s 178 (100ndash222)
201 (139ndash554)
00525
Clot formation time (ROTEM in-
tem) s 435
(330ndash111) 790
(350ndash882) 00209
Alpha angle (ROTEM in-tem) degrees 815 (710ndash830)
740 (240ndash830)
00191
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
62
Max clot firmness (ROTEM in-
tem) mm 785
(660ndash810) 690
(270ndash840) 00586
Max clot elasticity (ROTEM in-
tem) - 362
(192ndash434) 218
(370ndash507) 00559
Maximum lysis (ROTEM in-tem) 950 (200ndash190)
800 (100ndash180)
06890
4 Discussion
The main result of our study is that under the study conditions part of the dogs developed
CVC-associated thromboses However only a very low part of these thromboses were
obviously of a clinically significant degree regarding the amount of occlusion of the EJV
and none of the dogs had a symptomatic thrombosis whose definition includes presence
of clinical signs such as localised swelling pain and redness (Boersma et al 2008 Lee
and Kamphuisen 2012) In the present study we particularly refer to the extraluminal
thromboses with regard to the assessment of the antithrombotic management
One major problem of the statistical analysis and result presentation was that the two
methods used to define the occurrence and degree of thrombus formation (ultrasound and
ELMI) revealed discrepant results Therefore we performed a statistical analysis
independently regarding thrombus formation with either of the two methods It is well
known that ELMI which has already been used to detect CVC induced thromboses in
humans (Lucas et al 2017) and experimental animals (Xiang et al 1998 Xiang et al
2001) is a very sensitive diagnostic technique which allows the ex vivo diagnosis of very
small or fibrin rich thrombi that perhaps cannot be detected by ultrasonography or
venography during the in vivo evaluation of patients (Xiang et al 2001) A major reason
for the discrepant results between the two methods used to detect CVC associated
thromboses is that different CVC segments are evaluated Using ultrasonography it was
only possible to evaluate the EJV in the neck area of the patient and thereby only its
proximal third In contrast ELMI assessed mainly the CVC tip and a human study
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
63
revealed that even different positions of the catheter tip are associated with various risks
for development of CVC associated thrombosis (Ballard et al 2016) One additional point
to consider which also may contribute to the discrepancy is that it is possible that thrombi
developed on the CVC surface can be detached during the CVC removal from the vein
and thereby before its analysis by ELMI (Xiang et al 2001)
The contrast venography technique is used as gold standard to confirm thrombosis in
human medicine However this diagnostic technique is normally used only in the case of
a strong clinical suspicion of venous thrombosis that cannot be detected by
ultrasonography (Laurenson et al 2010 Langston and Eatroff 2018) Our study protocol
with privately owned clinic patients did not allow to use this method In a study performed
in 44 human patients requiring a CVC (Koumlksoy et al 1995) a sensitivity of 94 and a
specificity of 96 were determined for the colour Doppler ultrasound diagnosis when it
was compared with the diagnosis performed with the contrast venography technique and
that makes the colour Doppler ultrasound an accurate diagnosis method to detect CVC
related thrombosis even in humans (Koumlksoy et al 1995 van Rooden et al 2003)
The incidences of CVC related thrombosis detected by colour Doppler ultrasound of the
internal jugular vein reported in 3 human studies were 2 30 and 56 (Harter et al
2002 Lordick et al 2003 Wu et al 1999) and although with a wide range the median
value of these rates (30 ) is very well suited to the incidence of 28 (829) which we
calculated for our canine study based on sonographic findings The wide variation of
incidences of CVC related thrombosis in different human studies can in general be
explained by differences regarding the type and entry site of the CVC (jugular femoral
subclavian axillary cephalic vein) the time period with the CVC in place the thrombus
detection technique (venography or ultrasonography comment in the cited 3 studies
consistently the last-mentioned method was used) and criteria for diagnosis of
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
64
thrombosis the duration of follow up diseases and presence of predisposing factors in
these patients (eg previous venous thrombosis history presence of cancer) and finally
prophylactic treatment with anticoagulants (van Rooden et al 2005 Geerts 2014 Liu et
al 2015)
Different pathomechanisms contribute to the development of CVC induced thromboses
During the CVC insertion the puncture of the skin on the CVC insertion site and the
rupture of the blood vessels integrity activate the coagulation cascade and the CVC acts
as a matrix for adherence and accumulation of fibrin and cellular components (Smith et
al 2012) The attachment of proteins and blood cells to the internal andor external wall
of the CVC can lead to a partial or complete occlusion of the catheter lumen and
sometimes to a partial or complete obstruction of the blood flow andor
thromboembolism (Langston and Eatroff 2018) There is a strong interrelationship
between thrombus and biofilm formation (Smith et al 2012) Prevention of thrombus
formation has thereby also relevance regarding the increased risk for microbial
colonisation of the catheter (Boersma et al 2008) leading at least in humans to an
increased number of CVC related systemic infections (Lordick et al 2003)
In this context it has to be considered that CVC produced from polyurethane which are
routinely used in our clinic and thereby in the study are usually associated with a
relatively high thrombosis potential and thereby definitely provide an effective test for
an antithrombotic regimen Silicone catheters have a significantly reduced risk for
thrombotic complications but this advantage has to be purchased by a higher rate of
mechanical failure (eg rupture of the catheter) (Wildgruber et al 2016) Promising
experimental approaches work on surface modifications of the standard catheters For
example one group was able to demonstrate that a coating of the surface of the catheter
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
65
with a zwitterionic polymeric sulfobetaine showed a significant reduction of thrombus
formation in vitro with human blood and in an in vivo canine model (Smith et al 2012)
Due to ethical reasons we did not performed a placebo-controlled study However
interestingly not all human studies report systemic anticoagulation and part of the human
studies was even placebo-controlled (review Lee and Kamphuisen 2012) Obviously
there is no consensus in the available human literature regarding the beneficial use of
antithrombotic prophylaxis to prevent CVC related thrombosis Several studies did not
find a statistically significant difference in the incidence of CVC related thrombosis
between patients receiving the antithrombotic prophylaxis compared with the control
groups without systemic anticoagulation (Verso et al 2005 P = 035 Karthaus et al
2006 P gt 005 Niers et al 2007 P = 049) whereas in other studies a lower incidence
of CVC related thrombosis was found in the group receiving anticoagulants as
prophylaxis compared with the control groups (Abdelfeki et al 2004 150 vs 126
P = 003 De Cicco et al 2009 400 vs 526 P = 005)
Although it is well known that subcutaneously administered UFH has a limited and
variable bioavailability and thereby leads to unpredictable blood activities (Hirsh and
Raschke 2004) the extremely wide range of blood activities after a defined dosage in our
study was relatively unexpected Blood was collected 2 hours after the first UFH
injection and thereby at the time of suspected maximum heparin activity considering the
relatively low UGH dosage (Mischke and Jacobs 2001) The cited study was performed
on healthy experimental dogs Possible additional influencing factors in critical ill
patients include circulatory insufficiency which may reduce and delay absorption from
subcutanenous tissue decreased clearance function of the liver and kidneys as well as
altered blood plasma concentrations of heparin binding plasma proteins such as
fibrinogen and platelet factor 4 and alterations of the heparin binding capacity of
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
66
endothelial cells which altogether are reasons for a great inter- but also intrapatient
variability (Hirsh and Raschke 2004) In individual patients this can lead to heparin
resistance It has also to be considered that antithrombin activities which are necessary
for an adequate heparin effect (Bjoumlrk and Lindahl 1982) were reduced in our patients
Availability of approved blood tests which can predict the risk of development of CVC
associated thrombosis already at the moment of the CVC placement are important
(Boersma et al 2016) One of the most interesting results was that dogs which developed
sonographically detectable thrombi had nearly twofold higher fibrinogen levels Well in
agreement with our results one study performed in anticoagulated human patients
demonstrated that high fibrinogen levels were associated with an increased risk of
perioperative venous thromboembolism (P = 0004) (Kato et al 2016) The cited study
does not exclusively refer to CVC associated thrombosis but 76 (1621) of the venous
thromboses were suggested to be associated with CVCs Fibrinogen measurement a test
which is widely available is therefore a useful and practicable indicator of an increased
risk for CVC induced thromboses and may advice the clinician to treat these patients
especially carefully (eg with low molecular weight heparin) In the study performed by
Boersma et al (2016) in 168 human patients without indication of anticoagulation
prophylaxis elevated leukocyte counts high levels of plasminogen activator inhibitor
and of coagulation factor VIII were associated with an increased incidence of
symptomatic CVC related thrombosis diagnosed by ultrasonography A limitation of that
study is that all measurements were performed on blood samples taken after CVC
placement Interestingly in our study ROTEM results were partly associated with
thrombus formation which reflects the well-known fact that results of viscoelastic
measurements are a good indicator of hypercoagulability and thromboembolic events
(Harahsheh and Ho 2018) It is of further interest and possibly unexpeted that on the final
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
67
day of examination dogs with ELMI signs of thromboses had partly lower coagulation
activities than dogs without thromboses Due to the low number of dogs without
detectable thrombi using ELMI this implausible result has to be interpreted with caution
One possible explanation could be that temporary hypercoagulability in these dogs may
have finally resulted in consumption of haemostasis substrates
Limitations of the present study were as a consequence of the study design based on
clinical patients the relatively low total number of patients inclusion of different UFH
treatment regimens and the lack of a control group
5 Conclusions
The results of the present study indicate that the used UFH regimen was not completely
effective to prevent CVC-associated thromboses which were detectable in 28 [829]
of the patients by ultrasound However severe thromboses (high grades) are relatively
rare in intensive care canine patients receiving a routine anticoagulant treatment Because
heparin plasma activities were not different in dogs with or without CVC-associated
thrombus formation other local and systemic factors must play a significant role as well
A high fibrinogen concentration and low maximum lysis (ROTEM ex-tem) may be
useful indicators for an increased thrombotic risk in individual patients which may
require special anticoagulatory treatment
6 Acknowledgments
Jose Mauricio Pereira received a scholarship from the Universidad Nacional de Costa
Rica (reference code JB-C-1107-2016)
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
68
7 References
1 Abdelkefi A Ben Othman T Kammoun L Chelli M Ben Romdhane N Kriaa A Ladeb S
Torjman L Lakhal A Achour W Ben Hassen A Hsaiumlri M Ladeb F Ben Abdeladhim A
2004 Prevention of central venous line-related thrombosis by continuous infusion of low-dose
unfractionated heparin in patients with haemato-oncological disease A randomized controlled trial
Thromb Haemost 92 654ndash661
2 Adamantos S Brodbelt D Moores A L 2010 Prospective evaluation of complications associated
with jugular venous catheter use in a veterinary hospital J Small Anim Pract 51 254ndash257
3 Bagge A Schoumltt U Kander T 2016 Effects of naturopathic medicines on Multiplate and ROTEM
a prospective experimental pilot study in healthy volunteers BMC Complement Altern Med 16 64
4 Ballard DH Samra NS Gifford KM Roller R Wolfe BM Owings JT 2016 Distance of the
internal central venous catheter tip from the right atrium is positively correlated with central venous
thrombosis Emerg Radiol 23 269ndash273
5 Boersma RS Jie KS Verbon A van Pampus EC Schouten HC 2008 Thrombotic and
infectious complications of central venous catheters in patients with hematological malignancies Ann
Oncol 19 433ndash442
6 Boersma R S Hamulyak K van Oerle R Tuinenburg A Ten Cate-Hoek AJ Schouten HC
2016 Biomarkers for prediction of central venous catheter related-thrombosis in patients with
hematological malignancies Clin Appl Thromb Hemost 22 779ndash784
7 Bjoumlrk I Lindahl U 1982 Mechanism of the anticoagulant action of heparin Mol Cell Biochem
48 161ndash182
8 Claude A Riedesel D Riedesel E 2010 Electrocardiography-guided and retrospective analysis of
central venous catheter placement in the dog Vet Anaesth Analg 37 97ndash105
9 Comerlato PH Rebelatto TF Santiago de Almeida FA Klein LB Boniatti M M Schaan
DB Rados VD 2017 Complications of central venous catheter insertion in a teaching hospital
Rev Assoc Med Bras 63 613ndash620
10 Crochemore T Piza FMT Rodriacutegues RDR Guerra JCC Ferraz LJR Correcirca TD 2017
A new era of thromboelastometry Einstein (Sao Paulo) 15 380ndash385
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
69
11 De Cicco M Matovic M Balestreri L Steffan A Pacenzia R Malafronte M Fantin D
Bertuzzi CA Fabiani F Morassut S Bidoli E Veronesi A 2009 Early and short‐term
acenocumarine or dalteparin for the prevention of central vein catheter‐related thrombosis in cancer
patients a randomized controlled study based on serial venographies Ann Oncol 20 1936ndash1942
12 Evans NS Ratchford EV 2018 Catheter related-venous thrombosis Vasc Med 23 411ndash413
13 Geerts W 2014 Central venous catheterndashrelated thrombosis Hematology Am Soc Hematol Educ
Program 2014 306ndash311
14 Harahsheh Y Ho KM 2018 Use of viscoelastic tests to predict clinical thromboembolic events A
systematic review and meta‐analysis Eur J Haematol 100 113ndash123
15 Harter C Salwender HJ Bach A Egerer G Goldschmidt H Ho AD 2002 Catheter-related
infection and thrombosis of the internal jugular vein in hematologic-oncologic patients undergoing
chemotherapy a prospective comparison of silver-coated and uncoated catheters Cancer 94 245ndash251
16 Hirsh J Raschke R 2004 Heparin and low-molecular-weight heparin The Seventh ACCP
Conference on Antithrombotic and Thrombolytic Therapy Chest 126 (Suppl) 188S-203S
17 Hundley D Brooks A Thomovsky E Jhonson P Freeman L Schafbuch R Heng HG Moore
G 2018 Comparison of ultrasound-guided and landmark-based techniques for central venous
catheterization via the external jugular vein in healthy anesthetized dogs Am J Vet Res 79 628ndash
636
18 Kato F Takeuchi H Matsuda S Kawakubo H Omori T Kitagawa Y 2016 Incidence of and
risk factors for venous thromboembolism during surgical treatment for esophageal cancer a single-
institution study Surg Today 46 445ndash452
19 Karthaus M Kretzschmar A Kroning H Biakhov M Irwin D Marschner N Slabber C
Fountzilas G Garin A Abecasis NG Baronius W Steger GG Sudhoff T Giorgetti C
Reichardt P 2006 Dalteparin for prevention of catheter‐related complications in cancer patients with
central venous catheters final results of a double‐blind placebo‐controlled phase III trial Ann Oncol
17 289ndash296
20 Koumlksoy C Kuzu A Kutlay J Erden I Ozcan H Ergicircn K 1995 The diagnostic value of colour
Doppler ultrasound in central venous catheter related thrombosis Clin Radiol 50 687-689
21 Kucher N 2011 Clinical practice Deep-vein thrombosis of the upper extremities N Engl J Med
364 861ndash869
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
70
22 Langston CE Eatroff AE 2018 Hemodialysis catheter-associated fibrin sheath in a dog J Vet
Emerg Crit Care 28 366ndash371
23 Laurenson MP Hopper K Herrera MA Johnson EG 2010 Concurrent diseases and conditions
in dogs with splenic vein thrombosis J Vet Intern Med 24 1298ndash1304
24 Lee AYY Kamphuisen PW 2012 Epidemiology and prevention of catheter‐related thrombosis in
patients with cancer J Thromb Haemost 10 1491ndash1499
25 Liu G Fu ZQ Zhu P Li SJ 2015 Central venous catheter-related thrombosis in senile male
patients New risk factors and predictors J Huazhong Univ Sci Technolog Med Sci 35 445ndash449
26 Lordick F Hentrich M Decker T Hennig M Pohlmann H Hartenstein R Peschel C 2003
Ultrasound screening for internal jugular vein thrombosis aids the detection of central venous catheter-
related infections in patients with haemato-oncological diseases a prospective observational study Br
J Haematol 120 1073ndash1078
27 Lucas TC Silva EED Souza DOD Santos ARD Lara MO 2017 Microstructural
evaluation by confocal and electron microscopy in thrombi developed in central venous catheters Rev
Esc Enferm USP 51 1ndash9
28 Malick L Wilson R Stetson D 1976 Modified thiocarbo- hydrazide procedure for scanning
electron microscopy routine use for normal pathological and experimental tissue Stain Technol 50
265ndash269
29 Mischke R Jacobs C 2001 The monitoring of heparin administration by screening tests in
experimental dogs Res Vet Sci 70 101ndash108
30 Niers TM Di Nisio M Klerk CP Baarslag HJ Buller HR Biemond BJ 2007 Prevention
of catheter‐related venous thrombosis with nadroparin in patients receiving chemotherapy for
hematologic malignancies a randomized placebo‐controlled study J Thromb Haemost 5 1878ndash
1882
31 Reminga C Silverstein D Drobatz K Clarke D 2018 Evaluation of the placement and
maintenance of central venous jugular catheters in critically ill dogs and cats J Vet Emerg Crit Care
28 232ndash243
32 Smith RS Zhang Z Bouchard M Li J Lapp HS Brotske GR Lucchino DL Weaver D
Roth LA Coury A Biggerstaff J Sukavaneshvar S Langer R Loose C 2012 Vascular
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
71
catheters with a nonleaching poly-sulfobetaine surface modification reduce thrombus formation and
microbial attachment Sci Transl Med 4 153ra132 doi101126scitranslmed3004120
33 Toskala E Nuutinen J Rautiainen M 1995 Scanning electron microscopy findings on human
respiratory cilia in chronic sinusitis and in recurrent respiratory infections J Laryngol Otol 109 509ndash
514
34 Van Rooden CJ Tesselaar ME Osanto S Rosendaal FR Huisman MV 2005 Deep vein
thrombosis associated with central venous catheters - a review J Thromb Haemost 3 2409ndash2419
35 Verso M Agnelli G Bertoglio S Di Somma FC Paoletti F Ageno W Bazzan M Parise P
Quintavalle R Naglieri E Santoro A Imberti D Soraru M Mosca S 2005 Enoxaparin for the
prevention of venous thromboembolism associated with central vein catheter a double‐blind placebo‐
controlled randomized study in cancer patients J Clin Oncol 23 4057ndash4062
36 Villalta-Garciacutea P Loacutepez-Herranz M Mazo-Pascual S Honrubia-Fernaacutendez T Jaacutentildeez-Escalada
L Fernaacutendez-Peacuterez C 2015 Reliability of blood test results in samples obtained using a 2-mL discard
volume from the proximal lumen of a triple-lumen central venous catheter in the critically ill patient
Nurs Crit Care 22 298ndash304
37 Vose J Odunayo A Price JM Daves M Schildt JC Tolbert MK 2019 Comparison of
heparinized saline and 09 sodium chloride for maintaining central venous catheter patency in healthy
dogs PeerJ 7 e7072 httpsdoiorg107717peerj7072
38 Wildgruber M Lueg C Borgmeyer S Karimov I Braun U Kiechle M Meier R Koehler M
Ettl J Berger H 2016 Polyurethane versus silicone catheters for central venous port devices
implanted at the forearm Eur J Cancer 59 113ndash124 doi101016jejca201602011
39 Wu X Studer W Skarvan K Seeberger MD 1999 High incidence of intravenous thrombi after
short-term central venous catheterization of the internal jugular vein J Clin Anesth 11 482ndash485
40 Xiang DZ Verbeken EK Van Lommel AT Stas M De Wever I 1998 Composition and
formation of the sleeve enveloping a central venous catheter J Vasc Surg 28 260ndash271
41 Xiang DZ Verbeken EK Van Lommel AT Stas M De Wever I 2001 Sleeve-related
thrombosis a new form of catheter-related thrombosis Thromb Res 104 7ndash14
Chapter 4 General discussion
72
Chapter 4 General discussion
Incidence of CVC related thrombosis
In our study the incidence of CVC related thrombosis detected by ultrasound in critical
care canine patients was 28 To the best of the authoracutes knowledge similar studies
performed in dogs are rare in the available veterinary literature In one study performed
in 81 dogs and 12 cats (Adamantos et al 2010) an incidence of 2 was found for vein
thrombosis related to CVC This low incidence can be due to a lacking of systematic
ultrasound evaluations of the jugular vein containing the CVC In the mentioned study in
dogs and cats the vein thrombosis was confirmed by palpation or ultrasound presumably
after the presentation of clinical symptoms however in many cases as in our study
thrombus into the jugular vein can be developed without manifestation of clinical
symptoms what makes a routinely ultrasound evaluation of the vein containing the CVC
very important Our results are well aligned with the findings in humans in which a
median incidence of 30 was found for thrombosis related to CVC based on 25 studies
performed in patients requiring a CVC (review article van Rooden et al 2005) In the
mentioned 25 human studies the incidence of CVC related thrombosis varied within a
wide range (2ndash67 ) according to factors related to (1) the CVC (eg material type
entry site [jugular femoral subclavian axillary cephalic vein] time period in place (2)
diagnostic thrombus detection technique (ultrasonography or venography) and criteria
(3) patient collective (diseases and presence of predisposing factors [eg previous venous
thrombosis history presence of cancer] and (4) prophylactic antithrombotic treatment
(Geerts 2014 Liu et al 2015 van Rooden et al 2005)
The incidence of CVC related thrombosis detected by colour Doppler ultrasound in our
study fits also with the median incidence (30 ) found in three studies performed in
humans in which colour Doppler ultrasound evaluations of the jugular vein containing a
Chapter 4 General discussion
73
CVC were performed (Harter et al 2002 Lordick et al 2003 Wu et al 1999) In these
three studies the individual incidence of CVC related thrombosis was 2 (Harter et al
2002) 30 (Lordick et al 2003) and 56 (Wu et al 1999) The high thrombus
incidence reported in the study performed by Wu et al (1999) could be due to the criteria
for diagnosis of thrombosis because in that study both fibrin sleeves and compact
thrombi were considered as cases of thrombosis related to CVC The incidence for
compact thrombi was 25 which is more similar to our results In the study performed
by Harter et al (2002) the CVC related thrombosis incidence (2 ) was very low
compared with the incidence found in our study In that study the jugular veins were
examined just before or less than 24 hours after CVC removal (median period of CVC
use = 1025 days) In our study the ultrasound evaluations were performed before the
CVC placement (day 0) and then on days 1 3 5 etc until the CVC removal (median
period of CVC use = 7 days median interval until thrombus detection = 7 days) In our
study we detected thrombus formation as early as on the 3rd day so it could be possible
that in the cited study possibly only temporarily existing thrombi could have been missed
by ultrasonography in the final stage and thereby for the incidence calculation However
this scenario is not very likely because results of the present study show that
sonographically detectable thrombi during the observation period had in all cases their
maximum grade on the final day
In the study performed by Lordick et al (2003) the CVC related thrombosis incidence
determined for 43 human patients was very similar to our study and in both studies only
compact thrombi detectable by ultrasound were considered for assessment of the
incidence In the mentioned study all the human patients were screened for the presence
of thrombosis every 4 days after CVC insertion with real time B-mode ultrasound
(Lordick et al 2003)
Chapter 4 General discussion
74
In our study 70 of CVCs analysed by ELMI showed thrombi of grade 1ndash3 on the
external CVC wall To the best of the authoracutes knowledge studies about incidence of CVC
related thrombosis on the outside surface of the CVC detected by ELMI cannot be found
in the human and small animal literature In one study performed in humans (Lucas et al
2017) thrombus formation was analysed by ELMI in 78 central venous catheters That
study focussed on microstructural assessment of thrombus formation inside the distal
openings of the CVC which were found in 85 of the evaluated CVCs The percentage
of occurrence of thrombosis in the mentioned study in humans was nearly similar to the
incidence of intraluminal thrombus formation as detected in the presented study by ELMI
(74 ) In general intraluminal thrombi were not specially considered in the present
study because they mainly express the efficacy of the flushing technique rather than the
effectivity of the antithrombotic treatment In one study performed in rats the
microstructure of thrombi developed in the jugular vein containing the CVC was also
evaluated by ELMI (Xiang et al 1998) In the mentioned study pericatheter thrombosis
was detected on the proximal section of the CVC in 100 of rats (n = 12) after 1 and 3
days of catheterisation The analyses of the CVCs placed in rats were performed with the
catheter inside the vein in contrast to our study where the CVC was removed from the
jugular vein for the respective analysis Xiang et al (2001) hypothesised in one study
performed in rabbits and rats in which CVCs were placed in the jugular vein that fibrin
rich thrombi associated to the CVC can be detached during the catheter removal and this
could explain the difference between the thrombosis incidence assessed in our study and
that of the study performed by Xiang et al (1998)
In our study the incidence of CVC related thrombosis detected by ultrasonography in the
lumen of the EJV did not correlate with the incidence of CVC related thrombosis detected
by ELMI on the external wall of the CVC This was mainly due to the fact that the CVC
Chapter 4 General discussion
75
segment evaluated with both methods was different Using ultrasonography it was only
possible to evaluate the EJV in the neck area of the patient and thereby only the proximal
third of the CVC In contrast with the ELMI analysis only the distal section of the CVC
(last third) was evaluated because macroscopically detected thrombi mainly developed
near the CVC tip which was routinely positioned at the junction of the cranial cava vein
with the right atrium In a retrospective review of medical records of 169 human patients
requiring a CVC (Ballard et al 2016) the position of the CVC tip was determined by
radiologic evaluation and it was determined that the catheter tip position can be a
predisposing factor for central venous thrombosis development Thereby it could be of
interest to evaluate in further studies in dogs if a high incidence of thrombosis detected
by ELMI as was shown in our study could be related to the CVC tip position
In our study polyurethane catheters were tested which are widely used and are the
standard CVCs used in the Small Animal Clinic University of Veterinary Medicine
Hannover In vivo and in vitro studies have assessed the thrombogenicity of different
catheter materials (Borow and Crowley 1985 Smith et al 2012) In general terms
polyurethane catheters (central or peripheral) had shown a higher thrombogenicity
compared with coated (eg with hydromer) and silicone catheters Due to this property
of polyurethane catheters they can be supposed as a good test system for antithrombotic
regimens as evaluated in our study On the other hand despite silicone catheters have been
associated with a lower thrombogenicity their mechanical flexibility could be a
disadvantage in small clinical practice due to the increased risk of rupture detected even
in humans (Wildgruber et al 2016)
Chapter 4 General discussion
76
Heparin thromboprophylaxis for CVC related thrombosis
The occurence of CVC related thrombosis detected by colour Doppler ultrasound and
ELMI in our study demonstrates that the UFH in the given dosage was not completely
effective to prevent CVC related thrombosis in canine intensive care patients In human
medicine LMWH is commonly used in prophylactic anticoagulation regimens however
studies performed in human patients requiring a CVC and receiving a prophylactic
anticoagulation (with LMWH or UFH) did not achieve consensus regarding the efficacy
of prophylactic heparin to prevent CVC related thrombosis (Abdelfeki et al 2004
Lordick et al 2003 Wu et al 1999) To the best of the authoracutes knowledge studies
performed in dogs evaluating systemic prophylactic anticoagulation to prevent CVC
related thrombosis cannot be found However according to the evidence found in the
human literature and the results of our study further investigations using comparative
groups with different drugs dosages andor administration routes are required to establish
an effective antithrombotic treatment to prevent CVC related thrombosis
The plasma heparin activities measured in canine patients in our study varied significantly
after a defined subcutaneous dosage The limited and variable bioavailability of SC
administered UFH is well documented in humans (Hirsh and Raschke 2004)
Considerable variations of the circulatory status of the liver- and kidney function and
concentration of heparin-binding proteins in intensive care patients obviously amplify
this phenomenon leading to an extremely high inter-individual and intra-individual
variation of the heparin blood levels in our patients A different application route (IV)
may have reduced the influence of part of these factors and therefore could have led to
more predictable UFH blood levels However continuous infusion requires more
technical equipment and is also associated with a wide range of heparin activities in
canine patients (Scott et al 2009)
Chapter 4 General discussion
77
Prediction of CVC related thrombosis
Of 7 haemostasis tests (including ROTEM with 12 different parameters [6 for each
reagent ex-tem and in-tem]) evaluated as possible predictors of CVC related thrombosis
in our study initial values of high fibrinogen and low maximum lysis (ROTEM ex-tem)
were associated with sonographically detectable thrombus formation Some studies in
humans have established the association of laboratory biomarkers as predictors of risk
thrombosis (Boersma et al 2016 Cheng et al 2013 Kato et al 2016 Liu et al 2015)
One study performed in anticoagulated human patients showed ndash well in agreement with
our results ndash that high fibrinogen levels were associated with an increased risk of
perioperative venous thromboembolism (P = 0004) (Kato et al 2016) The cited study
does not exclusively refer to CVC associated thrombosis but 762 (1621) of the
venous thromboses were suggested to be associated with CVCs Fibrinogen concentration
measurements are normally available in the human and veterinary practice and the
association of this parameter with a high risk of development of CVC related thrombosis
can be useful to choose special antithrombotic treatment in individual critical care patients
to prevent such severe complications
Thrombin generation assay (TGA) abnormalities such as higher velocity and endogenous
thrombin potential (ETP) values measured initially ie before any anticoagulation
therapy have been associated with a higher thromboembolism risk in general (ie without
specific association with CVCs) in one study performed on 105 human patients (Espitia
et al 2017) In contrast in our study the initial TGA assay results did not show an
association with formation of CVC related thrombosis
The ROTEM parameters evaluated in the present study were partially associated with
formation of CVC thrombosis detected by colour Doppler ultrasound This finding
confirms the results obtained in humans in which ROTEM measurements are regarded as
Chapter 4 General discussion
78
a valuable indicator of hypercoagulability (Harahsheh and Ho 2018 Tuumlrk et al 2018)
However in our study individual ROTEM parameters showed partly lower coagulation
activities in dogs with CVC related thrombosis detected by ELMI at the final day of
evaluation Because the number of patients without signs of thrombosis detected by ELMI
was low compared with those who showed signs these implausible results should be
interpreted carefully The usefulness of ROTEM analysis which has also many other
indications demonstrates that is was worth to perform a detailed methodological approval
of the device including the establishment of reference values in the preliminary study
Conclusion
In conclusion the prethrombotic or thrombotic changes detected in our study did not
correlate between ultrasonography and ELMI however both methods show accurately
signs of CVC related thrombosis The UFH in the given dosage was not completely
effective to prevent CVC induced thrombus formation in canine intensive care patients
Plasma heparin activities varied significantly after a defined subcutaneous dosage
However plasma heparin activities were not different between dogs with and without
detectable CVC associated thromboses Initial high fibrinogen values and low maximum
lysis (ROTEM ex-tem) values were associated with sonographically detectable thrombi
and may be useful as indicators for an increased thrombotic risk
Finally our study in canine intensive care patients is to the best of the authoracutes
knowledge the first study in the available veterinary literature performed to evaluate the
efficacy of an antithrombotic treatment with UFH to prevent CVC related thrombosis
Chapter 5 Summary
79
Chapter 5 Summary
Central venous catheter induced thrombosis in dogs ndash occurrence under an
antithrombotic treatment regimen and possible indicators
Joseacute Mauricio Pereira Mora
Central venous catheters (CVC) are of importance for intensive care management of
canine patients Thrombosis of the jugular vein is a severe complication related to the
CVC use However only little is known about the incidence of CVC-associated
thromboses in dogs and the necessity and efficacy of prophylactic antithrombotic
regimens
Therefore the aims of the present study were (1) to determine the incidence of catheter-
induced thrombosis in canine intensive care patients (2) to evaluate the efficacy of a
prophylactic heparin regimen routinely used in the Small Animal Clinic of the University
of Veterinary Medicine Hannover and (3) to assess whether selected haemostasis test
results are predictive for an increased risk for thrombosis formation
The thesis also includes a preliminary methodological study on the use of rotational
elastometry in dogs using the ROTEM delta device In this preliminary study reference
intervals were determined based on 125 healthy dogs for different clot activator reagents
and for measurements without activator Coefficients of variation (CVs) were calculated
based on results of fourfold measurements of 3 normal and 3 abnormal blood samples in
order to assess precision of the method Influence of age groups sex neuter status and
size were also assessed The median CVs for most of the parameters evaluated in the
precision analysis were less than 10 and this confirmed that the ROTEM delta analyser
delivers repeatable results Regarding the influence of age sex neuter status and animal
Chapter 5 Summary
80
size only neuter status in female dogs appeared to be a relevant influencing factor on
most of important ROTEM parameters
The main study of the thesis comprises 29 patients hospitalised at the Small Animal Clinic
requiring a CVC for medical reasons These patients received a prophylactic
anticoagulation treatment with unfractionated heparin (UFH 150 IU [surgical dogs
received 75 IU]kg BW TID subcutaneously) A colour Doppler ultrasound of the
respective external jugular vein (EJV) in order to detect thrombus formation in the lumen
of the EJV and blood collection for haemostasis tests were performed directly before the
installation procedure (day 0) and on days 1 3 5 7 and so on after the CVC insertion
Haemostasis tests included prothrombin time activated partial thromboplastin time
thrombin time antithrombin activity D-dimer concentration thrombin generation and
rotational elastometry as well as the heparin activity using a chromogenic anti-factor Xa
test Finally when the CVC was removed electron microscopy (ELMI) of sections of the
CVC with macroscopically visible thrombi in the last third (5ndash10 cm) of the CVC was
performed 3 different grades were defined for CVC-associated (extraluminal) thrombi
detected by either ultrasound or ELMI respectively
829 patients evaluated by colour Doppler ultrasound during the study developed thrombi
into the lumen of EJV (4 x grade 1 3 x grade 2 and 1 x grade 3) 1927 CVCs assessed
using ELMI showed detectable thrombi on the external CVC surface (7 x grade 1 4 x
grade 2 and 8 x grade 3) There was no significant correlation between thrombus
formation detected by ultrasound or ELMI Thus statistical group comparisons of
patients with or without CVC-associated thrombus formation was performed
independently for both diagnostic procedures The plasmatic heparin activities showed
great variability but no significant differences were found between dogs with or without
CVC-associated thrombus formation regardless whether this was based on sonographic
Chapter 5 Summary
81
or ELMI findings From the initially performed haemostasis tests high fibrinogen
concentrations and low maximum lysis values as measured by the ROTEM delta device
(using the ex-tem reagent) were associated with sonographically detectable thrombi
In conclusion the preliminary methodological study on the ROTEM delta in dogs shows
that the method delivers repeatable results in canine citrated whole blood and the
reference values obtained should offer a reliable basis for the interpretation of test results
of this device in dogs with suspected haemostasis disorders The results of the main study
indicate that CVC-associated thromboses occur (eg 28 [829] of the patients as
detected by ultrasound) although severe thromboses (high grades) are relatively rare in
intensive care canine patients receiving a routine anticoagulant treatment Thereby
prophylactic antithrombotic protocol (unfractionated heparin [UFH]) in the given dosage
was not completely effective to prevent CVC induced thrombus formation in canine
intensive care patients A high fibrinogen concentration and low maximum lysis
(ROTEM ex-tem) may be useful indicators for an increased thrombotic risk in individual
patients which may require special anticoagulatory treatment Further studies are
required to evaluate whether different thromboprophylactic treatment regimens eg with
low molecular weight heparin are more effective in preventing CVC-associated
thromboses in dogs
Chapter 6 Zusammenfassung
82
Chapter 6 Zusammenfassung
Zentralvenenkatheter-induzierte Thrombose bei Hunden - Auftreten unter einem
antithrombotischen Behandlungsschema und moumlgliche Indikatoren
Joseacute Mauricio Pereira Mora
Zentralvenenkatheter (ZVK) sind fuumlr das Intensivmanagement von Hunden von
Bedeutung Die Entstehung einer Thrombose der Vena jugularis ist eine schwerwiegende
Komplikation im Zusammenhang mit der Verwendung eines ZVKs Uumlber die Inzidenz
von ZVK-assoziierten Thrombosen bei Hunden und die Notwendigkeit und Wirksamkeit
prophylaktischer antithrombotischer Protokolle liegen jedoch nur wenige fundierte
Angaben in der Literatur vor
Daher waren die Ziele der vorliegenden Studie (1) die Inzidenz der Katheter-induzierten
Thrombose bei caninen Intensivpatienten zu bestimmen (2) die Wirksamkeit eines
prophylaktischen Heparin-Regimes zu bewerten das routinemaumlszligig in der Klinik fuumlr
Kleintiere der Stiftung Tieraumlrztliche Hochschule Hannover angewendet wird und (3) um
zu beurteilen ob ausgewaumlhlte Haumlmostasetestergebnisse ein erhoumlhtes Risiko fuumlr die
Thrombosebildung vorhersagen koumlnnen
Die PhD-Arbeit enthaumllt zudem eine vorbereitende methodische Studie zur Verwendung
der Rotationselastometrie bei Hunden mit Hilfe des ROTEM delta-Geraumltes In dieser
Vorstudie wurden Referenzintervalle basierend auf 125 gesunden Hunden fuumlr
verschiedene Gerinnungsaktivatorreagenzien und fuumlr Messungen ohne Aktivator
bestimmt Um die Genauigkeit der Methode einzustufen wurden Variationskoeffizienten
(VKs) basierend auf den Ergebnissen von Vierfachmessungen von drei normalen und drei
abnormalen Blutproben berechnet Der Einfluss von Alter Geschlecht Kastration und
Groumlszlige wurde ebenfalls getestet Die Medianwerte der VKs fuumlr die meisten in der
Chapter 6 Zusammenfassung
83
Praumlzisionsanalyse bewerteten Parameter betrugen weniger als 10 und dies bestaumltigte
dass das ROTEM delta-Analysegeraumlt wiederholbare Ergebnisse liefert In Bezug auf den
Einfluss von Alter Geschlecht Kastration und Tiergroumlszlige erwies sich nur eine Kastration
bei Huumlndinnen als relevanter Einflussfaktor fuumlr die meisten wichtigen ROTEM-
Parameter
Die Hauptstudie der Arbeit umfasst 29 Patienten welche zum Zeitpunkt der
Untersuchung stationaumlr in der Kleintierklinik aufgenommen worden waren und aus
medizinischer Indikation einen ZVK benoumltigten Diese Patienten erhielten eine
prophylaktische Antikoagulation mit unfraktioniertem Heparin (UFH 150 IE
[chirurgische Hunde erhielten 75 IE]kg KG 3 x taumlglich subkutan) Bildgebende
Untersuchungen mittels Farbdoppler-Ultraschall der jeweiligen Vena jugularis externa
(VJE) zum Nachweis der Thrombusbildung im Lumen der VJE und Blutentnahmen fuumlr
Haumlmostasetests wurden jeweils direkt vor dem Installationsvorgang (Tag 0) sowie an den
Tagen 1 3 5 und 7 nach Legen des ZVKs durchgefuumlhrt Haumlmostasetests umfassten die
Prothrombinzeit die aktivierte partielle Thromboplastinzeit die Thrombinzeit die
Antithrombinaktivitaumlt die D-Dimer-Konzentration die Thrombingenerierung und die
Rotationselastometrie sowie die Heparinaktivitaumlt unter Verwendung eines chromogenen
Anti-Faktor-Xa-Tests Schlieszliglich wurde nach Entfernung des ZVKs eine
Elektronenmikroskopie (ELMI) von Abschnitten aus dem letzten Drittel (5ndash10 cm) des
ZVKs mit makroskopisch sichtbaren Thromben durchgefuumlhrt Fuumlr ZVK-assoziierte
(extraluminale) Thromben die entweder durch Ultraschall oder ELMI nachgewiesen
wurden wurden drei verschiedene Grade definiert
829 Patienten die waumlhrend der Studie mittels Farbdoppler-Ultraschall untersucht
wurden entwickelten Thromben im Lumen der VJE (4 x Grad 1 3 x Grad 2 und 1 x Grad
3) 1927 ZVKs die unter Verwendung von ELMI bewertet wurden zeigten
Chapter 6 Zusammenfassung
84
nachweisbare Thromben auf der aumluszligeren ZVK-Oberflaumlche (7 x Grad 1 4 x Grad 2 und 8
x Grad 3) Es gab keine signifikante Korrelation zwischen der durch Ultraschall oder
ELMI festgestellten Thrombusbildung Daher wurden statistische Gruppenvergleiche
von Patienten mit oder ohne ZVK-assoziierter Thrombusbildung fuumlr beide diagnostische
Verfahren unabhaumlngig voneinander durchgefuumlhrt Die plasmatischen Heparinaktivitaumlten
zeigten eine groszlige Variabilitaumlt es wurden jedoch keine signifikanten Unterschiede
zwischen Hunden mit oder ohne ZVK-assoziierter Thrombusbildung gefunden
unabhaumlngig davon ob diese auf sonographischen oder ELMI-Befunden beruhte Von den
initial durchgefuumlhrten Haumlmostasetests waren hohe Fibrinogenkonzentrationen und
niedrige maximale Lyse-Werte der Messungen mit ROTEM delta (ex-tem Reagenz) mit
sonographisch nachweisbaren Thromben assoziiert
Zusammenfassend zeigt die vorbereitende methodische Studie zum ROTEM delta bei
Hunden dass die Methode wiederholbare Ergebnisse in mit Zitrat
antikoaguliertemVollblut liefert Die erarbeiteten Referenzwerte sollten eine zuverlaumlssige
Grundlage fuumlr die Interpretation der Testergebnisse dieses Geraumlts bei Hunden mit
Verdacht auf Haumlmostasestoumlrungen bieten Die Ergebnisse der Hauptstudie zeigen dass
ZVK-assoziierte Thrombosen auftreten (z B 28 [829] der Patienten bezogen auf den
Ultraschall-gestuumltzten Nachweis) obwohl schwere Thrombosen (hohe Grade) bei
intensivmedizinisch versorgten Hundepatienten die eine Routineantikoagulation
erhalten relativ selten sind Somit war das prophylaktische antithrombotische Protokoll
(unfraktioniertes Heparin [UFH]) in der angewendeten Dosierung nicht vollstaumlndig
wirksam um die ZVK-induzierte Thrombusbildung bei caninen Intensivpatienten zu
verhindern Eine hohe Fibrinogenkonzentration und eine niedrige maximale Lyse
(ROTEM ex-tem) koumlnnen als Indikatoren eines erhoumlhten Thromboserisikos bei einzelnen
Patienten nuumltzlich sein fuumlr die moumlglicherweise eine spezielle gerinnungshemmende
Chapter 6 Zusammenfassung
85
Behandlung notwendig ist Weitere Studien sind erforderlich um zu untersuchen ob
verschiedene thromboprophylaktische Behandlungsschemata z B mit
niedermolekularem Heparin bezuumlglich der Verhinderung von ZVK-assoziierten
Thrombosen bei Hunden wirksamer sind
Chapter 7 References
86
Chapter 7 References
1 Abdelkefi A Ben Othman T Kammoun L Chelli M Ben Romdhane N Kriaa A Ladeb
S Torjman L Lakhal A Achour W Ben Hassen A Hsaiumlri M Ladeb F Ben Abdeladhim
A 2004 Prevention of central venous line-related thrombosis by continuous infusion of low-dose
unfractionated heparin in patients with haemato-oncological disease A randomized controlled
trial Thromb Haemost 92 654ndash661
2 Adamantos S Brodbelt D Moores A L 2010 Prospective evaluation of complications
associated with jugular venous catheter use in a veterinary hospital J Small Anim Pract 51 254ndash
257
3 Ballard D H Samra N S Gifford K M Roller R Wolfe B M Owings J T 2016
Distance of the internal central venous catheter tip from the right atrium is positively correlated
with central venous thrombosis Emerg Radiol 23 269ndash273
4 Baumann-Kreuziger L Onwuemene O Kolesar E Crowther M Lim W 2015 Systematic
review of anticoagulant treatment of catheter-related thrombosis Thromb Res 136 1103ndash1109
5 Bliss SP Bliss SK Harvey HJ 2002 Use of recombinant tissue‐plasminogen activator in a
dog with chylothorax secondary to catheter‐associated thrombosis of the cranial vena cava J Am
Anim Hosp Assoc 38 431ndash435
6 Boersma RS Hamulyak K van Oerle R Tuinenburg A Ten Cate-Hoek AJ Schouten
HC 2016 Biomarkers for prediction of central venous catheter related-thrombosis in patients
with hematological malignancies Clin Appl Thromb Hemost 22 779ndash784
7 Boersma RS Jie KS Verbon A van Pampus EC Schouten HC 2008 Thrombotic and
infectious complications of central venous catheters in patients with hematological
malignancies Ann Oncol 19 433ndash442
8 Bonnet F Loriferne JF Texier JP Texier M Salvat A Vasile N 1989 Evaluation of
Doppler examination for diagnosis of catheter-related deep vein thrombosis Intensive Care Med
15 238ndash240
9 Borow M Crowley J G 1985 Evaluation of central venous catheter thrombogenicity Acta
Anaesthesiol Scand Suppl 81 59ndash64
10 Cheng Y Cui T Fu P Liu F Zhou L 2013 Dyslipidemia is associated with tunneled-cuffed
catheter-related central venous thrombosis in hemodialysis patients a retrospective multicenter
Chapter 7 References
87
study Artif Organs 37 155ndash161
11 Comerlato PH Rebelatto TF Santiago de Almeida FA Klein LB Boniatti MM
Schaan BD Rados DV 2017 Complications of central venous catheter insertion in a teaching
hospital Rev Assoc Med Bras 63 613ndash620
12 De Cicco M Matovic M Balestreri L Steffan A Pacenzia R Malafronte M Fantin
D Bertuzzi CA Fabiani F Morassut S Bidoli E Veronesi A 2009 Early and short‐term
acenocumarine or dalteparin for the prevention of central vein catheter‐related thrombosis in
cancer patients a randomized controlled study based on serial venographies Ann
Oncol 20 1936ndash1942
13 Espitia O Fouassier M Hardouin JB Pistorius MA Agard C Planchon B Trossaert M
Pottier P 2017 Thrombin generation assay in hospitalized nonsurgical patients A new tool to
assess venous thromboembolism risk Clin Appl Thromb Hemost 23 45ndash51
14 Evans NS Ratchford EV 2018 Catheter related-venous thrombosis Vasc Med 23 411ndash413
15 Geerts W 2014 Central Venous Catheter-related thrombosis Hematology Am Soc Hematol
Educ Program 2014 306ndash311
16 Harahsheh Y Ho KM 2018 Use of viscoelastic tests to predict clinical thromboembolic
events A systematic review and meta‐analysis Eur J Haematol 100 113ndash123
17 Harter C Salwender HJ Bach A Egerer G Goldschmidt H Ho AD 2002 Catheter-
related infection and thrombosis of the internal jugular vein in hematologic-oncologic patients
undergoing chemotherapy a prospective comparison of silver-coated and uncoated
catheters Cancer 94 245ndash251
18 Hirsh J Raschke R 2004 Heparin and low-molecular-weight heparin The Seventh ACCP
Conference on Antithrombotic and Thrombolytic Therapy Chest 126 (Suppl) 188Sndash203S
19 Karthaus M Kretzschmar A Kroning H Biakhov M Irwin D Marschner N Slabber
C Fountzilas G Garin A Abecasis NG Baronius W Steger GG Sudhoff T Giorgetti
C Reichardt P 2006 Dalteparin for prevention of catheter‐related complications in cancer
patients with central venous catheters final results of a double‐blind placebo‐controlled phase III
trial Ann Oncol 17 289ndash296
20 Kato F Takeuchi H Matsuda S Kawakubo H Omori T Kitagawa Y 2016 Incidence of
and risk factors for venous thromboembolism during surgical treatment for esophageal cancer a
Chapter 7 References
88
single-institution study Surg Today 46 445ndash452
21 Kohler TR Kirkman TR 1998 Central venous catheter failure is induced by injury and can
be prevented by stabilizing the catheter tip J Vasc Surg 28 59ndash66
22 Koumlksoy C Kuzu A Kutlay J Erden I Ozcan H Ergicircn K 1995 The diagnostic value of
colour Doppler ultrasound in central venous catheter related thrombosis Clin Radiol 50 687ndash
689
23 Kucher N 2011 Clinical practice Deep-vein thrombosis of the upper extremities N Engl J
Med 364 861ndash869
24 Langston CE Eatroff AE 2018 Hemodialysis catheter-associated fibrin sheath in a dog J
Vet Emerg Crit Care 28 366ndash371
25 Langston CE Eatroff AE Poeppel K 2014 Use of tissue plasminogen activator in catheters
used for extracorporeal renal replacement therapy J Vet Intern Med 28 270ndash276
26 Laurenson MP Hopper K Herrera MA Johnson EG 2010 Concurrent diseases and
conditions in dogs with splenic vein thrombosis J Vet Intern Med 24 1298ndash1304
27 Lee AYY Kamphuisen PW 2012 Epidemiology and prevention of catheter‐related
thrombosis in patients with cancer J Thromb Haemost 10 1491ndash1499
28 Liu G Fu ZQ Zhu P Li SJ 2015 Central venous catheter-related thrombosis in senile male
patients New risk factors and predictors J Huazhong Univ Sci Technolog Med Sci 35 445ndash
449
29 Lordick F Hentrich M Decker T Hennig M Pohlmann H Hartenstein R Peschel C
2003 Ultrasound screening for internal jugular vein thrombosis aids the detection of central
venous catheter-related infections in patients with haemato-oncological diseases a prospective
observational study Br J Haematol 120 1073ndash1078
30 Lucas TC Silva EED Souza DOD Santos ARD Lara MO 2017 Microstructural
evaluation by confocal and electron microscopy in thrombi developed in central venous catheters
Rev Esc Enferm USP 51 1ndash9
31 Mismetti P Mille D Laporte S Charlet V Buchmuller‐Cordier A Jacquin JP Fournel
P Dutrey‐Dupagne C Decousus H 2003 Low‐molecular‐weight heparin (nadroparin) and
very low doses of warfarin in the prevention of upper extremity thrombosis in cancer patients with
indwelling long‐term central venous catheters a pilot randomized trial Haematologica 88 67ndash
Chapter 7 References
89
73
32 Niers TM Di Nisio M Klerk CP Baarslag HJ Buller HR Biemond BJ 2007
Prevention of catheter‐related venous thrombosis with nadroparin in patients receiving
chemotherapy for hematologic malignancies a randomized placebo‐controlled study J Thromb
Haemost 5 1878ndash1882
33 Scott K C Hansen B D DeFrancesco T C (2009) Coagulation effects of low molecular
weight heparin compared with heparin in dogs considered to be at risk for clinically significant
venous thrombosis J Vet Emerg Crit Care 19 74ndash80
34 Smith RS Zhang Z Bouchard M Li J Lapp HS Brotske GR Lucchino DL Weaver
D Roth LA Coury A Biggerstaff J Sukavaneshvar S Langer R Loose C 2012
Vascular catheters with a nonleaching poly-sulfobetaine surface modification reduce thrombus
formation and microbial attachment Sci Transl Med 4 153
35 Tuumlrk S M Cansu D Uuml Teke H Uuml Kaşifoğlu T Meltem Akay O Bilgin M amp Korkmaz
C 2018 Can we predict thrombotic tendency in rheumatoid arthritis A thromboelastographic
analysis (with ROTEM) Clin Rheumatol 37 2341ndash2349
36 van Rooden CJ Rosendaal FR Barge RM van Oostayen JA van der Meer FJM
Meinders AE Huisman MV 2003 Central venous catheter related thrombosis in haematology
patients and prediction of risk by screening with Doppler-ultrasound Br J Haematol 123 507ndash
512
37 van Rooden CJ Tesselaar ME Osanto S Rosendaal FR Huisman MV 2005 Deep vein
thrombosis associated with central venous catheters - a review J Thromb Haemost 3 2409ndash
2419
38 Verso M Agnelli G Bertoglio S Di Somma FC Paoletti F Ageno W Bazzan M Parise
P Quintavalle R Naglieri E Santoro A Imberti D Soraru M Mosca S 2005 Enoxaparin
for the prevention of venous thromboembolism associated with central vein catheter a double‐
blind placebo‐controlled randomized study in cancer patients J Clin Oncol 23 4057ndash4062
39 Vose J Odunayo A Price JM Daves M Schildt JC Tolbert MK 2019 Comparison of
heparinized saline and 09 sodium chloride for maintaining central venous catheter patency in
healthy dogs PeerJ 7e7072 httpdoi107717peerj7072
40 Wang LH Wei F Jia L Zhi Lu Wang B Dong HY Yu HY Sun GJ YangJ Li B
Chapter 7 References
90
Meng J Zhang RN Bi XQ Chen HY Jiang AL 2015 Fibrin sheath formation and
intimal thickening after catheter placement in dog model role of hemodynamic wall shear stress J
Vasc Access 16 275ndash284
41 Wildgruber M Lueg C Borgmeyer S Karimov I Braun U Kiechle M Meier R Koehler
M Ettl J Berger H 2016 Polyurethane versus silicone catheters for central venous port devices
implanted at the forearm Eur J Cancer 59 113ndash124
42 Whitesell RT Steenburg SD 2014 Imaging findings of acute intravascular thrombus on non-
enhanced computed tomography Emerg Radiol 21 271ndash277
43 Wu X Studer W Skarvan K Seeberger MD 1999 High incidence of intravenous thrombi
after short-term central venous catheterization of the internal jugular vein J Clin Anesth 11
482ndash485
44 Xiang DZ Verbeken EK Van Lommel AT Stas M De Wever I 1998 Composition and
formation of the sleeve enveloping a central venous catheter J Vasc Surg 28 260ndash271
45 Xiang DZ Verbeken EK Van Lommel AT Stas M De Wever I 2001 Sleeve-related
thrombosis a new form of catheter-related thrombosis Thromb Res 104 7ndash14
91
Acknowledgements
First I thank God for every day in my life for His guide and because He was is
and will be always with me and that certainty gives me strength to take a step
forward
My sincere thanks to Prof Dr Reinhard Mischke for the opportunity to perform
my PhD under his supervision for his support and guidance for all that I learned
with him and for his friendship
I also thank to Prof Dr Manfred Kietzmann and PD Dr Mario von Depka
Prondzinski for being part of my supervision group for their time and beneficial
advices and discussion during my PhD
My gratitude to Prof Hewicker-Trautwein and Mrs Rohn for their valuable work
and availability to help me
I thank the Werlhof Institute Medical Care Center Hannover for the valuable
cooperation especially to Mrs Ober-Cassebaum Anne and Franziska for all their
help
I thank all the staff of the Small Animal Clinic of the University of Veterinary
Medicine Hannover (TiHo) My special gratitude to the internal medicine and
critical care surgery neurology ultrasound and anesthesia departments and to
the TiHo students all their cooperation made possible my research
I thank the haematology clinical chemistry and haemostasis laboratory
department of the Small Animal Clinic TiHo especially to Dirk because he was
always available for me thanks for his guide and friendship all his explanations
in the laboratory were of great value but also the conversations about different
topics of the life were of big value for me
92
I thank the Hannover Graduate School for Veterinary Pathobiology
Neuroinfectology and translational Medicine (HGNI) for the well-organized
PhD program and especially thanks to Prof Dr Beatrice Grummer Dr Tina
Selle and Mrs Tania Czeslik for his guide and help during my studies
My sincere thanks to Mrs Maritta Ledwoch for her guidance at the International
office of the TiHo ldquoI remember you since my externship in Germanyrdquo
I also thank the Student Union Hannover for all the help with the accommodation
for me and my family
I want to thank the Universidad Nacional de Costa Rica and therefore to Costa
Rica (my loved country) for the financial support to perform my PhD studies
but also for my professional formation as a veterinarian Special thanks to the
Veterinary Medicine School from Universidad Nacional de Costa Rica To all the
academic and administrative staff for their support and for believing in me
Sincerely thanks to Dr Mauricio Jimenez Soto my mentor my adviser but most
important my friend Thanks for believing in me and for always being by my side
ldquoAgradezco profundamente a mis papaacutes ldquodon Tito (Papillo) y dontildea Eida (Mami)rdquo
Todo lo bueno que hay en mi proviene de Dios y de ellos Gracias a mis hermanos
(as) cuntildeados (as) sobrinos y a toda la familia relacionada a ellos Siempre han
sido un gran apoyo y los amordquo
ldquoGracias a dontildea Alba Fran y David Gracias por toda la ayuda y por estar siempre
pendientesrdquo
I would like to thank all my friends that during a short part or during my whole
life have been with me For all the good time shared really thanks
To Willy thanks ldquogordordquo
93
I sincerely thank to Adriano and Johanna for their help and hospitality Thanks
Adriano for your guidance and support during my beginning at the TiHo for make
me feel in Costa Rica with your friendship for the tourist walks and for the bike
training I missed the coffee times
I want to thank Ina the first person to give me a smile at the start of my PhD
Thanks for your hugs and friendship
I also thank Franz for his help and for being a very nice partner Thanks for help
me always when I requested you
ldquoFinalmente quiero expresar mi gratitud y amor a Karo Faacutetima y Abigaiacutel A Karo
por tu sacrificio y soporte por las palabras de aliento por estar a mi lado en las
buenas pero en especial en las malas A Faacute por tu sacrificio por tus sonrisas por
tus abrazos y besos por bailar con migo siempre por ser mi modelo e inspiracioacuten
A Abi por ser una nueva razoacuten in mi vida para seguir adelante por todo lo que
deseo compartir con tigo por mostrarme junto con Faacutetima la felicidad en una
sonrisardquo
Chapter 1 Introduction
1
Chapter 1 Introduction
Central venous catheters (CVC) are mainly used at critical care units in human and small
animal patients requiring infusion of large amounts of fluids or blood parenteral nutrition
support routine drug therapy haemodialysis chemotherapy plasmapheresis or
periodical blood sampling (Geerts 2014 Vose et al 2019) CVC related thrombosis is
one of the most severe complications (apart from infections and pneumothorax) of CVCs
in humans (Comerlato et al 2017 Evans and Ratchford 2018 Geerts 2014) leading to
patientrsquos distress CVC dysfunction venous stenosis and in some cases to the
development of thromboembolism (Geerts 2014) In the human literature there are
various studies dealing with epidemiologic and prophylactic aspects which are described
below in detail In contrast to the best of the authoracutes knowledge studies about extra
luminal CVC related thrombosis in dogs are rare in the available veterinary literature
Studies are mainly dealing with development of intraluminal CVC thrombi occurring
frequently in dogs and cats (Langston et al 2014) and leading to CVC malfunction (Vose
et al 2019)
Epidemiology of CVC related thrombosis
CVC related thrombosis represents 70ndash80 of all upper extremity thrombosis cases and
approximately 10 of thromboembolism cases in humans (Kucher 2011) The incidence
of CVC related thrombosis reported in humans based in 25 studies ranged widely from
2 to 67 (median = 30 ) (van Rooden et al 2005) The incidence of CVC related
thrombosis detected by colour Doppler ultrasound of the internal jugular vein reported in
3 studies performed in humans was 2 (Harter et al 2002) 30 (Lordick et al 2003)
and 56 (Wu et al 1999)
Chapter 1 Introduction
2
The incidence of CVC related thrombosis in humans can vary between studies according
to the CVC type used entry site of the CVC (jugular femoral subclavian axillary
cephalic vein) and time period with the CVC in place the diagnostic thrombus detection
technique (venography or ultrasonography) and criteria for diagnosis duration of follow
up diseases and presence of predisposing factors (eg previous venous thrombosis
history presence of cancer) and finally prophylactic anticoagulation treatment (Geerts
2014 Liu et al 2015 van Rooden et al 2005)
To the best of the authoracutes knowledge data about the incidence of CVC related
thrombosis detected by colour Doppler ultrasound into the lumen of the jugular vein in
dogs are rare in the available veterinary literature In one study performed in 81 dogs and
12 cats (Adamantos et al 2010) the incidence of vein thrombosis detected by palpation
or ultrasound was 2 and the incidence of intraluminal CVC thrombosis (presumed by
the inability to draw blood back) was 15 Additionally regarding to intraluminal CVC
thrombosis an incidence of 8 was reported in one case control study performed in 24
dogs receiving CVC flushing with heparinised solution (Vose et al 2019) Data about
incidence in other animal species was also found In another case-control study performed
on 37 pigs thrombosis in the caval vein related to CVC placed in the external jugular
vein (EJV) was evaluated by histological analysis however no ultrasound detection was
performed (Kohler and Kirkman 1998) In this study in pigs 94 of patients with CVC
developed thrombi in the vena cava based on sections at the site of the end of the catheter
tip and 05 and 1 cm proximal of the first section (Kohler and Kirkman 1998)
Pathogenesis of CVC related thrombosis
Two types of CVC related thrombosis are distinguished in the human medicine literature
(Geerts 2014 van Rooden et al 2005) First symptomatic thrombosis is defined as
Chapter 1 Introduction
3
thrombosis detected by diagnostic images (ultrasonography venography) when
symptoms and signs (eg localised swelling pain redness) are present (Lee and
Kamphuisen 2012) Second subclinical thrombosis is defined as thrombosis
demonstrated by the previously mentioned diagnostic images without overt symptoms or
signs (Boersma et al 2008) Additionally the CVC related thrombi can be classified as
occlusive thrombus into the CVC lumen peri-catheter related fibrin sheaths and mural
thrombi (Geerts 2014)
During the CVC insertion the puncture of the skin on the CVC insertion site and the
rupture of the blood vessels integrity activate the coagulation cascade and the CVC acts
as a matrix for adherence and accumulation of fibrin and cellular components (Smith et
al 2012) The attachment of proteins and blood cells to the internal andor external wall
of the CVC can lead to (1) (partial or complete) occlusion of the catheter lumen and (2)
sometimes if a thrombus formation starts into the lumen of the vein (attached to the vein
wall or to the CVC) a partial or complete obstruction of the blood flow andor embolism
can develop (Langston and Eatroff 2018) Additionally a study performed in humans
(Lordick et al 2003) demonstrated a relationship between CVC related thrombosis and
microbial colonisation of the catheter leading to CVC related systemic infections This
relationship was explained among others by the ease of microorganisms like
Staphylococcus aureus and Staphylococcus epidermidis to adhere to CVC related
thrombin sheaths (Boersma et al 2008)
Diagnosis of CVC related thrombosis
Venous thrombosis is commonly diagnosed in human and small animal medicine
(especially in dogs) with vascular ultrasonography with or without use of the colour
Doppler technique which is normally used to evaluate the blood flow through the blood
Chapter 1 Introduction
4
vessels (Evans and Ratchford 2018 Laurenson et al 2010) According to the literature
in humans cited below the CVC related thrombosis detected by colour Doppler
ultrasound is present if any of the following aspects are observed (1) well defined
thrombus visualised in the lumen of the vein (2) impossibility to perform compression
of the vein by slight pressure with the ultrasound probe andor (3) no detectable blood
flow signal by colour Doppler ultrasound (Bonnet et al 1989 Koumlksoy et al 1995)
Similarly in dogs the CVC related thrombosis is usually diagnosed by colour Doppler
ultrasonography through the detection of the thrombus within the lumen of the vein
andor the obstruction (partial or complete) of the normal blood flow (Bliss et al 2002)
Especially in humans the contrast venography technique is used as gold standard to
confirm thrombosis For this purpose a contrast solution is injected through a peripheral
vein to assess its passage through the bloodstream by x-ray However this diagnostic
technique is normally used only in the case of a strong clinical suspicion of venous
thrombosis that cannot be detected by ultrasonography (Laurenson et al 2010 Langston
and Eatroff 2018) In a study performed in 44 human patients requiring a CVC (Koumlksoy
et al 1995) a sensitivity of 94 and a specificity of 96 were determined for the colour
Doppler ultrasound diagnosis when it was compared with the diagnosis performed with
the contrast venography technique That results make the colour Doppler ultrasound an
accurate diagnosis method to detect CVC related thrombosis in humans (Koumlksoy et al
1995 van Rooden et al 2003)
Advanced diagnostic image techniques like computed tomography or magnetic resonance
tomography are also accurate to perform the diagnosis of CVC related thrombosis in
humans (Whitesell and Steenburg 2014) In dogs multidetector computed tomography
venography was used in one study for the diagnosis of CVC related thrombosis allowing
the identification of catheter related sheaths around the CVC external wall (Wang at al
Chapter 1 Introduction
5
2015) However the high costs of these advanced diagnostic imaging procedures and the
requirement to perform at least sedation or general anesthesia are disadvantages when
compared with ultrasonography
Finally electron microscopy (ELMI) is a diagnostic technique that has been used in
humans (Lucas et al 2017) and experimental animals (Xiang et al 1998 Xiang et al
2001) mainly to perform a microstructural evaluation of thrombi developed into the
lumen of the CVC or in the vein containing the CVC ELMI allows the ex vivo diagnosis
of very small thrombi or sleeve related thrombosis that perhaps cannot be detected by
ultrasonography or venography during the evaluation of patients with CVCs (Xiang et al
2001) However one point to consider is that it is possible that compact thrombi
developed on the CVC can be detached during the CVC removal from the vein and
thereby before its analysis by ELMI (Xiang et al 2001)
Heparin thromboprophylaxis for CVC related thrombosis
Application of CVCs in human medicine is commonly accompanied by prophylactic
anticoagulation in order to decrease the risk of development of partial or complete vein
occlusion andor thromboembolism and thereby to improve the patientrsquos comfort
(Baumann-Kreuziger et al 2015) Apart from vitamin K antagonists (Mismetti et al
2003) unfractionated heparin (UFH) (Abdelfeki et al 2004) and low molecular weight
heparin (LMWH) (Karthaus et al 2006) are anticoagulants that have been used on
studies in humans administrated by intravenous (IV) infusion or by subcutaneous (SC)
injection to prevent thrombi development into the lumen of the veins containing the CVC
(review article Baumann-Kreuziger et al 2015) Different heparin dosages have been
used as thromboprophylactic anticoagulation in humans with CVC (review article Lee
and Kamphuisen 2012) For example in five different case-control studies performed in
Chapter 1 Introduction
6
human haemato-oncological patients UFH at a daily dosage of 100 IUkg given by IV
infusion (Abdelfeki et al 2004) and LMWH at SC daily dosages of 40 mg enoxaparin
(Verso et al 2005) 5000 IU AXa dalteparin (Karthaus et al 2006 De Cicco et al 2009)
and 2850 IU AXa nadroparin (Niers et al 2007) were used Only in part of these studies
it is stated that systemic heparin was combined with flushing of the CVC using
heparinised saline solution to reduce the risk of catheter occlusion (Karthaus et al 2006)
In 3 of the 5 previously mentioned studies no statistically significant difference was found
in the incidence of CVC related thrombosis between patients receiving the
anticoagulation prophylaxis compared with the control groups without systemic
anticoagulation (Verso et al 2005 p = 035 Karthaus et al 2006 p gt 005 Niers et al
2007 p = 049) In contrast in the residual two studies a lower incidence of CVC related
thrombosis was found in the group receiving an anticoagulation prophylaxis compared
with the control groups (Abdelfeki et al 2004 150 vs 126 p = 003 De Cicco et
al 2009 400 vs 526 p = 005)
Obviously there is no consensus in the available human literature regarding the beneficial
use of anticoagulation prophylaxis to prevent CVC related thrombosis and this may
reflect great variations for example according to the study design period trial drug
administration route (Lee and Kamphuisen 2012) and the moment of the initial
anticoagulant dosage administration (Decicco et al 2009)
To the best of the authoracutes knowledge studies about thromboprophylaxis for extraluminal
CVC related thrombosis in dogs cannot be found in the available veterinary literature
However similarly to humans prophylactic anticoagulation with heparinised saline
(administrated through CVC flushing) to prevent CVC occlusion by intraluminal thrombi
was used in one case-control study performed in 24 dogs (Vose et al 2019) In the cited
study 12 dogs received dosages of 3 ml of heparinised saline solution (10 IU UFHml) to
Chapter 1 Introduction
7
flush the catheter every 6 hours during 72 hours whereas 12 control dogs received
flushing with the same volume of non-heparinised saline solution administered at the
same frequency and no statistically significant difference was found
Prediction of CVC related thrombosis
Studies regarding the relationship between laboratory biomarkers and CVC related
thrombosis are available in the human literature (Boersma et al 2016 Cheng et al 2013
Liu et al 2015) however to the best of the authoracutes knowledge literature about laboratory
tests used as predictors for CVC related thrombosis in small animals cannot be found at
all Availability of approved blood tests which can predict the risk of thrombosis
development associated to CVC already at the moment of the CVC placement are
important in order to identify an increased risk of CVC related thrombosis (Boersma et
al 2016) In the study performed by Boersma et al (2016) in 168 human patients without
indication of anticoagulation prophylaxis elevated leukocyte counts high levels of
plasminogen activator inhibitor and of coagulation factor VIII (all measured on blood
samples taken after CVC placement) were associated with an increased incidence of
symptomatic CVC-related thrombosis diagnosed by ultrasonography In another study
(Liu et al 2015) high blood levels of lactate dehydrogenase and low concentrations of
HDL and albumin were associated with the incidence for CVC related thrombosis
diagnosed by ultrasound in 324 senile patients which was in total 617 Additionally
dyslipidemia (high triglycerides low HDL and high LDLHDL ratio) was associated
with CVC related thrombosis (diagnosed by ultrasound and computer tomography
angiography) in human patients undergoing haemodialysis (Cheng et al 2013) Studies
about predictive tests for CVC related thrombosis in dogs were not found in the available
literature to the best of the authoracutes knowledge
Chapter 1 Introduction
8
Aims of the study
The aims of the preliminary methodological study of the present scientific project were
(1) to define reference intervals for viscoelastic analyses of canine haemostasis using the
ROTEMreg delta analyser and (2) to determine the precision (repeatability) of this
method And the aims of the main study of the present scientific project were (1) to
determine the incidence of catheter-induced thrombosis in canine intensive care patients
(2) to evaluate the efficacy of a prophylactic heparin regimen routinely used in the Small
Animal Clinic of the University of Veterinary Medicine Hannover and (3) to assess
whether selected haemostasis test results are predictive for an increased risk for
thrombosis formation
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
9
Chapter 2 Reference intervals for rotational thromboelastometry measurements
using the ROTEMreg delta device in dogs
Pereira JM Rohn K Mischke R
Contributions of the authors
Pereira JM performed (1) the patients data and blood samples collection (2)
haematology and ROTEMreg analysis (3) generated the reference intervals and
coefficients of variation included in the study and (4) wrote the manuscript
Rohn K performed the statistical analysis
Mischke R performed the clinical chemistry and coagulation tests and was involved in
designing and supervision of the study analysis and discussion of the data and its
statistical analysis and revision edition and correction of the manuscript
Original source Pereira J M Rohn K amp Mischke R 2020 Reference intervals
for rotational thromboelastometry measurements using the ROTEMreg delta device
in dogs Res Vet Sci 130 26ndash32 httpsdoiorg101016jrvsc202001019
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
10
Reference intervals for rotational thromboelastometry measurements using the
ROTEMreg delta device in dogs
Pereira JMa Rohn Kb Mischke Ra
aSmall Animal Clinic University of Veterinary Medicine Hannover Buumlnteweg 9 D-
30559 Hannover Germany
bInstitute of Biometry Epidemiology and Information Processing University of
Veterinary Medicine Hannover Buumlnteweg 2 30559 Hannover Germany
Corresponding author
E-mail ReinhardMischketiho-hannoverde
Original source Pereira J M Rohn K amp Mischke R 2020 Reference intervals
for rotational thromboelastometry measurements using the ROTEMreg delta device
in dogs Res Vet Sci 130 26ndash32 httpsdoiorg101016jrvsc202001019
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
11
ABSTRACT
Aims of the present study were to define reference intervals for viscoelastic analyses of
canine haemostasis using the ROTEMreg delta analyser and as a secondary aspect to
determine the precision (repeatability) of this method Blood samples from 125 clinically
healthy dogs were included Measurements were performed with commercially available
activating reagents (ex-tem in-tem and kaolin solution) as well as without activation
Additional fourfold measurements were done in 3 of the normal blood samples and in 3
samples with haemostatic alterations to evaluate the precision of the method
Coefficients of Variation (CVs) for most of the ROTEM variables were lt 10 Clot
formation time and maximum clot elasticity showed a wide inter-individual variation in
comparison with alpha angle and maximum clot firmness A multivariate analysis on
various ROTEM parameters revealed particularly a significant influence of neuter status
and a significant interrelationship between the factors sex and neuter status for
measurements with different activating reagents These results reflected the fact that
significant differences occurred only between intact and neutered females but not in
males No or only occasionally significant differences were found between groups of sex
age and size
In conclusion CVs demonstrated that the method delivers repeatable results in canine
citrated whole blood Established reference intervals should deliver valuable orientation
for the evaluation of viscoelastic properties of clotting whole blood in dogs using the
ROTEM delta analyser Neuter status in females appeared to be the most relevant
influencing factor and should be considered for the interpretation of ROTEM delta test
results
Keywords Viscoelastic measurements reference values canine age sex neuter status
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
12
1 Introduction
Heamostasis is a physiologic and dynamic process in which plasma soluble coagulation
factors blood cells and blood vessels are involved Clot formation and possible
subsequent clot lysis as well as vascular tissue repair aim to prevent haemorrhages on the
one hand and thromboses on the other hand (Kol and Borjesson 2010 Versteeg et al
2013) Conventional assays of coagulation such as prothrombin time (PT) activated
partial thromboplastin time (aPTT) and thrombin time TT have limitations especially
cellular elements are not incorporated into the test and thereby these tests reflect only
limitedly in vivo functionality of haemostasis (McMichael and Smith 2011 Crochemore
et al 2018)
The rotational thromboelastometry (ROTEMreg) currently termed as thromboelastometry
(TEM) and thromboelastography (TEG) are methods for the rapid evaluation of the
overall haemostasis capacity through the measurement of viscoelastic properties of
clotting whole blood (Kol and Borjesson 2010 Theusinger et al 2010 Sankarankutty
et al 2012 Versteeg et al 2013) In TEG the cup with the blood sample is rotating
whereas the torsion wire is fixed In ROTEMTEM the cup is fixed whereas the pin is
rotating Changes in torque ie resistance to rotation by the fibrin bridge created between
pin and cup in the clotting blood are detected electromechanically in TEG and optically
in ROTEM (Bollinger et al 2012) and displayed as a graph that indicates the time for the
formation of blood clot and its strength (Bagge et al 2016) The resulting graph reflects
the initiation and propagation of the coagulation system fibrin-platelet interactions and
clot lysis
TEG was first described in 1948 by Hellmut Harten (Hartert 1948) as an in vitro method
to evaluate the clot formation process while resembling the in vivo blood flow The
development of the actually used instrument types TEG and ROTEMregTEM were based
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
13
on that original principle Viscoelastic measurements have been widely used since the
1980s in human medicine to evaluate haemostasis and hypercoagulable states in surgical
patients (eg cardiac and hepatic surgeries) and in general in critical care units (Kol and
Borjesson 2010 Nogami 2016 Wikkelso et al 2016 Crochemore et al 2017) In
veterinary medicine viscoelastic measurements using TEG and ROTEMTEM have been
validated for use in dogs cats and horses and used to study haemostasis in different
diseases (eg infectious diseases liver diseases) and medical conditions (eg
orthopaedic surgery hyperfibrinolysis) and also to investigate the effect of
anticoagulants and other drugs (eg corticosteroids) on haemostasis (Kol and Borjesson
2010 Bruno et al 2015a 2015b Woodman et al 2015 Conversy et al 2017 Sigrist et
al 2018 Turner et al 2019) The methodology seems to be especially suitable to detect
hypercoagulability (Wiinberg et al 2008 Kol and Borjesson 2010 Marschner et al
2018)
In the available veterinary literature information about reference values for TEG were
found using kaolin as activation reagent (Bauer et al 2009) Falco et al (2012)
determined reference values for the ROTEM analyser in dogs using ex-tem in-tem and
fib-tem reagents for selected ROTEM parameters in a study on forty five healthy dogs
However data related to reference intervals obtained in an adequate number of samples
(at least 120) according to the American Society for Veterinary Clinical Pathology
(ASVCP) (Friedrichs et al 2012) and the National Committee for Clinical Laboratory
Standards (NCCLS) (Horn and Pesce 2003) could not be found in the available literature
The aim of this study is to determine reference values for the ROTEMreg delta device in
dogs using citrated whole blood with different clot activator reagents (ex-tem in-tem
kaolin) and without activator
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
14
2 Materials and methods
21 Study design
Reference intervals Single thromboelastometric measurements were performed in 125
clinically healthy dogs using the ROTEMreg analyser and different activating reagents (ex-
tem in-tem and kaolin) as well as without activation Reference values were calculated
and influences of age groups (young adult and senior) sex neuter status and size
(groups small medium and large) were assessed
Precision analysis Additional fourfold measurements using ex-tem or in-tem activating
reagents respectively were performed on 3 of the normal and also in 3 abnormal blood
samples each in order to assess within-run imprecision for these measurements
22 Animals
The clinically healthy dogs with a body weight of at least 5 kg were recruited by launching
a call in the University of Veterinary Medicine Hannover network for students and staff
members Interested dog owners received a free health check of their animals (clinical
examination and blood tests specified below) in combination with the blood collection
In addition individual privately owned patient dogs of the Small Animal Clinic with
undisturbed general health were also included if blood was collected due to another
indication (eg general health check pre-anaesthetic examination test for hereditary
diseases etc) Before taking the blood samples all dogs were fasted for at least 12 h with
free access to water 125 animals for which the examination of the health status (clinical
examination routine haematology and clinical chemistry basis coagulation profile) did
not reveal any deviations from the reference were included in the study whereas 3 of the
128 initially recruited animals had to be excluded due to laboratory abnormalities
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
15
Haematological and clinical chemistry profiles performed in order to confirm the health
status included white and red cell count haematocrit platelet counts and reticulocytes
plasma activities of alanine aminotransferase glutamate dehydrogenase alkaline
phosphatase and pseudocholinesterase as well as concentrations of total bilirubin urea
creatinine glucose total protein albumin cholesterol and ionised calcium A basis
coagulation profile including PT PTT and TT was also performed to exclude significant
abnormalities within the blood coagulation system
The 125 healthy dogs enclosed in the establishment of reference values included 48 mixed
breed dogs and the following pedigree dogs 13 Labrador Retrievers 6 Australian
Shepherds 4 Havanese dogs 3 from each of the following breeds American
Staffordshire Terriers and Cocker Spaniels 2 from each of the following breeds Beagles
Border Collies Dalmatians Elo dogs (breed not recognised by Feacutedeacuteration cynologique
international [FCI]) German Longhaired Pointers German Shorthaired Pointers Giant
Schnauzers and Golden Retrievers 1 from each of the following breeds Belgian
Malinois Bolonka Zwetna Briard Brussels Griffon American English Coonhound
Italian Mastiff Flat Coated Retriever French Bulldog German Shepherd Great Dane
Greater Swiss Mountain Dog Giant Spitz Goldendoodle Hovawart Hungarian Vizsla
Italian Bloodhound Lycanis Wolfdog Miniature American Shepherd Miniature
Schnauzer Old German Shepherd Podenco Poodle Pyrenean Shepherd Rhodesian
Ridgeback Rottweiler Schafpudel (breed not recognised by FCI) Small
Munsterlander Spanish Mastiff Tibet Terrier Weimaraner West Highland White
Terrier and Wirehaired Pointer
These dogs were aged between 6 and 154 months (median = 410 months) and had body
weights ranging from 50 to 707 kg (median = 226 kg) 30 were castrated males 33
intact males 41 spayed females and 21 intact females According to the height at withers
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
16
the dogs were divided into 3 different size groups small (le40 cm n = 22) medium (41ndash
60 cm n = 77) and large (gt60 cm n = 26) Dogs were also divided in three different age
groups young (le 24 months n = 34) adult (25ndash84 months n = 59) and senior dogs (gt84
months n = 32)
The three blood samples with haemostatic abnormalities were taken from two dogs with
complex haemostatic disorders (Disseminated Intravascular Coagulation [DIC])
including reduced platelet count and abnormal PT and APTT and one dog with
thrombocytopenia
Animals were treated in accordance with the German Animal Welfare Law The
experimental design was approved by the Animal Welfare officer of the University of
Veterinary Medicine Hannover Foundation and by the ethics committee of the
responsible authority (Lower Saxony State Office for Consumer Protection and Food
Safety reference number 17A101)
23 Blood sampling
The venipuncture area was shaved and then disinfected with alcohol The saphenous or
cephalic vein was visualised with slight pressure and punctured with disposable cannulas
(diameter 09 or 10 mm) The continuously running blood was directly collected from
the needle into the tubes containing the respective anticoagulants From each dog
approximately 10 ml of blood were collected including approximately 8 ml of citrated
blood for the measurement of haemostasis tests (ROTEM PT aPTT TT) in tubes
containing sodium citrate 313 (Eifelfangoreg GmbH amp Co KG Bad Neuenahr-
Ahrweiler Germany) in a ratio of 1 volume9 volumes of blood In addition
approximately 1 ml of EDTA blood for haematological analysis and approximately 1 ml
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
17
of lithium heparin blood for clinical chemical measurements were collected into
commercial 13 ml tubes (Sarstedt AG amp Co Nuumlmbrecht Germany)
Citrated blood was initially kept at room temperature (20-25 C) in order to avoid errors
within the preanalytical phase (Toulon et al 2017) Two ml of citrated blood were used
for the ROTEMreg analysis the residual citrate blood was centrifuged for 10 min at 3800
rpm and 5 degC then the plasma was separated by careful pipetting and it was centrifuged
a second time under the same conditions to get platelet poor plasma which was stored in
aliquots of 500 microl and 250 microl at -80 degC for coagulation tests
24 Rotational elastometry
Viscoelastic measurements using the ROTEMreg delta for the establishment of reference
intervals were performed approximately 15 min after the blood collection using activating
reagents (liquid reagents) provided by the manufacturer of the instrument (r-ex-temreg in-
temreg) and a kaolin solution (5 g1 as part of the PTT reagent CK Prest [Diagnostica
Stago SAS Asniegraveres sur Seine Cedex France]) In addition non-activated analyses
were performed using isotonic NaCl solution instead of the activating reagent in the fourth
channel of the device Fourfold measurements for precision analysis were performed
simultaneously in the four channels of the device in separate runs
According to the standard test protocol provided by the manufacturer of the ROTEMreg
delta (Werfen GmbHInstrumentation Laboratory Munich Germany) 20 μl of the
reagent for recalcification (star-temreg20) and 20 μl of the activating reagent were pipetted
into a cuvette using the automated pipette provided with the device Immediately
thereafter 300 μl of citrated whole blood (prewarmed to 37 degC for five minutes in the
heating device of the instrument and gently rocked to resuspend possibly sedimented
cells) were added to the test preparation in the cuvette using the automated pipette and
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
18
then the cuvette was laced carefully in the ROTEMreg device whereby the rotating pin
entered into the test approach
Different parameters are provided automatically by the ROTEMreg delta device and the
following variables (described according to the Pentapharm GmbH ROTEMreg delta
Manual [2009]) were used in the study
Clotting time (CT) the time (in s) from the beginning of the test (by adding the clot
activator) until the formation of fibrin starts (defined as the moment when the curve
achieves an amplitude of 2 mm) This Parameter facilitates the decision to replace clotting
factors when it is required
Clot formation time (CFT) describes the next phase of clotting by the formation of a
stable clot through the activation of thrombocytes and fibrin formation It is defined as
the time (in s) between the 2 mm amplitude and 20 mm amplitude of the curve
Alpha angle (α angle) is the angle (in degrees) between the middle axis and the tangent
to the clotting curve through the 2 mm amplitude point
Amplitude after ldquoXXrdquo minutes (eg A30 after 30 min) is the amplitude (in mm) at a
certain time after the CT has finished
Maximum clot firmness (MCF) corresponds to the maximum amplitude (in mm) reached
before the clot is dissolved by fibrinolysis This parameter measures the firmness of the
clot and its quality Like CFT and α angle it facilitates the decision for substitution therapy
with platelet concentrate andor fibrinogen especially in humans
Maximum clot elasticity (MCE) is a parameter calculated from MCF and may be superior
at high amplitudes as compared to the MCF
Lysis index at 30 45 and 60 min (LI30 LI45 LI60) represents the fibrinolysis (in
percentage) 30 45 and 60 min after the CT It may facilitate the decision for or against a
therapy with anti-fibrinolytic drugs
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
19
Maximum lysis (ML) describes the degree of fibrinolysis relative to the MCF achieved
during the measurement (percent loss of clot firmness)
25 Statistical methods
Original data obtained from the ROTEMreg delta device software were transferred into the
Microsoftreg Excelreg formal (Microsoft Office Professional Plus 2013) The Statistical
analyses were performed using the SAS Enterprise Guide Version 71 (SAS Institute Inc
Cary NC USA)
Coefficients of Variation (CVs) for selected relevant parameters of the ROTEM delta
analysis using ex-tem and in-tem regents were calculated for the fourfold measurements
on 3 normal and 3 abnormal blood samples in order to assess within-run imprecision CVs
for the results of each sample were calculated by dividing the standard deviation of the
data set by its arithmetic mean Finally the median of the CVs for each group of 3 samples
was calculated The reference values were calculated according to recommendations of
the American College of Veterinary Clinical Pathology (ACVCP) based on non-
parametric methods (25 and 975 quantiles) supplemented by 95 confidence
intervals (Friedrichs et al 2012)
Data were analysed for Standard normal distribution by Kolmogorov Smirnov test and
assessment of residuals of the analysis of variance (ANOVA) Because these tests
indicated almost normally distributed values for the studied ROTEM variables data were
analysed by a fourway ANOVA stratified by the activating reagent (ex-tem in-tem
kaolin or without activation) to test the influence of size age sex and neuter status on
several parameters and taking all interactions into account Post hoc t-tests were
performed when indicated The significance level was set to P lt 05
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
20
3 Results
31 Precision of the method
For all the parameters evaluated in the study the CVs for the fourfold measurement in
healthy dogs were less than 10 (Table 1) In the case of dogs with haemostatic
abnormalities CVs of the vast majority of ROTEM variables were also less than 10
except the variables CFT and ML irrespective whether ex-tem or in-tem reagents were
used (Table 2)
Table 1 Coefficients of variation () for rotational thromboelastometry using the
ROTEMreg delta analyser in normal blood samples
Activator CT (s) CFT (s) α (deg) A30 (mm) MCF (mm) MCE(mm) LI30 () LI45 () LI60 () ML ()
Ex-temreg 875 629 262 213 179 634 000 020 037 534
In-temreg 245 596 166 117 135 299 000 000 046 216
Abbreviations CT ndash clotting time CFT ndash clot formation time α ndash alpha angle A30 ndash
amplitude after 30 min MCF ndash maximum clot firmness MCE ndash maximum clot elasticity
LI 30 LI 45 LI 60 ndash Lysis index at 30 45 and 60 min ML ndash maximum lysis
Median values of the measurements of 3 samples
Table 2 Coefficients of variation () for rotational thromboelastometry using the
ROTEMreg delta analyser in canine blood samples with haemostatic alterations
Activator CT (s) CFT (s) α (deg) A30 (mm) MCF (mm) MCE(mm) LI30 () LI45 () LI60 () ML ()
Ex-temreg 668 1478 393 218 268 582 0 018 000 848
In-temreg 228 799 461 219 251 371 0 0 017 283
Median values of the measurements of 3 samples from patients with DIC (n = 2)
thrombocytopenia (n = 1) abbreviations please see legend to Table 1
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
21
32 Reference intervals
Activation by ex-tem reagent (tissue factor) resulted in faster CTs when compared to
activation via the intrinsic pathway using ellagic acid (in-tem) or kaolin reagents whereas
the CFT was much less affected by the activating reagent (Table 3) Especially CFT and
MCE showed a wide inter-individual variation in comparison for example with α angle
and MCF regardless of the used activating reagent (Table 3)
33 Influence of age sex neuter status and size
ANOVA did not reveal statistically significant differences in ROTEM delta parameters
between dog groups of variable size or sex (P gt 05) Age had influence only on CT and
only when the in-tem reagent was used (P = 0169 ANOVA) but not when using the ex-
tem reagent (P = 7105) Post hoc comparisons using t-test of the in-tem results between
age groups revealed shorter CTs in the group of senior dogs when compared with young
dogs (P = 0194) but not between adult dogs and these two groups ANOVA showed
particularly influences of the neuter status and a significant interrelationship between the
factors sex and neuter status on different ROTEM delta parameters using ex-tem and in-
tem respectively as activator (Tables 4 and 5) ANOVA revealed also a significant
influence of neuter status on different ROTEM parameters when kaolin was used as
activating reagent (CFT α angle MCF MCE) whereas non-activated measurements
were not subject to any significant influence of the assessed factors (results not shown)
The interrelationship between sex and neuter status mentioned above reflected the fact
that significant differences between neutered and not neutered dogs were only found in
females The statistical analysis using the t-test to compare intact vs neutered dogs
stratified by sex showed statistically significantly (P lt 05) longer CTs and CFTs and
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
22
lower α angle A30 MCF and MCE values in spayed when compared to intact females
(Tables 4 and 5)
Chapter 2 Reference intervals for rotational thromboelastometry measurements using the ROTEMreg delta device in dogs
23
Table 3 Reference values (median reference interval based on 25 - and 975 - quantiles) for thromboelastometric measurements with the ROTEMreg delta analyser in
dogs (n=125 95 confidence intervals in brackets) Abbreviations please see legend to Table 1
Activator
Statistical
para-meter
CT
(s)
CFT
(s)
Α
(deg)
A30
(mm)
MCF
(mm)
MCE
(mm)
LI30
()
LI45
()
LI60
()
ML
()
Ex-temreg
Median 450
[430ndash470]
110
[105ndash115]
680
[670ndash690]
600
[580ndash610]
610
[600ndash620]
154
[147ndash164]
100
[960ndash100]
100
[100ndash100]
970
[960ndash980]
220
[210ndash240]
25 340
[270ndash350]
650
[490ndash710]
540
[420ndash560]
460
[410ndash480]
460
[420-490]
870
[720ndash950]
100
[100-100]
870
[800ndash960]
700
[640ndash820]
800
[100ndash110]
975 830
[790ndash990]
220
[201ndash315]
790
[770ndash800]
770
[690ndash770]
730
[700ndash770]
264
[230-334]
100
[100ndash100]
100
[100ndash100]
100
[990ndash100]
590
[530ndash680]
In-tem
Median 190
[187ndash201]
116
[111ndash125]
680
[670ndash690]
570
[560ndash580]
590
[580ndash600]
143
[135ndash149]
100
[100ndash100]
100
[100ndash100]
980
[980ndash980]
180
[170ndash190]
25 135
[112ndash142]
730
[590ndash800]
510
[460ndash570]
450
[410ndash470]
480
[410ndash500]
920
[710ndash101]
100
[950ndash100]
990
[820ndash990]
930
[740ndash930]
500
[100ndash700]
975 283
[268ndash291]
251
[198ndash227]
750
[750ndash780]
660
[660ndash690]
680
[660ndash690]
210
[195ndash227]
100
[100ndash100]
100
[100ndash100]
100
[100ndash100]
270
[260ndash380]
Activator
Statistical
para-meter
CT
(s)
CFT
(s)
Α
(deg)
A30
(mm)
MCF
(mm)
MCE
(mm)
LI30
()
LI45
()
LI60
()
ML
()
Kaolin
Median 162
[154ndash173]
870
[820ndash910]
740
[720ndash750]
500
[470ndash530]
550
[530ndash560]
121
[115ndash127]
960
[940ndash980]
840
[790ndash880]
710
[640ndash770]
500
[450ndash540]
25
109
[990ndash115]
470
[440ndash580]
560
[490ndash600]
110
[700ndash230]
390
[380ndash400]
640
[600ndash680]
270
[140ndash480]
110
[800ndash260]
800
[400ndash170]
200
[160ndash240]
975 246
[230ndash337]
214
[185ndash290]
810
[790ndash810]
640
[630ndash690]
650
[650ndash690]
187
[182ndash223]
100
[100ndash100]
990
[980ndash100]
940
[930ndash990]
970
[930ndash100]
Without activation
Median 607
[495ndash662]
221
[194ndash265]
510
[460ndash550]
520
[500ndash530]
540
[520ndash550]
115
[109ndash123]
100
[100ndash100]
100
[100ndash100]
980
[980ndash990]
170
[160ndash190]
25 203
[123ndash215]
860
[550ndash930]
190
[120ndash230]
350
[230ndash370]
370
[260ndash400]
600
[350ndash670]
100
[950ndash100]
980
[890ndash990]
930
[840ndash950]
400
[000ndash500]
975 1356
[1305ndash1721]
811
[688ndash1333]
730
[710ndash790]
660
[640ndash720]
660
[640ndash730]
197
[179ndash265]
100
[100ndash100]
100
[100ndash100]
100
[100ndash100]
290
[280ndash320]
Chapter 2 Reference intervals for rotational thromboelastometry measurements using the ROTEMreg delta device in dogs
24
Table 4 Median values (minimum and maximum in brackets) for different ROTEMreg variables using ex-tem reagent stratified by sex and neuter status
Group
CT
(s)
CFT
(s)
Α
(deg)
A30
(mm)
MCF
(mm)
MCE
(mm)
LI30
()
LI45
()
LI60
()
ML
()
Female intact 430
(320ndash530)
960
(590ndash198)
710
(570ndash800)
620
(480ndash730)
640
(510ndash740)
179
(103ndash282)
100
(100ndash100)
100
(980ndash100)
960
(800ndash100)
230
(100ndash640)
Female spayed 460
(270ndash830)
121
(650ndash224)
660
(520ndash770)
580
(430ndash690)
590
(440ndash700)
146
(790ndash230)
100
(960ndash100)
100
(800ndash100)
970
(690ndash100)
240
(100ndash590)
p-value (t-test)
Female
intact vs spayed
00025 00063 00049 00023 00016 00015 na na na na
Male intact 450
(320ndash990)
105
(650ndash315)
690
(420ndash790)
590
(420ndash700)
610
(440ndash710)
154
(770ndash244)
100
(100ndash100)
100
(870ndash100)
970
(650ndash100)
220
(700ndash680)
Male castrated 460
(340ndash930)
111
(490ndash255)
685
(480ndash800)
590
(410ndash770)
600
(420ndash770)
152
(720ndash334)
100
(100ndash100)
100
(830ndash100)
980
(640ndash100)
205
(500ndash590)
p-value (t-test)
Male
Intact vs castrated
08326 05106 04756 01799 01568 02163 na na na na
p-values (ANOVA)
Factor Sex
05428 04847 06612 02206 01887 01630 08489 07893 07689 02758
Factor neuter status 01872 00167 00173 00039 00037 00028 07141 04737 09356 05948
Interrelationship
sexneuter status 00383 00968 00782 00488 00472 00319 04943 08611 05422 00160
na ndash not analysed all significant P-values (lt 005) are highlighted in bold print
Chapter 2 Reference intervals for rotational thromboelastometry measurements using the ROTEMreg delta device in dogs
25
Table 5 Median values (minimum and maximum in brackets) for different ROTEMreg variables using the in-tem reagent stratified by sex and neutering status
Group
CT
(s)
CFT
(s)
Α
(deg)
A30
(mm)
MCF
(mm)
MCE
(mm)
LI30
()
LI45
()
LI60
()
ML
()
Female intact 186
(126ndash238)
940
(590ndash164)
720
(620ndash780)
610
(490ndash690)
620
(530ndash690)
166
(113ndash227)
100
(100ndash100)
100
(990ndash100)
980
(930ndash100)
170
(100ndash300)
Female spayed 201
(149ndash291)
1220
(820ndash262)
670
(500ndash740)
550
(450ndash660)
570
(480ndash680)
133
(910ndash214)
100
(950ndash100)
100
(820ndash100)
980
(740ndash100)
190
(500ndash380)
p-value (t-test)
Female
intact vs spayed
na na lt0001 00009 00005 00005 na na na na
Male intact 199
(117ndash283)
122
(730ndash277)
680
(460ndash750)
570
(430ndash660)
590
(450ndash660)
146
(810ndash192)
100
(100ndash100)
100
(990ndash100)
980
(930ndash100)
180
(600ndash270)
Male castrated 181
(112ndash288)
123
(820ndash222)
670
(540ndash740)
565
(410ndash630)
585
(410ndash640)
138
(710ndash180)
100
(100ndash100)
100
(970ndash100)
980
(890ndash100)
180
(800ndash260)
p-value (t-test)
Male
intact vs castrated
na na 08232 01401 01162 00849 na na na na
p-values (ANOVA)
Factor Sex
02105 03317 03370 00877 01258 00510 09564 07832 08117 03638
Factor neuter status 04289 00660 00619 00107 00088 00054 07152 09795 07056 08299
Interrelationship
sexneuter status
04310 00550 00408 00707 01733 00996 05240 05982 08047 04578
na ndash not analysed all significant P-values (lt 005) are highlighted in bold print
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
26
4 Discussion
41 Coefficients of variation
In accordance with the literature CVs below 10 indicate adequate repeatability in intra-
assays evaluations (Steiner et al 2003 Hua et al 2018) According to our results
obtained from the fourfold measurements in citrated whole blood ROTEM delta
measurements on healthy dogs and dogs with haemostatic alterations fulfil these
requirements with only a few exceptions concerning CFT and ML values in abnormal
samples
Our results regarding repeatability are similar to those obtained for several TEM
parameters in previous studies performed in animals In one study the imprecision of the
ROTEM analyser was evaluated by duplicate measurements in 10 blood samples of
healthy dogs and as result of that evaluation a good precision (CVs below 10 ) was
calculated for CT CFT MCF and α angle but in contrary to our results a low
repeatability was found for ML (CVs of 233 [ex-tem] and 285 [in-tem] own values
534 and 216 ) (Falco et al 2012)
In a previous feline study using ex-tem and in-tem as activating reagents for the ROTEM
delta repeatability was assessed similarly to the present study based on fourfold
measurements of blood samples of two healthy individuals (Doumlderlein and Mischke
2015) The results also revealed CVs below 10 for most of the parameters evaluated
The partially low repeatability of ML values may reflect the vulnerability of this variable
which is not only influenced by the complex clot formation process but also by its
subsequent lysis Reproducibility of ex-tem or in-tem activated measurements of the
ROTEM delta analyser which was not investigated in our study was studied in blood
from healthy and unhealthy horses by two different operators using three different
ROTEM devices Results indicated acceptable reproducibility (Junge et al 2016)
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
27
42 Reference intervals
The number of 125 healthy dogs used in the present study to create reference intervals is
regarded adequate according to the guidelines suggested by the American Society of
Veterinary Clinical Pathology (ASVCP) and the National Committee for Clinical
Laboratory Standards (NCCLS) who recommend to include at least 120 individuals
(Friedrichs et al 2012) To the best of our knowledge reference intervals for the most
relevant ROTEMreg delta parameters obtained in an adequate number of samples of whole
citrated canine blood have not been published so far A previous study was performed on
a smaller number of 45 dogs (Falco et al 2012) The median values for reference
intervals obtained in the present study are partially similar to those calculated in the
mentioned study based on 45 healthy dogs (eg ex- tem CT 610 s450 s [Falco et al
2012own results] CFT 150 s 110 s α angle 620deg680deg MCF 530 m 610 mm ML
180 220 in-tem CT 235 s190 s CFT 136 s116 s α angle 660deg680deg MCF
560 mm590 mm ML 30 180 ) However in comparison to our results the
reference intervals in the mentioned study are slightly wider (eg ex-tem CT 29ndash92
s34ndash83 s [Falco et al 2012own results] CFT 540ndash275 s650ndash220 s α angle 470ndash
790deg540ndash790deg MCF 360ndash730 mm460ndash730 mm ML 000ndash560 800ndash590
in-tem CT 126ndash363 s135ndash283 s CFT 470ndash224 s730ndash251 s aα angle 550ndash
810deg510ndash750deg MCF 500-ndash750 mm480ndash680 mm ML 000ndash400 500ndash270 )
although the previous study used the 5 and 95 quantiles to define limits of the
reference range whereas we calculated the commonly used 25 and 975 quantiles
to capture the central 95 of reference values (Friedrichs et al 2012)
Although it is desirable that each laboratory should specifically determine its own
reference intervals the results obtained in this study may provide an orientation for other
laboratories to interpret ROTEM delta parameters analysed in citrated canine blood In
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
28
order to test transference of the reference ranges to another laboratory a validation
procedure with measurement of 20 samples should be performed (Friedrichs et al 2012)
Although intra-individual control values (subject-based reference values) are regarded
superior to population based reference values (Wiinber et al 2007) the latter have to be
routinely used in clinical patients due to lacking individual controls especially for specific
tests such as ROTEM analyses In addition the calculated reference values cannot be
used for the fully automated device ROTEM sigma because - according to an actual
human study - values of part of the parameters do not correlate well between both
instruments (Gillissen et al 2019)
43 Influence of size age sex and neuter status
In previous studies in humans (Attard et al 2013) and animals (Mischke 1994 Borrelli
et al 2017) age has been described as an influencing factor for different haemostasis
parameters We also found an influence of age on the CT with shorter times in senior
dogs (gt 84 months) compared with young dogs (up to 24 months) which was limited to
measurements activated by the in-tem reagent
This age dependency confirms in principle a previous study on 20 healthy dogs
(Barthelemy et al 2015) which found shorter CTs in elderly dogs and results of a human
study where individuals with an age gt 45 years showed shorter CTs compared with
individuals lt 45 years old (Sucher et al 2011) However in the cited studies this effect
was only seen when ex-tem but not when the in-tem reagent was used
Based on the age distribution of our dogs which were primarily recruited to establish
reference intervals we cannot deliver information regarding very young dogs A human
study focusing on ROTEM reference ranges in children demonstrated significant
differences for nearly all ROTEM parameters with the most striking differences in
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
29
subjects aged 0-3 months (Oswald et al 2010) The limited number of dogs with an age
lt 1 year which did not allow to consider these separately in the present study may also
be one reason for the different results in a feline study (Doumlderlein and Mischke 2015) In
the cited study on healthy cats age showed an influence on CT but contrary to our study
the CT was shorter in young individuals (6-12 months) compared with older ones In
addition this was only the case in measurements using kaolin and in non-activated
measurements When compared to humans and cats uni- or multivariate analyses
considering factors age and breed or size based on do populations including different
breeds have the limitation that they can be biased by the variate life expectancy between
small medium and large dog breeds (Willems et al 2017) Apart from age we found a
significant influence only for the neuter status in bitches reflected by a significant
interaction effect between factors neuter status and sex in the ANOVA analysis
Especially for CFT values (eg activated by ex-tem median values of 121 s [spayed] vs
96 s [intact]) this difference seems to be also clinically relevant with respect to the
interpretation of ROTEM results In contrast although also significantly different
absolute and relative differences of CT values were small (eg activated by ex-tem 46 s
[spayed] vs 43 s [intact]) These differences may not only be considered for the result
interpretation of clinical patients but indicate also the necessity for sex-matched patient
groups in scientific studies
The obviously more active haemostasis system in intact females is probably related to an
influence of sex hormones (specifically estrogens) on the coagulation system
Accordingly ex vivo and in vivo studies demonstrated the activating properties of
endogenous estrogens on the haemostasis system In one ex vivo study addition of
endogenous estrogens (estrone [E1] 17szlig-estradiol [E2] and estriol [E3]) to blood samples
from men resulted in shorter mean values of the reaction time in TEG analyses
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
30
corresponding to CT in ROTEM measurements (452-537 s vs 698 s [control]) and of
the TEG variable kinetics (corresponding to the CFT in ROTEM analyses) (Swanepoel
et al 2017) In a further study subcutaneous injection of E2 into male rats induced a
significant increase in activities of coagulation factors X (by 8 ) and XI (by 10-24 )
however did not show any significant effect on PT aPTT and factor VII activity (Franco-
Murillo and Jaimez 2017)
In the cited studies also the effects of the synthetic estrogen ethinylestradiol [EE] were
studied which interestingly seems to have an inhibiting effect as indicated by prolonged
reaction time and kinetics (TEG) compared with the control in the human ex vivo study
and significantly longer PTs and aPTTs and lower factor VII and X activities in the study
on rats As expected we did not find a significant influence of size on ROTEM parameters
in dogs To the best of the authoracutes knowledge size has not been examined as an
influencing factor on viscoelastic measurements or any other haemostatic variable in
dogs Apart from Greyhounds there is limited literature on breed comparisons of
haemostatic variables (Clemons and Meyers 1984 Mischke 1994 Nielsen et al 2007
Nielsen et al 2010 Boyd et al 2018) indicating very limited breed differences for
example for factor VII (Mischke 1994) However these also do not indicate systematic
influence of size and in addition possible breed-specific effects have to be considered
As already stated one limitation of the study is the limited number of young animals
which does not allow to verify reference values for animals in this stage of life In
addition with respect to the main aim of the study possible influencing factors such as
platelet count and haematocrit were not considered in the statistical analysis Finally the
relatively small number of dogs used for the precision analysis which express the fact
that this was only a secondary aspect of the study may be regarded as a limitation At the
same time these considerations provide space for further investigations
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
31
5 Conclusions
Results of the precision analysis revealed acceptable results and thereby confirm that the
ROTEMreg delta analyser delivers repeatable results to measure overall haemostasis in
canine citrated blood samples The established reference values should offer a reliable
basis for the interpretation of thromboelastometric measurements in whole blood using
the device ROTEMreg delta in dogs with suspected haemostasis disorders Neuter status
in female dogs appeared to be a relevant influencing factor on most important ROTEMreg
parameters and should be considered for result interpretation and with respect to
experimental designs
6 Acknowledgments
Jose Mauricio Pereira received a scholarship from the Universidad Nacional de Costa
Rica (reference code JB-C-1107-2016)
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
32
7 References
1 Attard C van der Straaten T Karlafitis V Monagle P Ignjativic V 2013 Developmental
hemostasis age-specific differences in the levels of hemostatic proteins J Thromb and
Haemost 11 1850ndash1854
2 Bagge A Schoumltt U Kander T 2016 Effects of naturopathic medicines on Multiplate and
ROTEMreg A prospective experimental pilot study in healthy volunteers BMC Complemen
Altern Med 17 (16) 64
3 Bartheacutelemy A Rannou B Forterre M Verwaerde P Bonnet-Garin JM Pouzot-Nevoret
C Goy-Thollot I 2015 Differences between coagulation and cytokine profiles in dogs of
different ages Vet J 205 410ndash412
4 Bauer N Eralp O Moritz A 2009 Establishment of reference intervals for kaolin-activated
thromboelastography in dogs including an assessment of the effects of sex and anticoagulat use
J Vet Diagn Investi 21 641ndash648
5 Bollinger D Seeberger MD Tanaka KA 2012 Principles and practice of
thrromboelastography in clinical coagulation management and transfusio practice Transfus Med
Rev 26 1ndash13
6 Borrelli A Botto A Maurella C Falco S Pagani E Miniscaldo B Tarducci A Bruno
B 2017 Thromboelastometric assesment of hemostasis in newborn Piemontese calves J Vet
Diagn Invest 29 293ndash297
7 Boyd CJ Claus MA Raisis AL Hosqood G Sharp CR SmartL 2018
Hypocoagulability and platelet dysfunction are exacerbated by synthetic colloids in a canine
hemorrhagic shock model Front Vet Sci 5 279
8 Bruno B Maurella C Falco S Tarducci A Zanatta R DrsquoAngelo A Borrelli A 2015a
Thromboelastometric evaluation of hemostasis in dogs infected with Leishmania infantum J Vet
Emerg Crit Care (San Antonio) 25 502ndash511
9 Bruno B Maurella C Falco S Tarducci A Zanatta R Gianella P DrsquoAngelo A Piras L
Di Bella A Borrelli A 2015b Assessment of coagulation utilizing thromboelastometry in dogs
undergoing orthopedic surgery J Vet Emerg Crit Care (San Antonio) 25 358ndash363
10 Clemons RM Meyers KM 1984 Acquisition and aggregation of canine blood platelets Basic
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
33
mechanisms of function and differences because of breed origin Am J Vet Res 45 137ndash144
11 Conversy B Blais MC Dunn M Gara-Boivin C del Castillo JRE 2017 Anticoagulant
activity of oral rivaroxaban in healthy dogs Vet J 223 5ndash11
12 Crochemore T Piza FMT Rodriacuteguez RDR Guerra JCC Ferraz LJR Correcirca TD
2017 A new era of thromboelastometry Einstein (Sao Paulo) 15 380ndash385
13 Crochemore T Correcirca TD Lance MD Solomon C Neto AS Guerra JCC Lellis PS
Bernz LM Nunes N Mancio CM Yokoyama APH Silva E 2018 Thromboelastometry
profile in critically ill patients A single-center retrospective observational study PLoS One 13
e0192965
14 Doumlderlein E Mischke R 2015 Reference intervals for thromboelastometry with the ROTEMreg
delta in cats Res Vet Sci 100 271ndash276
15 Falco S Bruno B Maurella C Bellino C DacuteAngello A Gianella P Tarducci A Zannata
R Borrelli A 2012 In vitro evaluation of canine hemostasis following dilution with
hydroxyethyl starch (13004) via thromboelastometry J Vet Emerg Crit Care (San Antonio)
22 640ndash645
16 Franco-Murillo Y Jaimez R 2017 Inhibitory effect of ethinylestradiol on coagulation factors
in rats Exp Anim 66 107ndash113
17 Friedrichs KR Harr KE Freeman KP Szladovits B Walton RM Barnhart KF Blanco-
Chavez J 2012 ASVCP reference interval guidelines Determination of de novo reference
intervals in veterinary species and other related topics Vet Clin Pathol 41 441ndash453
18 Gillissen A va den Akker T Caram-Deelder C Henriquez DDCA Bloemenkamp
RWM Eikenboom J van der Bom JG de Maat MPM 2019 Comparison of
thromboelastometry by ROTEMreg Delta and ROTEMreg Sigma in women with postpartum
haemorrhage Scand J Clin Lab Invest 79 32ndash38
19 Harttert H 1948 Blutgerinnungstudien mit der Thrombelastographie einem neuen
Untersuchungsverfahren Klin Wochenschr 26 577ndash583
20 Horn P Pesce A 2003 Reference intervals and update Clin Chim Acta 334 5ndash23
21 Hua Yanhun Qiu W Xiao Q Wu Q 2018 Precision (repeatability and reproducibility) of
ocular parameters obtained by the Tomey OA-2000 biometer compared to the IOLMaster in
healthy eyes PLoS One 13 e0193023
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
34
22 Junge H Ringer S Mayer N Schwarzwald C 2016 Assesment of method reliability and
determination of reference intervals for rotational thromboelastometry in horses J Vet Emerg
Crit Care (San Antonio) 26 691ndash703
23 Kol A Borjesson DL 2010 Application of thrombelastographythromboelastometry to
veterinary medicine Vet Clin Pathol 39 405ndash416
24 Marschner CB Wiinberg B Tarnow I Markussen B Kuumlhnel L Bochsen L Kristensen
AT 2018 The influence of inflammation and hematocrit on clot strength in canine
thromboelastographic hypercoagulability J Vet Emerg Crit Care (San Antonio) 28 20ndash30
25 McMichael MA Smith SA 2011 Viscoelastic coagulation testing technology applications
and limitations Vet Clin Pathol 40 140ndash153
26 Mischke R 1994 Activity of coagulation factors II V VII and X in healthy dogs- dependence
on age sex and breed Berl Muumlnch Tieraumlrztl Wochenschr 107 289ndash294
27 Nielsen A Zois N Pedersen H Olse L Tarnow I 2007 Platelet function in dogs bread
differences and effect of acetylsalicylic acid administration Vet Clin Pathol 36 267ndash273
28 Nielsen L Kjelgaard‐Hansen M Jensen A Kristensen A 2010 Breed‐specific variation of
hematologic and biochemical analytes in healthy adult Bernese Mountain dogs Vet Clin Pathol
39 20ndash28
29 Nogami K 2016 The utility of thromboelastography in inherited and acquired bleeding
disorders Br J Haematol 174 503ndash514
30 Oswald E Stalzer B Heitz E Weiss M Schmugge M Strasak A Innerhofer P Haas T
2010 Thromboelastometry (ROTEMreg) in children Age-related reference ranges and correlations
with standard coagulation tests Br J Anaesth 105 827ndash835
31 Sankarankutty A Nascimento B Teodoro da Luz L Rizoli S 2012 TEGreg and ROTEMreg
in trauma similar test but different results World J Emerg Surg 7 (Suppl 1) S3
32 Sigrist NE Schefer RJJ Kutter APN 2018 Characteristics of hyperfibrinolysis in dogs and
cats demonstrated by rotational Thromboelastometry (ROTEM) Vet J 242 67-73
33 Steiner J Teague S Williams D 2003 Development and analytic validation of an enzime-
linked immunosorbent assay for the measurement of canine pancreatic lipase immunoreactivity in
serum Can J Vet Res 68 161ndash168
34 Sucker C Tharra K Lithmate J Scharf RE Zotz RB 2011 Rotation thromboelastography
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
35
(ROTEM) parameters are influenced by age gender and oral contraception Perfusion 26 334ndash
340
35 Swanepoel A Emmerson O Pretorius E 2017 The effect of endogenous and synthetic
estrogens on whole blood clot formation and erythrocyte structure Microsc Microanal 23 599ndash
606
36 Theusinger OM Nuumlrnberg J Asmis LM Seifert B Spahn DR 2010 Rotation
thromboelastometry (ROTEM) stability and reproducibility over time Eur J Cardiothorac Surg
37 677ndash683
37 Toulon P Metge S Hangard M Zwahlen S Piaulenne S Besson V 2017 Impact of
different storage times at room temperature of unspun citrated blood samples on routine
coagulation tests results Results of a bicenter study and review of the literature Int J Lab
Hematol 3 458ndash468
38 Turner JS Kutter APN Sigrist NE 2019 Correlation of rotational thrromboelastometry
(ROTEM) parameters with platelet count and their ability to predict thrombocytopenia in dogs
Res Vet Sci 126 45ndash50
39 Versteeg HH Heemskerk JWM Levi M Reitsma PH 2013 New Fundamentals in
Hemostasis Physiol Rev 93 327ndash358
40 Wiinberg B Jensen AL Kjelgaard-Hansen M Rojkjaer R Johansson PI Gade LP
Gram DX Kristensen AT 2007 Study on biological variation of haemostatic parameters in
clinically healthy dogs Vet J 174 62ndash68
41 Wiinberg B Jensen AL Johansson PL Rozanski E Tranholm M Kristensen AT 2008
Thromboelastographic evaluation of hemostatic function in dogs with disseminated intravascular
coagulation J Vet Intern Med 22 357ndash365
42 Wikkelsoslash A Wetterslev J Moslashller AM Afshari A 2016 Thromboelastography (TEG) or
thromboelastometry (ROTEMreg) to monitor haemostatic treatment versus usual care in adults or
children with bleeding Cochrane Database Syst Rev Art
httpsdoiorg10100214651858CD007871pub3 No CD007871
43 Willems A Paepe D Marynissen S Smets P Van de Maele I Picavet P Duchateau L
Daminet S 2017 Results of screening of apparently healthy senior and geriatric dogs J Vet
Intern Med 31 81ndash92
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
36
44 Woodman J Wagg CR Boysen SR Leguillete R Mizen K Roy MF 2015 Evaluation
of coagulation via thromboelastography in healthy horses administered dexamethasone Can Vet
J 56 1271ndash1274
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
37
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of
standard heparin prophylaxis and correlation to haemostatic changes
Pereira JM Hewicker-Trautwein M Rohn K von Depka Prondzinski M
Mischke R
Contributions of the authors
Pereira JM performed (1) the ultrasound evaluations and blood sampling of the
patients under study (2) haematology analysis (3) injection of the anticoagulation
prophylaxis (partially) to the patients under study (4) analysis of samples (collaboration)
at Werlhof Institute Medical Care Center (5) analysis of some haemostasis test
(collaboration) (6) statistical analysis (partially) (7) data documentation (8) and wrote
the manuscript
Hewicker-Trautwein M performed the electron microscopy analysis
Rohn K performed the statistical analysis
von Depka Prondzinski M performed some haemostasis test (thrombin generation
assay and endogenous thrombin potential)
Mischke R performed the coagulation and haemostasis test was involved in designing
and supervision of the study analysis and discussion of the data and its statistical analysis
and revision edition and correction of the manuscript
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
38
Central venous catheter induced thrombosis in dogs ndash efficacy of standard heparin
prophylaxis and correlation to haemostatic changes
Joseacute Mauricio Pereira1 Marion Hewicker-Trautwein2 Karl Rohn3 Mario von Depka
Prondzinski4 Reinhard Mischke1
1Small Animal Clinic University of Veterinary Medicine Hannover Foundation
Buumlnteweg 9 D-30559 Hannover Germany
2Institute for Pathology University of Veterinary Medicine Hannover Foundation
Buumlnteweg 17 D-30559 Hannover Germany
3Institute of Biometry Epidemiology and Information Processing University of
Veterinary Medicine Hannover Foundation Buumlnteweg 2 30559 Hannover Germany
4Werlhof Institute Schillerstrasse 23 30159 Hannover Germany
Abbreviations anti-Xa anti-factor Xa BW body weight CVC central venous catheter
EJV external jugular vein ELMI electron microscopy IV intravenous JV jugular vein
LMWH low molecular weight heparin ML maximum lysis (ROTEM) PIVC
peripheral intravenous catheter ROTEM rotational elastometry SC subcutaneous
TGA thrombin generation assay TID three times a day TT thrombin time UFH
unfractionated heparin
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
39
Corresponding author
E-mail addresses matiscrgmailcom (JM Pereira) MarionHewicker-
Trautweintiho-hannoverde (M Hewicker-Trautwein) KarlRohntiho-hannoverde
(K Rohn) mailwerlhof-institutde (M von Depka Prondzinski)
ReinhardMischketiho-hannoverde (R Mischke)
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
40
ABSTRACT
Central venous catheters (CVC) are of importance for intensive care management of
canine patients Thrombosis of the jugular vein is a severe complication related to the
CVC use However only little is known about the incidence of CVC-associated
thromboses in dogs and the necessity and efficacy of prophylactic antithrombotic
regimens The objectives of the present study were (1) to determine the incidence of
catheter-induced thrombosis in canine intensive care patients and thereby (2) to evaluate
the efficacy of a routine prophylactic heparin treatment and finally (3) to assess whether
initial changes of selected haemostasis parameters are predictive for an increased risk for
thrombosis formation 29 dogs hospitalised in the Small Animal Clinic University of
Veterinary Medicine Hannover Foundation and receiving a central venous catheter
(CVC) in the external jugular vein (EJV) for medical reasons were included in the final
assessment 17 dogs received the standard antithrombotic dosage regimen of
unfractionated heparin (UFH 150 IUKG BW TID sc) and 12 dogs (usually surgical
patients) the reduced dosage (75 IUKG BW TID sc) A colour Doppler ultrasound of
the respective EJV in order to detect thrombus formation and blood collection for
haemostasis tests were performed immediately before the installation procedure (day 0)
and on days 1 3 5 7 and so on after the CVC insertion Haemostasis tests included
prothrombin time activated partial thromboplastin time thrombin time antithrombin
activity D-dimer concentration thrombin generation and rotational elastometry as well
as the heparin activity using a chromogenic anti-factor Xa test Finally electron
microscopy (ELMI) of sections of the removed CVC was performed In 8 dogs
sonographic examination revealed thrombus formation in the lumen of EJV and in 1927
CVCs assessed using ELMI thrombi on the external CVC surface were detected without
significant correlation between both methods Plasmatic heparin activities showed great
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
41
variability but no significant differences were found between dogs with or without CVC-
associated thrombus formation From the initially performed haemostasis tests high
fibrinogen concentrations and low maximum lysis values as measured by the ROTEM
delta device (ex-tem reagent) were associated with sonographically detectable thrombi
The results of the present study indicate that CVC-associated thromboses occur (eg 28
[829] of the patients as detected by ultrasound) although severe thromboses (high
grades) are relatively rare in intensive care canine patients receiving a routine
anticoagulant treatment A high fibrinogen concentration and low maximum lysis
(ROTEM ex-tem) may be useful indicators for an increased thrombotic risk in individual
patients which may require special anticoagulatory treatment
Keywords CVC complication venal occlusion antithrombotic treatment ultrasound
electron microscopy rotational elastometry
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
42
1 Introduction
Central venous catheters (CVCs) are very important tools for care of patients in critical
care units allowing parenteral nutrition support therapy administration of high volumes
of intravenous fluids safe drugs administration that cannot be given through peripheral
veins plasmapheresis haemodialysis blood transfusions as well as periodical blood
sampling (Geerts 2014 Vose et al 2019) CVC related thrombosis is one of the most
severe complications (apart from infections and pneumothorax) of CVCs in humans
(Geerts 2014 Comerlato et al 2017 Evans and Ratchford 2018) leading to patientrsquos
distress CVC dysfunction venous stenosis and in some cases to the development of
thromboembolism (Geerts 2014)
In the human literature there are various studies dealing with epidemiology with reference
to different anticoagulatory regimens including placebo-controlled studies (Lee and
Kamphuisen 2012) In humans CVC related thromboses represent 70ndash80 of all upper
extremity thrombosis cases and approximately 10 of thromboembolism cases in
humans (Kucher 2011) The incidence of CVC related thrombosis reported in humans
based on 25 studies ranged widely from 2 to 67 (median = 30 ) (van Rooden et al
2005 review article) This wide range of calculated incidences of CVC related
thromboses in humans reflects the use of different CVC types different locations of entry
site of the CVC (jugular femoral subclavian axillary cephalic vein) different thrombus
detection technique (venography or ultrasonography) unequal durations of follow up
varying diseases of patients (eg haematological oncological) and presence of
predisposing factors (eg previous venous thrombosis history presence of cancer) (van
Rooden et al 2005 Geerts 2014 Liu et al 2015)
Part of the authors empirically observed CVC-associated thrombosis of the external
jugular vein (EJV) as a relevant complication in their canine intensive care patients which
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
43
awakened their interest in that topic To the best of the authoracutes knowledge studies about
extra luminal CVC related thromboses in dogs are rare in the available veterinary
literature One study evaluating the complications associated with 100 CVCs placed in
the jugular vein of dogs and cats reported only 2 cases of thrombosis (confirmed by
palpation or ultrasound) but obviously did not assess the appearance of subclinical CVC
related thromboses (Adamantos et al 2010) Thereby the incidence of CVC-induced
thromboses in dogs is unknown and systematic studies regarding an effective
anticoagulatory management are lacking
In this context it is of interest whether a possible development of thrombosis or an
increased risk (despite the prevention by a standard application of unfractionated heparin
[UFH]) can be predicted by means of changes of haemostasis related parameters during
the initial examination In one study on 168 human patients without indication of
anticoagulation prophylaxis for example elevated leukocyte counts high plasminogen
activator inhibitor and high coagulation factor VIII levels (all measured on blood samples
taken after CVC placement) were associated with an increased incidence of symptomatic
CVC-related thrombosis diagnosed by ultrasonography (Boersma et al 2016)
Therefore aims of the present study were (1) to determine the incidence of catheter-
induced thrombosis in canine intensive care patients (as detected by ultrasonography and
electron microscopy ELMI) and thereby (2) to evaluate the efficacy of a standard
antithrombotic regimen with unfractionated heparin (UFH) routinely used in the Small
Animal Clinic of the University of Veterinary Medicine Hannover and (3) to assess
whether selected haemostasis test results are predictive for an increased risk for
thrombosis formation
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
44
2 Material and methods
21 Study design
Canine in-patients of the Small Animal Clinic University of Veterinary Medicine
Hannover receiving a CVC in the EJV for medical reasons from March 2017 to
December 2019 were included Anticoagulatory treatment was performed according to
the standard protocol of the clinic Colour Doppler ultrasonographic controls of the EJV
and blood samplings (for heparin activity basic haematology and haemostasis tests) were
done immediately before the catheter was placed (day 0) and on days 1 3 5 etc (2 hours
after the first daily UFH injection) until the CVC was removed Only patients were
included which could be examined at least until day 3 Haemostasis tests included
prothrombin time activated partial thromboplastin time thrombin time fibrinogen
concentration antithrombin activity fibrin monomers D-dimers thrombin generation
assay (TGA) and rotational elastometry as well as the heparin activity using a
chromogenic anti-factor Xa test Finally ELMI of sections of the CVC after its removal
was performed
22 Animals
Originally 32 patients hospitalised in the Small Animal Clinic and receiving a CVC in the
EJV for medical reasons entered the study Three of them were subsequently excluded
because they did not reach the defined minimum study period of 3 days Two were
euthanised and one referred back to the referring vet before day 3 22 dogs weighing more
than 15 kg were included so that the withdrawal of about 10 ml of blood on several days
was not a significant burden for the patient (lt 1 of the total blood volume) Five dogs
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
45
with a body weight of less than 15 kg but at least 8 kg were included with special
consideration of the amount of blood taken (no more than 6 ml on individual days)
All animals received standard anticoagulatory treatment with UFH 17 dogs with internal
diseases received 150 IUkg BW TID subcutaneously and 12 dogs (post surgery) received
a reduced dosage of 75 IUkg BW TID subcutaneously
The 29 dogs enclosed in the study included 4 mixed breed dogs and the following
pedigree dogs 5 Labrador Retrievers 3 German Shepherds 2 Australian Shepherds and
one from each of the following breeds Akita Inu Asian Shepherd Bavarian Mountain
Hound Beagle Belgian Shepherd Briard Canaan dog Dalmatian Golden Retriever
Havanese dog Hovawart Rhodesian Ridgeback Schnauzer Shar Pei and Wolfspitz
These dogs were aged between 2 and 164 months (median 77 months) and had body
weights ranging from 81 and 63 kg (median 27 kg) 9 were castrated males 8 intact
males 7 spayed females and 5 intact females
The diseases of the 29 patients requiring a CVC included 3 from each of the following
diseases hepatopathy and esophagus dilatation 2 from each of the following diseases
bacterial cystitis diabetes mellitus and 1 from each of the following diseases abdominal
wall perforation aspiration pneumonia cystitis intestinal round cells sarcoma carcinoma
of the nasal cavity chronic renal insufficiency cystic duct rupture heartworm disease
ivermectine intoxication lymphoma multifocal alveolar histiocytosis pancreatitis post-
surgical bleeding stomach dilatation stomach foreign body tetanus thoracic
pyogranulomatous inflammation thrombocytopenia and trigeminal nerve paresis
Animals were treated in accordance with the German Animal Welfare Law The
experimental design was approved by the Animal Welfare officer of the University of
Veterinary Medicine and reported to the responsible authority (Lower Saxony State
Office for Consumer Protection and Food Safety reference number 17A101)
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
46
23 Positioning and handling of the central venous catheter
Two types of single lumen radiopaque polyurethane CVC (different on length and
diameter) were used during the study The CVC type I (n = 2529) was Cavafixreg Certoreg
Splittocanreg CVC 32 cm length 11 x 17 mm diameter16 G (BBraun Melsungen AG)
the CVC type II used (n = 429) was Certofixreg Mono S 215 15 cm length 08 x 14 mm
diameter18G (BBraun Melsungen AG) Each CVC was inserted into the EJV using a
modified Seldinger technique as follows the skin of the neck was shaved and disinfected
with antiseptic soap alcohol and iodine Local anesthesia (Lidocaine HCl 2) was
administrated subcutaneously on the catheter insertion site (a middle point in the lateral
neck over the jugular sulcus) A small skin incision was performed in the insertion site
and one peripheral plastic cannula with a metallic needle inside (14 G for CVC type I
and 20 G for CVC type II) was inserted in the EJV in cranio-caudal orientation (Hundley
et al 2018) Once the EJV was reached the needle was removed and a flexible wire
guide was inserted through the peripheral cannula following an external landmark
(4thintercostal space) (Reminga et al 2018) The peripheral cannula was removed and
the respective CVC was inserted slowly and with gentle pressure into the EJV guided
with the flexible wire and following the same external landmark to reach the right
position After the insertion of the CVC the flexible wire guide was removed The correct
positioning of the CVC was checked with a latero-lateral thorax x-ray ensuring that the
CVC tip was placed at the junction of the cranial cava vein with the right atrium Each
CVC was fixed to the neck skin (adjacent to the insertion point) with a monofilament
non-absorbable suture (gauge 3-0) Finally a bandage elaborated with Povidone-Iodine
gauze (Betaisodonareg) padded synthetic band (Artiflexreg Natur) and elastic band
(Elastomullreg) was used to protect the CVC output (Claude et al 2010) and changed
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
47
daily Several times day the catheter was flushed with isotonic sodium chloride
containing 1 IUml UFH
24 Heparin preparation and administration
Heparin-sodium-25000-ratiopharm (injection solution containing 5000 IUml sodium
heparin [mucosa]) was used SC injection was performed over the lateral thorax wall
using 1 mL syringes with fine graduation marks
25 Ultrasonography
Colour Doppler ultrasonographic examinations were performed in lateral position using
a LO-GIQ E9 ultrasound device (GE Healthcare Chicago Illinois USA) with a 15
Megahertz linear ultrasound probe The development of a thrombus or fibrin debris in the
EJV lumen around the CVC or inside it was assessed at each control Once the
ultrasound was finished a new protective bandage was placed around the neck of the dog
According to the percentage of lumen occlusion of the EJV (evaluated in cross sectional
views during the ultrasound scanning) the thrombi were classified in four different grades
(Fig 1)
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
48
of the residual vein lumen C ndash catheter T ndash thrombus
Fig 1 Definition of different grades of central venous catheter induced thrombosis of the
jugular vein as detected using colour Doppler ultrasound
26 Blood collection
At each time point approx 6ndash10 ml of blood (according to the body weight) was collected
This included approx 1 ml of EDTA blood for hematological analysis and approx 5ndash9
ml of citrated blood (tubes containing one part of 011 molL [38 ] sodium citrate to
nine parts of blood) for the measurement of hemostasis tests in citrated blood (ROTEM)
or citrated plasma Blood samples were collected by puncture of peripheral veins with
disposable cannulas (09 x 40 mm [20G] or 10 x 40mm [21G]) directly into sample tubes
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
49
or from the CVC using the three-syringe-technique (Villalta-Garciacutea et al 2015) in
situations when the peripheral venipuncture was not indicated due to phlebitis or irritation
andor inflammation of the surrounding skin Briefly 2ndash3 ml of blood were taken in a
sterile disposable syringe and immediately afterwards the blood sample was taken in a
new disposable syringe and transferred into the test tubes containing the respective
anticoagulants finally the first 2-3 ml taken were re-infused to the dog through the CVC
followed by flushing with a solution of sodium chlorideUFH (02) Immediately after
blood collection each sample tube was swayed gently until blood and anticoagulant had
been mixed thoroughly To prepare citrated platelet poor plasma citrated blood was
centrifuged at 16000 g for 10 min at room temperature using a microcentrifuge The
plasma was transferred into plastic tubes and the centrifugation procedure was repeated
This platelet poor plasma was then frozen in aliquots at -70 degC until used for analysis
Directly before analysis the plastic tubes were thawed at 37 degC in a water bath
27 Laboratory tests
Haematological analyses were made automatically with Adviareg 2120i (Siemens
Healthcare GmbH Germany)
Prothrombin time activated partial thromboplastin time and thrombin time were
measured with the autoanalyser Amax Destiny Plus (Tcoag Deutschland GmbH
Germany) and commercial reagents (Thromborel S Siemens Healthcare Diagnostics
Products GmbH Marburg Germany CK Prestreg and Test Thrombin Reagent
Diagnostica Stago SAS Asniegraveres sur Seine Cedex France) Whereas the last two tests
were measured according to the manufacturer`s instructions a modified test optimised
for canine plasma was used to measure prothrombin time (25 microl citrated plasma 120 with
imidazole buffer + 25 microl of a human fibrinogen solution 2 gl prewarmed for 120 s at 37
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
50
oC addition of 25 microl of activating reagent Thromborel S) and the results converted into a
percentage activity based on a calibration curve prepared with canine pooled plasma The
fibrinogen concentration was measured with the method according to Clauss using a test
kit from Stago Diagnostica SAS D-dimer concentration was measured with a
quantitative commercial human latex turbidimetric method in the autoanalyser Cobas
c311 using a completely assembled kit and human standards (Roche Diagnostics
Mannheim Germany) Antithrombin activity was also measured with a completely
assembled chromogenic test kit in the autoanalyser Cobas c311 and calibrated with a
canine pool plasma (n = 100)
The thrombin generation assay (Technothrombinreg TGA (Technoclone GbmH Vienna
Austria) determines thrombin generation based on monitoring of the fluorescence
generated by cleavage of a fluorogenic substrate by thrombin over time after activation
of the coagulation cascade by different concentrations of tissue factor and negatively
charged phospholipid in platelet poor plasma From the fluorescence development over
time the concentration of thrombin in the sample can be calculated using a thrombin
calibration curve The increase in thrombin concentration with time then allows to
calculate generation of thrombin in the sample and to plot thrombin values over time for
the whole coagulation process
The rotational thromboelastometry analysis with ROTEM delta device (Werfen Munich
Germany) was started approx 15 minutes after the blood collection To perform the
analysis and according to the automated ROTEM software 20 microl of recalcification reagent
(200 mmoll calcium chloride solution star-tem) and 20 microl of the respective activating
reagent (ex-tem in-tem) were pipetted into and carefully mixed in a pre-warmed cuvette
using the automated pipette provided in the device immediately afterwards 300 microl of
citrated whole blood (previously gently revolved) were added and mixed with the
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
51
reagents previously deposited Finally the cuvette was carefully placed and fixed to an
oscillating pin (oscillation movements on left and right through an angle of 475deg) in the
ROTEM delta device The interaction between blood cells and subsequently the bond
between fibrin and platelets promoted by the biomechanical changes in pH electrolytes
and temperature start the clot formation with its subsequent retraction and lysis The
magnitude of the resistance produced by the clot over the oscillating pin is quantitatively
represented by different parameters measured during the analysis and also qualitatively
in a graphic illustration (Bagge et al 2016 Crochemore et al 2017)
A chromogenic substrate test (Coatest Heparin Chromogenix-Instrumentation
Laboratory Haemochrom Diagnostica Essen Germany) was used for measurements of
UFH in the auto-analyser Cobas c311 The test application was created based on
manufacturers` instructions Different dilutions of the same batch of the commercial
sodium UFH preparation used in the experiment with normal canine pool plasma
(prepared from identical aliquots of 10 healthy adult dogs) served as standards for the
calibration of the measurement
28 Electron microscopy
Selected areas on the last third of the CVC (5ndash10 cm according with the CVC) with
macroscopically detectable thrombi were analysed and some of the CVCs were cut in
two different places The cut samples were preserved in a fixative medium composed of
1 glutaraldehyde and 4 formalin Once the sample was fixed it was prepared
according to the osmium thiocarbohydrazide procedure (OTOTO-procedure) (Malick et
al 1976) The fixed CVCs were dehydrated in seven series as follow first three series of
120 minutes each one on 30 50 and 70 ethanol respectively followed by two series
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
52
of 120 min each one on 90 ethanol and then two series of 24 hours each one on 100
ethanol After the dehydration process the samples were placed two times during 24
hours each one on isoamyl acetate and then placed on in a critical point drier Finally the
samples were coated with gold in a splutter-coater with 25 kV acceleration voltage in an
argon atmosphere with a current of 20 mA for one minute The samples were examined
in an EVO 15 scanning electron microscope (ZEISS International Carl Zeiss Jena
Germany) and photographed with magnifications of 600x and 6000x avoiding mechanical
artifacts (Toskala et al 1995) Thrombi on the external wall of the CVC were analysed
and measured using the software ImageJ (National Institutes of Health and the Laboratory
for Optical and Computational Instrumentation University of Wisconsin USA)
According to their extension (surface size) and thickness (related to the CVC wall
thickness in a cross sectional view) thrombi on the external wall and additionally within
the lumen of the CVC were each classified in four grades (Fig 2 Fig 1S [suppl
material]) In case of two cuts the highest degree of thrombosis detected at one of the two
sites was used for the final assessment
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
53
surface sizepercent of CVC wall thickness
Fig 2 Definition and illustration of different grades of central venous catheter induced
(extraluminal) thrombosis as detected using electron microscopy
Fig 1S (suppl material) Definition and illustration of different grades of intraluminal
thrombosis in central venous catheters as detected using electron microscopy
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
54
29 Statistical methods
Comparison between thrombus graduation either based on sonographic and ELMI
findings respectively was performed using the McNemar test with calculation of the
concordance index (κ) Standard normal distribution was assessed using the
Kolomogorov-Smirnov test
Due to non-normal distribution of part of data sets heparin activities and results of
haemostasis assays are reported using non-parametric variables (median minimumndash
maximum box and whisker plots) Heparin activities and results of haemostasis tests
between dogs with or without thrombosis were compared using Mann-Whitney-U test
3 Results
31 Sonographic detection of thrombus formation
Thrombus formation was sonographically detected in 829 patients during the study
period Based on examination on the final day in half of these eight patients (n = 4) the
thrombus formation was scored grade 1 in 3 dogs grade 2 and only in one grade 3 In all
of these 8 dogs this was the maximum grade during the study period One dog with fibrin
precipitates was excluded from further analysis because it could not be clearly assigned
to one of the groups In 78 cases thrombi were attached mainly to the venous wall
whereas only in one case was attached to the CVC external wall Seven of these 8 dogs
which developed thrombi received the standard heparin dosage (150 IUkg TID 41
[717] of dogs receiving this dosage) and only one the reduced heparin dosage (75 IUkg
TID 8 [112] of dogs receiving this dosage)
The time point of first detection of thrombus formation varied between days 3 and 11
(median day 8) In addition detritus (presumably composed mainly of fibrin) was
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
55
detectable in the lumen of the EJV in one further dog and within the lumen of the CVC
in further 5 dogs
32 Thrombus formation by electron microscopy
27 from 29 CVC catheter tips were analysed by electron microscopy 827 samples were
without detectable thrombus (grade 0) and 1927 with detectable thrombi 72 showed
thrombus formation of grade 1 427 of grade 2 and 827 of grade 3 There was no
concordance between thrombus graduation based on sonographic and the ELMI
graduation respectively The concordance index (McNemar test) was negative (κ = -
00718) indicating that the degree of agreement is less than by chance
Of the in total 19 dogs with detectable thrombus formation by electron microscopy 11
dogs received 150 IUkg TID (in total 16 dogs of these 27 dogs received that heparin
dosage) and 8 received 75 IUkg TID (in total 11 of these 27 dogs received that heparin
dosage 73 811) and Of the 12 patients with grade 2 and 3 thrombus formation 5 dogs
received 75 IUkg TID and 7 dogs 150 IUkg
The supplementary analysis of intraluminal thrombus formation revealed no detectable
thrombi in 7 cases and thrombus formation of grade 1 (lt 25 occlusion) in 11 cases of
grade 2 (25ndash50 ) in 3 cases and of grade 3 (gt 50 ) in 6 cases
33 Heparin activities and relationship to thrombus formation
Heparin activities varied remarkably in both dosage groups (Fig 3 ab) There was no
significant difference of heparin activities between blood samples collected via
venipuncture or from the CVC (Table 1S supplementary material) In addition there was
no significant difference between heparin activities in dogs with or without
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
56
sonographically or electron microscopically detectable thrombosis (Tables 1 and 2)
Table 1 refers to sonographically detectable thromboses and is based on median heparin
activities calculated from the measurements on day 1 and following days per patient
firstly for the total number of patients and secondly for the patients receiving 150 IUkg
TID In addition in the eight dogs heparin activities on the day of first thrombus detection
(023 IUml 019ndash036 IUml) were not significantly different from the median values
(Table 1 P = 05214 Wilcoxon test) Table 2 compares heparin activities (median values
of all days and results on the final day) between dogs with and without electron
microscopically detectable CVC-associated thrombi
Table 1S (Supplementary material) Heparin activities (IUml median values
minimumndashmaximum) measured in 27 dogs receiving 75 or 150 IUkg unfractionated
(standard) heparin TID subcutaneously 2 hours after the first injection on the day
Comparison of samples collected via puncture of the peripheral veins and those collected
from the central venous catheter (CVC) using the three-syringe-technique
Peripheral vein
puncture CVC
Mann-Whitney-U
test
Total number
(75 and 150
IUkg TID)
019
(00ndash108)
(n = 65)
023
(000ndash105)
(n = 65)
P = 03033
75 IUkg TID
010
(000ndash108)
(n = 25)
012
(000ndash023)
(n = 21)
P = 06907
150 IUkg
TID
030
(000ndash096)
(n = 40)
036
(000ndash105)
(n = 44)
P = 01223
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
57
Fig 3ab Heparin activities (box- and whisker plot arithmetic means) in dogs receiving
unfractionated heparin at a dosage of 75 IUkg BW tid (a) or 150 IUkg BW tid (b) two
hours after the first daily heparin administration
day 1 represents the first day after initiation of heparin treatment
only days were considered with at least results from 5 patients
Table 1 Heparin activities (IUml median values minimumndashmaximum) in 28 dogs
receiving 75 or 150 IUkg unfractionated (standard) heparin TID subcutaneously with
(grades 1ndash3) or without (grade 0) sonographically detectable thrombus Blood collections
were performed 2 hours after the first daily injection
Grade 0
(sonography)
Grades 1ndash3
(sonography)
Mann-Whitney U
test
Total number
(75 and 150 IUkg
TID)
020 (000ndash072)
(n = 20)
027 (018ndash059)
(n = 8)
P = 03212
150 IUkg TID 042 (000ndash072)
(n = 10)
031 (018ndash059)
(n = 7)
P = 04593
one dog with fibrin precipitates in the lumen of the jugular vein but without defined thrombus formation
was not considered because it could not be clearly assigned to one of the two groups
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
58
Table 2 Heparin activities (IUml median values minimumndashmaximum) in 27 dogs
receiving 75 or 150 IUkg unfractionated (standard) heparin TID subcutaneously with
(score 1ndash3) or without (score 0) detectable thrombus via electron microscopy
Grade 0
(electron
microscop)
(n = 8)
Grades 1ndash3
(electron
microscop)
(n = 19)
Mann-Whitney U
test
Median value of
different days 028 (005ndash060)
022 (000ndash072)
P = 05770
Final day 020 (001ndash045)
015 (000ndash105)
P = 06706
34 Relationship between haemostasis tests and thrombus formation
From the initially performed haemostasis tests high fibrinogen concentrations and low
maximum lysis values as measured by the ROTEM delta device (using the ex-tem
reagent) were associated with sonographically detectable thrombi (Tab 3) Additional
analyses based on the median values of different days for individual patients revealed that
low maximum lysis values as measured by the ROTEM delta device (using the in-tem
reagent) were associated with sonographically detectable thrombi (maximum lysis score
0 850 [100ndash175 ] score 1ndash3 375 [100ndash900 ] median [minimumndash
maximum] P = 00166) but no other test Comparison of the results of haemostasis tests
on the final day between dogs with and without sonographically detectable thromboses
did not show any significant difference (P gt 005)
Comparison of results of haemostasis tests on the final day in dogs with detectable
thrombosis via ELMI and dogs without significant thrombus formation showed several
significant differences including lower fibrinogen concentrations and longer clotting
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
59
times (ex-tem reagent) and clot formation times (ex-tem and in-tem reagents) using the
ROTEM delta analyser in general expressing a less coagulable status (Tab 4) If values
of dogs graded 0 and 1 (n =15) are compared to those of grades 2 and 3 (n = 12) the last-
mentioned had longer clotting times (ROTEM delta in-tem reagent) as well as longer clot
formation times and lower alpha angles (ROTEM delta in-tem reagent) (P lt 005 results
not shown)
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
60
Table 3 Comparison of initial results of haemostasis parameters (median values
minimumndashmaximum in dogs with (grades 1ndash3) and without (grade 0) sonographically
detectable central venous catheter-induced thromboses
Parameter Unit
Sonographic
thrombus grade
P-value
(Mann-Whitney
U test)
0
(n = 20)
123
(n = 8)
Haematocrit 355 (228ndash641)
382 (245ndash499)
06111
Platelet count x103microl 292 (420ndash483)
218 (860ndash458)
02320
Prothrombin time 944 (359ndash169)
896 (471ndash136)
05758
Activated partial thromboplastin
time s 128
(955ndash249) 140
(117ndash164) 02322
Thrombin time s 129 (121ndash178)
141 (650ndash160)
02028
Fibrinogen gl 448 (153ndash770)
747 (153ndash116)
00235
Antithrombin activity 639 (289ndash107)
670 (464ndash746)
05249
D-dimers microgml 006 (000ndash148)
015 (003ndash372)
03997
Thrombin generation assay nmol 130 (000ndash365)
912 (190ndash277)
08787
Clotting time (ROTEM ex-tem) s 420 (290ndash930)
430 (170ndash159)
07989
Clot formation time (ROTEM ex-
tem) s 665
(310ndash143) 470
(240ndash800) 00633
Alpha angle (ROTEM ex-tem) degrees 795 (620ndash840)
815 (750ndash860)
01667
Max clot firmness (ROTEM ex-
tem) mm 750
(570ndash860) 775
(700ndash830) 01753
Max clot elasticity (ROTEM ex-
tem) - 299
(131ndash619) 347
(235ndash484) 01544
Maximum lysis (ROTEM ex-tem) 135 (300ndash290)
400 (000ndash800)
00037
Clotting time (ROTEM in-tem) s 208 (840ndash810)
249 (720ndash371)
05085
Clot formation time (ROTEM in-
tem) s 835
(370ndash634) 745
(390ndash235) 08988
Alpha angle (ROTEM in-tem) degrees 745 (250ndash820)
750 (500ndash820)
09797
Max clot firmness (ROTEM in-
tem) mm 700
(540ndash810) 740
(610ndash770) 03587
Max clot elasticity (ROTEM in-
tem) - 235
(118ndash424) 287
(156ndash332) 04764
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
61
Maximum lysis (ROTEM in-tem) 850 (000ndash210)
350 (000ndash220)
01327
Table 4 Comparison of results of haemostasis parameters (median values minimumndash
maximum) on the last day in dogs with (grades 1ndash3) and without (grade 0) central venous
catheter-associated thrombus formation as detected by electron microscopy
Parameter Unit
Thrombus grade
(ELMI)
P-value
(Mann Whitney
U test)
0
(n = 8)
123
(n = 19)
Haematocrit 322 (241ndash454)
330 (216ndash510)
05953
Platelet count 103microl 446 (321ndash592)
268 (101ndash675)
00558
Prothrombin time 145 (104ndash264)
120 (561ndash148)
00462
Activated partial thromboplastin
time s 123
(109ndash140) 136
(109ndash188) 00665
Thrombin time s 129 (121ndash178)
141 (650ndash160)
02028
Fibrinogen gl 465 (275ndash866)
347 (189ndash992)
00491
Antithrombin activity 793 (448ndash981)
692 (398ndash889)
04572
D-dimers microgml 018 (000ndash173)
007 (000ndash118)
01920
Thrombin generation assay nmol 501 (000ndash160)
93 (000ndash287)
09562
Clotting time (ROTEM ex-tem) s 360
(270ndash410) 400
(310ndash620) 00238
Clot formation time (ROTEM ex-
tem) s 395
(120ndash740) 650
(240ndash174) 00275
Alpha angle (ROTEM ex-tem) degrees 835 (760ndash880)
790 (580ndash850)
00484
Max clot firmness (ROTEM ex-
tem) mm 780
(700ndash850) 720
(550ndash860) 00586
Max clot elasticity (ROTEM ex-
tem) - 346
(239ndash555) 254
(121ndash637) 00495
Maximum lysis (ROTEM ex-tem) 105 (200ndash190)
110 (000ndash380)
03796
Clotting time (ROTEM in-tem) s 178 (100ndash222)
201 (139ndash554)
00525
Clot formation time (ROTEM in-
tem) s 435
(330ndash111) 790
(350ndash882) 00209
Alpha angle (ROTEM in-tem) degrees 815 (710ndash830)
740 (240ndash830)
00191
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
62
Max clot firmness (ROTEM in-
tem) mm 785
(660ndash810) 690
(270ndash840) 00586
Max clot elasticity (ROTEM in-
tem) - 362
(192ndash434) 218
(370ndash507) 00559
Maximum lysis (ROTEM in-tem) 950 (200ndash190)
800 (100ndash180)
06890
4 Discussion
The main result of our study is that under the study conditions part of the dogs developed
CVC-associated thromboses However only a very low part of these thromboses were
obviously of a clinically significant degree regarding the amount of occlusion of the EJV
and none of the dogs had a symptomatic thrombosis whose definition includes presence
of clinical signs such as localised swelling pain and redness (Boersma et al 2008 Lee
and Kamphuisen 2012) In the present study we particularly refer to the extraluminal
thromboses with regard to the assessment of the antithrombotic management
One major problem of the statistical analysis and result presentation was that the two
methods used to define the occurrence and degree of thrombus formation (ultrasound and
ELMI) revealed discrepant results Therefore we performed a statistical analysis
independently regarding thrombus formation with either of the two methods It is well
known that ELMI which has already been used to detect CVC induced thromboses in
humans (Lucas et al 2017) and experimental animals (Xiang et al 1998 Xiang et al
2001) is a very sensitive diagnostic technique which allows the ex vivo diagnosis of very
small or fibrin rich thrombi that perhaps cannot be detected by ultrasonography or
venography during the in vivo evaluation of patients (Xiang et al 2001) A major reason
for the discrepant results between the two methods used to detect CVC associated
thromboses is that different CVC segments are evaluated Using ultrasonography it was
only possible to evaluate the EJV in the neck area of the patient and thereby only its
proximal third In contrast ELMI assessed mainly the CVC tip and a human study
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
63
revealed that even different positions of the catheter tip are associated with various risks
for development of CVC associated thrombosis (Ballard et al 2016) One additional point
to consider which also may contribute to the discrepancy is that it is possible that thrombi
developed on the CVC surface can be detached during the CVC removal from the vein
and thereby before its analysis by ELMI (Xiang et al 2001)
The contrast venography technique is used as gold standard to confirm thrombosis in
human medicine However this diagnostic technique is normally used only in the case of
a strong clinical suspicion of venous thrombosis that cannot be detected by
ultrasonography (Laurenson et al 2010 Langston and Eatroff 2018) Our study protocol
with privately owned clinic patients did not allow to use this method In a study performed
in 44 human patients requiring a CVC (Koumlksoy et al 1995) a sensitivity of 94 and a
specificity of 96 were determined for the colour Doppler ultrasound diagnosis when it
was compared with the diagnosis performed with the contrast venography technique and
that makes the colour Doppler ultrasound an accurate diagnosis method to detect CVC
related thrombosis even in humans (Koumlksoy et al 1995 van Rooden et al 2003)
The incidences of CVC related thrombosis detected by colour Doppler ultrasound of the
internal jugular vein reported in 3 human studies were 2 30 and 56 (Harter et al
2002 Lordick et al 2003 Wu et al 1999) and although with a wide range the median
value of these rates (30 ) is very well suited to the incidence of 28 (829) which we
calculated for our canine study based on sonographic findings The wide variation of
incidences of CVC related thrombosis in different human studies can in general be
explained by differences regarding the type and entry site of the CVC (jugular femoral
subclavian axillary cephalic vein) the time period with the CVC in place the thrombus
detection technique (venography or ultrasonography comment in the cited 3 studies
consistently the last-mentioned method was used) and criteria for diagnosis of
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
64
thrombosis the duration of follow up diseases and presence of predisposing factors in
these patients (eg previous venous thrombosis history presence of cancer) and finally
prophylactic treatment with anticoagulants (van Rooden et al 2005 Geerts 2014 Liu et
al 2015)
Different pathomechanisms contribute to the development of CVC induced thromboses
During the CVC insertion the puncture of the skin on the CVC insertion site and the
rupture of the blood vessels integrity activate the coagulation cascade and the CVC acts
as a matrix for adherence and accumulation of fibrin and cellular components (Smith et
al 2012) The attachment of proteins and blood cells to the internal andor external wall
of the CVC can lead to a partial or complete occlusion of the catheter lumen and
sometimes to a partial or complete obstruction of the blood flow andor
thromboembolism (Langston and Eatroff 2018) There is a strong interrelationship
between thrombus and biofilm formation (Smith et al 2012) Prevention of thrombus
formation has thereby also relevance regarding the increased risk for microbial
colonisation of the catheter (Boersma et al 2008) leading at least in humans to an
increased number of CVC related systemic infections (Lordick et al 2003)
In this context it has to be considered that CVC produced from polyurethane which are
routinely used in our clinic and thereby in the study are usually associated with a
relatively high thrombosis potential and thereby definitely provide an effective test for
an antithrombotic regimen Silicone catheters have a significantly reduced risk for
thrombotic complications but this advantage has to be purchased by a higher rate of
mechanical failure (eg rupture of the catheter) (Wildgruber et al 2016) Promising
experimental approaches work on surface modifications of the standard catheters For
example one group was able to demonstrate that a coating of the surface of the catheter
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
65
with a zwitterionic polymeric sulfobetaine showed a significant reduction of thrombus
formation in vitro with human blood and in an in vivo canine model (Smith et al 2012)
Due to ethical reasons we did not performed a placebo-controlled study However
interestingly not all human studies report systemic anticoagulation and part of the human
studies was even placebo-controlled (review Lee and Kamphuisen 2012) Obviously
there is no consensus in the available human literature regarding the beneficial use of
antithrombotic prophylaxis to prevent CVC related thrombosis Several studies did not
find a statistically significant difference in the incidence of CVC related thrombosis
between patients receiving the antithrombotic prophylaxis compared with the control
groups without systemic anticoagulation (Verso et al 2005 P = 035 Karthaus et al
2006 P gt 005 Niers et al 2007 P = 049) whereas in other studies a lower incidence
of CVC related thrombosis was found in the group receiving anticoagulants as
prophylaxis compared with the control groups (Abdelfeki et al 2004 150 vs 126
P = 003 De Cicco et al 2009 400 vs 526 P = 005)
Although it is well known that subcutaneously administered UFH has a limited and
variable bioavailability and thereby leads to unpredictable blood activities (Hirsh and
Raschke 2004) the extremely wide range of blood activities after a defined dosage in our
study was relatively unexpected Blood was collected 2 hours after the first UFH
injection and thereby at the time of suspected maximum heparin activity considering the
relatively low UGH dosage (Mischke and Jacobs 2001) The cited study was performed
on healthy experimental dogs Possible additional influencing factors in critical ill
patients include circulatory insufficiency which may reduce and delay absorption from
subcutanenous tissue decreased clearance function of the liver and kidneys as well as
altered blood plasma concentrations of heparin binding plasma proteins such as
fibrinogen and platelet factor 4 and alterations of the heparin binding capacity of
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
66
endothelial cells which altogether are reasons for a great inter- but also intrapatient
variability (Hirsh and Raschke 2004) In individual patients this can lead to heparin
resistance It has also to be considered that antithrombin activities which are necessary
for an adequate heparin effect (Bjoumlrk and Lindahl 1982) were reduced in our patients
Availability of approved blood tests which can predict the risk of development of CVC
associated thrombosis already at the moment of the CVC placement are important
(Boersma et al 2016) One of the most interesting results was that dogs which developed
sonographically detectable thrombi had nearly twofold higher fibrinogen levels Well in
agreement with our results one study performed in anticoagulated human patients
demonstrated that high fibrinogen levels were associated with an increased risk of
perioperative venous thromboembolism (P = 0004) (Kato et al 2016) The cited study
does not exclusively refer to CVC associated thrombosis but 76 (1621) of the venous
thromboses were suggested to be associated with CVCs Fibrinogen measurement a test
which is widely available is therefore a useful and practicable indicator of an increased
risk for CVC induced thromboses and may advice the clinician to treat these patients
especially carefully (eg with low molecular weight heparin) In the study performed by
Boersma et al (2016) in 168 human patients without indication of anticoagulation
prophylaxis elevated leukocyte counts high levels of plasminogen activator inhibitor
and of coagulation factor VIII were associated with an increased incidence of
symptomatic CVC related thrombosis diagnosed by ultrasonography A limitation of that
study is that all measurements were performed on blood samples taken after CVC
placement Interestingly in our study ROTEM results were partly associated with
thrombus formation which reflects the well-known fact that results of viscoelastic
measurements are a good indicator of hypercoagulability and thromboembolic events
(Harahsheh and Ho 2018) It is of further interest and possibly unexpeted that on the final
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
67
day of examination dogs with ELMI signs of thromboses had partly lower coagulation
activities than dogs without thromboses Due to the low number of dogs without
detectable thrombi using ELMI this implausible result has to be interpreted with caution
One possible explanation could be that temporary hypercoagulability in these dogs may
have finally resulted in consumption of haemostasis substrates
Limitations of the present study were as a consequence of the study design based on
clinical patients the relatively low total number of patients inclusion of different UFH
treatment regimens and the lack of a control group
5 Conclusions
The results of the present study indicate that the used UFH regimen was not completely
effective to prevent CVC-associated thromboses which were detectable in 28 [829]
of the patients by ultrasound However severe thromboses (high grades) are relatively
rare in intensive care canine patients receiving a routine anticoagulant treatment Because
heparin plasma activities were not different in dogs with or without CVC-associated
thrombus formation other local and systemic factors must play a significant role as well
A high fibrinogen concentration and low maximum lysis (ROTEM ex-tem) may be
useful indicators for an increased thrombotic risk in individual patients which may
require special anticoagulatory treatment
6 Acknowledgments
Jose Mauricio Pereira received a scholarship from the Universidad Nacional de Costa
Rica (reference code JB-C-1107-2016)
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
68
7 References
1 Abdelkefi A Ben Othman T Kammoun L Chelli M Ben Romdhane N Kriaa A Ladeb S
Torjman L Lakhal A Achour W Ben Hassen A Hsaiumlri M Ladeb F Ben Abdeladhim A
2004 Prevention of central venous line-related thrombosis by continuous infusion of low-dose
unfractionated heparin in patients with haemato-oncological disease A randomized controlled trial
Thromb Haemost 92 654ndash661
2 Adamantos S Brodbelt D Moores A L 2010 Prospective evaluation of complications associated
with jugular venous catheter use in a veterinary hospital J Small Anim Pract 51 254ndash257
3 Bagge A Schoumltt U Kander T 2016 Effects of naturopathic medicines on Multiplate and ROTEM
a prospective experimental pilot study in healthy volunteers BMC Complement Altern Med 16 64
4 Ballard DH Samra NS Gifford KM Roller R Wolfe BM Owings JT 2016 Distance of the
internal central venous catheter tip from the right atrium is positively correlated with central venous
thrombosis Emerg Radiol 23 269ndash273
5 Boersma RS Jie KS Verbon A van Pampus EC Schouten HC 2008 Thrombotic and
infectious complications of central venous catheters in patients with hematological malignancies Ann
Oncol 19 433ndash442
6 Boersma R S Hamulyak K van Oerle R Tuinenburg A Ten Cate-Hoek AJ Schouten HC
2016 Biomarkers for prediction of central venous catheter related-thrombosis in patients with
hematological malignancies Clin Appl Thromb Hemost 22 779ndash784
7 Bjoumlrk I Lindahl U 1982 Mechanism of the anticoagulant action of heparin Mol Cell Biochem
48 161ndash182
8 Claude A Riedesel D Riedesel E 2010 Electrocardiography-guided and retrospective analysis of
central venous catheter placement in the dog Vet Anaesth Analg 37 97ndash105
9 Comerlato PH Rebelatto TF Santiago de Almeida FA Klein LB Boniatti M M Schaan
DB Rados VD 2017 Complications of central venous catheter insertion in a teaching hospital
Rev Assoc Med Bras 63 613ndash620
10 Crochemore T Piza FMT Rodriacutegues RDR Guerra JCC Ferraz LJR Correcirca TD 2017
A new era of thromboelastometry Einstein (Sao Paulo) 15 380ndash385
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
69
11 De Cicco M Matovic M Balestreri L Steffan A Pacenzia R Malafronte M Fantin D
Bertuzzi CA Fabiani F Morassut S Bidoli E Veronesi A 2009 Early and short‐term
acenocumarine or dalteparin for the prevention of central vein catheter‐related thrombosis in cancer
patients a randomized controlled study based on serial venographies Ann Oncol 20 1936ndash1942
12 Evans NS Ratchford EV 2018 Catheter related-venous thrombosis Vasc Med 23 411ndash413
13 Geerts W 2014 Central venous catheterndashrelated thrombosis Hematology Am Soc Hematol Educ
Program 2014 306ndash311
14 Harahsheh Y Ho KM 2018 Use of viscoelastic tests to predict clinical thromboembolic events A
systematic review and meta‐analysis Eur J Haematol 100 113ndash123
15 Harter C Salwender HJ Bach A Egerer G Goldschmidt H Ho AD 2002 Catheter-related
infection and thrombosis of the internal jugular vein in hematologic-oncologic patients undergoing
chemotherapy a prospective comparison of silver-coated and uncoated catheters Cancer 94 245ndash251
16 Hirsh J Raschke R 2004 Heparin and low-molecular-weight heparin The Seventh ACCP
Conference on Antithrombotic and Thrombolytic Therapy Chest 126 (Suppl) 188S-203S
17 Hundley D Brooks A Thomovsky E Jhonson P Freeman L Schafbuch R Heng HG Moore
G 2018 Comparison of ultrasound-guided and landmark-based techniques for central venous
catheterization via the external jugular vein in healthy anesthetized dogs Am J Vet Res 79 628ndash
636
18 Kato F Takeuchi H Matsuda S Kawakubo H Omori T Kitagawa Y 2016 Incidence of and
risk factors for venous thromboembolism during surgical treatment for esophageal cancer a single-
institution study Surg Today 46 445ndash452
19 Karthaus M Kretzschmar A Kroning H Biakhov M Irwin D Marschner N Slabber C
Fountzilas G Garin A Abecasis NG Baronius W Steger GG Sudhoff T Giorgetti C
Reichardt P 2006 Dalteparin for prevention of catheter‐related complications in cancer patients with
central venous catheters final results of a double‐blind placebo‐controlled phase III trial Ann Oncol
17 289ndash296
20 Koumlksoy C Kuzu A Kutlay J Erden I Ozcan H Ergicircn K 1995 The diagnostic value of colour
Doppler ultrasound in central venous catheter related thrombosis Clin Radiol 50 687-689
21 Kucher N 2011 Clinical practice Deep-vein thrombosis of the upper extremities N Engl J Med
364 861ndash869
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
70
22 Langston CE Eatroff AE 2018 Hemodialysis catheter-associated fibrin sheath in a dog J Vet
Emerg Crit Care 28 366ndash371
23 Laurenson MP Hopper K Herrera MA Johnson EG 2010 Concurrent diseases and conditions
in dogs with splenic vein thrombosis J Vet Intern Med 24 1298ndash1304
24 Lee AYY Kamphuisen PW 2012 Epidemiology and prevention of catheter‐related thrombosis in
patients with cancer J Thromb Haemost 10 1491ndash1499
25 Liu G Fu ZQ Zhu P Li SJ 2015 Central venous catheter-related thrombosis in senile male
patients New risk factors and predictors J Huazhong Univ Sci Technolog Med Sci 35 445ndash449
26 Lordick F Hentrich M Decker T Hennig M Pohlmann H Hartenstein R Peschel C 2003
Ultrasound screening for internal jugular vein thrombosis aids the detection of central venous catheter-
related infections in patients with haemato-oncological diseases a prospective observational study Br
J Haematol 120 1073ndash1078
27 Lucas TC Silva EED Souza DOD Santos ARD Lara MO 2017 Microstructural
evaluation by confocal and electron microscopy in thrombi developed in central venous catheters Rev
Esc Enferm USP 51 1ndash9
28 Malick L Wilson R Stetson D 1976 Modified thiocarbo- hydrazide procedure for scanning
electron microscopy routine use for normal pathological and experimental tissue Stain Technol 50
265ndash269
29 Mischke R Jacobs C 2001 The monitoring of heparin administration by screening tests in
experimental dogs Res Vet Sci 70 101ndash108
30 Niers TM Di Nisio M Klerk CP Baarslag HJ Buller HR Biemond BJ 2007 Prevention
of catheter‐related venous thrombosis with nadroparin in patients receiving chemotherapy for
hematologic malignancies a randomized placebo‐controlled study J Thromb Haemost 5 1878ndash
1882
31 Reminga C Silverstein D Drobatz K Clarke D 2018 Evaluation of the placement and
maintenance of central venous jugular catheters in critically ill dogs and cats J Vet Emerg Crit Care
28 232ndash243
32 Smith RS Zhang Z Bouchard M Li J Lapp HS Brotske GR Lucchino DL Weaver D
Roth LA Coury A Biggerstaff J Sukavaneshvar S Langer R Loose C 2012 Vascular
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
71
catheters with a nonleaching poly-sulfobetaine surface modification reduce thrombus formation and
microbial attachment Sci Transl Med 4 153ra132 doi101126scitranslmed3004120
33 Toskala E Nuutinen J Rautiainen M 1995 Scanning electron microscopy findings on human
respiratory cilia in chronic sinusitis and in recurrent respiratory infections J Laryngol Otol 109 509ndash
514
34 Van Rooden CJ Tesselaar ME Osanto S Rosendaal FR Huisman MV 2005 Deep vein
thrombosis associated with central venous catheters - a review J Thromb Haemost 3 2409ndash2419
35 Verso M Agnelli G Bertoglio S Di Somma FC Paoletti F Ageno W Bazzan M Parise P
Quintavalle R Naglieri E Santoro A Imberti D Soraru M Mosca S 2005 Enoxaparin for the
prevention of venous thromboembolism associated with central vein catheter a double‐blind placebo‐
controlled randomized study in cancer patients J Clin Oncol 23 4057ndash4062
36 Villalta-Garciacutea P Loacutepez-Herranz M Mazo-Pascual S Honrubia-Fernaacutendez T Jaacutentildeez-Escalada
L Fernaacutendez-Peacuterez C 2015 Reliability of blood test results in samples obtained using a 2-mL discard
volume from the proximal lumen of a triple-lumen central venous catheter in the critically ill patient
Nurs Crit Care 22 298ndash304
37 Vose J Odunayo A Price JM Daves M Schildt JC Tolbert MK 2019 Comparison of
heparinized saline and 09 sodium chloride for maintaining central venous catheter patency in healthy
dogs PeerJ 7 e7072 httpsdoiorg107717peerj7072
38 Wildgruber M Lueg C Borgmeyer S Karimov I Braun U Kiechle M Meier R Koehler M
Ettl J Berger H 2016 Polyurethane versus silicone catheters for central venous port devices
implanted at the forearm Eur J Cancer 59 113ndash124 doi101016jejca201602011
39 Wu X Studer W Skarvan K Seeberger MD 1999 High incidence of intravenous thrombi after
short-term central venous catheterization of the internal jugular vein J Clin Anesth 11 482ndash485
40 Xiang DZ Verbeken EK Van Lommel AT Stas M De Wever I 1998 Composition and
formation of the sleeve enveloping a central venous catheter J Vasc Surg 28 260ndash271
41 Xiang DZ Verbeken EK Van Lommel AT Stas M De Wever I 2001 Sleeve-related
thrombosis a new form of catheter-related thrombosis Thromb Res 104 7ndash14
Chapter 4 General discussion
72
Chapter 4 General discussion
Incidence of CVC related thrombosis
In our study the incidence of CVC related thrombosis detected by ultrasound in critical
care canine patients was 28 To the best of the authoracutes knowledge similar studies
performed in dogs are rare in the available veterinary literature In one study performed
in 81 dogs and 12 cats (Adamantos et al 2010) an incidence of 2 was found for vein
thrombosis related to CVC This low incidence can be due to a lacking of systematic
ultrasound evaluations of the jugular vein containing the CVC In the mentioned study in
dogs and cats the vein thrombosis was confirmed by palpation or ultrasound presumably
after the presentation of clinical symptoms however in many cases as in our study
thrombus into the jugular vein can be developed without manifestation of clinical
symptoms what makes a routinely ultrasound evaluation of the vein containing the CVC
very important Our results are well aligned with the findings in humans in which a
median incidence of 30 was found for thrombosis related to CVC based on 25 studies
performed in patients requiring a CVC (review article van Rooden et al 2005) In the
mentioned 25 human studies the incidence of CVC related thrombosis varied within a
wide range (2ndash67 ) according to factors related to (1) the CVC (eg material type
entry site [jugular femoral subclavian axillary cephalic vein] time period in place (2)
diagnostic thrombus detection technique (ultrasonography or venography) and criteria
(3) patient collective (diseases and presence of predisposing factors [eg previous venous
thrombosis history presence of cancer] and (4) prophylactic antithrombotic treatment
(Geerts 2014 Liu et al 2015 van Rooden et al 2005)
The incidence of CVC related thrombosis detected by colour Doppler ultrasound in our
study fits also with the median incidence (30 ) found in three studies performed in
humans in which colour Doppler ultrasound evaluations of the jugular vein containing a
Chapter 4 General discussion
73
CVC were performed (Harter et al 2002 Lordick et al 2003 Wu et al 1999) In these
three studies the individual incidence of CVC related thrombosis was 2 (Harter et al
2002) 30 (Lordick et al 2003) and 56 (Wu et al 1999) The high thrombus
incidence reported in the study performed by Wu et al (1999) could be due to the criteria
for diagnosis of thrombosis because in that study both fibrin sleeves and compact
thrombi were considered as cases of thrombosis related to CVC The incidence for
compact thrombi was 25 which is more similar to our results In the study performed
by Harter et al (2002) the CVC related thrombosis incidence (2 ) was very low
compared with the incidence found in our study In that study the jugular veins were
examined just before or less than 24 hours after CVC removal (median period of CVC
use = 1025 days) In our study the ultrasound evaluations were performed before the
CVC placement (day 0) and then on days 1 3 5 etc until the CVC removal (median
period of CVC use = 7 days median interval until thrombus detection = 7 days) In our
study we detected thrombus formation as early as on the 3rd day so it could be possible
that in the cited study possibly only temporarily existing thrombi could have been missed
by ultrasonography in the final stage and thereby for the incidence calculation However
this scenario is not very likely because results of the present study show that
sonographically detectable thrombi during the observation period had in all cases their
maximum grade on the final day
In the study performed by Lordick et al (2003) the CVC related thrombosis incidence
determined for 43 human patients was very similar to our study and in both studies only
compact thrombi detectable by ultrasound were considered for assessment of the
incidence In the mentioned study all the human patients were screened for the presence
of thrombosis every 4 days after CVC insertion with real time B-mode ultrasound
(Lordick et al 2003)
Chapter 4 General discussion
74
In our study 70 of CVCs analysed by ELMI showed thrombi of grade 1ndash3 on the
external CVC wall To the best of the authoracutes knowledge studies about incidence of CVC
related thrombosis on the outside surface of the CVC detected by ELMI cannot be found
in the human and small animal literature In one study performed in humans (Lucas et al
2017) thrombus formation was analysed by ELMI in 78 central venous catheters That
study focussed on microstructural assessment of thrombus formation inside the distal
openings of the CVC which were found in 85 of the evaluated CVCs The percentage
of occurrence of thrombosis in the mentioned study in humans was nearly similar to the
incidence of intraluminal thrombus formation as detected in the presented study by ELMI
(74 ) In general intraluminal thrombi were not specially considered in the present
study because they mainly express the efficacy of the flushing technique rather than the
effectivity of the antithrombotic treatment In one study performed in rats the
microstructure of thrombi developed in the jugular vein containing the CVC was also
evaluated by ELMI (Xiang et al 1998) In the mentioned study pericatheter thrombosis
was detected on the proximal section of the CVC in 100 of rats (n = 12) after 1 and 3
days of catheterisation The analyses of the CVCs placed in rats were performed with the
catheter inside the vein in contrast to our study where the CVC was removed from the
jugular vein for the respective analysis Xiang et al (2001) hypothesised in one study
performed in rabbits and rats in which CVCs were placed in the jugular vein that fibrin
rich thrombi associated to the CVC can be detached during the catheter removal and this
could explain the difference between the thrombosis incidence assessed in our study and
that of the study performed by Xiang et al (1998)
In our study the incidence of CVC related thrombosis detected by ultrasonography in the
lumen of the EJV did not correlate with the incidence of CVC related thrombosis detected
by ELMI on the external wall of the CVC This was mainly due to the fact that the CVC
Chapter 4 General discussion
75
segment evaluated with both methods was different Using ultrasonography it was only
possible to evaluate the EJV in the neck area of the patient and thereby only the proximal
third of the CVC In contrast with the ELMI analysis only the distal section of the CVC
(last third) was evaluated because macroscopically detected thrombi mainly developed
near the CVC tip which was routinely positioned at the junction of the cranial cava vein
with the right atrium In a retrospective review of medical records of 169 human patients
requiring a CVC (Ballard et al 2016) the position of the CVC tip was determined by
radiologic evaluation and it was determined that the catheter tip position can be a
predisposing factor for central venous thrombosis development Thereby it could be of
interest to evaluate in further studies in dogs if a high incidence of thrombosis detected
by ELMI as was shown in our study could be related to the CVC tip position
In our study polyurethane catheters were tested which are widely used and are the
standard CVCs used in the Small Animal Clinic University of Veterinary Medicine
Hannover In vivo and in vitro studies have assessed the thrombogenicity of different
catheter materials (Borow and Crowley 1985 Smith et al 2012) In general terms
polyurethane catheters (central or peripheral) had shown a higher thrombogenicity
compared with coated (eg with hydromer) and silicone catheters Due to this property
of polyurethane catheters they can be supposed as a good test system for antithrombotic
regimens as evaluated in our study On the other hand despite silicone catheters have been
associated with a lower thrombogenicity their mechanical flexibility could be a
disadvantage in small clinical practice due to the increased risk of rupture detected even
in humans (Wildgruber et al 2016)
Chapter 4 General discussion
76
Heparin thromboprophylaxis for CVC related thrombosis
The occurence of CVC related thrombosis detected by colour Doppler ultrasound and
ELMI in our study demonstrates that the UFH in the given dosage was not completely
effective to prevent CVC related thrombosis in canine intensive care patients In human
medicine LMWH is commonly used in prophylactic anticoagulation regimens however
studies performed in human patients requiring a CVC and receiving a prophylactic
anticoagulation (with LMWH or UFH) did not achieve consensus regarding the efficacy
of prophylactic heparin to prevent CVC related thrombosis (Abdelfeki et al 2004
Lordick et al 2003 Wu et al 1999) To the best of the authoracutes knowledge studies
performed in dogs evaluating systemic prophylactic anticoagulation to prevent CVC
related thrombosis cannot be found However according to the evidence found in the
human literature and the results of our study further investigations using comparative
groups with different drugs dosages andor administration routes are required to establish
an effective antithrombotic treatment to prevent CVC related thrombosis
The plasma heparin activities measured in canine patients in our study varied significantly
after a defined subcutaneous dosage The limited and variable bioavailability of SC
administered UFH is well documented in humans (Hirsh and Raschke 2004)
Considerable variations of the circulatory status of the liver- and kidney function and
concentration of heparin-binding proteins in intensive care patients obviously amplify
this phenomenon leading to an extremely high inter-individual and intra-individual
variation of the heparin blood levels in our patients A different application route (IV)
may have reduced the influence of part of these factors and therefore could have led to
more predictable UFH blood levels However continuous infusion requires more
technical equipment and is also associated with a wide range of heparin activities in
canine patients (Scott et al 2009)
Chapter 4 General discussion
77
Prediction of CVC related thrombosis
Of 7 haemostasis tests (including ROTEM with 12 different parameters [6 for each
reagent ex-tem and in-tem]) evaluated as possible predictors of CVC related thrombosis
in our study initial values of high fibrinogen and low maximum lysis (ROTEM ex-tem)
were associated with sonographically detectable thrombus formation Some studies in
humans have established the association of laboratory biomarkers as predictors of risk
thrombosis (Boersma et al 2016 Cheng et al 2013 Kato et al 2016 Liu et al 2015)
One study performed in anticoagulated human patients showed ndash well in agreement with
our results ndash that high fibrinogen levels were associated with an increased risk of
perioperative venous thromboembolism (P = 0004) (Kato et al 2016) The cited study
does not exclusively refer to CVC associated thrombosis but 762 (1621) of the
venous thromboses were suggested to be associated with CVCs Fibrinogen concentration
measurements are normally available in the human and veterinary practice and the
association of this parameter with a high risk of development of CVC related thrombosis
can be useful to choose special antithrombotic treatment in individual critical care patients
to prevent such severe complications
Thrombin generation assay (TGA) abnormalities such as higher velocity and endogenous
thrombin potential (ETP) values measured initially ie before any anticoagulation
therapy have been associated with a higher thromboembolism risk in general (ie without
specific association with CVCs) in one study performed on 105 human patients (Espitia
et al 2017) In contrast in our study the initial TGA assay results did not show an
association with formation of CVC related thrombosis
The ROTEM parameters evaluated in the present study were partially associated with
formation of CVC thrombosis detected by colour Doppler ultrasound This finding
confirms the results obtained in humans in which ROTEM measurements are regarded as
Chapter 4 General discussion
78
a valuable indicator of hypercoagulability (Harahsheh and Ho 2018 Tuumlrk et al 2018)
However in our study individual ROTEM parameters showed partly lower coagulation
activities in dogs with CVC related thrombosis detected by ELMI at the final day of
evaluation Because the number of patients without signs of thrombosis detected by ELMI
was low compared with those who showed signs these implausible results should be
interpreted carefully The usefulness of ROTEM analysis which has also many other
indications demonstrates that is was worth to perform a detailed methodological approval
of the device including the establishment of reference values in the preliminary study
Conclusion
In conclusion the prethrombotic or thrombotic changes detected in our study did not
correlate between ultrasonography and ELMI however both methods show accurately
signs of CVC related thrombosis The UFH in the given dosage was not completely
effective to prevent CVC induced thrombus formation in canine intensive care patients
Plasma heparin activities varied significantly after a defined subcutaneous dosage
However plasma heparin activities were not different between dogs with and without
detectable CVC associated thromboses Initial high fibrinogen values and low maximum
lysis (ROTEM ex-tem) values were associated with sonographically detectable thrombi
and may be useful as indicators for an increased thrombotic risk
Finally our study in canine intensive care patients is to the best of the authoracutes
knowledge the first study in the available veterinary literature performed to evaluate the
efficacy of an antithrombotic treatment with UFH to prevent CVC related thrombosis
Chapter 5 Summary
79
Chapter 5 Summary
Central venous catheter induced thrombosis in dogs ndash occurrence under an
antithrombotic treatment regimen and possible indicators
Joseacute Mauricio Pereira Mora
Central venous catheters (CVC) are of importance for intensive care management of
canine patients Thrombosis of the jugular vein is a severe complication related to the
CVC use However only little is known about the incidence of CVC-associated
thromboses in dogs and the necessity and efficacy of prophylactic antithrombotic
regimens
Therefore the aims of the present study were (1) to determine the incidence of catheter-
induced thrombosis in canine intensive care patients (2) to evaluate the efficacy of a
prophylactic heparin regimen routinely used in the Small Animal Clinic of the University
of Veterinary Medicine Hannover and (3) to assess whether selected haemostasis test
results are predictive for an increased risk for thrombosis formation
The thesis also includes a preliminary methodological study on the use of rotational
elastometry in dogs using the ROTEM delta device In this preliminary study reference
intervals were determined based on 125 healthy dogs for different clot activator reagents
and for measurements without activator Coefficients of variation (CVs) were calculated
based on results of fourfold measurements of 3 normal and 3 abnormal blood samples in
order to assess precision of the method Influence of age groups sex neuter status and
size were also assessed The median CVs for most of the parameters evaluated in the
precision analysis were less than 10 and this confirmed that the ROTEM delta analyser
delivers repeatable results Regarding the influence of age sex neuter status and animal
Chapter 5 Summary
80
size only neuter status in female dogs appeared to be a relevant influencing factor on
most of important ROTEM parameters
The main study of the thesis comprises 29 patients hospitalised at the Small Animal Clinic
requiring a CVC for medical reasons These patients received a prophylactic
anticoagulation treatment with unfractionated heparin (UFH 150 IU [surgical dogs
received 75 IU]kg BW TID subcutaneously) A colour Doppler ultrasound of the
respective external jugular vein (EJV) in order to detect thrombus formation in the lumen
of the EJV and blood collection for haemostasis tests were performed directly before the
installation procedure (day 0) and on days 1 3 5 7 and so on after the CVC insertion
Haemostasis tests included prothrombin time activated partial thromboplastin time
thrombin time antithrombin activity D-dimer concentration thrombin generation and
rotational elastometry as well as the heparin activity using a chromogenic anti-factor Xa
test Finally when the CVC was removed electron microscopy (ELMI) of sections of the
CVC with macroscopically visible thrombi in the last third (5ndash10 cm) of the CVC was
performed 3 different grades were defined for CVC-associated (extraluminal) thrombi
detected by either ultrasound or ELMI respectively
829 patients evaluated by colour Doppler ultrasound during the study developed thrombi
into the lumen of EJV (4 x grade 1 3 x grade 2 and 1 x grade 3) 1927 CVCs assessed
using ELMI showed detectable thrombi on the external CVC surface (7 x grade 1 4 x
grade 2 and 8 x grade 3) There was no significant correlation between thrombus
formation detected by ultrasound or ELMI Thus statistical group comparisons of
patients with or without CVC-associated thrombus formation was performed
independently for both diagnostic procedures The plasmatic heparin activities showed
great variability but no significant differences were found between dogs with or without
CVC-associated thrombus formation regardless whether this was based on sonographic
Chapter 5 Summary
81
or ELMI findings From the initially performed haemostasis tests high fibrinogen
concentrations and low maximum lysis values as measured by the ROTEM delta device
(using the ex-tem reagent) were associated with sonographically detectable thrombi
In conclusion the preliminary methodological study on the ROTEM delta in dogs shows
that the method delivers repeatable results in canine citrated whole blood and the
reference values obtained should offer a reliable basis for the interpretation of test results
of this device in dogs with suspected haemostasis disorders The results of the main study
indicate that CVC-associated thromboses occur (eg 28 [829] of the patients as
detected by ultrasound) although severe thromboses (high grades) are relatively rare in
intensive care canine patients receiving a routine anticoagulant treatment Thereby
prophylactic antithrombotic protocol (unfractionated heparin [UFH]) in the given dosage
was not completely effective to prevent CVC induced thrombus formation in canine
intensive care patients A high fibrinogen concentration and low maximum lysis
(ROTEM ex-tem) may be useful indicators for an increased thrombotic risk in individual
patients which may require special anticoagulatory treatment Further studies are
required to evaluate whether different thromboprophylactic treatment regimens eg with
low molecular weight heparin are more effective in preventing CVC-associated
thromboses in dogs
Chapter 6 Zusammenfassung
82
Chapter 6 Zusammenfassung
Zentralvenenkatheter-induzierte Thrombose bei Hunden - Auftreten unter einem
antithrombotischen Behandlungsschema und moumlgliche Indikatoren
Joseacute Mauricio Pereira Mora
Zentralvenenkatheter (ZVK) sind fuumlr das Intensivmanagement von Hunden von
Bedeutung Die Entstehung einer Thrombose der Vena jugularis ist eine schwerwiegende
Komplikation im Zusammenhang mit der Verwendung eines ZVKs Uumlber die Inzidenz
von ZVK-assoziierten Thrombosen bei Hunden und die Notwendigkeit und Wirksamkeit
prophylaktischer antithrombotischer Protokolle liegen jedoch nur wenige fundierte
Angaben in der Literatur vor
Daher waren die Ziele der vorliegenden Studie (1) die Inzidenz der Katheter-induzierten
Thrombose bei caninen Intensivpatienten zu bestimmen (2) die Wirksamkeit eines
prophylaktischen Heparin-Regimes zu bewerten das routinemaumlszligig in der Klinik fuumlr
Kleintiere der Stiftung Tieraumlrztliche Hochschule Hannover angewendet wird und (3) um
zu beurteilen ob ausgewaumlhlte Haumlmostasetestergebnisse ein erhoumlhtes Risiko fuumlr die
Thrombosebildung vorhersagen koumlnnen
Die PhD-Arbeit enthaumllt zudem eine vorbereitende methodische Studie zur Verwendung
der Rotationselastometrie bei Hunden mit Hilfe des ROTEM delta-Geraumltes In dieser
Vorstudie wurden Referenzintervalle basierend auf 125 gesunden Hunden fuumlr
verschiedene Gerinnungsaktivatorreagenzien und fuumlr Messungen ohne Aktivator
bestimmt Um die Genauigkeit der Methode einzustufen wurden Variationskoeffizienten
(VKs) basierend auf den Ergebnissen von Vierfachmessungen von drei normalen und drei
abnormalen Blutproben berechnet Der Einfluss von Alter Geschlecht Kastration und
Groumlszlige wurde ebenfalls getestet Die Medianwerte der VKs fuumlr die meisten in der
Chapter 6 Zusammenfassung
83
Praumlzisionsanalyse bewerteten Parameter betrugen weniger als 10 und dies bestaumltigte
dass das ROTEM delta-Analysegeraumlt wiederholbare Ergebnisse liefert In Bezug auf den
Einfluss von Alter Geschlecht Kastration und Tiergroumlszlige erwies sich nur eine Kastration
bei Huumlndinnen als relevanter Einflussfaktor fuumlr die meisten wichtigen ROTEM-
Parameter
Die Hauptstudie der Arbeit umfasst 29 Patienten welche zum Zeitpunkt der
Untersuchung stationaumlr in der Kleintierklinik aufgenommen worden waren und aus
medizinischer Indikation einen ZVK benoumltigten Diese Patienten erhielten eine
prophylaktische Antikoagulation mit unfraktioniertem Heparin (UFH 150 IE
[chirurgische Hunde erhielten 75 IE]kg KG 3 x taumlglich subkutan) Bildgebende
Untersuchungen mittels Farbdoppler-Ultraschall der jeweiligen Vena jugularis externa
(VJE) zum Nachweis der Thrombusbildung im Lumen der VJE und Blutentnahmen fuumlr
Haumlmostasetests wurden jeweils direkt vor dem Installationsvorgang (Tag 0) sowie an den
Tagen 1 3 5 und 7 nach Legen des ZVKs durchgefuumlhrt Haumlmostasetests umfassten die
Prothrombinzeit die aktivierte partielle Thromboplastinzeit die Thrombinzeit die
Antithrombinaktivitaumlt die D-Dimer-Konzentration die Thrombingenerierung und die
Rotationselastometrie sowie die Heparinaktivitaumlt unter Verwendung eines chromogenen
Anti-Faktor-Xa-Tests Schlieszliglich wurde nach Entfernung des ZVKs eine
Elektronenmikroskopie (ELMI) von Abschnitten aus dem letzten Drittel (5ndash10 cm) des
ZVKs mit makroskopisch sichtbaren Thromben durchgefuumlhrt Fuumlr ZVK-assoziierte
(extraluminale) Thromben die entweder durch Ultraschall oder ELMI nachgewiesen
wurden wurden drei verschiedene Grade definiert
829 Patienten die waumlhrend der Studie mittels Farbdoppler-Ultraschall untersucht
wurden entwickelten Thromben im Lumen der VJE (4 x Grad 1 3 x Grad 2 und 1 x Grad
3) 1927 ZVKs die unter Verwendung von ELMI bewertet wurden zeigten
Chapter 6 Zusammenfassung
84
nachweisbare Thromben auf der aumluszligeren ZVK-Oberflaumlche (7 x Grad 1 4 x Grad 2 und 8
x Grad 3) Es gab keine signifikante Korrelation zwischen der durch Ultraschall oder
ELMI festgestellten Thrombusbildung Daher wurden statistische Gruppenvergleiche
von Patienten mit oder ohne ZVK-assoziierter Thrombusbildung fuumlr beide diagnostische
Verfahren unabhaumlngig voneinander durchgefuumlhrt Die plasmatischen Heparinaktivitaumlten
zeigten eine groszlige Variabilitaumlt es wurden jedoch keine signifikanten Unterschiede
zwischen Hunden mit oder ohne ZVK-assoziierter Thrombusbildung gefunden
unabhaumlngig davon ob diese auf sonographischen oder ELMI-Befunden beruhte Von den
initial durchgefuumlhrten Haumlmostasetests waren hohe Fibrinogenkonzentrationen und
niedrige maximale Lyse-Werte der Messungen mit ROTEM delta (ex-tem Reagenz) mit
sonographisch nachweisbaren Thromben assoziiert
Zusammenfassend zeigt die vorbereitende methodische Studie zum ROTEM delta bei
Hunden dass die Methode wiederholbare Ergebnisse in mit Zitrat
antikoaguliertemVollblut liefert Die erarbeiteten Referenzwerte sollten eine zuverlaumlssige
Grundlage fuumlr die Interpretation der Testergebnisse dieses Geraumlts bei Hunden mit
Verdacht auf Haumlmostasestoumlrungen bieten Die Ergebnisse der Hauptstudie zeigen dass
ZVK-assoziierte Thrombosen auftreten (z B 28 [829] der Patienten bezogen auf den
Ultraschall-gestuumltzten Nachweis) obwohl schwere Thrombosen (hohe Grade) bei
intensivmedizinisch versorgten Hundepatienten die eine Routineantikoagulation
erhalten relativ selten sind Somit war das prophylaktische antithrombotische Protokoll
(unfraktioniertes Heparin [UFH]) in der angewendeten Dosierung nicht vollstaumlndig
wirksam um die ZVK-induzierte Thrombusbildung bei caninen Intensivpatienten zu
verhindern Eine hohe Fibrinogenkonzentration und eine niedrige maximale Lyse
(ROTEM ex-tem) koumlnnen als Indikatoren eines erhoumlhten Thromboserisikos bei einzelnen
Patienten nuumltzlich sein fuumlr die moumlglicherweise eine spezielle gerinnungshemmende
Chapter 6 Zusammenfassung
85
Behandlung notwendig ist Weitere Studien sind erforderlich um zu untersuchen ob
verschiedene thromboprophylaktische Behandlungsschemata z B mit
niedermolekularem Heparin bezuumlglich der Verhinderung von ZVK-assoziierten
Thrombosen bei Hunden wirksamer sind
Chapter 7 References
86
Chapter 7 References
1 Abdelkefi A Ben Othman T Kammoun L Chelli M Ben Romdhane N Kriaa A Ladeb
S Torjman L Lakhal A Achour W Ben Hassen A Hsaiumlri M Ladeb F Ben Abdeladhim
A 2004 Prevention of central venous line-related thrombosis by continuous infusion of low-dose
unfractionated heparin in patients with haemato-oncological disease A randomized controlled
trial Thromb Haemost 92 654ndash661
2 Adamantos S Brodbelt D Moores A L 2010 Prospective evaluation of complications
associated with jugular venous catheter use in a veterinary hospital J Small Anim Pract 51 254ndash
257
3 Ballard D H Samra N S Gifford K M Roller R Wolfe B M Owings J T 2016
Distance of the internal central venous catheter tip from the right atrium is positively correlated
with central venous thrombosis Emerg Radiol 23 269ndash273
4 Baumann-Kreuziger L Onwuemene O Kolesar E Crowther M Lim W 2015 Systematic
review of anticoagulant treatment of catheter-related thrombosis Thromb Res 136 1103ndash1109
5 Bliss SP Bliss SK Harvey HJ 2002 Use of recombinant tissue‐plasminogen activator in a
dog with chylothorax secondary to catheter‐associated thrombosis of the cranial vena cava J Am
Anim Hosp Assoc 38 431ndash435
6 Boersma RS Hamulyak K van Oerle R Tuinenburg A Ten Cate-Hoek AJ Schouten
HC 2016 Biomarkers for prediction of central venous catheter related-thrombosis in patients
with hematological malignancies Clin Appl Thromb Hemost 22 779ndash784
7 Boersma RS Jie KS Verbon A van Pampus EC Schouten HC 2008 Thrombotic and
infectious complications of central venous catheters in patients with hematological
malignancies Ann Oncol 19 433ndash442
8 Bonnet F Loriferne JF Texier JP Texier M Salvat A Vasile N 1989 Evaluation of
Doppler examination for diagnosis of catheter-related deep vein thrombosis Intensive Care Med
15 238ndash240
9 Borow M Crowley J G 1985 Evaluation of central venous catheter thrombogenicity Acta
Anaesthesiol Scand Suppl 81 59ndash64
10 Cheng Y Cui T Fu P Liu F Zhou L 2013 Dyslipidemia is associated with tunneled-cuffed
catheter-related central venous thrombosis in hemodialysis patients a retrospective multicenter
Chapter 7 References
87
study Artif Organs 37 155ndash161
11 Comerlato PH Rebelatto TF Santiago de Almeida FA Klein LB Boniatti MM
Schaan BD Rados DV 2017 Complications of central venous catheter insertion in a teaching
hospital Rev Assoc Med Bras 63 613ndash620
12 De Cicco M Matovic M Balestreri L Steffan A Pacenzia R Malafronte M Fantin
D Bertuzzi CA Fabiani F Morassut S Bidoli E Veronesi A 2009 Early and short‐term
acenocumarine or dalteparin for the prevention of central vein catheter‐related thrombosis in
cancer patients a randomized controlled study based on serial venographies Ann
Oncol 20 1936ndash1942
13 Espitia O Fouassier M Hardouin JB Pistorius MA Agard C Planchon B Trossaert M
Pottier P 2017 Thrombin generation assay in hospitalized nonsurgical patients A new tool to
assess venous thromboembolism risk Clin Appl Thromb Hemost 23 45ndash51
14 Evans NS Ratchford EV 2018 Catheter related-venous thrombosis Vasc Med 23 411ndash413
15 Geerts W 2014 Central Venous Catheter-related thrombosis Hematology Am Soc Hematol
Educ Program 2014 306ndash311
16 Harahsheh Y Ho KM 2018 Use of viscoelastic tests to predict clinical thromboembolic
events A systematic review and meta‐analysis Eur J Haematol 100 113ndash123
17 Harter C Salwender HJ Bach A Egerer G Goldschmidt H Ho AD 2002 Catheter-
related infection and thrombosis of the internal jugular vein in hematologic-oncologic patients
undergoing chemotherapy a prospective comparison of silver-coated and uncoated
catheters Cancer 94 245ndash251
18 Hirsh J Raschke R 2004 Heparin and low-molecular-weight heparin The Seventh ACCP
Conference on Antithrombotic and Thrombolytic Therapy Chest 126 (Suppl) 188Sndash203S
19 Karthaus M Kretzschmar A Kroning H Biakhov M Irwin D Marschner N Slabber
C Fountzilas G Garin A Abecasis NG Baronius W Steger GG Sudhoff T Giorgetti
C Reichardt P 2006 Dalteparin for prevention of catheter‐related complications in cancer
patients with central venous catheters final results of a double‐blind placebo‐controlled phase III
trial Ann Oncol 17 289ndash296
20 Kato F Takeuchi H Matsuda S Kawakubo H Omori T Kitagawa Y 2016 Incidence of
and risk factors for venous thromboembolism during surgical treatment for esophageal cancer a
Chapter 7 References
88
single-institution study Surg Today 46 445ndash452
21 Kohler TR Kirkman TR 1998 Central venous catheter failure is induced by injury and can
be prevented by stabilizing the catheter tip J Vasc Surg 28 59ndash66
22 Koumlksoy C Kuzu A Kutlay J Erden I Ozcan H Ergicircn K 1995 The diagnostic value of
colour Doppler ultrasound in central venous catheter related thrombosis Clin Radiol 50 687ndash
689
23 Kucher N 2011 Clinical practice Deep-vein thrombosis of the upper extremities N Engl J
Med 364 861ndash869
24 Langston CE Eatroff AE 2018 Hemodialysis catheter-associated fibrin sheath in a dog J
Vet Emerg Crit Care 28 366ndash371
25 Langston CE Eatroff AE Poeppel K 2014 Use of tissue plasminogen activator in catheters
used for extracorporeal renal replacement therapy J Vet Intern Med 28 270ndash276
26 Laurenson MP Hopper K Herrera MA Johnson EG 2010 Concurrent diseases and
conditions in dogs with splenic vein thrombosis J Vet Intern Med 24 1298ndash1304
27 Lee AYY Kamphuisen PW 2012 Epidemiology and prevention of catheter‐related
thrombosis in patients with cancer J Thromb Haemost 10 1491ndash1499
28 Liu G Fu ZQ Zhu P Li SJ 2015 Central venous catheter-related thrombosis in senile male
patients New risk factors and predictors J Huazhong Univ Sci Technolog Med Sci 35 445ndash
449
29 Lordick F Hentrich M Decker T Hennig M Pohlmann H Hartenstein R Peschel C
2003 Ultrasound screening for internal jugular vein thrombosis aids the detection of central
venous catheter-related infections in patients with haemato-oncological diseases a prospective
observational study Br J Haematol 120 1073ndash1078
30 Lucas TC Silva EED Souza DOD Santos ARD Lara MO 2017 Microstructural
evaluation by confocal and electron microscopy in thrombi developed in central venous catheters
Rev Esc Enferm USP 51 1ndash9
31 Mismetti P Mille D Laporte S Charlet V Buchmuller‐Cordier A Jacquin JP Fournel
P Dutrey‐Dupagne C Decousus H 2003 Low‐molecular‐weight heparin (nadroparin) and
very low doses of warfarin in the prevention of upper extremity thrombosis in cancer patients with
indwelling long‐term central venous catheters a pilot randomized trial Haematologica 88 67ndash
Chapter 7 References
89
73
32 Niers TM Di Nisio M Klerk CP Baarslag HJ Buller HR Biemond BJ 2007
Prevention of catheter‐related venous thrombosis with nadroparin in patients receiving
chemotherapy for hematologic malignancies a randomized placebo‐controlled study J Thromb
Haemost 5 1878ndash1882
33 Scott K C Hansen B D DeFrancesco T C (2009) Coagulation effects of low molecular
weight heparin compared with heparin in dogs considered to be at risk for clinically significant
venous thrombosis J Vet Emerg Crit Care 19 74ndash80
34 Smith RS Zhang Z Bouchard M Li J Lapp HS Brotske GR Lucchino DL Weaver
D Roth LA Coury A Biggerstaff J Sukavaneshvar S Langer R Loose C 2012
Vascular catheters with a nonleaching poly-sulfobetaine surface modification reduce thrombus
formation and microbial attachment Sci Transl Med 4 153
35 Tuumlrk S M Cansu D Uuml Teke H Uuml Kaşifoğlu T Meltem Akay O Bilgin M amp Korkmaz
C 2018 Can we predict thrombotic tendency in rheumatoid arthritis A thromboelastographic
analysis (with ROTEM) Clin Rheumatol 37 2341ndash2349
36 van Rooden CJ Rosendaal FR Barge RM van Oostayen JA van der Meer FJM
Meinders AE Huisman MV 2003 Central venous catheter related thrombosis in haematology
patients and prediction of risk by screening with Doppler-ultrasound Br J Haematol 123 507ndash
512
37 van Rooden CJ Tesselaar ME Osanto S Rosendaal FR Huisman MV 2005 Deep vein
thrombosis associated with central venous catheters - a review J Thromb Haemost 3 2409ndash
2419
38 Verso M Agnelli G Bertoglio S Di Somma FC Paoletti F Ageno W Bazzan M Parise
P Quintavalle R Naglieri E Santoro A Imberti D Soraru M Mosca S 2005 Enoxaparin
for the prevention of venous thromboembolism associated with central vein catheter a double‐
blind placebo‐controlled randomized study in cancer patients J Clin Oncol 23 4057ndash4062
39 Vose J Odunayo A Price JM Daves M Schildt JC Tolbert MK 2019 Comparison of
heparinized saline and 09 sodium chloride for maintaining central venous catheter patency in
healthy dogs PeerJ 7e7072 httpdoi107717peerj7072
40 Wang LH Wei F Jia L Zhi Lu Wang B Dong HY Yu HY Sun GJ YangJ Li B
Chapter 7 References
90
Meng J Zhang RN Bi XQ Chen HY Jiang AL 2015 Fibrin sheath formation and
intimal thickening after catheter placement in dog model role of hemodynamic wall shear stress J
Vasc Access 16 275ndash284
41 Wildgruber M Lueg C Borgmeyer S Karimov I Braun U Kiechle M Meier R Koehler
M Ettl J Berger H 2016 Polyurethane versus silicone catheters for central venous port devices
implanted at the forearm Eur J Cancer 59 113ndash124
42 Whitesell RT Steenburg SD 2014 Imaging findings of acute intravascular thrombus on non-
enhanced computed tomography Emerg Radiol 21 271ndash277
43 Wu X Studer W Skarvan K Seeberger MD 1999 High incidence of intravenous thrombi
after short-term central venous catheterization of the internal jugular vein J Clin Anesth 11
482ndash485
44 Xiang DZ Verbeken EK Van Lommel AT Stas M De Wever I 1998 Composition and
formation of the sleeve enveloping a central venous catheter J Vasc Surg 28 260ndash271
45 Xiang DZ Verbeken EK Van Lommel AT Stas M De Wever I 2001 Sleeve-related
thrombosis a new form of catheter-related thrombosis Thromb Res 104 7ndash14
91
Acknowledgements
First I thank God for every day in my life for His guide and because He was is
and will be always with me and that certainty gives me strength to take a step
forward
My sincere thanks to Prof Dr Reinhard Mischke for the opportunity to perform
my PhD under his supervision for his support and guidance for all that I learned
with him and for his friendship
I also thank to Prof Dr Manfred Kietzmann and PD Dr Mario von Depka
Prondzinski for being part of my supervision group for their time and beneficial
advices and discussion during my PhD
My gratitude to Prof Hewicker-Trautwein and Mrs Rohn for their valuable work
and availability to help me
I thank the Werlhof Institute Medical Care Center Hannover for the valuable
cooperation especially to Mrs Ober-Cassebaum Anne and Franziska for all their
help
I thank all the staff of the Small Animal Clinic of the University of Veterinary
Medicine Hannover (TiHo) My special gratitude to the internal medicine and
critical care surgery neurology ultrasound and anesthesia departments and to
the TiHo students all their cooperation made possible my research
I thank the haematology clinical chemistry and haemostasis laboratory
department of the Small Animal Clinic TiHo especially to Dirk because he was
always available for me thanks for his guide and friendship all his explanations
in the laboratory were of great value but also the conversations about different
topics of the life were of big value for me
92
I thank the Hannover Graduate School for Veterinary Pathobiology
Neuroinfectology and translational Medicine (HGNI) for the well-organized
PhD program and especially thanks to Prof Dr Beatrice Grummer Dr Tina
Selle and Mrs Tania Czeslik for his guide and help during my studies
My sincere thanks to Mrs Maritta Ledwoch for her guidance at the International
office of the TiHo ldquoI remember you since my externship in Germanyrdquo
I also thank the Student Union Hannover for all the help with the accommodation
for me and my family
I want to thank the Universidad Nacional de Costa Rica and therefore to Costa
Rica (my loved country) for the financial support to perform my PhD studies
but also for my professional formation as a veterinarian Special thanks to the
Veterinary Medicine School from Universidad Nacional de Costa Rica To all the
academic and administrative staff for their support and for believing in me
Sincerely thanks to Dr Mauricio Jimenez Soto my mentor my adviser but most
important my friend Thanks for believing in me and for always being by my side
ldquoAgradezco profundamente a mis papaacutes ldquodon Tito (Papillo) y dontildea Eida (Mami)rdquo
Todo lo bueno que hay en mi proviene de Dios y de ellos Gracias a mis hermanos
(as) cuntildeados (as) sobrinos y a toda la familia relacionada a ellos Siempre han
sido un gran apoyo y los amordquo
ldquoGracias a dontildea Alba Fran y David Gracias por toda la ayuda y por estar siempre
pendientesrdquo
I would like to thank all my friends that during a short part or during my whole
life have been with me For all the good time shared really thanks
To Willy thanks ldquogordordquo
93
I sincerely thank to Adriano and Johanna for their help and hospitality Thanks
Adriano for your guidance and support during my beginning at the TiHo for make
me feel in Costa Rica with your friendship for the tourist walks and for the bike
training I missed the coffee times
I want to thank Ina the first person to give me a smile at the start of my PhD
Thanks for your hugs and friendship
I also thank Franz for his help and for being a very nice partner Thanks for help
me always when I requested you
ldquoFinalmente quiero expresar mi gratitud y amor a Karo Faacutetima y Abigaiacutel A Karo
por tu sacrificio y soporte por las palabras de aliento por estar a mi lado en las
buenas pero en especial en las malas A Faacute por tu sacrificio por tus sonrisas por
tus abrazos y besos por bailar con migo siempre por ser mi modelo e inspiracioacuten
A Abi por ser una nueva razoacuten in mi vida para seguir adelante por todo lo que
deseo compartir con tigo por mostrarme junto con Faacutetima la felicidad en una
sonrisardquo
Chapter 1 Introduction
2
The incidence of CVC related thrombosis in humans can vary between studies according
to the CVC type used entry site of the CVC (jugular femoral subclavian axillary
cephalic vein) and time period with the CVC in place the diagnostic thrombus detection
technique (venography or ultrasonography) and criteria for diagnosis duration of follow
up diseases and presence of predisposing factors (eg previous venous thrombosis
history presence of cancer) and finally prophylactic anticoagulation treatment (Geerts
2014 Liu et al 2015 van Rooden et al 2005)
To the best of the authoracutes knowledge data about the incidence of CVC related
thrombosis detected by colour Doppler ultrasound into the lumen of the jugular vein in
dogs are rare in the available veterinary literature In one study performed in 81 dogs and
12 cats (Adamantos et al 2010) the incidence of vein thrombosis detected by palpation
or ultrasound was 2 and the incidence of intraluminal CVC thrombosis (presumed by
the inability to draw blood back) was 15 Additionally regarding to intraluminal CVC
thrombosis an incidence of 8 was reported in one case control study performed in 24
dogs receiving CVC flushing with heparinised solution (Vose et al 2019) Data about
incidence in other animal species was also found In another case-control study performed
on 37 pigs thrombosis in the caval vein related to CVC placed in the external jugular
vein (EJV) was evaluated by histological analysis however no ultrasound detection was
performed (Kohler and Kirkman 1998) In this study in pigs 94 of patients with CVC
developed thrombi in the vena cava based on sections at the site of the end of the catheter
tip and 05 and 1 cm proximal of the first section (Kohler and Kirkman 1998)
Pathogenesis of CVC related thrombosis
Two types of CVC related thrombosis are distinguished in the human medicine literature
(Geerts 2014 van Rooden et al 2005) First symptomatic thrombosis is defined as
Chapter 1 Introduction
3
thrombosis detected by diagnostic images (ultrasonography venography) when
symptoms and signs (eg localised swelling pain redness) are present (Lee and
Kamphuisen 2012) Second subclinical thrombosis is defined as thrombosis
demonstrated by the previously mentioned diagnostic images without overt symptoms or
signs (Boersma et al 2008) Additionally the CVC related thrombi can be classified as
occlusive thrombus into the CVC lumen peri-catheter related fibrin sheaths and mural
thrombi (Geerts 2014)
During the CVC insertion the puncture of the skin on the CVC insertion site and the
rupture of the blood vessels integrity activate the coagulation cascade and the CVC acts
as a matrix for adherence and accumulation of fibrin and cellular components (Smith et
al 2012) The attachment of proteins and blood cells to the internal andor external wall
of the CVC can lead to (1) (partial or complete) occlusion of the catheter lumen and (2)
sometimes if a thrombus formation starts into the lumen of the vein (attached to the vein
wall or to the CVC) a partial or complete obstruction of the blood flow andor embolism
can develop (Langston and Eatroff 2018) Additionally a study performed in humans
(Lordick et al 2003) demonstrated a relationship between CVC related thrombosis and
microbial colonisation of the catheter leading to CVC related systemic infections This
relationship was explained among others by the ease of microorganisms like
Staphylococcus aureus and Staphylococcus epidermidis to adhere to CVC related
thrombin sheaths (Boersma et al 2008)
Diagnosis of CVC related thrombosis
Venous thrombosis is commonly diagnosed in human and small animal medicine
(especially in dogs) with vascular ultrasonography with or without use of the colour
Doppler technique which is normally used to evaluate the blood flow through the blood
Chapter 1 Introduction
4
vessels (Evans and Ratchford 2018 Laurenson et al 2010) According to the literature
in humans cited below the CVC related thrombosis detected by colour Doppler
ultrasound is present if any of the following aspects are observed (1) well defined
thrombus visualised in the lumen of the vein (2) impossibility to perform compression
of the vein by slight pressure with the ultrasound probe andor (3) no detectable blood
flow signal by colour Doppler ultrasound (Bonnet et al 1989 Koumlksoy et al 1995)
Similarly in dogs the CVC related thrombosis is usually diagnosed by colour Doppler
ultrasonography through the detection of the thrombus within the lumen of the vein
andor the obstruction (partial or complete) of the normal blood flow (Bliss et al 2002)
Especially in humans the contrast venography technique is used as gold standard to
confirm thrombosis For this purpose a contrast solution is injected through a peripheral
vein to assess its passage through the bloodstream by x-ray However this diagnostic
technique is normally used only in the case of a strong clinical suspicion of venous
thrombosis that cannot be detected by ultrasonography (Laurenson et al 2010 Langston
and Eatroff 2018) In a study performed in 44 human patients requiring a CVC (Koumlksoy
et al 1995) a sensitivity of 94 and a specificity of 96 were determined for the colour
Doppler ultrasound diagnosis when it was compared with the diagnosis performed with
the contrast venography technique That results make the colour Doppler ultrasound an
accurate diagnosis method to detect CVC related thrombosis in humans (Koumlksoy et al
1995 van Rooden et al 2003)
Advanced diagnostic image techniques like computed tomography or magnetic resonance
tomography are also accurate to perform the diagnosis of CVC related thrombosis in
humans (Whitesell and Steenburg 2014) In dogs multidetector computed tomography
venography was used in one study for the diagnosis of CVC related thrombosis allowing
the identification of catheter related sheaths around the CVC external wall (Wang at al
Chapter 1 Introduction
5
2015) However the high costs of these advanced diagnostic imaging procedures and the
requirement to perform at least sedation or general anesthesia are disadvantages when
compared with ultrasonography
Finally electron microscopy (ELMI) is a diagnostic technique that has been used in
humans (Lucas et al 2017) and experimental animals (Xiang et al 1998 Xiang et al
2001) mainly to perform a microstructural evaluation of thrombi developed into the
lumen of the CVC or in the vein containing the CVC ELMI allows the ex vivo diagnosis
of very small thrombi or sleeve related thrombosis that perhaps cannot be detected by
ultrasonography or venography during the evaluation of patients with CVCs (Xiang et al
2001) However one point to consider is that it is possible that compact thrombi
developed on the CVC can be detached during the CVC removal from the vein and
thereby before its analysis by ELMI (Xiang et al 2001)
Heparin thromboprophylaxis for CVC related thrombosis
Application of CVCs in human medicine is commonly accompanied by prophylactic
anticoagulation in order to decrease the risk of development of partial or complete vein
occlusion andor thromboembolism and thereby to improve the patientrsquos comfort
(Baumann-Kreuziger et al 2015) Apart from vitamin K antagonists (Mismetti et al
2003) unfractionated heparin (UFH) (Abdelfeki et al 2004) and low molecular weight
heparin (LMWH) (Karthaus et al 2006) are anticoagulants that have been used on
studies in humans administrated by intravenous (IV) infusion or by subcutaneous (SC)
injection to prevent thrombi development into the lumen of the veins containing the CVC
(review article Baumann-Kreuziger et al 2015) Different heparin dosages have been
used as thromboprophylactic anticoagulation in humans with CVC (review article Lee
and Kamphuisen 2012) For example in five different case-control studies performed in
Chapter 1 Introduction
6
human haemato-oncological patients UFH at a daily dosage of 100 IUkg given by IV
infusion (Abdelfeki et al 2004) and LMWH at SC daily dosages of 40 mg enoxaparin
(Verso et al 2005) 5000 IU AXa dalteparin (Karthaus et al 2006 De Cicco et al 2009)
and 2850 IU AXa nadroparin (Niers et al 2007) were used Only in part of these studies
it is stated that systemic heparin was combined with flushing of the CVC using
heparinised saline solution to reduce the risk of catheter occlusion (Karthaus et al 2006)
In 3 of the 5 previously mentioned studies no statistically significant difference was found
in the incidence of CVC related thrombosis between patients receiving the
anticoagulation prophylaxis compared with the control groups without systemic
anticoagulation (Verso et al 2005 p = 035 Karthaus et al 2006 p gt 005 Niers et al
2007 p = 049) In contrast in the residual two studies a lower incidence of CVC related
thrombosis was found in the group receiving an anticoagulation prophylaxis compared
with the control groups (Abdelfeki et al 2004 150 vs 126 p = 003 De Cicco et
al 2009 400 vs 526 p = 005)
Obviously there is no consensus in the available human literature regarding the beneficial
use of anticoagulation prophylaxis to prevent CVC related thrombosis and this may
reflect great variations for example according to the study design period trial drug
administration route (Lee and Kamphuisen 2012) and the moment of the initial
anticoagulant dosage administration (Decicco et al 2009)
To the best of the authoracutes knowledge studies about thromboprophylaxis for extraluminal
CVC related thrombosis in dogs cannot be found in the available veterinary literature
However similarly to humans prophylactic anticoagulation with heparinised saline
(administrated through CVC flushing) to prevent CVC occlusion by intraluminal thrombi
was used in one case-control study performed in 24 dogs (Vose et al 2019) In the cited
study 12 dogs received dosages of 3 ml of heparinised saline solution (10 IU UFHml) to
Chapter 1 Introduction
7
flush the catheter every 6 hours during 72 hours whereas 12 control dogs received
flushing with the same volume of non-heparinised saline solution administered at the
same frequency and no statistically significant difference was found
Prediction of CVC related thrombosis
Studies regarding the relationship between laboratory biomarkers and CVC related
thrombosis are available in the human literature (Boersma et al 2016 Cheng et al 2013
Liu et al 2015) however to the best of the authoracutes knowledge literature about laboratory
tests used as predictors for CVC related thrombosis in small animals cannot be found at
all Availability of approved blood tests which can predict the risk of thrombosis
development associated to CVC already at the moment of the CVC placement are
important in order to identify an increased risk of CVC related thrombosis (Boersma et
al 2016) In the study performed by Boersma et al (2016) in 168 human patients without
indication of anticoagulation prophylaxis elevated leukocyte counts high levels of
plasminogen activator inhibitor and of coagulation factor VIII (all measured on blood
samples taken after CVC placement) were associated with an increased incidence of
symptomatic CVC-related thrombosis diagnosed by ultrasonography In another study
(Liu et al 2015) high blood levels of lactate dehydrogenase and low concentrations of
HDL and albumin were associated with the incidence for CVC related thrombosis
diagnosed by ultrasound in 324 senile patients which was in total 617 Additionally
dyslipidemia (high triglycerides low HDL and high LDLHDL ratio) was associated
with CVC related thrombosis (diagnosed by ultrasound and computer tomography
angiography) in human patients undergoing haemodialysis (Cheng et al 2013) Studies
about predictive tests for CVC related thrombosis in dogs were not found in the available
literature to the best of the authoracutes knowledge
Chapter 1 Introduction
8
Aims of the study
The aims of the preliminary methodological study of the present scientific project were
(1) to define reference intervals for viscoelastic analyses of canine haemostasis using the
ROTEMreg delta analyser and (2) to determine the precision (repeatability) of this
method And the aims of the main study of the present scientific project were (1) to
determine the incidence of catheter-induced thrombosis in canine intensive care patients
(2) to evaluate the efficacy of a prophylactic heparin regimen routinely used in the Small
Animal Clinic of the University of Veterinary Medicine Hannover and (3) to assess
whether selected haemostasis test results are predictive for an increased risk for
thrombosis formation
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
9
Chapter 2 Reference intervals for rotational thromboelastometry measurements
using the ROTEMreg delta device in dogs
Pereira JM Rohn K Mischke R
Contributions of the authors
Pereira JM performed (1) the patients data and blood samples collection (2)
haematology and ROTEMreg analysis (3) generated the reference intervals and
coefficients of variation included in the study and (4) wrote the manuscript
Rohn K performed the statistical analysis
Mischke R performed the clinical chemistry and coagulation tests and was involved in
designing and supervision of the study analysis and discussion of the data and its
statistical analysis and revision edition and correction of the manuscript
Original source Pereira J M Rohn K amp Mischke R 2020 Reference intervals
for rotational thromboelastometry measurements using the ROTEMreg delta device
in dogs Res Vet Sci 130 26ndash32 httpsdoiorg101016jrvsc202001019
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
10
Reference intervals for rotational thromboelastometry measurements using the
ROTEMreg delta device in dogs
Pereira JMa Rohn Kb Mischke Ra
aSmall Animal Clinic University of Veterinary Medicine Hannover Buumlnteweg 9 D-
30559 Hannover Germany
bInstitute of Biometry Epidemiology and Information Processing University of
Veterinary Medicine Hannover Buumlnteweg 2 30559 Hannover Germany
Corresponding author
E-mail ReinhardMischketiho-hannoverde
Original source Pereira J M Rohn K amp Mischke R 2020 Reference intervals
for rotational thromboelastometry measurements using the ROTEMreg delta device
in dogs Res Vet Sci 130 26ndash32 httpsdoiorg101016jrvsc202001019
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
11
ABSTRACT
Aims of the present study were to define reference intervals for viscoelastic analyses of
canine haemostasis using the ROTEMreg delta analyser and as a secondary aspect to
determine the precision (repeatability) of this method Blood samples from 125 clinically
healthy dogs were included Measurements were performed with commercially available
activating reagents (ex-tem in-tem and kaolin solution) as well as without activation
Additional fourfold measurements were done in 3 of the normal blood samples and in 3
samples with haemostatic alterations to evaluate the precision of the method
Coefficients of Variation (CVs) for most of the ROTEM variables were lt 10 Clot
formation time and maximum clot elasticity showed a wide inter-individual variation in
comparison with alpha angle and maximum clot firmness A multivariate analysis on
various ROTEM parameters revealed particularly a significant influence of neuter status
and a significant interrelationship between the factors sex and neuter status for
measurements with different activating reagents These results reflected the fact that
significant differences occurred only between intact and neutered females but not in
males No or only occasionally significant differences were found between groups of sex
age and size
In conclusion CVs demonstrated that the method delivers repeatable results in canine
citrated whole blood Established reference intervals should deliver valuable orientation
for the evaluation of viscoelastic properties of clotting whole blood in dogs using the
ROTEM delta analyser Neuter status in females appeared to be the most relevant
influencing factor and should be considered for the interpretation of ROTEM delta test
results
Keywords Viscoelastic measurements reference values canine age sex neuter status
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
12
1 Introduction
Heamostasis is a physiologic and dynamic process in which plasma soluble coagulation
factors blood cells and blood vessels are involved Clot formation and possible
subsequent clot lysis as well as vascular tissue repair aim to prevent haemorrhages on the
one hand and thromboses on the other hand (Kol and Borjesson 2010 Versteeg et al
2013) Conventional assays of coagulation such as prothrombin time (PT) activated
partial thromboplastin time (aPTT) and thrombin time TT have limitations especially
cellular elements are not incorporated into the test and thereby these tests reflect only
limitedly in vivo functionality of haemostasis (McMichael and Smith 2011 Crochemore
et al 2018)
The rotational thromboelastometry (ROTEMreg) currently termed as thromboelastometry
(TEM) and thromboelastography (TEG) are methods for the rapid evaluation of the
overall haemostasis capacity through the measurement of viscoelastic properties of
clotting whole blood (Kol and Borjesson 2010 Theusinger et al 2010 Sankarankutty
et al 2012 Versteeg et al 2013) In TEG the cup with the blood sample is rotating
whereas the torsion wire is fixed In ROTEMTEM the cup is fixed whereas the pin is
rotating Changes in torque ie resistance to rotation by the fibrin bridge created between
pin and cup in the clotting blood are detected electromechanically in TEG and optically
in ROTEM (Bollinger et al 2012) and displayed as a graph that indicates the time for the
formation of blood clot and its strength (Bagge et al 2016) The resulting graph reflects
the initiation and propagation of the coagulation system fibrin-platelet interactions and
clot lysis
TEG was first described in 1948 by Hellmut Harten (Hartert 1948) as an in vitro method
to evaluate the clot formation process while resembling the in vivo blood flow The
development of the actually used instrument types TEG and ROTEMregTEM were based
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
13
on that original principle Viscoelastic measurements have been widely used since the
1980s in human medicine to evaluate haemostasis and hypercoagulable states in surgical
patients (eg cardiac and hepatic surgeries) and in general in critical care units (Kol and
Borjesson 2010 Nogami 2016 Wikkelso et al 2016 Crochemore et al 2017) In
veterinary medicine viscoelastic measurements using TEG and ROTEMTEM have been
validated for use in dogs cats and horses and used to study haemostasis in different
diseases (eg infectious diseases liver diseases) and medical conditions (eg
orthopaedic surgery hyperfibrinolysis) and also to investigate the effect of
anticoagulants and other drugs (eg corticosteroids) on haemostasis (Kol and Borjesson
2010 Bruno et al 2015a 2015b Woodman et al 2015 Conversy et al 2017 Sigrist et
al 2018 Turner et al 2019) The methodology seems to be especially suitable to detect
hypercoagulability (Wiinberg et al 2008 Kol and Borjesson 2010 Marschner et al
2018)
In the available veterinary literature information about reference values for TEG were
found using kaolin as activation reagent (Bauer et al 2009) Falco et al (2012)
determined reference values for the ROTEM analyser in dogs using ex-tem in-tem and
fib-tem reagents for selected ROTEM parameters in a study on forty five healthy dogs
However data related to reference intervals obtained in an adequate number of samples
(at least 120) according to the American Society for Veterinary Clinical Pathology
(ASVCP) (Friedrichs et al 2012) and the National Committee for Clinical Laboratory
Standards (NCCLS) (Horn and Pesce 2003) could not be found in the available literature
The aim of this study is to determine reference values for the ROTEMreg delta device in
dogs using citrated whole blood with different clot activator reagents (ex-tem in-tem
kaolin) and without activator
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
14
2 Materials and methods
21 Study design
Reference intervals Single thromboelastometric measurements were performed in 125
clinically healthy dogs using the ROTEMreg analyser and different activating reagents (ex-
tem in-tem and kaolin) as well as without activation Reference values were calculated
and influences of age groups (young adult and senior) sex neuter status and size
(groups small medium and large) were assessed
Precision analysis Additional fourfold measurements using ex-tem or in-tem activating
reagents respectively were performed on 3 of the normal and also in 3 abnormal blood
samples each in order to assess within-run imprecision for these measurements
22 Animals
The clinically healthy dogs with a body weight of at least 5 kg were recruited by launching
a call in the University of Veterinary Medicine Hannover network for students and staff
members Interested dog owners received a free health check of their animals (clinical
examination and blood tests specified below) in combination with the blood collection
In addition individual privately owned patient dogs of the Small Animal Clinic with
undisturbed general health were also included if blood was collected due to another
indication (eg general health check pre-anaesthetic examination test for hereditary
diseases etc) Before taking the blood samples all dogs were fasted for at least 12 h with
free access to water 125 animals for which the examination of the health status (clinical
examination routine haematology and clinical chemistry basis coagulation profile) did
not reveal any deviations from the reference were included in the study whereas 3 of the
128 initially recruited animals had to be excluded due to laboratory abnormalities
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
15
Haematological and clinical chemistry profiles performed in order to confirm the health
status included white and red cell count haematocrit platelet counts and reticulocytes
plasma activities of alanine aminotransferase glutamate dehydrogenase alkaline
phosphatase and pseudocholinesterase as well as concentrations of total bilirubin urea
creatinine glucose total protein albumin cholesterol and ionised calcium A basis
coagulation profile including PT PTT and TT was also performed to exclude significant
abnormalities within the blood coagulation system
The 125 healthy dogs enclosed in the establishment of reference values included 48 mixed
breed dogs and the following pedigree dogs 13 Labrador Retrievers 6 Australian
Shepherds 4 Havanese dogs 3 from each of the following breeds American
Staffordshire Terriers and Cocker Spaniels 2 from each of the following breeds Beagles
Border Collies Dalmatians Elo dogs (breed not recognised by Feacutedeacuteration cynologique
international [FCI]) German Longhaired Pointers German Shorthaired Pointers Giant
Schnauzers and Golden Retrievers 1 from each of the following breeds Belgian
Malinois Bolonka Zwetna Briard Brussels Griffon American English Coonhound
Italian Mastiff Flat Coated Retriever French Bulldog German Shepherd Great Dane
Greater Swiss Mountain Dog Giant Spitz Goldendoodle Hovawart Hungarian Vizsla
Italian Bloodhound Lycanis Wolfdog Miniature American Shepherd Miniature
Schnauzer Old German Shepherd Podenco Poodle Pyrenean Shepherd Rhodesian
Ridgeback Rottweiler Schafpudel (breed not recognised by FCI) Small
Munsterlander Spanish Mastiff Tibet Terrier Weimaraner West Highland White
Terrier and Wirehaired Pointer
These dogs were aged between 6 and 154 months (median = 410 months) and had body
weights ranging from 50 to 707 kg (median = 226 kg) 30 were castrated males 33
intact males 41 spayed females and 21 intact females According to the height at withers
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
16
the dogs were divided into 3 different size groups small (le40 cm n = 22) medium (41ndash
60 cm n = 77) and large (gt60 cm n = 26) Dogs were also divided in three different age
groups young (le 24 months n = 34) adult (25ndash84 months n = 59) and senior dogs (gt84
months n = 32)
The three blood samples with haemostatic abnormalities were taken from two dogs with
complex haemostatic disorders (Disseminated Intravascular Coagulation [DIC])
including reduced platelet count and abnormal PT and APTT and one dog with
thrombocytopenia
Animals were treated in accordance with the German Animal Welfare Law The
experimental design was approved by the Animal Welfare officer of the University of
Veterinary Medicine Hannover Foundation and by the ethics committee of the
responsible authority (Lower Saxony State Office for Consumer Protection and Food
Safety reference number 17A101)
23 Blood sampling
The venipuncture area was shaved and then disinfected with alcohol The saphenous or
cephalic vein was visualised with slight pressure and punctured with disposable cannulas
(diameter 09 or 10 mm) The continuously running blood was directly collected from
the needle into the tubes containing the respective anticoagulants From each dog
approximately 10 ml of blood were collected including approximately 8 ml of citrated
blood for the measurement of haemostasis tests (ROTEM PT aPTT TT) in tubes
containing sodium citrate 313 (Eifelfangoreg GmbH amp Co KG Bad Neuenahr-
Ahrweiler Germany) in a ratio of 1 volume9 volumes of blood In addition
approximately 1 ml of EDTA blood for haematological analysis and approximately 1 ml
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
17
of lithium heparin blood for clinical chemical measurements were collected into
commercial 13 ml tubes (Sarstedt AG amp Co Nuumlmbrecht Germany)
Citrated blood was initially kept at room temperature (20-25 C) in order to avoid errors
within the preanalytical phase (Toulon et al 2017) Two ml of citrated blood were used
for the ROTEMreg analysis the residual citrate blood was centrifuged for 10 min at 3800
rpm and 5 degC then the plasma was separated by careful pipetting and it was centrifuged
a second time under the same conditions to get platelet poor plasma which was stored in
aliquots of 500 microl and 250 microl at -80 degC for coagulation tests
24 Rotational elastometry
Viscoelastic measurements using the ROTEMreg delta for the establishment of reference
intervals were performed approximately 15 min after the blood collection using activating
reagents (liquid reagents) provided by the manufacturer of the instrument (r-ex-temreg in-
temreg) and a kaolin solution (5 g1 as part of the PTT reagent CK Prest [Diagnostica
Stago SAS Asniegraveres sur Seine Cedex France]) In addition non-activated analyses
were performed using isotonic NaCl solution instead of the activating reagent in the fourth
channel of the device Fourfold measurements for precision analysis were performed
simultaneously in the four channels of the device in separate runs
According to the standard test protocol provided by the manufacturer of the ROTEMreg
delta (Werfen GmbHInstrumentation Laboratory Munich Germany) 20 μl of the
reagent for recalcification (star-temreg20) and 20 μl of the activating reagent were pipetted
into a cuvette using the automated pipette provided with the device Immediately
thereafter 300 μl of citrated whole blood (prewarmed to 37 degC for five minutes in the
heating device of the instrument and gently rocked to resuspend possibly sedimented
cells) were added to the test preparation in the cuvette using the automated pipette and
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
18
then the cuvette was laced carefully in the ROTEMreg device whereby the rotating pin
entered into the test approach
Different parameters are provided automatically by the ROTEMreg delta device and the
following variables (described according to the Pentapharm GmbH ROTEMreg delta
Manual [2009]) were used in the study
Clotting time (CT) the time (in s) from the beginning of the test (by adding the clot
activator) until the formation of fibrin starts (defined as the moment when the curve
achieves an amplitude of 2 mm) This Parameter facilitates the decision to replace clotting
factors when it is required
Clot formation time (CFT) describes the next phase of clotting by the formation of a
stable clot through the activation of thrombocytes and fibrin formation It is defined as
the time (in s) between the 2 mm amplitude and 20 mm amplitude of the curve
Alpha angle (α angle) is the angle (in degrees) between the middle axis and the tangent
to the clotting curve through the 2 mm amplitude point
Amplitude after ldquoXXrdquo minutes (eg A30 after 30 min) is the amplitude (in mm) at a
certain time after the CT has finished
Maximum clot firmness (MCF) corresponds to the maximum amplitude (in mm) reached
before the clot is dissolved by fibrinolysis This parameter measures the firmness of the
clot and its quality Like CFT and α angle it facilitates the decision for substitution therapy
with platelet concentrate andor fibrinogen especially in humans
Maximum clot elasticity (MCE) is a parameter calculated from MCF and may be superior
at high amplitudes as compared to the MCF
Lysis index at 30 45 and 60 min (LI30 LI45 LI60) represents the fibrinolysis (in
percentage) 30 45 and 60 min after the CT It may facilitate the decision for or against a
therapy with anti-fibrinolytic drugs
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
19
Maximum lysis (ML) describes the degree of fibrinolysis relative to the MCF achieved
during the measurement (percent loss of clot firmness)
25 Statistical methods
Original data obtained from the ROTEMreg delta device software were transferred into the
Microsoftreg Excelreg formal (Microsoft Office Professional Plus 2013) The Statistical
analyses were performed using the SAS Enterprise Guide Version 71 (SAS Institute Inc
Cary NC USA)
Coefficients of Variation (CVs) for selected relevant parameters of the ROTEM delta
analysis using ex-tem and in-tem regents were calculated for the fourfold measurements
on 3 normal and 3 abnormal blood samples in order to assess within-run imprecision CVs
for the results of each sample were calculated by dividing the standard deviation of the
data set by its arithmetic mean Finally the median of the CVs for each group of 3 samples
was calculated The reference values were calculated according to recommendations of
the American College of Veterinary Clinical Pathology (ACVCP) based on non-
parametric methods (25 and 975 quantiles) supplemented by 95 confidence
intervals (Friedrichs et al 2012)
Data were analysed for Standard normal distribution by Kolmogorov Smirnov test and
assessment of residuals of the analysis of variance (ANOVA) Because these tests
indicated almost normally distributed values for the studied ROTEM variables data were
analysed by a fourway ANOVA stratified by the activating reagent (ex-tem in-tem
kaolin or without activation) to test the influence of size age sex and neuter status on
several parameters and taking all interactions into account Post hoc t-tests were
performed when indicated The significance level was set to P lt 05
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
20
3 Results
31 Precision of the method
For all the parameters evaluated in the study the CVs for the fourfold measurement in
healthy dogs were less than 10 (Table 1) In the case of dogs with haemostatic
abnormalities CVs of the vast majority of ROTEM variables were also less than 10
except the variables CFT and ML irrespective whether ex-tem or in-tem reagents were
used (Table 2)
Table 1 Coefficients of variation () for rotational thromboelastometry using the
ROTEMreg delta analyser in normal blood samples
Activator CT (s) CFT (s) α (deg) A30 (mm) MCF (mm) MCE(mm) LI30 () LI45 () LI60 () ML ()
Ex-temreg 875 629 262 213 179 634 000 020 037 534
In-temreg 245 596 166 117 135 299 000 000 046 216
Abbreviations CT ndash clotting time CFT ndash clot formation time α ndash alpha angle A30 ndash
amplitude after 30 min MCF ndash maximum clot firmness MCE ndash maximum clot elasticity
LI 30 LI 45 LI 60 ndash Lysis index at 30 45 and 60 min ML ndash maximum lysis
Median values of the measurements of 3 samples
Table 2 Coefficients of variation () for rotational thromboelastometry using the
ROTEMreg delta analyser in canine blood samples with haemostatic alterations
Activator CT (s) CFT (s) α (deg) A30 (mm) MCF (mm) MCE(mm) LI30 () LI45 () LI60 () ML ()
Ex-temreg 668 1478 393 218 268 582 0 018 000 848
In-temreg 228 799 461 219 251 371 0 0 017 283
Median values of the measurements of 3 samples from patients with DIC (n = 2)
thrombocytopenia (n = 1) abbreviations please see legend to Table 1
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
21
32 Reference intervals
Activation by ex-tem reagent (tissue factor) resulted in faster CTs when compared to
activation via the intrinsic pathway using ellagic acid (in-tem) or kaolin reagents whereas
the CFT was much less affected by the activating reagent (Table 3) Especially CFT and
MCE showed a wide inter-individual variation in comparison for example with α angle
and MCF regardless of the used activating reagent (Table 3)
33 Influence of age sex neuter status and size
ANOVA did not reveal statistically significant differences in ROTEM delta parameters
between dog groups of variable size or sex (P gt 05) Age had influence only on CT and
only when the in-tem reagent was used (P = 0169 ANOVA) but not when using the ex-
tem reagent (P = 7105) Post hoc comparisons using t-test of the in-tem results between
age groups revealed shorter CTs in the group of senior dogs when compared with young
dogs (P = 0194) but not between adult dogs and these two groups ANOVA showed
particularly influences of the neuter status and a significant interrelationship between the
factors sex and neuter status on different ROTEM delta parameters using ex-tem and in-
tem respectively as activator (Tables 4 and 5) ANOVA revealed also a significant
influence of neuter status on different ROTEM parameters when kaolin was used as
activating reagent (CFT α angle MCF MCE) whereas non-activated measurements
were not subject to any significant influence of the assessed factors (results not shown)
The interrelationship between sex and neuter status mentioned above reflected the fact
that significant differences between neutered and not neutered dogs were only found in
females The statistical analysis using the t-test to compare intact vs neutered dogs
stratified by sex showed statistically significantly (P lt 05) longer CTs and CFTs and
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
22
lower α angle A30 MCF and MCE values in spayed when compared to intact females
(Tables 4 and 5)
Chapter 2 Reference intervals for rotational thromboelastometry measurements using the ROTEMreg delta device in dogs
23
Table 3 Reference values (median reference interval based on 25 - and 975 - quantiles) for thromboelastometric measurements with the ROTEMreg delta analyser in
dogs (n=125 95 confidence intervals in brackets) Abbreviations please see legend to Table 1
Activator
Statistical
para-meter
CT
(s)
CFT
(s)
Α
(deg)
A30
(mm)
MCF
(mm)
MCE
(mm)
LI30
()
LI45
()
LI60
()
ML
()
Ex-temreg
Median 450
[430ndash470]
110
[105ndash115]
680
[670ndash690]
600
[580ndash610]
610
[600ndash620]
154
[147ndash164]
100
[960ndash100]
100
[100ndash100]
970
[960ndash980]
220
[210ndash240]
25 340
[270ndash350]
650
[490ndash710]
540
[420ndash560]
460
[410ndash480]
460
[420-490]
870
[720ndash950]
100
[100-100]
870
[800ndash960]
700
[640ndash820]
800
[100ndash110]
975 830
[790ndash990]
220
[201ndash315]
790
[770ndash800]
770
[690ndash770]
730
[700ndash770]
264
[230-334]
100
[100ndash100]
100
[100ndash100]
100
[990ndash100]
590
[530ndash680]
In-tem
Median 190
[187ndash201]
116
[111ndash125]
680
[670ndash690]
570
[560ndash580]
590
[580ndash600]
143
[135ndash149]
100
[100ndash100]
100
[100ndash100]
980
[980ndash980]
180
[170ndash190]
25 135
[112ndash142]
730
[590ndash800]
510
[460ndash570]
450
[410ndash470]
480
[410ndash500]
920
[710ndash101]
100
[950ndash100]
990
[820ndash990]
930
[740ndash930]
500
[100ndash700]
975 283
[268ndash291]
251
[198ndash227]
750
[750ndash780]
660
[660ndash690]
680
[660ndash690]
210
[195ndash227]
100
[100ndash100]
100
[100ndash100]
100
[100ndash100]
270
[260ndash380]
Activator
Statistical
para-meter
CT
(s)
CFT
(s)
Α
(deg)
A30
(mm)
MCF
(mm)
MCE
(mm)
LI30
()
LI45
()
LI60
()
ML
()
Kaolin
Median 162
[154ndash173]
870
[820ndash910]
740
[720ndash750]
500
[470ndash530]
550
[530ndash560]
121
[115ndash127]
960
[940ndash980]
840
[790ndash880]
710
[640ndash770]
500
[450ndash540]
25
109
[990ndash115]
470
[440ndash580]
560
[490ndash600]
110
[700ndash230]
390
[380ndash400]
640
[600ndash680]
270
[140ndash480]
110
[800ndash260]
800
[400ndash170]
200
[160ndash240]
975 246
[230ndash337]
214
[185ndash290]
810
[790ndash810]
640
[630ndash690]
650
[650ndash690]
187
[182ndash223]
100
[100ndash100]
990
[980ndash100]
940
[930ndash990]
970
[930ndash100]
Without activation
Median 607
[495ndash662]
221
[194ndash265]
510
[460ndash550]
520
[500ndash530]
540
[520ndash550]
115
[109ndash123]
100
[100ndash100]
100
[100ndash100]
980
[980ndash990]
170
[160ndash190]
25 203
[123ndash215]
860
[550ndash930]
190
[120ndash230]
350
[230ndash370]
370
[260ndash400]
600
[350ndash670]
100
[950ndash100]
980
[890ndash990]
930
[840ndash950]
400
[000ndash500]
975 1356
[1305ndash1721]
811
[688ndash1333]
730
[710ndash790]
660
[640ndash720]
660
[640ndash730]
197
[179ndash265]
100
[100ndash100]
100
[100ndash100]
100
[100ndash100]
290
[280ndash320]
Chapter 2 Reference intervals for rotational thromboelastometry measurements using the ROTEMreg delta device in dogs
24
Table 4 Median values (minimum and maximum in brackets) for different ROTEMreg variables using ex-tem reagent stratified by sex and neuter status
Group
CT
(s)
CFT
(s)
Α
(deg)
A30
(mm)
MCF
(mm)
MCE
(mm)
LI30
()
LI45
()
LI60
()
ML
()
Female intact 430
(320ndash530)
960
(590ndash198)
710
(570ndash800)
620
(480ndash730)
640
(510ndash740)
179
(103ndash282)
100
(100ndash100)
100
(980ndash100)
960
(800ndash100)
230
(100ndash640)
Female spayed 460
(270ndash830)
121
(650ndash224)
660
(520ndash770)
580
(430ndash690)
590
(440ndash700)
146
(790ndash230)
100
(960ndash100)
100
(800ndash100)
970
(690ndash100)
240
(100ndash590)
p-value (t-test)
Female
intact vs spayed
00025 00063 00049 00023 00016 00015 na na na na
Male intact 450
(320ndash990)
105
(650ndash315)
690
(420ndash790)
590
(420ndash700)
610
(440ndash710)
154
(770ndash244)
100
(100ndash100)
100
(870ndash100)
970
(650ndash100)
220
(700ndash680)
Male castrated 460
(340ndash930)
111
(490ndash255)
685
(480ndash800)
590
(410ndash770)
600
(420ndash770)
152
(720ndash334)
100
(100ndash100)
100
(830ndash100)
980
(640ndash100)
205
(500ndash590)
p-value (t-test)
Male
Intact vs castrated
08326 05106 04756 01799 01568 02163 na na na na
p-values (ANOVA)
Factor Sex
05428 04847 06612 02206 01887 01630 08489 07893 07689 02758
Factor neuter status 01872 00167 00173 00039 00037 00028 07141 04737 09356 05948
Interrelationship
sexneuter status 00383 00968 00782 00488 00472 00319 04943 08611 05422 00160
na ndash not analysed all significant P-values (lt 005) are highlighted in bold print
Chapter 2 Reference intervals for rotational thromboelastometry measurements using the ROTEMreg delta device in dogs
25
Table 5 Median values (minimum and maximum in brackets) for different ROTEMreg variables using the in-tem reagent stratified by sex and neutering status
Group
CT
(s)
CFT
(s)
Α
(deg)
A30
(mm)
MCF
(mm)
MCE
(mm)
LI30
()
LI45
()
LI60
()
ML
()
Female intact 186
(126ndash238)
940
(590ndash164)
720
(620ndash780)
610
(490ndash690)
620
(530ndash690)
166
(113ndash227)
100
(100ndash100)
100
(990ndash100)
980
(930ndash100)
170
(100ndash300)
Female spayed 201
(149ndash291)
1220
(820ndash262)
670
(500ndash740)
550
(450ndash660)
570
(480ndash680)
133
(910ndash214)
100
(950ndash100)
100
(820ndash100)
980
(740ndash100)
190
(500ndash380)
p-value (t-test)
Female
intact vs spayed
na na lt0001 00009 00005 00005 na na na na
Male intact 199
(117ndash283)
122
(730ndash277)
680
(460ndash750)
570
(430ndash660)
590
(450ndash660)
146
(810ndash192)
100
(100ndash100)
100
(990ndash100)
980
(930ndash100)
180
(600ndash270)
Male castrated 181
(112ndash288)
123
(820ndash222)
670
(540ndash740)
565
(410ndash630)
585
(410ndash640)
138
(710ndash180)
100
(100ndash100)
100
(970ndash100)
980
(890ndash100)
180
(800ndash260)
p-value (t-test)
Male
intact vs castrated
na na 08232 01401 01162 00849 na na na na
p-values (ANOVA)
Factor Sex
02105 03317 03370 00877 01258 00510 09564 07832 08117 03638
Factor neuter status 04289 00660 00619 00107 00088 00054 07152 09795 07056 08299
Interrelationship
sexneuter status
04310 00550 00408 00707 01733 00996 05240 05982 08047 04578
na ndash not analysed all significant P-values (lt 005) are highlighted in bold print
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
26
4 Discussion
41 Coefficients of variation
In accordance with the literature CVs below 10 indicate adequate repeatability in intra-
assays evaluations (Steiner et al 2003 Hua et al 2018) According to our results
obtained from the fourfold measurements in citrated whole blood ROTEM delta
measurements on healthy dogs and dogs with haemostatic alterations fulfil these
requirements with only a few exceptions concerning CFT and ML values in abnormal
samples
Our results regarding repeatability are similar to those obtained for several TEM
parameters in previous studies performed in animals In one study the imprecision of the
ROTEM analyser was evaluated by duplicate measurements in 10 blood samples of
healthy dogs and as result of that evaluation a good precision (CVs below 10 ) was
calculated for CT CFT MCF and α angle but in contrary to our results a low
repeatability was found for ML (CVs of 233 [ex-tem] and 285 [in-tem] own values
534 and 216 ) (Falco et al 2012)
In a previous feline study using ex-tem and in-tem as activating reagents for the ROTEM
delta repeatability was assessed similarly to the present study based on fourfold
measurements of blood samples of two healthy individuals (Doumlderlein and Mischke
2015) The results also revealed CVs below 10 for most of the parameters evaluated
The partially low repeatability of ML values may reflect the vulnerability of this variable
which is not only influenced by the complex clot formation process but also by its
subsequent lysis Reproducibility of ex-tem or in-tem activated measurements of the
ROTEM delta analyser which was not investigated in our study was studied in blood
from healthy and unhealthy horses by two different operators using three different
ROTEM devices Results indicated acceptable reproducibility (Junge et al 2016)
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
27
42 Reference intervals
The number of 125 healthy dogs used in the present study to create reference intervals is
regarded adequate according to the guidelines suggested by the American Society of
Veterinary Clinical Pathology (ASVCP) and the National Committee for Clinical
Laboratory Standards (NCCLS) who recommend to include at least 120 individuals
(Friedrichs et al 2012) To the best of our knowledge reference intervals for the most
relevant ROTEMreg delta parameters obtained in an adequate number of samples of whole
citrated canine blood have not been published so far A previous study was performed on
a smaller number of 45 dogs (Falco et al 2012) The median values for reference
intervals obtained in the present study are partially similar to those calculated in the
mentioned study based on 45 healthy dogs (eg ex- tem CT 610 s450 s [Falco et al
2012own results] CFT 150 s 110 s α angle 620deg680deg MCF 530 m 610 mm ML
180 220 in-tem CT 235 s190 s CFT 136 s116 s α angle 660deg680deg MCF
560 mm590 mm ML 30 180 ) However in comparison to our results the
reference intervals in the mentioned study are slightly wider (eg ex-tem CT 29ndash92
s34ndash83 s [Falco et al 2012own results] CFT 540ndash275 s650ndash220 s α angle 470ndash
790deg540ndash790deg MCF 360ndash730 mm460ndash730 mm ML 000ndash560 800ndash590
in-tem CT 126ndash363 s135ndash283 s CFT 470ndash224 s730ndash251 s aα angle 550ndash
810deg510ndash750deg MCF 500-ndash750 mm480ndash680 mm ML 000ndash400 500ndash270 )
although the previous study used the 5 and 95 quantiles to define limits of the
reference range whereas we calculated the commonly used 25 and 975 quantiles
to capture the central 95 of reference values (Friedrichs et al 2012)
Although it is desirable that each laboratory should specifically determine its own
reference intervals the results obtained in this study may provide an orientation for other
laboratories to interpret ROTEM delta parameters analysed in citrated canine blood In
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
28
order to test transference of the reference ranges to another laboratory a validation
procedure with measurement of 20 samples should be performed (Friedrichs et al 2012)
Although intra-individual control values (subject-based reference values) are regarded
superior to population based reference values (Wiinber et al 2007) the latter have to be
routinely used in clinical patients due to lacking individual controls especially for specific
tests such as ROTEM analyses In addition the calculated reference values cannot be
used for the fully automated device ROTEM sigma because - according to an actual
human study - values of part of the parameters do not correlate well between both
instruments (Gillissen et al 2019)
43 Influence of size age sex and neuter status
In previous studies in humans (Attard et al 2013) and animals (Mischke 1994 Borrelli
et al 2017) age has been described as an influencing factor for different haemostasis
parameters We also found an influence of age on the CT with shorter times in senior
dogs (gt 84 months) compared with young dogs (up to 24 months) which was limited to
measurements activated by the in-tem reagent
This age dependency confirms in principle a previous study on 20 healthy dogs
(Barthelemy et al 2015) which found shorter CTs in elderly dogs and results of a human
study where individuals with an age gt 45 years showed shorter CTs compared with
individuals lt 45 years old (Sucher et al 2011) However in the cited studies this effect
was only seen when ex-tem but not when the in-tem reagent was used
Based on the age distribution of our dogs which were primarily recruited to establish
reference intervals we cannot deliver information regarding very young dogs A human
study focusing on ROTEM reference ranges in children demonstrated significant
differences for nearly all ROTEM parameters with the most striking differences in
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
29
subjects aged 0-3 months (Oswald et al 2010) The limited number of dogs with an age
lt 1 year which did not allow to consider these separately in the present study may also
be one reason for the different results in a feline study (Doumlderlein and Mischke 2015) In
the cited study on healthy cats age showed an influence on CT but contrary to our study
the CT was shorter in young individuals (6-12 months) compared with older ones In
addition this was only the case in measurements using kaolin and in non-activated
measurements When compared to humans and cats uni- or multivariate analyses
considering factors age and breed or size based on do populations including different
breeds have the limitation that they can be biased by the variate life expectancy between
small medium and large dog breeds (Willems et al 2017) Apart from age we found a
significant influence only for the neuter status in bitches reflected by a significant
interaction effect between factors neuter status and sex in the ANOVA analysis
Especially for CFT values (eg activated by ex-tem median values of 121 s [spayed] vs
96 s [intact]) this difference seems to be also clinically relevant with respect to the
interpretation of ROTEM results In contrast although also significantly different
absolute and relative differences of CT values were small (eg activated by ex-tem 46 s
[spayed] vs 43 s [intact]) These differences may not only be considered for the result
interpretation of clinical patients but indicate also the necessity for sex-matched patient
groups in scientific studies
The obviously more active haemostasis system in intact females is probably related to an
influence of sex hormones (specifically estrogens) on the coagulation system
Accordingly ex vivo and in vivo studies demonstrated the activating properties of
endogenous estrogens on the haemostasis system In one ex vivo study addition of
endogenous estrogens (estrone [E1] 17szlig-estradiol [E2] and estriol [E3]) to blood samples
from men resulted in shorter mean values of the reaction time in TEG analyses
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
30
corresponding to CT in ROTEM measurements (452-537 s vs 698 s [control]) and of
the TEG variable kinetics (corresponding to the CFT in ROTEM analyses) (Swanepoel
et al 2017) In a further study subcutaneous injection of E2 into male rats induced a
significant increase in activities of coagulation factors X (by 8 ) and XI (by 10-24 )
however did not show any significant effect on PT aPTT and factor VII activity (Franco-
Murillo and Jaimez 2017)
In the cited studies also the effects of the synthetic estrogen ethinylestradiol [EE] were
studied which interestingly seems to have an inhibiting effect as indicated by prolonged
reaction time and kinetics (TEG) compared with the control in the human ex vivo study
and significantly longer PTs and aPTTs and lower factor VII and X activities in the study
on rats As expected we did not find a significant influence of size on ROTEM parameters
in dogs To the best of the authoracutes knowledge size has not been examined as an
influencing factor on viscoelastic measurements or any other haemostatic variable in
dogs Apart from Greyhounds there is limited literature on breed comparisons of
haemostatic variables (Clemons and Meyers 1984 Mischke 1994 Nielsen et al 2007
Nielsen et al 2010 Boyd et al 2018) indicating very limited breed differences for
example for factor VII (Mischke 1994) However these also do not indicate systematic
influence of size and in addition possible breed-specific effects have to be considered
As already stated one limitation of the study is the limited number of young animals
which does not allow to verify reference values for animals in this stage of life In
addition with respect to the main aim of the study possible influencing factors such as
platelet count and haematocrit were not considered in the statistical analysis Finally the
relatively small number of dogs used for the precision analysis which express the fact
that this was only a secondary aspect of the study may be regarded as a limitation At the
same time these considerations provide space for further investigations
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
31
5 Conclusions
Results of the precision analysis revealed acceptable results and thereby confirm that the
ROTEMreg delta analyser delivers repeatable results to measure overall haemostasis in
canine citrated blood samples The established reference values should offer a reliable
basis for the interpretation of thromboelastometric measurements in whole blood using
the device ROTEMreg delta in dogs with suspected haemostasis disorders Neuter status
in female dogs appeared to be a relevant influencing factor on most important ROTEMreg
parameters and should be considered for result interpretation and with respect to
experimental designs
6 Acknowledgments
Jose Mauricio Pereira received a scholarship from the Universidad Nacional de Costa
Rica (reference code JB-C-1107-2016)
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
32
7 References
1 Attard C van der Straaten T Karlafitis V Monagle P Ignjativic V 2013 Developmental
hemostasis age-specific differences in the levels of hemostatic proteins J Thromb and
Haemost 11 1850ndash1854
2 Bagge A Schoumltt U Kander T 2016 Effects of naturopathic medicines on Multiplate and
ROTEMreg A prospective experimental pilot study in healthy volunteers BMC Complemen
Altern Med 17 (16) 64
3 Bartheacutelemy A Rannou B Forterre M Verwaerde P Bonnet-Garin JM Pouzot-Nevoret
C Goy-Thollot I 2015 Differences between coagulation and cytokine profiles in dogs of
different ages Vet J 205 410ndash412
4 Bauer N Eralp O Moritz A 2009 Establishment of reference intervals for kaolin-activated
thromboelastography in dogs including an assessment of the effects of sex and anticoagulat use
J Vet Diagn Investi 21 641ndash648
5 Bollinger D Seeberger MD Tanaka KA 2012 Principles and practice of
thrromboelastography in clinical coagulation management and transfusio practice Transfus Med
Rev 26 1ndash13
6 Borrelli A Botto A Maurella C Falco S Pagani E Miniscaldo B Tarducci A Bruno
B 2017 Thromboelastometric assesment of hemostasis in newborn Piemontese calves J Vet
Diagn Invest 29 293ndash297
7 Boyd CJ Claus MA Raisis AL Hosqood G Sharp CR SmartL 2018
Hypocoagulability and platelet dysfunction are exacerbated by synthetic colloids in a canine
hemorrhagic shock model Front Vet Sci 5 279
8 Bruno B Maurella C Falco S Tarducci A Zanatta R DrsquoAngelo A Borrelli A 2015a
Thromboelastometric evaluation of hemostasis in dogs infected with Leishmania infantum J Vet
Emerg Crit Care (San Antonio) 25 502ndash511
9 Bruno B Maurella C Falco S Tarducci A Zanatta R Gianella P DrsquoAngelo A Piras L
Di Bella A Borrelli A 2015b Assessment of coagulation utilizing thromboelastometry in dogs
undergoing orthopedic surgery J Vet Emerg Crit Care (San Antonio) 25 358ndash363
10 Clemons RM Meyers KM 1984 Acquisition and aggregation of canine blood platelets Basic
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
33
mechanisms of function and differences because of breed origin Am J Vet Res 45 137ndash144
11 Conversy B Blais MC Dunn M Gara-Boivin C del Castillo JRE 2017 Anticoagulant
activity of oral rivaroxaban in healthy dogs Vet J 223 5ndash11
12 Crochemore T Piza FMT Rodriacuteguez RDR Guerra JCC Ferraz LJR Correcirca TD
2017 A new era of thromboelastometry Einstein (Sao Paulo) 15 380ndash385
13 Crochemore T Correcirca TD Lance MD Solomon C Neto AS Guerra JCC Lellis PS
Bernz LM Nunes N Mancio CM Yokoyama APH Silva E 2018 Thromboelastometry
profile in critically ill patients A single-center retrospective observational study PLoS One 13
e0192965
14 Doumlderlein E Mischke R 2015 Reference intervals for thromboelastometry with the ROTEMreg
delta in cats Res Vet Sci 100 271ndash276
15 Falco S Bruno B Maurella C Bellino C DacuteAngello A Gianella P Tarducci A Zannata
R Borrelli A 2012 In vitro evaluation of canine hemostasis following dilution with
hydroxyethyl starch (13004) via thromboelastometry J Vet Emerg Crit Care (San Antonio)
22 640ndash645
16 Franco-Murillo Y Jaimez R 2017 Inhibitory effect of ethinylestradiol on coagulation factors
in rats Exp Anim 66 107ndash113
17 Friedrichs KR Harr KE Freeman KP Szladovits B Walton RM Barnhart KF Blanco-
Chavez J 2012 ASVCP reference interval guidelines Determination of de novo reference
intervals in veterinary species and other related topics Vet Clin Pathol 41 441ndash453
18 Gillissen A va den Akker T Caram-Deelder C Henriquez DDCA Bloemenkamp
RWM Eikenboom J van der Bom JG de Maat MPM 2019 Comparison of
thromboelastometry by ROTEMreg Delta and ROTEMreg Sigma in women with postpartum
haemorrhage Scand J Clin Lab Invest 79 32ndash38
19 Harttert H 1948 Blutgerinnungstudien mit der Thrombelastographie einem neuen
Untersuchungsverfahren Klin Wochenschr 26 577ndash583
20 Horn P Pesce A 2003 Reference intervals and update Clin Chim Acta 334 5ndash23
21 Hua Yanhun Qiu W Xiao Q Wu Q 2018 Precision (repeatability and reproducibility) of
ocular parameters obtained by the Tomey OA-2000 biometer compared to the IOLMaster in
healthy eyes PLoS One 13 e0193023
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
34
22 Junge H Ringer S Mayer N Schwarzwald C 2016 Assesment of method reliability and
determination of reference intervals for rotational thromboelastometry in horses J Vet Emerg
Crit Care (San Antonio) 26 691ndash703
23 Kol A Borjesson DL 2010 Application of thrombelastographythromboelastometry to
veterinary medicine Vet Clin Pathol 39 405ndash416
24 Marschner CB Wiinberg B Tarnow I Markussen B Kuumlhnel L Bochsen L Kristensen
AT 2018 The influence of inflammation and hematocrit on clot strength in canine
thromboelastographic hypercoagulability J Vet Emerg Crit Care (San Antonio) 28 20ndash30
25 McMichael MA Smith SA 2011 Viscoelastic coagulation testing technology applications
and limitations Vet Clin Pathol 40 140ndash153
26 Mischke R 1994 Activity of coagulation factors II V VII and X in healthy dogs- dependence
on age sex and breed Berl Muumlnch Tieraumlrztl Wochenschr 107 289ndash294
27 Nielsen A Zois N Pedersen H Olse L Tarnow I 2007 Platelet function in dogs bread
differences and effect of acetylsalicylic acid administration Vet Clin Pathol 36 267ndash273
28 Nielsen L Kjelgaard‐Hansen M Jensen A Kristensen A 2010 Breed‐specific variation of
hematologic and biochemical analytes in healthy adult Bernese Mountain dogs Vet Clin Pathol
39 20ndash28
29 Nogami K 2016 The utility of thromboelastography in inherited and acquired bleeding
disorders Br J Haematol 174 503ndash514
30 Oswald E Stalzer B Heitz E Weiss M Schmugge M Strasak A Innerhofer P Haas T
2010 Thromboelastometry (ROTEMreg) in children Age-related reference ranges and correlations
with standard coagulation tests Br J Anaesth 105 827ndash835
31 Sankarankutty A Nascimento B Teodoro da Luz L Rizoli S 2012 TEGreg and ROTEMreg
in trauma similar test but different results World J Emerg Surg 7 (Suppl 1) S3
32 Sigrist NE Schefer RJJ Kutter APN 2018 Characteristics of hyperfibrinolysis in dogs and
cats demonstrated by rotational Thromboelastometry (ROTEM) Vet J 242 67-73
33 Steiner J Teague S Williams D 2003 Development and analytic validation of an enzime-
linked immunosorbent assay for the measurement of canine pancreatic lipase immunoreactivity in
serum Can J Vet Res 68 161ndash168
34 Sucker C Tharra K Lithmate J Scharf RE Zotz RB 2011 Rotation thromboelastography
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
35
(ROTEM) parameters are influenced by age gender and oral contraception Perfusion 26 334ndash
340
35 Swanepoel A Emmerson O Pretorius E 2017 The effect of endogenous and synthetic
estrogens on whole blood clot formation and erythrocyte structure Microsc Microanal 23 599ndash
606
36 Theusinger OM Nuumlrnberg J Asmis LM Seifert B Spahn DR 2010 Rotation
thromboelastometry (ROTEM) stability and reproducibility over time Eur J Cardiothorac Surg
37 677ndash683
37 Toulon P Metge S Hangard M Zwahlen S Piaulenne S Besson V 2017 Impact of
different storage times at room temperature of unspun citrated blood samples on routine
coagulation tests results Results of a bicenter study and review of the literature Int J Lab
Hematol 3 458ndash468
38 Turner JS Kutter APN Sigrist NE 2019 Correlation of rotational thrromboelastometry
(ROTEM) parameters with platelet count and their ability to predict thrombocytopenia in dogs
Res Vet Sci 126 45ndash50
39 Versteeg HH Heemskerk JWM Levi M Reitsma PH 2013 New Fundamentals in
Hemostasis Physiol Rev 93 327ndash358
40 Wiinberg B Jensen AL Kjelgaard-Hansen M Rojkjaer R Johansson PI Gade LP
Gram DX Kristensen AT 2007 Study on biological variation of haemostatic parameters in
clinically healthy dogs Vet J 174 62ndash68
41 Wiinberg B Jensen AL Johansson PL Rozanski E Tranholm M Kristensen AT 2008
Thromboelastographic evaluation of hemostatic function in dogs with disseminated intravascular
coagulation J Vet Intern Med 22 357ndash365
42 Wikkelsoslash A Wetterslev J Moslashller AM Afshari A 2016 Thromboelastography (TEG) or
thromboelastometry (ROTEMreg) to monitor haemostatic treatment versus usual care in adults or
children with bleeding Cochrane Database Syst Rev Art
httpsdoiorg10100214651858CD007871pub3 No CD007871
43 Willems A Paepe D Marynissen S Smets P Van de Maele I Picavet P Duchateau L
Daminet S 2017 Results of screening of apparently healthy senior and geriatric dogs J Vet
Intern Med 31 81ndash92
Chapter 2 Reference intervals for rotational thromboelastometry measurements using
the ROTEMreg delta device in dogs
36
44 Woodman J Wagg CR Boysen SR Leguillete R Mizen K Roy MF 2015 Evaluation
of coagulation via thromboelastography in healthy horses administered dexamethasone Can Vet
J 56 1271ndash1274
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
37
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of
standard heparin prophylaxis and correlation to haemostatic changes
Pereira JM Hewicker-Trautwein M Rohn K von Depka Prondzinski M
Mischke R
Contributions of the authors
Pereira JM performed (1) the ultrasound evaluations and blood sampling of the
patients under study (2) haematology analysis (3) injection of the anticoagulation
prophylaxis (partially) to the patients under study (4) analysis of samples (collaboration)
at Werlhof Institute Medical Care Center (5) analysis of some haemostasis test
(collaboration) (6) statistical analysis (partially) (7) data documentation (8) and wrote
the manuscript
Hewicker-Trautwein M performed the electron microscopy analysis
Rohn K performed the statistical analysis
von Depka Prondzinski M performed some haemostasis test (thrombin generation
assay and endogenous thrombin potential)
Mischke R performed the coagulation and haemostasis test was involved in designing
and supervision of the study analysis and discussion of the data and its statistical analysis
and revision edition and correction of the manuscript
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
38
Central venous catheter induced thrombosis in dogs ndash efficacy of standard heparin
prophylaxis and correlation to haemostatic changes
Joseacute Mauricio Pereira1 Marion Hewicker-Trautwein2 Karl Rohn3 Mario von Depka
Prondzinski4 Reinhard Mischke1
1Small Animal Clinic University of Veterinary Medicine Hannover Foundation
Buumlnteweg 9 D-30559 Hannover Germany
2Institute for Pathology University of Veterinary Medicine Hannover Foundation
Buumlnteweg 17 D-30559 Hannover Germany
3Institute of Biometry Epidemiology and Information Processing University of
Veterinary Medicine Hannover Foundation Buumlnteweg 2 30559 Hannover Germany
4Werlhof Institute Schillerstrasse 23 30159 Hannover Germany
Abbreviations anti-Xa anti-factor Xa BW body weight CVC central venous catheter
EJV external jugular vein ELMI electron microscopy IV intravenous JV jugular vein
LMWH low molecular weight heparin ML maximum lysis (ROTEM) PIVC
peripheral intravenous catheter ROTEM rotational elastometry SC subcutaneous
TGA thrombin generation assay TID three times a day TT thrombin time UFH
unfractionated heparin
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
39
Corresponding author
E-mail addresses matiscrgmailcom (JM Pereira) MarionHewicker-
Trautweintiho-hannoverde (M Hewicker-Trautwein) KarlRohntiho-hannoverde
(K Rohn) mailwerlhof-institutde (M von Depka Prondzinski)
ReinhardMischketiho-hannoverde (R Mischke)
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
40
ABSTRACT
Central venous catheters (CVC) are of importance for intensive care management of
canine patients Thrombosis of the jugular vein is a severe complication related to the
CVC use However only little is known about the incidence of CVC-associated
thromboses in dogs and the necessity and efficacy of prophylactic antithrombotic
regimens The objectives of the present study were (1) to determine the incidence of
catheter-induced thrombosis in canine intensive care patients and thereby (2) to evaluate
the efficacy of a routine prophylactic heparin treatment and finally (3) to assess whether
initial changes of selected haemostasis parameters are predictive for an increased risk for
thrombosis formation 29 dogs hospitalised in the Small Animal Clinic University of
Veterinary Medicine Hannover Foundation and receiving a central venous catheter
(CVC) in the external jugular vein (EJV) for medical reasons were included in the final
assessment 17 dogs received the standard antithrombotic dosage regimen of
unfractionated heparin (UFH 150 IUKG BW TID sc) and 12 dogs (usually surgical
patients) the reduced dosage (75 IUKG BW TID sc) A colour Doppler ultrasound of
the respective EJV in order to detect thrombus formation and blood collection for
haemostasis tests were performed immediately before the installation procedure (day 0)
and on days 1 3 5 7 and so on after the CVC insertion Haemostasis tests included
prothrombin time activated partial thromboplastin time thrombin time antithrombin
activity D-dimer concentration thrombin generation and rotational elastometry as well
as the heparin activity using a chromogenic anti-factor Xa test Finally electron
microscopy (ELMI) of sections of the removed CVC was performed In 8 dogs
sonographic examination revealed thrombus formation in the lumen of EJV and in 1927
CVCs assessed using ELMI thrombi on the external CVC surface were detected without
significant correlation between both methods Plasmatic heparin activities showed great
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
41
variability but no significant differences were found between dogs with or without CVC-
associated thrombus formation From the initially performed haemostasis tests high
fibrinogen concentrations and low maximum lysis values as measured by the ROTEM
delta device (ex-tem reagent) were associated with sonographically detectable thrombi
The results of the present study indicate that CVC-associated thromboses occur (eg 28
[829] of the patients as detected by ultrasound) although severe thromboses (high
grades) are relatively rare in intensive care canine patients receiving a routine
anticoagulant treatment A high fibrinogen concentration and low maximum lysis
(ROTEM ex-tem) may be useful indicators for an increased thrombotic risk in individual
patients which may require special anticoagulatory treatment
Keywords CVC complication venal occlusion antithrombotic treatment ultrasound
electron microscopy rotational elastometry
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
42
1 Introduction
Central venous catheters (CVCs) are very important tools for care of patients in critical
care units allowing parenteral nutrition support therapy administration of high volumes
of intravenous fluids safe drugs administration that cannot be given through peripheral
veins plasmapheresis haemodialysis blood transfusions as well as periodical blood
sampling (Geerts 2014 Vose et al 2019) CVC related thrombosis is one of the most
severe complications (apart from infections and pneumothorax) of CVCs in humans
(Geerts 2014 Comerlato et al 2017 Evans and Ratchford 2018) leading to patientrsquos
distress CVC dysfunction venous stenosis and in some cases to the development of
thromboembolism (Geerts 2014)
In the human literature there are various studies dealing with epidemiology with reference
to different anticoagulatory regimens including placebo-controlled studies (Lee and
Kamphuisen 2012) In humans CVC related thromboses represent 70ndash80 of all upper
extremity thrombosis cases and approximately 10 of thromboembolism cases in
humans (Kucher 2011) The incidence of CVC related thrombosis reported in humans
based on 25 studies ranged widely from 2 to 67 (median = 30 ) (van Rooden et al
2005 review article) This wide range of calculated incidences of CVC related
thromboses in humans reflects the use of different CVC types different locations of entry
site of the CVC (jugular femoral subclavian axillary cephalic vein) different thrombus
detection technique (venography or ultrasonography) unequal durations of follow up
varying diseases of patients (eg haematological oncological) and presence of
predisposing factors (eg previous venous thrombosis history presence of cancer) (van
Rooden et al 2005 Geerts 2014 Liu et al 2015)
Part of the authors empirically observed CVC-associated thrombosis of the external
jugular vein (EJV) as a relevant complication in their canine intensive care patients which
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
43
awakened their interest in that topic To the best of the authoracutes knowledge studies about
extra luminal CVC related thromboses in dogs are rare in the available veterinary
literature One study evaluating the complications associated with 100 CVCs placed in
the jugular vein of dogs and cats reported only 2 cases of thrombosis (confirmed by
palpation or ultrasound) but obviously did not assess the appearance of subclinical CVC
related thromboses (Adamantos et al 2010) Thereby the incidence of CVC-induced
thromboses in dogs is unknown and systematic studies regarding an effective
anticoagulatory management are lacking
In this context it is of interest whether a possible development of thrombosis or an
increased risk (despite the prevention by a standard application of unfractionated heparin
[UFH]) can be predicted by means of changes of haemostasis related parameters during
the initial examination In one study on 168 human patients without indication of
anticoagulation prophylaxis for example elevated leukocyte counts high plasminogen
activator inhibitor and high coagulation factor VIII levels (all measured on blood samples
taken after CVC placement) were associated with an increased incidence of symptomatic
CVC-related thrombosis diagnosed by ultrasonography (Boersma et al 2016)
Therefore aims of the present study were (1) to determine the incidence of catheter-
induced thrombosis in canine intensive care patients (as detected by ultrasonography and
electron microscopy ELMI) and thereby (2) to evaluate the efficacy of a standard
antithrombotic regimen with unfractionated heparin (UFH) routinely used in the Small
Animal Clinic of the University of Veterinary Medicine Hannover and (3) to assess
whether selected haemostasis test results are predictive for an increased risk for
thrombosis formation
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
44
2 Material and methods
21 Study design
Canine in-patients of the Small Animal Clinic University of Veterinary Medicine
Hannover receiving a CVC in the EJV for medical reasons from March 2017 to
December 2019 were included Anticoagulatory treatment was performed according to
the standard protocol of the clinic Colour Doppler ultrasonographic controls of the EJV
and blood samplings (for heparin activity basic haematology and haemostasis tests) were
done immediately before the catheter was placed (day 0) and on days 1 3 5 etc (2 hours
after the first daily UFH injection) until the CVC was removed Only patients were
included which could be examined at least until day 3 Haemostasis tests included
prothrombin time activated partial thromboplastin time thrombin time fibrinogen
concentration antithrombin activity fibrin monomers D-dimers thrombin generation
assay (TGA) and rotational elastometry as well as the heparin activity using a
chromogenic anti-factor Xa test Finally ELMI of sections of the CVC after its removal
was performed
22 Animals
Originally 32 patients hospitalised in the Small Animal Clinic and receiving a CVC in the
EJV for medical reasons entered the study Three of them were subsequently excluded
because they did not reach the defined minimum study period of 3 days Two were
euthanised and one referred back to the referring vet before day 3 22 dogs weighing more
than 15 kg were included so that the withdrawal of about 10 ml of blood on several days
was not a significant burden for the patient (lt 1 of the total blood volume) Five dogs
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
45
with a body weight of less than 15 kg but at least 8 kg were included with special
consideration of the amount of blood taken (no more than 6 ml on individual days)
All animals received standard anticoagulatory treatment with UFH 17 dogs with internal
diseases received 150 IUkg BW TID subcutaneously and 12 dogs (post surgery) received
a reduced dosage of 75 IUkg BW TID subcutaneously
The 29 dogs enclosed in the study included 4 mixed breed dogs and the following
pedigree dogs 5 Labrador Retrievers 3 German Shepherds 2 Australian Shepherds and
one from each of the following breeds Akita Inu Asian Shepherd Bavarian Mountain
Hound Beagle Belgian Shepherd Briard Canaan dog Dalmatian Golden Retriever
Havanese dog Hovawart Rhodesian Ridgeback Schnauzer Shar Pei and Wolfspitz
These dogs were aged between 2 and 164 months (median 77 months) and had body
weights ranging from 81 and 63 kg (median 27 kg) 9 were castrated males 8 intact
males 7 spayed females and 5 intact females
The diseases of the 29 patients requiring a CVC included 3 from each of the following
diseases hepatopathy and esophagus dilatation 2 from each of the following diseases
bacterial cystitis diabetes mellitus and 1 from each of the following diseases abdominal
wall perforation aspiration pneumonia cystitis intestinal round cells sarcoma carcinoma
of the nasal cavity chronic renal insufficiency cystic duct rupture heartworm disease
ivermectine intoxication lymphoma multifocal alveolar histiocytosis pancreatitis post-
surgical bleeding stomach dilatation stomach foreign body tetanus thoracic
pyogranulomatous inflammation thrombocytopenia and trigeminal nerve paresis
Animals were treated in accordance with the German Animal Welfare Law The
experimental design was approved by the Animal Welfare officer of the University of
Veterinary Medicine and reported to the responsible authority (Lower Saxony State
Office for Consumer Protection and Food Safety reference number 17A101)
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
46
23 Positioning and handling of the central venous catheter
Two types of single lumen radiopaque polyurethane CVC (different on length and
diameter) were used during the study The CVC type I (n = 2529) was Cavafixreg Certoreg
Splittocanreg CVC 32 cm length 11 x 17 mm diameter16 G (BBraun Melsungen AG)
the CVC type II used (n = 429) was Certofixreg Mono S 215 15 cm length 08 x 14 mm
diameter18G (BBraun Melsungen AG) Each CVC was inserted into the EJV using a
modified Seldinger technique as follows the skin of the neck was shaved and disinfected
with antiseptic soap alcohol and iodine Local anesthesia (Lidocaine HCl 2) was
administrated subcutaneously on the catheter insertion site (a middle point in the lateral
neck over the jugular sulcus) A small skin incision was performed in the insertion site
and one peripheral plastic cannula with a metallic needle inside (14 G for CVC type I
and 20 G for CVC type II) was inserted in the EJV in cranio-caudal orientation (Hundley
et al 2018) Once the EJV was reached the needle was removed and a flexible wire
guide was inserted through the peripheral cannula following an external landmark
(4thintercostal space) (Reminga et al 2018) The peripheral cannula was removed and
the respective CVC was inserted slowly and with gentle pressure into the EJV guided
with the flexible wire and following the same external landmark to reach the right
position After the insertion of the CVC the flexible wire guide was removed The correct
positioning of the CVC was checked with a latero-lateral thorax x-ray ensuring that the
CVC tip was placed at the junction of the cranial cava vein with the right atrium Each
CVC was fixed to the neck skin (adjacent to the insertion point) with a monofilament
non-absorbable suture (gauge 3-0) Finally a bandage elaborated with Povidone-Iodine
gauze (Betaisodonareg) padded synthetic band (Artiflexreg Natur) and elastic band
(Elastomullreg) was used to protect the CVC output (Claude et al 2010) and changed
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
47
daily Several times day the catheter was flushed with isotonic sodium chloride
containing 1 IUml UFH
24 Heparin preparation and administration
Heparin-sodium-25000-ratiopharm (injection solution containing 5000 IUml sodium
heparin [mucosa]) was used SC injection was performed over the lateral thorax wall
using 1 mL syringes with fine graduation marks
25 Ultrasonography
Colour Doppler ultrasonographic examinations were performed in lateral position using
a LO-GIQ E9 ultrasound device (GE Healthcare Chicago Illinois USA) with a 15
Megahertz linear ultrasound probe The development of a thrombus or fibrin debris in the
EJV lumen around the CVC or inside it was assessed at each control Once the
ultrasound was finished a new protective bandage was placed around the neck of the dog
According to the percentage of lumen occlusion of the EJV (evaluated in cross sectional
views during the ultrasound scanning) the thrombi were classified in four different grades
(Fig 1)
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
48
of the residual vein lumen C ndash catheter T ndash thrombus
Fig 1 Definition of different grades of central venous catheter induced thrombosis of the
jugular vein as detected using colour Doppler ultrasound
26 Blood collection
At each time point approx 6ndash10 ml of blood (according to the body weight) was collected
This included approx 1 ml of EDTA blood for hematological analysis and approx 5ndash9
ml of citrated blood (tubes containing one part of 011 molL [38 ] sodium citrate to
nine parts of blood) for the measurement of hemostasis tests in citrated blood (ROTEM)
or citrated plasma Blood samples were collected by puncture of peripheral veins with
disposable cannulas (09 x 40 mm [20G] or 10 x 40mm [21G]) directly into sample tubes
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
49
or from the CVC using the three-syringe-technique (Villalta-Garciacutea et al 2015) in
situations when the peripheral venipuncture was not indicated due to phlebitis or irritation
andor inflammation of the surrounding skin Briefly 2ndash3 ml of blood were taken in a
sterile disposable syringe and immediately afterwards the blood sample was taken in a
new disposable syringe and transferred into the test tubes containing the respective
anticoagulants finally the first 2-3 ml taken were re-infused to the dog through the CVC
followed by flushing with a solution of sodium chlorideUFH (02) Immediately after
blood collection each sample tube was swayed gently until blood and anticoagulant had
been mixed thoroughly To prepare citrated platelet poor plasma citrated blood was
centrifuged at 16000 g for 10 min at room temperature using a microcentrifuge The
plasma was transferred into plastic tubes and the centrifugation procedure was repeated
This platelet poor plasma was then frozen in aliquots at -70 degC until used for analysis
Directly before analysis the plastic tubes were thawed at 37 degC in a water bath
27 Laboratory tests
Haematological analyses were made automatically with Adviareg 2120i (Siemens
Healthcare GmbH Germany)
Prothrombin time activated partial thromboplastin time and thrombin time were
measured with the autoanalyser Amax Destiny Plus (Tcoag Deutschland GmbH
Germany) and commercial reagents (Thromborel S Siemens Healthcare Diagnostics
Products GmbH Marburg Germany CK Prestreg and Test Thrombin Reagent
Diagnostica Stago SAS Asniegraveres sur Seine Cedex France) Whereas the last two tests
were measured according to the manufacturer`s instructions a modified test optimised
for canine plasma was used to measure prothrombin time (25 microl citrated plasma 120 with
imidazole buffer + 25 microl of a human fibrinogen solution 2 gl prewarmed for 120 s at 37
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
50
oC addition of 25 microl of activating reagent Thromborel S) and the results converted into a
percentage activity based on a calibration curve prepared with canine pooled plasma The
fibrinogen concentration was measured with the method according to Clauss using a test
kit from Stago Diagnostica SAS D-dimer concentration was measured with a
quantitative commercial human latex turbidimetric method in the autoanalyser Cobas
c311 using a completely assembled kit and human standards (Roche Diagnostics
Mannheim Germany) Antithrombin activity was also measured with a completely
assembled chromogenic test kit in the autoanalyser Cobas c311 and calibrated with a
canine pool plasma (n = 100)
The thrombin generation assay (Technothrombinreg TGA (Technoclone GbmH Vienna
Austria) determines thrombin generation based on monitoring of the fluorescence
generated by cleavage of a fluorogenic substrate by thrombin over time after activation
of the coagulation cascade by different concentrations of tissue factor and negatively
charged phospholipid in platelet poor plasma From the fluorescence development over
time the concentration of thrombin in the sample can be calculated using a thrombin
calibration curve The increase in thrombin concentration with time then allows to
calculate generation of thrombin in the sample and to plot thrombin values over time for
the whole coagulation process
The rotational thromboelastometry analysis with ROTEM delta device (Werfen Munich
Germany) was started approx 15 minutes after the blood collection To perform the
analysis and according to the automated ROTEM software 20 microl of recalcification reagent
(200 mmoll calcium chloride solution star-tem) and 20 microl of the respective activating
reagent (ex-tem in-tem) were pipetted into and carefully mixed in a pre-warmed cuvette
using the automated pipette provided in the device immediately afterwards 300 microl of
citrated whole blood (previously gently revolved) were added and mixed with the
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
51
reagents previously deposited Finally the cuvette was carefully placed and fixed to an
oscillating pin (oscillation movements on left and right through an angle of 475deg) in the
ROTEM delta device The interaction between blood cells and subsequently the bond
between fibrin and platelets promoted by the biomechanical changes in pH electrolytes
and temperature start the clot formation with its subsequent retraction and lysis The
magnitude of the resistance produced by the clot over the oscillating pin is quantitatively
represented by different parameters measured during the analysis and also qualitatively
in a graphic illustration (Bagge et al 2016 Crochemore et al 2017)
A chromogenic substrate test (Coatest Heparin Chromogenix-Instrumentation
Laboratory Haemochrom Diagnostica Essen Germany) was used for measurements of
UFH in the auto-analyser Cobas c311 The test application was created based on
manufacturers` instructions Different dilutions of the same batch of the commercial
sodium UFH preparation used in the experiment with normal canine pool plasma
(prepared from identical aliquots of 10 healthy adult dogs) served as standards for the
calibration of the measurement
28 Electron microscopy
Selected areas on the last third of the CVC (5ndash10 cm according with the CVC) with
macroscopically detectable thrombi were analysed and some of the CVCs were cut in
two different places The cut samples were preserved in a fixative medium composed of
1 glutaraldehyde and 4 formalin Once the sample was fixed it was prepared
according to the osmium thiocarbohydrazide procedure (OTOTO-procedure) (Malick et
al 1976) The fixed CVCs were dehydrated in seven series as follow first three series of
120 minutes each one on 30 50 and 70 ethanol respectively followed by two series
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
52
of 120 min each one on 90 ethanol and then two series of 24 hours each one on 100
ethanol After the dehydration process the samples were placed two times during 24
hours each one on isoamyl acetate and then placed on in a critical point drier Finally the
samples were coated with gold in a splutter-coater with 25 kV acceleration voltage in an
argon atmosphere with a current of 20 mA for one minute The samples were examined
in an EVO 15 scanning electron microscope (ZEISS International Carl Zeiss Jena
Germany) and photographed with magnifications of 600x and 6000x avoiding mechanical
artifacts (Toskala et al 1995) Thrombi on the external wall of the CVC were analysed
and measured using the software ImageJ (National Institutes of Health and the Laboratory
for Optical and Computational Instrumentation University of Wisconsin USA)
According to their extension (surface size) and thickness (related to the CVC wall
thickness in a cross sectional view) thrombi on the external wall and additionally within
the lumen of the CVC were each classified in four grades (Fig 2 Fig 1S [suppl
material]) In case of two cuts the highest degree of thrombosis detected at one of the two
sites was used for the final assessment
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
53
surface sizepercent of CVC wall thickness
Fig 2 Definition and illustration of different grades of central venous catheter induced
(extraluminal) thrombosis as detected using electron microscopy
Fig 1S (suppl material) Definition and illustration of different grades of intraluminal
thrombosis in central venous catheters as detected using electron microscopy
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
54
29 Statistical methods
Comparison between thrombus graduation either based on sonographic and ELMI
findings respectively was performed using the McNemar test with calculation of the
concordance index (κ) Standard normal distribution was assessed using the
Kolomogorov-Smirnov test
Due to non-normal distribution of part of data sets heparin activities and results of
haemostasis assays are reported using non-parametric variables (median minimumndash
maximum box and whisker plots) Heparin activities and results of haemostasis tests
between dogs with or without thrombosis were compared using Mann-Whitney-U test
3 Results
31 Sonographic detection of thrombus formation
Thrombus formation was sonographically detected in 829 patients during the study
period Based on examination on the final day in half of these eight patients (n = 4) the
thrombus formation was scored grade 1 in 3 dogs grade 2 and only in one grade 3 In all
of these 8 dogs this was the maximum grade during the study period One dog with fibrin
precipitates was excluded from further analysis because it could not be clearly assigned
to one of the groups In 78 cases thrombi were attached mainly to the venous wall
whereas only in one case was attached to the CVC external wall Seven of these 8 dogs
which developed thrombi received the standard heparin dosage (150 IUkg TID 41
[717] of dogs receiving this dosage) and only one the reduced heparin dosage (75 IUkg
TID 8 [112] of dogs receiving this dosage)
The time point of first detection of thrombus formation varied between days 3 and 11
(median day 8) In addition detritus (presumably composed mainly of fibrin) was
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
55
detectable in the lumen of the EJV in one further dog and within the lumen of the CVC
in further 5 dogs
32 Thrombus formation by electron microscopy
27 from 29 CVC catheter tips were analysed by electron microscopy 827 samples were
without detectable thrombus (grade 0) and 1927 with detectable thrombi 72 showed
thrombus formation of grade 1 427 of grade 2 and 827 of grade 3 There was no
concordance between thrombus graduation based on sonographic and the ELMI
graduation respectively The concordance index (McNemar test) was negative (κ = -
00718) indicating that the degree of agreement is less than by chance
Of the in total 19 dogs with detectable thrombus formation by electron microscopy 11
dogs received 150 IUkg TID (in total 16 dogs of these 27 dogs received that heparin
dosage) and 8 received 75 IUkg TID (in total 11 of these 27 dogs received that heparin
dosage 73 811) and Of the 12 patients with grade 2 and 3 thrombus formation 5 dogs
received 75 IUkg TID and 7 dogs 150 IUkg
The supplementary analysis of intraluminal thrombus formation revealed no detectable
thrombi in 7 cases and thrombus formation of grade 1 (lt 25 occlusion) in 11 cases of
grade 2 (25ndash50 ) in 3 cases and of grade 3 (gt 50 ) in 6 cases
33 Heparin activities and relationship to thrombus formation
Heparin activities varied remarkably in both dosage groups (Fig 3 ab) There was no
significant difference of heparin activities between blood samples collected via
venipuncture or from the CVC (Table 1S supplementary material) In addition there was
no significant difference between heparin activities in dogs with or without
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
56
sonographically or electron microscopically detectable thrombosis (Tables 1 and 2)
Table 1 refers to sonographically detectable thromboses and is based on median heparin
activities calculated from the measurements on day 1 and following days per patient
firstly for the total number of patients and secondly for the patients receiving 150 IUkg
TID In addition in the eight dogs heparin activities on the day of first thrombus detection
(023 IUml 019ndash036 IUml) were not significantly different from the median values
(Table 1 P = 05214 Wilcoxon test) Table 2 compares heparin activities (median values
of all days and results on the final day) between dogs with and without electron
microscopically detectable CVC-associated thrombi
Table 1S (Supplementary material) Heparin activities (IUml median values
minimumndashmaximum) measured in 27 dogs receiving 75 or 150 IUkg unfractionated
(standard) heparin TID subcutaneously 2 hours after the first injection on the day
Comparison of samples collected via puncture of the peripheral veins and those collected
from the central venous catheter (CVC) using the three-syringe-technique
Peripheral vein
puncture CVC
Mann-Whitney-U
test
Total number
(75 and 150
IUkg TID)
019
(00ndash108)
(n = 65)
023
(000ndash105)
(n = 65)
P = 03033
75 IUkg TID
010
(000ndash108)
(n = 25)
012
(000ndash023)
(n = 21)
P = 06907
150 IUkg
TID
030
(000ndash096)
(n = 40)
036
(000ndash105)
(n = 44)
P = 01223
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
57
Fig 3ab Heparin activities (box- and whisker plot arithmetic means) in dogs receiving
unfractionated heparin at a dosage of 75 IUkg BW tid (a) or 150 IUkg BW tid (b) two
hours after the first daily heparin administration
day 1 represents the first day after initiation of heparin treatment
only days were considered with at least results from 5 patients
Table 1 Heparin activities (IUml median values minimumndashmaximum) in 28 dogs
receiving 75 or 150 IUkg unfractionated (standard) heparin TID subcutaneously with
(grades 1ndash3) or without (grade 0) sonographically detectable thrombus Blood collections
were performed 2 hours after the first daily injection
Grade 0
(sonography)
Grades 1ndash3
(sonography)
Mann-Whitney U
test
Total number
(75 and 150 IUkg
TID)
020 (000ndash072)
(n = 20)
027 (018ndash059)
(n = 8)
P = 03212
150 IUkg TID 042 (000ndash072)
(n = 10)
031 (018ndash059)
(n = 7)
P = 04593
one dog with fibrin precipitates in the lumen of the jugular vein but without defined thrombus formation
was not considered because it could not be clearly assigned to one of the two groups
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
58
Table 2 Heparin activities (IUml median values minimumndashmaximum) in 27 dogs
receiving 75 or 150 IUkg unfractionated (standard) heparin TID subcutaneously with
(score 1ndash3) or without (score 0) detectable thrombus via electron microscopy
Grade 0
(electron
microscop)
(n = 8)
Grades 1ndash3
(electron
microscop)
(n = 19)
Mann-Whitney U
test
Median value of
different days 028 (005ndash060)
022 (000ndash072)
P = 05770
Final day 020 (001ndash045)
015 (000ndash105)
P = 06706
34 Relationship between haemostasis tests and thrombus formation
From the initially performed haemostasis tests high fibrinogen concentrations and low
maximum lysis values as measured by the ROTEM delta device (using the ex-tem
reagent) were associated with sonographically detectable thrombi (Tab 3) Additional
analyses based on the median values of different days for individual patients revealed that
low maximum lysis values as measured by the ROTEM delta device (using the in-tem
reagent) were associated with sonographically detectable thrombi (maximum lysis score
0 850 [100ndash175 ] score 1ndash3 375 [100ndash900 ] median [minimumndash
maximum] P = 00166) but no other test Comparison of the results of haemostasis tests
on the final day between dogs with and without sonographically detectable thromboses
did not show any significant difference (P gt 005)
Comparison of results of haemostasis tests on the final day in dogs with detectable
thrombosis via ELMI and dogs without significant thrombus formation showed several
significant differences including lower fibrinogen concentrations and longer clotting
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
59
times (ex-tem reagent) and clot formation times (ex-tem and in-tem reagents) using the
ROTEM delta analyser in general expressing a less coagulable status (Tab 4) If values
of dogs graded 0 and 1 (n =15) are compared to those of grades 2 and 3 (n = 12) the last-
mentioned had longer clotting times (ROTEM delta in-tem reagent) as well as longer clot
formation times and lower alpha angles (ROTEM delta in-tem reagent) (P lt 005 results
not shown)
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
60
Table 3 Comparison of initial results of haemostasis parameters (median values
minimumndashmaximum in dogs with (grades 1ndash3) and without (grade 0) sonographically
detectable central venous catheter-induced thromboses
Parameter Unit
Sonographic
thrombus grade
P-value
(Mann-Whitney
U test)
0
(n = 20)
123
(n = 8)
Haematocrit 355 (228ndash641)
382 (245ndash499)
06111
Platelet count x103microl 292 (420ndash483)
218 (860ndash458)
02320
Prothrombin time 944 (359ndash169)
896 (471ndash136)
05758
Activated partial thromboplastin
time s 128
(955ndash249) 140
(117ndash164) 02322
Thrombin time s 129 (121ndash178)
141 (650ndash160)
02028
Fibrinogen gl 448 (153ndash770)
747 (153ndash116)
00235
Antithrombin activity 639 (289ndash107)
670 (464ndash746)
05249
D-dimers microgml 006 (000ndash148)
015 (003ndash372)
03997
Thrombin generation assay nmol 130 (000ndash365)
912 (190ndash277)
08787
Clotting time (ROTEM ex-tem) s 420 (290ndash930)
430 (170ndash159)
07989
Clot formation time (ROTEM ex-
tem) s 665
(310ndash143) 470
(240ndash800) 00633
Alpha angle (ROTEM ex-tem) degrees 795 (620ndash840)
815 (750ndash860)
01667
Max clot firmness (ROTEM ex-
tem) mm 750
(570ndash860) 775
(700ndash830) 01753
Max clot elasticity (ROTEM ex-
tem) - 299
(131ndash619) 347
(235ndash484) 01544
Maximum lysis (ROTEM ex-tem) 135 (300ndash290)
400 (000ndash800)
00037
Clotting time (ROTEM in-tem) s 208 (840ndash810)
249 (720ndash371)
05085
Clot formation time (ROTEM in-
tem) s 835
(370ndash634) 745
(390ndash235) 08988
Alpha angle (ROTEM in-tem) degrees 745 (250ndash820)
750 (500ndash820)
09797
Max clot firmness (ROTEM in-
tem) mm 700
(540ndash810) 740
(610ndash770) 03587
Max clot elasticity (ROTEM in-
tem) - 235
(118ndash424) 287
(156ndash332) 04764
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
61
Maximum lysis (ROTEM in-tem) 850 (000ndash210)
350 (000ndash220)
01327
Table 4 Comparison of results of haemostasis parameters (median values minimumndash
maximum) on the last day in dogs with (grades 1ndash3) and without (grade 0) central venous
catheter-associated thrombus formation as detected by electron microscopy
Parameter Unit
Thrombus grade
(ELMI)
P-value
(Mann Whitney
U test)
0
(n = 8)
123
(n = 19)
Haematocrit 322 (241ndash454)
330 (216ndash510)
05953
Platelet count 103microl 446 (321ndash592)
268 (101ndash675)
00558
Prothrombin time 145 (104ndash264)
120 (561ndash148)
00462
Activated partial thromboplastin
time s 123
(109ndash140) 136
(109ndash188) 00665
Thrombin time s 129 (121ndash178)
141 (650ndash160)
02028
Fibrinogen gl 465 (275ndash866)
347 (189ndash992)
00491
Antithrombin activity 793 (448ndash981)
692 (398ndash889)
04572
D-dimers microgml 018 (000ndash173)
007 (000ndash118)
01920
Thrombin generation assay nmol 501 (000ndash160)
93 (000ndash287)
09562
Clotting time (ROTEM ex-tem) s 360
(270ndash410) 400
(310ndash620) 00238
Clot formation time (ROTEM ex-
tem) s 395
(120ndash740) 650
(240ndash174) 00275
Alpha angle (ROTEM ex-tem) degrees 835 (760ndash880)
790 (580ndash850)
00484
Max clot firmness (ROTEM ex-
tem) mm 780
(700ndash850) 720
(550ndash860) 00586
Max clot elasticity (ROTEM ex-
tem) - 346
(239ndash555) 254
(121ndash637) 00495
Maximum lysis (ROTEM ex-tem) 105 (200ndash190)
110 (000ndash380)
03796
Clotting time (ROTEM in-tem) s 178 (100ndash222)
201 (139ndash554)
00525
Clot formation time (ROTEM in-
tem) s 435
(330ndash111) 790
(350ndash882) 00209
Alpha angle (ROTEM in-tem) degrees 815 (710ndash830)
740 (240ndash830)
00191
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
62
Max clot firmness (ROTEM in-
tem) mm 785
(660ndash810) 690
(270ndash840) 00586
Max clot elasticity (ROTEM in-
tem) - 362
(192ndash434) 218
(370ndash507) 00559
Maximum lysis (ROTEM in-tem) 950 (200ndash190)
800 (100ndash180)
06890
4 Discussion
The main result of our study is that under the study conditions part of the dogs developed
CVC-associated thromboses However only a very low part of these thromboses were
obviously of a clinically significant degree regarding the amount of occlusion of the EJV
and none of the dogs had a symptomatic thrombosis whose definition includes presence
of clinical signs such as localised swelling pain and redness (Boersma et al 2008 Lee
and Kamphuisen 2012) In the present study we particularly refer to the extraluminal
thromboses with regard to the assessment of the antithrombotic management
One major problem of the statistical analysis and result presentation was that the two
methods used to define the occurrence and degree of thrombus formation (ultrasound and
ELMI) revealed discrepant results Therefore we performed a statistical analysis
independently regarding thrombus formation with either of the two methods It is well
known that ELMI which has already been used to detect CVC induced thromboses in
humans (Lucas et al 2017) and experimental animals (Xiang et al 1998 Xiang et al
2001) is a very sensitive diagnostic technique which allows the ex vivo diagnosis of very
small or fibrin rich thrombi that perhaps cannot be detected by ultrasonography or
venography during the in vivo evaluation of patients (Xiang et al 2001) A major reason
for the discrepant results between the two methods used to detect CVC associated
thromboses is that different CVC segments are evaluated Using ultrasonography it was
only possible to evaluate the EJV in the neck area of the patient and thereby only its
proximal third In contrast ELMI assessed mainly the CVC tip and a human study
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
63
revealed that even different positions of the catheter tip are associated with various risks
for development of CVC associated thrombosis (Ballard et al 2016) One additional point
to consider which also may contribute to the discrepancy is that it is possible that thrombi
developed on the CVC surface can be detached during the CVC removal from the vein
and thereby before its analysis by ELMI (Xiang et al 2001)
The contrast venography technique is used as gold standard to confirm thrombosis in
human medicine However this diagnostic technique is normally used only in the case of
a strong clinical suspicion of venous thrombosis that cannot be detected by
ultrasonography (Laurenson et al 2010 Langston and Eatroff 2018) Our study protocol
with privately owned clinic patients did not allow to use this method In a study performed
in 44 human patients requiring a CVC (Koumlksoy et al 1995) a sensitivity of 94 and a
specificity of 96 were determined for the colour Doppler ultrasound diagnosis when it
was compared with the diagnosis performed with the contrast venography technique and
that makes the colour Doppler ultrasound an accurate diagnosis method to detect CVC
related thrombosis even in humans (Koumlksoy et al 1995 van Rooden et al 2003)
The incidences of CVC related thrombosis detected by colour Doppler ultrasound of the
internal jugular vein reported in 3 human studies were 2 30 and 56 (Harter et al
2002 Lordick et al 2003 Wu et al 1999) and although with a wide range the median
value of these rates (30 ) is very well suited to the incidence of 28 (829) which we
calculated for our canine study based on sonographic findings The wide variation of
incidences of CVC related thrombosis in different human studies can in general be
explained by differences regarding the type and entry site of the CVC (jugular femoral
subclavian axillary cephalic vein) the time period with the CVC in place the thrombus
detection technique (venography or ultrasonography comment in the cited 3 studies
consistently the last-mentioned method was used) and criteria for diagnosis of
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
64
thrombosis the duration of follow up diseases and presence of predisposing factors in
these patients (eg previous venous thrombosis history presence of cancer) and finally
prophylactic treatment with anticoagulants (van Rooden et al 2005 Geerts 2014 Liu et
al 2015)
Different pathomechanisms contribute to the development of CVC induced thromboses
During the CVC insertion the puncture of the skin on the CVC insertion site and the
rupture of the blood vessels integrity activate the coagulation cascade and the CVC acts
as a matrix for adherence and accumulation of fibrin and cellular components (Smith et
al 2012) The attachment of proteins and blood cells to the internal andor external wall
of the CVC can lead to a partial or complete occlusion of the catheter lumen and
sometimes to a partial or complete obstruction of the blood flow andor
thromboembolism (Langston and Eatroff 2018) There is a strong interrelationship
between thrombus and biofilm formation (Smith et al 2012) Prevention of thrombus
formation has thereby also relevance regarding the increased risk for microbial
colonisation of the catheter (Boersma et al 2008) leading at least in humans to an
increased number of CVC related systemic infections (Lordick et al 2003)
In this context it has to be considered that CVC produced from polyurethane which are
routinely used in our clinic and thereby in the study are usually associated with a
relatively high thrombosis potential and thereby definitely provide an effective test for
an antithrombotic regimen Silicone catheters have a significantly reduced risk for
thrombotic complications but this advantage has to be purchased by a higher rate of
mechanical failure (eg rupture of the catheter) (Wildgruber et al 2016) Promising
experimental approaches work on surface modifications of the standard catheters For
example one group was able to demonstrate that a coating of the surface of the catheter
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
65
with a zwitterionic polymeric sulfobetaine showed a significant reduction of thrombus
formation in vitro with human blood and in an in vivo canine model (Smith et al 2012)
Due to ethical reasons we did not performed a placebo-controlled study However
interestingly not all human studies report systemic anticoagulation and part of the human
studies was even placebo-controlled (review Lee and Kamphuisen 2012) Obviously
there is no consensus in the available human literature regarding the beneficial use of
antithrombotic prophylaxis to prevent CVC related thrombosis Several studies did not
find a statistically significant difference in the incidence of CVC related thrombosis
between patients receiving the antithrombotic prophylaxis compared with the control
groups without systemic anticoagulation (Verso et al 2005 P = 035 Karthaus et al
2006 P gt 005 Niers et al 2007 P = 049) whereas in other studies a lower incidence
of CVC related thrombosis was found in the group receiving anticoagulants as
prophylaxis compared with the control groups (Abdelfeki et al 2004 150 vs 126
P = 003 De Cicco et al 2009 400 vs 526 P = 005)
Although it is well known that subcutaneously administered UFH has a limited and
variable bioavailability and thereby leads to unpredictable blood activities (Hirsh and
Raschke 2004) the extremely wide range of blood activities after a defined dosage in our
study was relatively unexpected Blood was collected 2 hours after the first UFH
injection and thereby at the time of suspected maximum heparin activity considering the
relatively low UGH dosage (Mischke and Jacobs 2001) The cited study was performed
on healthy experimental dogs Possible additional influencing factors in critical ill
patients include circulatory insufficiency which may reduce and delay absorption from
subcutanenous tissue decreased clearance function of the liver and kidneys as well as
altered blood plasma concentrations of heparin binding plasma proteins such as
fibrinogen and platelet factor 4 and alterations of the heparin binding capacity of
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
66
endothelial cells which altogether are reasons for a great inter- but also intrapatient
variability (Hirsh and Raschke 2004) In individual patients this can lead to heparin
resistance It has also to be considered that antithrombin activities which are necessary
for an adequate heparin effect (Bjoumlrk and Lindahl 1982) were reduced in our patients
Availability of approved blood tests which can predict the risk of development of CVC
associated thrombosis already at the moment of the CVC placement are important
(Boersma et al 2016) One of the most interesting results was that dogs which developed
sonographically detectable thrombi had nearly twofold higher fibrinogen levels Well in
agreement with our results one study performed in anticoagulated human patients
demonstrated that high fibrinogen levels were associated with an increased risk of
perioperative venous thromboembolism (P = 0004) (Kato et al 2016) The cited study
does not exclusively refer to CVC associated thrombosis but 76 (1621) of the venous
thromboses were suggested to be associated with CVCs Fibrinogen measurement a test
which is widely available is therefore a useful and practicable indicator of an increased
risk for CVC induced thromboses and may advice the clinician to treat these patients
especially carefully (eg with low molecular weight heparin) In the study performed by
Boersma et al (2016) in 168 human patients without indication of anticoagulation
prophylaxis elevated leukocyte counts high levels of plasminogen activator inhibitor
and of coagulation factor VIII were associated with an increased incidence of
symptomatic CVC related thrombosis diagnosed by ultrasonography A limitation of that
study is that all measurements were performed on blood samples taken after CVC
placement Interestingly in our study ROTEM results were partly associated with
thrombus formation which reflects the well-known fact that results of viscoelastic
measurements are a good indicator of hypercoagulability and thromboembolic events
(Harahsheh and Ho 2018) It is of further interest and possibly unexpeted that on the final
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
67
day of examination dogs with ELMI signs of thromboses had partly lower coagulation
activities than dogs without thromboses Due to the low number of dogs without
detectable thrombi using ELMI this implausible result has to be interpreted with caution
One possible explanation could be that temporary hypercoagulability in these dogs may
have finally resulted in consumption of haemostasis substrates
Limitations of the present study were as a consequence of the study design based on
clinical patients the relatively low total number of patients inclusion of different UFH
treatment regimens and the lack of a control group
5 Conclusions
The results of the present study indicate that the used UFH regimen was not completely
effective to prevent CVC-associated thromboses which were detectable in 28 [829]
of the patients by ultrasound However severe thromboses (high grades) are relatively
rare in intensive care canine patients receiving a routine anticoagulant treatment Because
heparin plasma activities were not different in dogs with or without CVC-associated
thrombus formation other local and systemic factors must play a significant role as well
A high fibrinogen concentration and low maximum lysis (ROTEM ex-tem) may be
useful indicators for an increased thrombotic risk in individual patients which may
require special anticoagulatory treatment
6 Acknowledgments
Jose Mauricio Pereira received a scholarship from the Universidad Nacional de Costa
Rica (reference code JB-C-1107-2016)
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
68
7 References
1 Abdelkefi A Ben Othman T Kammoun L Chelli M Ben Romdhane N Kriaa A Ladeb S
Torjman L Lakhal A Achour W Ben Hassen A Hsaiumlri M Ladeb F Ben Abdeladhim A
2004 Prevention of central venous line-related thrombosis by continuous infusion of low-dose
unfractionated heparin in patients with haemato-oncological disease A randomized controlled trial
Thromb Haemost 92 654ndash661
2 Adamantos S Brodbelt D Moores A L 2010 Prospective evaluation of complications associated
with jugular venous catheter use in a veterinary hospital J Small Anim Pract 51 254ndash257
3 Bagge A Schoumltt U Kander T 2016 Effects of naturopathic medicines on Multiplate and ROTEM
a prospective experimental pilot study in healthy volunteers BMC Complement Altern Med 16 64
4 Ballard DH Samra NS Gifford KM Roller R Wolfe BM Owings JT 2016 Distance of the
internal central venous catheter tip from the right atrium is positively correlated with central venous
thrombosis Emerg Radiol 23 269ndash273
5 Boersma RS Jie KS Verbon A van Pampus EC Schouten HC 2008 Thrombotic and
infectious complications of central venous catheters in patients with hematological malignancies Ann
Oncol 19 433ndash442
6 Boersma R S Hamulyak K van Oerle R Tuinenburg A Ten Cate-Hoek AJ Schouten HC
2016 Biomarkers for prediction of central venous catheter related-thrombosis in patients with
hematological malignancies Clin Appl Thromb Hemost 22 779ndash784
7 Bjoumlrk I Lindahl U 1982 Mechanism of the anticoagulant action of heparin Mol Cell Biochem
48 161ndash182
8 Claude A Riedesel D Riedesel E 2010 Electrocardiography-guided and retrospective analysis of
central venous catheter placement in the dog Vet Anaesth Analg 37 97ndash105
9 Comerlato PH Rebelatto TF Santiago de Almeida FA Klein LB Boniatti M M Schaan
DB Rados VD 2017 Complications of central venous catheter insertion in a teaching hospital
Rev Assoc Med Bras 63 613ndash620
10 Crochemore T Piza FMT Rodriacutegues RDR Guerra JCC Ferraz LJR Correcirca TD 2017
A new era of thromboelastometry Einstein (Sao Paulo) 15 380ndash385
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
69
11 De Cicco M Matovic M Balestreri L Steffan A Pacenzia R Malafronte M Fantin D
Bertuzzi CA Fabiani F Morassut S Bidoli E Veronesi A 2009 Early and short‐term
acenocumarine or dalteparin for the prevention of central vein catheter‐related thrombosis in cancer
patients a randomized controlled study based on serial venographies Ann Oncol 20 1936ndash1942
12 Evans NS Ratchford EV 2018 Catheter related-venous thrombosis Vasc Med 23 411ndash413
13 Geerts W 2014 Central venous catheterndashrelated thrombosis Hematology Am Soc Hematol Educ
Program 2014 306ndash311
14 Harahsheh Y Ho KM 2018 Use of viscoelastic tests to predict clinical thromboembolic events A
systematic review and meta‐analysis Eur J Haematol 100 113ndash123
15 Harter C Salwender HJ Bach A Egerer G Goldschmidt H Ho AD 2002 Catheter-related
infection and thrombosis of the internal jugular vein in hematologic-oncologic patients undergoing
chemotherapy a prospective comparison of silver-coated and uncoated catheters Cancer 94 245ndash251
16 Hirsh J Raschke R 2004 Heparin and low-molecular-weight heparin The Seventh ACCP
Conference on Antithrombotic and Thrombolytic Therapy Chest 126 (Suppl) 188S-203S
17 Hundley D Brooks A Thomovsky E Jhonson P Freeman L Schafbuch R Heng HG Moore
G 2018 Comparison of ultrasound-guided and landmark-based techniques for central venous
catheterization via the external jugular vein in healthy anesthetized dogs Am J Vet Res 79 628ndash
636
18 Kato F Takeuchi H Matsuda S Kawakubo H Omori T Kitagawa Y 2016 Incidence of and
risk factors for venous thromboembolism during surgical treatment for esophageal cancer a single-
institution study Surg Today 46 445ndash452
19 Karthaus M Kretzschmar A Kroning H Biakhov M Irwin D Marschner N Slabber C
Fountzilas G Garin A Abecasis NG Baronius W Steger GG Sudhoff T Giorgetti C
Reichardt P 2006 Dalteparin for prevention of catheter‐related complications in cancer patients with
central venous catheters final results of a double‐blind placebo‐controlled phase III trial Ann Oncol
17 289ndash296
20 Koumlksoy C Kuzu A Kutlay J Erden I Ozcan H Ergicircn K 1995 The diagnostic value of colour
Doppler ultrasound in central venous catheter related thrombosis Clin Radiol 50 687-689
21 Kucher N 2011 Clinical practice Deep-vein thrombosis of the upper extremities N Engl J Med
364 861ndash869
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
70
22 Langston CE Eatroff AE 2018 Hemodialysis catheter-associated fibrin sheath in a dog J Vet
Emerg Crit Care 28 366ndash371
23 Laurenson MP Hopper K Herrera MA Johnson EG 2010 Concurrent diseases and conditions
in dogs with splenic vein thrombosis J Vet Intern Med 24 1298ndash1304
24 Lee AYY Kamphuisen PW 2012 Epidemiology and prevention of catheter‐related thrombosis in
patients with cancer J Thromb Haemost 10 1491ndash1499
25 Liu G Fu ZQ Zhu P Li SJ 2015 Central venous catheter-related thrombosis in senile male
patients New risk factors and predictors J Huazhong Univ Sci Technolog Med Sci 35 445ndash449
26 Lordick F Hentrich M Decker T Hennig M Pohlmann H Hartenstein R Peschel C 2003
Ultrasound screening for internal jugular vein thrombosis aids the detection of central venous catheter-
related infections in patients with haemato-oncological diseases a prospective observational study Br
J Haematol 120 1073ndash1078
27 Lucas TC Silva EED Souza DOD Santos ARD Lara MO 2017 Microstructural
evaluation by confocal and electron microscopy in thrombi developed in central venous catheters Rev
Esc Enferm USP 51 1ndash9
28 Malick L Wilson R Stetson D 1976 Modified thiocarbo- hydrazide procedure for scanning
electron microscopy routine use for normal pathological and experimental tissue Stain Technol 50
265ndash269
29 Mischke R Jacobs C 2001 The monitoring of heparin administration by screening tests in
experimental dogs Res Vet Sci 70 101ndash108
30 Niers TM Di Nisio M Klerk CP Baarslag HJ Buller HR Biemond BJ 2007 Prevention
of catheter‐related venous thrombosis with nadroparin in patients receiving chemotherapy for
hematologic malignancies a randomized placebo‐controlled study J Thromb Haemost 5 1878ndash
1882
31 Reminga C Silverstein D Drobatz K Clarke D 2018 Evaluation of the placement and
maintenance of central venous jugular catheters in critically ill dogs and cats J Vet Emerg Crit Care
28 232ndash243
32 Smith RS Zhang Z Bouchard M Li J Lapp HS Brotske GR Lucchino DL Weaver D
Roth LA Coury A Biggerstaff J Sukavaneshvar S Langer R Loose C 2012 Vascular
Chapter 3 Central venous catheter induced thrombosis in dogs ndash efficacy of standard
heparin prophylaxis and correlation to haemostatic changes
71
catheters with a nonleaching poly-sulfobetaine surface modification reduce thrombus formation and
microbial attachment Sci Transl Med 4 153ra132 doi101126scitranslmed3004120
33 Toskala E Nuutinen J Rautiainen M 1995 Scanning electron microscopy findings on human
respiratory cilia in chronic sinusitis and in recurrent respiratory infections J Laryngol Otol 109 509ndash
514
34 Van Rooden CJ Tesselaar ME Osanto S Rosendaal FR Huisman MV 2005 Deep vein
thrombosis associated with central venous catheters - a review J Thromb Haemost 3 2409ndash2419
35 Verso M Agnelli G Bertoglio S Di Somma FC Paoletti F Ageno W Bazzan M Parise P
Quintavalle R Naglieri E Santoro A Imberti D Soraru M Mosca S 2005 Enoxaparin for the
prevention of venous thromboembolism associated with central vein catheter a double‐blind placebo‐
controlled randomized study in cancer patients J Clin Oncol 23 4057ndash4062
36 Villalta-Garciacutea P Loacutepez-Herranz M Mazo-Pascual S Honrubia-Fernaacutendez T Jaacutentildeez-Escalada
L Fernaacutendez-Peacuterez C 2015 Reliability of blood test results in samples obtained using a 2-mL discard
volume from the proximal lumen of a triple-lumen central venous catheter in the critically ill patient
Nurs Crit Care 22 298ndash304
37 Vose J Odunayo A Price JM Daves M Schildt JC Tolbert MK 2019 Comparison of
heparinized saline and 09 sodium chloride for maintaining central venous catheter patency in healthy
dogs PeerJ 7 e7072 httpsdoiorg107717peerj7072
38 Wildgruber M Lueg C Borgmeyer S Karimov I Braun U Kiechle M Meier R Koehler M
Ettl J Berger H 2016 Polyurethane versus silicone catheters for central venous port devices
implanted at the forearm Eur J Cancer 59 113ndash124 doi101016jejca201602011
39 Wu X Studer W Skarvan K Seeberger MD 1999 High incidence of intravenous thrombi after
short-term central venous catheterization of the internal jugular vein J Clin Anesth 11 482ndash485
40 Xiang DZ Verbeken EK Van Lommel AT Stas M De Wever I 1998 Composition and
formation of the sleeve enveloping a central venous catheter J Vasc Surg 28 260ndash271
41 Xiang DZ Verbeken EK Van Lommel AT Stas M De Wever I 2001 Sleeve-related
thrombosis a new form of catheter-related thrombosis Thromb Res 104 7ndash14
Chapter 4 General discussion
72
Chapter 4 General discussion
Incidence of CVC related thrombosis
In our study the incidence of CVC related thrombosis detected by ultrasound in critical
care canine patients was 28 To the best of the authoracutes knowledge similar studies
performed in dogs are rare in the available veterinary literature In one study performed
in 81 dogs and 12 cats (Adamantos et al 2010) an incidence of 2 was found for vein
thrombosis related to CVC This low incidence can be due to a lacking of systematic
ultrasound evaluations of the jugular vein containing the CVC In the mentioned study in
dogs and cats the vein thrombosis was confirmed by palpation or ultrasound presumably
after the presentation of clinical symptoms however in many cases as in our study
thrombus into the jugular vein can be developed without manifestation of clinical
symptoms what makes a routinely ultrasound evaluation of the vein containing the CVC
very important Our results are well aligned with the findings in humans in which a
median incidence of 30 was found for thrombosis related to CVC based on 25 studies
performed in patients requiring a CVC (review article van Rooden et al 2005) In the
mentioned 25 human studies the incidence of CVC related thrombosis varied within a
wide range (2ndash67 ) according to factors related to (1) the CVC (eg material type
entry site [jugular femoral subclavian axillary cephalic vein] time period in place (2)
diagnostic thrombus detection technique (ultrasonography or venography) and criteria
(3) patient collective (diseases and presence of predisposing factors [eg previous venous
thrombosis history presence of cancer] and (4) prophylactic antithrombotic treatment
(Geerts 2014 Liu et al 2015 van Rooden et al 2005)
The incidence of CVC related thrombosis detected by colour Doppler ultrasound in our
study fits also with the median incidence (30 ) found in three studies performed in
humans in which colour Doppler ultrasound evaluations of the jugular vein containing a
Chapter 4 General discussion
73
CVC were performed (Harter et al 2002 Lordick et al 2003 Wu et al 1999) In these
three studies the individual incidence of CVC related thrombosis was 2 (Harter et al
2002) 30 (Lordick et al 2003) and 56 (Wu et al 1999) The high thrombus
incidence reported in the study performed by Wu et al (1999) could be due to the criteria
for diagnosis of thrombosis because in that study both fibrin sleeves and compact
thrombi were considered as cases of thrombosis related to CVC The incidence for
compact thrombi was 25 which is more similar to our results In the study performed
by Harter et al (2002) the CVC related thrombosis incidence (2 ) was very low
compared with the incidence found in our study In that study the jugular veins were
examined just before or less than 24 hours after CVC removal (median period of CVC
use = 1025 days) In our study the ultrasound evaluations were performed before the
CVC placement (day 0) and then on days 1 3 5 etc until the CVC removal (median
period of CVC use = 7 days median interval until thrombus detection = 7 days) In our
study we detected thrombus formation as early as on the 3rd day so it could be possible
that in the cited study possibly only temporarily existing thrombi could have been missed
by ultrasonography in the final stage and thereby for the incidence calculation However
this scenario is not very likely because results of the present study show that
sonographically detectable thrombi during the observation period had in all cases their
maximum grade on the final day
In the study performed by Lordick et al (2003) the CVC related thrombosis incidence
determined for 43 human patients was very similar to our study and in both studies only
compact thrombi detectable by ultrasound were considered for assessment of the
incidence In the mentioned study all the human patients were screened for the presence
of thrombosis every 4 days after CVC insertion with real time B-mode ultrasound
(Lordick et al 2003)
Chapter 4 General discussion
74
In our study 70 of CVCs analysed by ELMI showed thrombi of grade 1ndash3 on the
external CVC wall To the best of the authoracutes knowledge studies about incidence of CVC
related thrombosis on the outside surface of the CVC detected by ELMI cannot be found
in the human and small animal literature In one study performed in humans (Lucas et al
2017) thrombus formation was analysed by ELMI in 78 central venous catheters That
study focussed on microstructural assessment of thrombus formation inside the distal
openings of the CVC which were found in 85 of the evaluated CVCs The percentage
of occurrence of thrombosis in the mentioned study in humans was nearly similar to the
incidence of intraluminal thrombus formation as detected in the presented study by ELMI
(74 ) In general intraluminal thrombi were not specially considered in the present
study because they mainly express the efficacy of the flushing technique rather than the
effectivity of the antithrombotic treatment In one study performed in rats the
microstructure of thrombi developed in the jugular vein containing the CVC was also
evaluated by ELMI (Xiang et al 1998) In the mentioned study pericatheter thrombosis
was detected on the proximal section of the CVC in 100 of rats (n = 12) after 1 and 3
days of catheterisation The analyses of the CVCs placed in rats were performed with the
catheter inside the vein in contrast to our study where the CVC was removed from the
jugular vein for the respective analysis Xiang et al (2001) hypothesised in one study
performed in rabbits and rats in which CVCs were placed in the jugular vein that fibrin
rich thrombi associated to the CVC can be detached during the catheter removal and this
could explain the difference between the thrombosis incidence assessed in our study and
that of the study performed by Xiang et al (1998)
In our study the incidence of CVC related thrombosis detected by ultrasonography in the
lumen of the EJV did not correlate with the incidence of CVC related thrombosis detected
by ELMI on the external wall of the CVC This was mainly due to the fact that the CVC
Chapter 4 General discussion
75
segment evaluated with both methods was different Using ultrasonography it was only
possible to evaluate the EJV in the neck area of the patient and thereby only the proximal
third of the CVC In contrast with the ELMI analysis only the distal section of the CVC
(last third) was evaluated because macroscopically detected thrombi mainly developed
near the CVC tip which was routinely positioned at the junction of the cranial cava vein
with the right atrium In a retrospective review of medical records of 169 human patients
requiring a CVC (Ballard et al 2016) the position of the CVC tip was determined by
radiologic evaluation and it was determined that the catheter tip position can be a
predisposing factor for central venous thrombosis development Thereby it could be of
interest to evaluate in further studies in dogs if a high incidence of thrombosis detected
by ELMI as was shown in our study could be related to the CVC tip position
In our study polyurethane catheters were tested which are widely used and are the
standard CVCs used in the Small Animal Clinic University of Veterinary Medicine
Hannover In vivo and in vitro studies have assessed the thrombogenicity of different
catheter materials (Borow and Crowley 1985 Smith et al 2012) In general terms
polyurethane catheters (central or peripheral) had shown a higher thrombogenicity
compared with coated (eg with hydromer) and silicone catheters Due to this property
of polyurethane catheters they can be supposed as a good test system for antithrombotic
regimens as evaluated in our study On the other hand despite silicone catheters have been
associated with a lower thrombogenicity their mechanical flexibility could be a
disadvantage in small clinical practice due to the increased risk of rupture detected even
in humans (Wildgruber et al 2016)
Chapter 4 General discussion
76
Heparin thromboprophylaxis for CVC related thrombosis
The occurence of CVC related thrombosis detected by colour Doppler ultrasound and
ELMI in our study demonstrates that the UFH in the given dosage was not completely
effective to prevent CVC related thrombosis in canine intensive care patients In human
medicine LMWH is commonly used in prophylactic anticoagulation regimens however
studies performed in human patients requiring a CVC and receiving a prophylactic
anticoagulation (with LMWH or UFH) did not achieve consensus regarding the efficacy
of prophylactic heparin to prevent CVC related thrombosis (Abdelfeki et al 2004
Lordick et al 2003 Wu et al 1999) To the best of the authoracutes knowledge studies
performed in dogs evaluating systemic prophylactic anticoagulation to prevent CVC
related thrombosis cannot be found However according to the evidence found in the
human literature and the results of our study further investigations using comparative
groups with different drugs dosages andor administration routes are required to establish
an effective antithrombotic treatment to prevent CVC related thrombosis
The plasma heparin activities measured in canine patients in our study varied significantly
after a defined subcutaneous dosage The limited and variable bioavailability of SC
administered UFH is well documented in humans (Hirsh and Raschke 2004)
Considerable variations of the circulatory status of the liver- and kidney function and
concentration of heparin-binding proteins in intensive care patients obviously amplify
this phenomenon leading to an extremely high inter-individual and intra-individual
variation of the heparin blood levels in our patients A different application route (IV)
may have reduced the influence of part of these factors and therefore could have led to
more predictable UFH blood levels However continuous infusion requires more
technical equipment and is also associated with a wide range of heparin activities in
canine patients (Scott et al 2009)
Chapter 4 General discussion
77
Prediction of CVC related thrombosis
Of 7 haemostasis tests (including ROTEM with 12 different parameters [6 for each
reagent ex-tem and in-tem]) evaluated as possible predictors of CVC related thrombosis
in our study initial values of high fibrinogen and low maximum lysis (ROTEM ex-tem)
were associated with sonographically detectable thrombus formation Some studies in
humans have established the association of laboratory biomarkers as predictors of risk
thrombosis (Boersma et al 2016 Cheng et al 2013 Kato et al 2016 Liu et al 2015)
One study performed in anticoagulated human patients showed ndash well in agreement with
our results ndash that high fibrinogen levels were associated with an increased risk of
perioperative venous thromboembolism (P = 0004) (Kato et al 2016) The cited study
does not exclusively refer to CVC associated thrombosis but 762 (1621) of the
venous thromboses were suggested to be associated with CVCs Fibrinogen concentration
measurements are normally available in the human and veterinary practice and the
association of this parameter with a high risk of development of CVC related thrombosis
can be useful to choose special antithrombotic treatment in individual critical care patients
to prevent such severe complications
Thrombin generation assay (TGA) abnormalities such as higher velocity and endogenous
thrombin potential (ETP) values measured initially ie before any anticoagulation
therapy have been associated with a higher thromboembolism risk in general (ie without
specific association with CVCs) in one study performed on 105 human patients (Espitia
et al 2017) In contrast in our study the initial TGA assay results did not show an
association with formation of CVC related thrombosis
The ROTEM parameters evaluated in the present study were partially associated with
formation of CVC thrombosis detected by colour Doppler ultrasound This finding
confirms the results obtained in humans in which ROTEM measurements are regarded as
Chapter 4 General discussion
78
a valuable indicator of hypercoagulability (Harahsheh and Ho 2018 Tuumlrk et al 2018)
However in our study individual ROTEM parameters showed partly lower coagulation
activities in dogs with CVC related thrombosis detected by ELMI at the final day of
evaluation Because the number of patients without signs of thrombosis detected by ELMI
was low compared with those who showed signs these implausible results should be
interpreted carefully The usefulness of ROTEM analysis which has also many other
indications demonstrates that is was worth to perform a detailed methodological approval
of the device including the establishment of reference values in the preliminary study
Conclusion
In conclusion the prethrombotic or thrombotic changes detected in our study did not
correlate between ultrasonography and ELMI however both methods show accurately
signs of CVC related thrombosis The UFH in the given dosage was not completely
effective to prevent CVC induced thrombus formation in canine intensive care patients
Plasma heparin activities varied significantly after a defined subcutaneous dosage
However plasma heparin activities were not different between dogs with and without
detectable CVC associated thromboses Initial high fibrinogen values and low maximum
lysis (ROTEM ex-tem) values were associated with sonographically detectable thrombi
and may be useful as indicators for an increased thrombotic risk
Finally our study in canine intensive care patients is to the best of the authoracutes
knowledge the first study in the available veterinary literature performed to evaluate the
efficacy of an antithrombotic treatment with UFH to prevent CVC related thrombosis
Chapter 5 Summary
79
Chapter 5 Summary
Central venous catheter induced thrombosis in dogs ndash occurrence under an
antithrombotic treatment regimen and possible indicators
Joseacute Mauricio Pereira Mora
Central venous catheters (CVC) are of importance for intensive care management of
canine patients Thrombosis of the jugular vein is a severe complication related to the
CVC use However only little is known about the incidence of CVC-associated
thromboses in dogs and the necessity and efficacy of prophylactic antithrombotic
regimens
Therefore the aims of the present study were (1) to determine the incidence of catheter-
induced thrombosis in canine intensive care patients (2) to evaluate the efficacy of a
prophylactic heparin regimen routinely used in the Small Animal Clinic of the University
of Veterinary Medicine Hannover and (3) to assess whether selected haemostasis test
results are predictive for an increased risk for thrombosis formation
The thesis also includes a preliminary methodological study on the use of rotational
elastometry in dogs using the ROTEM delta device In this preliminary study reference
intervals were determined based on 125 healthy dogs for different clot activator reagents
and for measurements without activator Coefficients of variation (CVs) were calculated
based on results of fourfold measurements of 3 normal and 3 abnormal blood samples in
order to assess precision of the method Influence of age groups sex neuter status and
size were also assessed The median CVs for most of the parameters evaluated in the
precision analysis were less than 10 and this confirmed that the ROTEM delta analyser
delivers repeatable results Regarding the influence of age sex neuter status and animal
Chapter 5 Summary
80
size only neuter status in female dogs appeared to be a relevant influencing factor on
most of important ROTEM parameters
The main study of the thesis comprises 29 patients hospitalised at the Small Animal Clinic
requiring a CVC for medical reasons These patients received a prophylactic
anticoagulation treatment with unfractionated heparin (UFH 150 IU [surgical dogs
received 75 IU]kg BW TID subcutaneously) A colour Doppler ultrasound of the
respective external jugular vein (EJV) in order to detect thrombus formation in the lumen
of the EJV and blood collection for haemostasis tests were performed directly before the
installation procedure (day 0) and on days 1 3 5 7 and so on after the CVC insertion
Haemostasis tests included prothrombin time activated partial thromboplastin time
thrombin time antithrombin activity D-dimer concentration thrombin generation and
rotational elastometry as well as the heparin activity using a chromogenic anti-factor Xa
test Finally when the CVC was removed electron microscopy (ELMI) of sections of the
CVC with macroscopically visible thrombi in the last third (5ndash10 cm) of the CVC was
performed 3 different grades were defined for CVC-associated (extraluminal) thrombi
detected by either ultrasound or ELMI respectively
829 patients evaluated by colour Doppler ultrasound during the study developed thrombi
into the lumen of EJV (4 x grade 1 3 x grade 2 and 1 x grade 3) 1927 CVCs assessed
using ELMI showed detectable thrombi on the external CVC surface (7 x grade 1 4 x
grade 2 and 8 x grade 3) There was no significant correlation between thrombus
formation detected by ultrasound or ELMI Thus statistical group comparisons of
patients with or without CVC-associated thrombus formation was performed
independently for both diagnostic procedures The plasmatic heparin activities showed
great variability but no significant differences were found between dogs with or without
CVC-associated thrombus formation regardless whether this was based on sonographic
Chapter 5 Summary
81
or ELMI findings From the initially performed haemostasis tests high fibrinogen
concentrations and low maximum lysis values as measured by the ROTEM delta device
(using the ex-tem reagent) were associated with sonographically detectable thrombi
In conclusion the preliminary methodological study on the ROTEM delta in dogs shows
that the method delivers repeatable results in canine citrated whole blood and the
reference values obtained should offer a reliable basis for the interpretation of test results
of this device in dogs with suspected haemostasis disorders The results of the main study
indicate that CVC-associated thromboses occur (eg 28 [829] of the patients as
detected by ultrasound) although severe thromboses (high grades) are relatively rare in
intensive care canine patients receiving a routine anticoagulant treatment Thereby
prophylactic antithrombotic protocol (unfractionated heparin [UFH]) in the given dosage
was not completely effective to prevent CVC induced thrombus formation in canine
intensive care patients A high fibrinogen concentration and low maximum lysis
(ROTEM ex-tem) may be useful indicators for an increased thrombotic risk in individual
patients which may require special anticoagulatory treatment Further studies are
required to evaluate whether different thromboprophylactic treatment regimens eg with
low molecular weight heparin are more effective in preventing CVC-associated
thromboses in dogs
Chapter 6 Zusammenfassung
82
Chapter 6 Zusammenfassung
Zentralvenenkatheter-induzierte Thrombose bei Hunden - Auftreten unter einem
antithrombotischen Behandlungsschema und moumlgliche Indikatoren
Joseacute Mauricio Pereira Mora
Zentralvenenkatheter (ZVK) sind fuumlr das Intensivmanagement von Hunden von
Bedeutung Die Entstehung einer Thrombose der Vena jugularis ist eine schwerwiegende
Komplikation im Zusammenhang mit der Verwendung eines ZVKs Uumlber die Inzidenz
von ZVK-assoziierten Thrombosen bei Hunden und die Notwendigkeit und Wirksamkeit
prophylaktischer antithrombotischer Protokolle liegen jedoch nur wenige fundierte
Angaben in der Literatur vor
Daher waren die Ziele der vorliegenden Studie (1) die Inzidenz der Katheter-induzierten
Thrombose bei caninen Intensivpatienten zu bestimmen (2) die Wirksamkeit eines
prophylaktischen Heparin-Regimes zu bewerten das routinemaumlszligig in der Klinik fuumlr
Kleintiere der Stiftung Tieraumlrztliche Hochschule Hannover angewendet wird und (3) um
zu beurteilen ob ausgewaumlhlte Haumlmostasetestergebnisse ein erhoumlhtes Risiko fuumlr die
Thrombosebildung vorhersagen koumlnnen
Die PhD-Arbeit enthaumllt zudem eine vorbereitende methodische Studie zur Verwendung
der Rotationselastometrie bei Hunden mit Hilfe des ROTEM delta-Geraumltes In dieser
Vorstudie wurden Referenzintervalle basierend auf 125 gesunden Hunden fuumlr
verschiedene Gerinnungsaktivatorreagenzien und fuumlr Messungen ohne Aktivator
bestimmt Um die Genauigkeit der Methode einzustufen wurden Variationskoeffizienten
(VKs) basierend auf den Ergebnissen von Vierfachmessungen von drei normalen und drei
abnormalen Blutproben berechnet Der Einfluss von Alter Geschlecht Kastration und
Groumlszlige wurde ebenfalls getestet Die Medianwerte der VKs fuumlr die meisten in der
Chapter 6 Zusammenfassung
83
Praumlzisionsanalyse bewerteten Parameter betrugen weniger als 10 und dies bestaumltigte
dass das ROTEM delta-Analysegeraumlt wiederholbare Ergebnisse liefert In Bezug auf den
Einfluss von Alter Geschlecht Kastration und Tiergroumlszlige erwies sich nur eine Kastration
bei Huumlndinnen als relevanter Einflussfaktor fuumlr die meisten wichtigen ROTEM-
Parameter
Die Hauptstudie der Arbeit umfasst 29 Patienten welche zum Zeitpunkt der
Untersuchung stationaumlr in der Kleintierklinik aufgenommen worden waren und aus
medizinischer Indikation einen ZVK benoumltigten Diese Patienten erhielten eine
prophylaktische Antikoagulation mit unfraktioniertem Heparin (UFH 150 IE
[chirurgische Hunde erhielten 75 IE]kg KG 3 x taumlglich subkutan) Bildgebende
Untersuchungen mittels Farbdoppler-Ultraschall der jeweiligen Vena jugularis externa
(VJE) zum Nachweis der Thrombusbildung im Lumen der VJE und Blutentnahmen fuumlr
Haumlmostasetests wurden jeweils direkt vor dem Installationsvorgang (Tag 0) sowie an den
Tagen 1 3 5 und 7 nach Legen des ZVKs durchgefuumlhrt Haumlmostasetests umfassten die
Prothrombinzeit die aktivierte partielle Thromboplastinzeit die Thrombinzeit die
Antithrombinaktivitaumlt die D-Dimer-Konzentration die Thrombingenerierung und die
Rotationselastometrie sowie die Heparinaktivitaumlt unter Verwendung eines chromogenen
Anti-Faktor-Xa-Tests Schlieszliglich wurde nach Entfernung des ZVKs eine
Elektronenmikroskopie (ELMI) von Abschnitten aus dem letzten Drittel (5ndash10 cm) des
ZVKs mit makroskopisch sichtbaren Thromben durchgefuumlhrt Fuumlr ZVK-assoziierte
(extraluminale) Thromben die entweder durch Ultraschall oder ELMI nachgewiesen
wurden wurden drei verschiedene Grade definiert
829 Patienten die waumlhrend der Studie mittels Farbdoppler-Ultraschall untersucht
wurden entwickelten Thromben im Lumen der VJE (4 x Grad 1 3 x Grad 2 und 1 x Grad
3) 1927 ZVKs die unter Verwendung von ELMI bewertet wurden zeigten
Chapter 6 Zusammenfassung
84
nachweisbare Thromben auf der aumluszligeren ZVK-Oberflaumlche (7 x Grad 1 4 x Grad 2 und 8
x Grad 3) Es gab keine signifikante Korrelation zwischen der durch Ultraschall oder
ELMI festgestellten Thrombusbildung Daher wurden statistische Gruppenvergleiche
von Patienten mit oder ohne ZVK-assoziierter Thrombusbildung fuumlr beide diagnostische
Verfahren unabhaumlngig voneinander durchgefuumlhrt Die plasmatischen Heparinaktivitaumlten
zeigten eine groszlige Variabilitaumlt es wurden jedoch keine signifikanten Unterschiede
zwischen Hunden mit oder ohne ZVK-assoziierter Thrombusbildung gefunden
unabhaumlngig davon ob diese auf sonographischen oder ELMI-Befunden beruhte Von den
initial durchgefuumlhrten Haumlmostasetests waren hohe Fibrinogenkonzentrationen und
niedrige maximale Lyse-Werte der Messungen mit ROTEM delta (ex-tem Reagenz) mit
sonographisch nachweisbaren Thromben assoziiert
Zusammenfassend zeigt die vorbereitende methodische Studie zum ROTEM delta bei
Hunden dass die Methode wiederholbare Ergebnisse in mit Zitrat
antikoaguliertemVollblut liefert Die erarbeiteten Referenzwerte sollten eine zuverlaumlssige
Grundlage fuumlr die Interpretation der Testergebnisse dieses Geraumlts bei Hunden mit
Verdacht auf Haumlmostasestoumlrungen bieten Die Ergebnisse der Hauptstudie zeigen dass
ZVK-assoziierte Thrombosen auftreten (z B 28 [829] der Patienten bezogen auf den
Ultraschall-gestuumltzten Nachweis) obwohl schwere Thrombosen (hohe Grade) bei
intensivmedizinisch versorgten Hundepatienten die eine Routineantikoagulation
erhalten relativ selten sind Somit war das prophylaktische antithrombotische Protokoll
(unfraktioniertes Heparin [UFH]) in der angewendeten Dosierung nicht vollstaumlndig
wirksam um die ZVK-induzierte Thrombusbildung bei caninen Intensivpatienten zu
verhindern Eine hohe Fibrinogenkonzentration und eine niedrige maximale Lyse
(ROTEM ex-tem) koumlnnen als Indikatoren eines erhoumlhten Thromboserisikos bei einzelnen
Patienten nuumltzlich sein fuumlr die moumlglicherweise eine spezielle gerinnungshemmende
Chapter 6 Zusammenfassung
85
Behandlung notwendig ist Weitere Studien sind erforderlich um zu untersuchen ob
verschiedene thromboprophylaktische Behandlungsschemata z B mit
niedermolekularem Heparin bezuumlglich der Verhinderung von ZVK-assoziierten
Thrombosen bei Hunden wirksamer sind
Chapter 7 References
86
Chapter 7 References
1 Abdelkefi A Ben Othman T Kammoun L Chelli M Ben Romdhane N Kriaa A Ladeb
S Torjman L Lakhal A Achour W Ben Hassen A Hsaiumlri M Ladeb F Ben Abdeladhim
A 2004 Prevention of central venous line-related thrombosis by continuous infusion of low-dose
unfractionated heparin in patients with haemato-oncological disease A randomized controlled
trial Thromb Haemost 92 654ndash661
2 Adamantos S Brodbelt D Moores A L 2010 Prospective evaluation of complications
associated with jugular venous catheter use in a veterinary hospital J Small Anim Pract 51 254ndash
257
3 Ballard D H Samra N S Gifford K M Roller R Wolfe B M Owings J T 2016
Distance of the internal central venous catheter tip from the right atrium is positively correlated
with central venous thrombosis Emerg Radiol 23 269ndash273
4 Baumann-Kreuziger L Onwuemene O Kolesar E Crowther M Lim W 2015 Systematic
review of anticoagulant treatment of catheter-related thrombosis Thromb Res 136 1103ndash1109
5 Bliss SP Bliss SK Harvey HJ 2002 Use of recombinant tissue‐plasminogen activator in a
dog with chylothorax secondary to catheter‐associated thrombosis of the cranial vena cava J Am
Anim Hosp Assoc 38 431ndash435
6 Boersma RS Hamulyak K van Oerle R Tuinenburg A Ten Cate-Hoek AJ Schouten
HC 2016 Biomarkers for prediction of central venous catheter related-thrombosis in patients
with hematological malignancies Clin Appl Thromb Hemost 22 779ndash784
7 Boersma RS Jie KS Verbon A van Pampus EC Schouten HC 2008 Thrombotic and
infectious complications of central venous catheters in patients with hematological
malignancies Ann Oncol 19 433ndash442
8 Bonnet F Loriferne JF Texier JP Texier M Salvat A Vasile N 1989 Evaluation of
Doppler examination for diagnosis of catheter-related deep vein thrombosis Intensive Care Med
15 238ndash240
9 Borow M Crowley J G 1985 Evaluation of central venous catheter thrombogenicity Acta
Anaesthesiol Scand Suppl 81 59ndash64
10 Cheng Y Cui T Fu P Liu F Zhou L 2013 Dyslipidemia is associated with tunneled-cuffed
catheter-related central venous thrombosis in hemodialysis patients a retrospective multicenter
Chapter 7 References
87
study Artif Organs 37 155ndash161
11 Comerlato PH Rebelatto TF Santiago de Almeida FA Klein LB Boniatti MM
Schaan BD Rados DV 2017 Complications of central venous catheter insertion in a teaching
hospital Rev Assoc Med Bras 63 613ndash620
12 De Cicco M Matovic M Balestreri L Steffan A Pacenzia R Malafronte M Fantin
D Bertuzzi CA Fabiani F Morassut S Bidoli E Veronesi A 2009 Early and short‐term
acenocumarine or dalteparin for the prevention of central vein catheter‐related thrombosis in
cancer patients a randomized controlled study based on serial venographies Ann
Oncol 20 1936ndash1942
13 Espitia O Fouassier M Hardouin JB Pistorius MA Agard C Planchon B Trossaert M
Pottier P 2017 Thrombin generation assay in hospitalized nonsurgical patients A new tool to
assess venous thromboembolism risk Clin Appl Thromb Hemost 23 45ndash51
14 Evans NS Ratchford EV 2018 Catheter related-venous thrombosis Vasc Med 23 411ndash413
15 Geerts W 2014 Central Venous Catheter-related thrombosis Hematology Am Soc Hematol
Educ Program 2014 306ndash311
16 Harahsheh Y Ho KM 2018 Use of viscoelastic tests to predict clinical thromboembolic
events A systematic review and meta‐analysis Eur J Haematol 100 113ndash123
17 Harter C Salwender HJ Bach A Egerer G Goldschmidt H Ho AD 2002 Catheter-
related infection and thrombosis of the internal jugular vein in hematologic-oncologic patients
undergoing chemotherapy a prospective comparison of silver-coated and uncoated
catheters Cancer 94 245ndash251
18 Hirsh J Raschke R 2004 Heparin and low-molecular-weight heparin The Seventh ACCP
Conference on Antithrombotic and Thrombolytic Therapy Chest 126 (Suppl) 188Sndash203S
19 Karthaus M Kretzschmar A Kroning H Biakhov M Irwin D Marschner N Slabber
C Fountzilas G Garin A Abecasis NG Baronius W Steger GG Sudhoff T Giorgetti
C Reichardt P 2006 Dalteparin for prevention of catheter‐related complications in cancer
patients with central venous catheters final results of a double‐blind placebo‐controlled phase III
trial Ann Oncol 17 289ndash296
20 Kato F Takeuchi H Matsuda S Kawakubo H Omori T Kitagawa Y 2016 Incidence of
and risk factors for venous thromboembolism during surgical treatment for esophageal cancer a
Chapter 7 References
88
single-institution study Surg Today 46 445ndash452
21 Kohler TR Kirkman TR 1998 Central venous catheter failure is induced by injury and can
be prevented by stabilizing the catheter tip J Vasc Surg 28 59ndash66
22 Koumlksoy C Kuzu A Kutlay J Erden I Ozcan H Ergicircn K 1995 The diagnostic value of
colour Doppler ultrasound in central venous catheter related thrombosis Clin Radiol 50 687ndash
689
23 Kucher N 2011 Clinical practice Deep-vein thrombosis of the upper extremities N Engl J
Med 364 861ndash869
24 Langston CE Eatroff AE 2018 Hemodialysis catheter-associated fibrin sheath in a dog J
Vet Emerg Crit Care 28 366ndash371
25 Langston CE Eatroff AE Poeppel K 2014 Use of tissue plasminogen activator in catheters
used for extracorporeal renal replacement therapy J Vet Intern Med 28 270ndash276
26 Laurenson MP Hopper K Herrera MA Johnson EG 2010 Concurrent diseases and
conditions in dogs with splenic vein thrombosis J Vet Intern Med 24 1298ndash1304
27 Lee AYY Kamphuisen PW 2012 Epidemiology and prevention of catheter‐related
thrombosis in patients with cancer J Thromb Haemost 10 1491ndash1499
28 Liu G Fu ZQ Zhu P Li SJ 2015 Central venous catheter-related thrombosis in senile male
patients New risk factors and predictors J Huazhong Univ Sci Technolog Med Sci 35 445ndash
449
29 Lordick F Hentrich M Decker T Hennig M Pohlmann H Hartenstein R Peschel C
2003 Ultrasound screening for internal jugular vein thrombosis aids the detection of central
venous catheter-related infections in patients with haemato-oncological diseases a prospective
observational study Br J Haematol 120 1073ndash1078
30 Lucas TC Silva EED Souza DOD Santos ARD Lara MO 2017 Microstructural
evaluation by confocal and electron microscopy in thrombi developed in central venous catheters
Rev Esc Enferm USP 51 1ndash9
31 Mismetti P Mille D Laporte S Charlet V Buchmuller‐Cordier A Jacquin JP Fournel
P Dutrey‐Dupagne C Decousus H 2003 Low‐molecular‐weight heparin (nadroparin) and
very low doses of warfarin in the prevention of upper extremity thrombosis in cancer patients with
indwelling long‐term central venous catheters a pilot randomized trial Haematologica 88 67ndash
Chapter 7 References
89
73
32 Niers TM Di Nisio M Klerk CP Baarslag HJ Buller HR Biemond BJ 2007
Prevention of catheter‐related venous thrombosis with nadroparin in patients receiving
chemotherapy for hematologic malignancies a randomized placebo‐controlled study J Thromb
Haemost 5 1878ndash1882
33 Scott K C Hansen B D DeFrancesco T C (2009) Coagulation effects of low molecular
weight heparin compared with heparin in dogs considered to be at risk for clinically significant
venous thrombosis J Vet Emerg Crit Care 19 74ndash80
34 Smith RS Zhang Z Bouchard M Li J Lapp HS Brotske GR Lucchino DL Weaver
D Roth LA Coury A Biggerstaff J Sukavaneshvar S Langer R Loose C 2012
Vascular catheters with a nonleaching poly-sulfobetaine surface modification reduce thrombus
formation and microbial attachment Sci Transl Med 4 153
35 Tuumlrk S M Cansu D Uuml Teke H Uuml Kaşifoğlu T Meltem Akay O Bilgin M amp Korkmaz
C 2018 Can we predict thrombotic tendency in rheumatoid arthritis A thromboelastographic
analysis (with ROTEM) Clin Rheumatol 37 2341ndash2349
36 van Rooden CJ Rosendaal FR Barge RM van Oostayen JA van der Meer FJM
Meinders AE Huisman MV 2003 Central venous catheter related thrombosis in haematology
patients and prediction of risk by screening with Doppler-ultrasound Br J Haematol 123 507ndash
512
37 van Rooden CJ Tesselaar ME Osanto S Rosendaal FR Huisman MV 2005 Deep vein
thrombosis associated with central venous catheters - a review J Thromb Haemost 3 2409ndash
2419
38 Verso M Agnelli G Bertoglio S Di Somma FC Paoletti F Ageno W Bazzan M Parise
P Quintavalle R Naglieri E Santoro A Imberti D Soraru M Mosca S 2005 Enoxaparin
for the prevention of venous thromboembolism associated with central vein catheter a double‐
blind placebo‐controlled randomized study in cancer patients J Clin Oncol 23 4057ndash4062
39 Vose J Odunayo A Price JM Daves M Schildt JC Tolbert MK 2019 Comparison of
heparinized saline and 09 sodium chloride for maintaining central venous catheter patency in
healthy dogs PeerJ 7e7072 httpdoi107717peerj7072
40 Wang LH Wei F Jia L Zhi Lu Wang B Dong HY Yu HY Sun GJ YangJ Li B
Chapter 7 References
90
Meng J Zhang RN Bi XQ Chen HY Jiang AL 2015 Fibrin sheath formation and
intimal thickening after catheter placement in dog model role of hemodynamic wall shear stress J
Vasc Access 16 275ndash284
41 Wildgruber M Lueg C Borgmeyer S Karimov I Braun U Kiechle M Meier R Koehler
M Ettl J Berger H 2016 Polyurethane versus silicone catheters for central venous port devices
implanted at the forearm Eur J Cancer 59 113ndash124
42 Whitesell RT Steenburg SD 2014 Imaging findings of acute intravascular thrombus on non-
enhanced computed tomography Emerg Radiol 21 271ndash277
43 Wu X Studer W Skarvan K Seeberger MD 1999 High incidence of intravenous thrombi
after short-term central venous catheterization of the internal jugular vein J Clin Anesth 11
482ndash485
44 Xiang DZ Verbeken EK Van Lommel AT Stas M De Wever I 1998 Composition and
formation of the sleeve enveloping a central venous catheter J Vasc Surg 28 260ndash271
45 Xiang DZ Verbeken EK Van Lommel AT Stas M De Wever I 2001 Sleeve-related
thrombosis a new form of catheter-related thrombosis Thromb Res 104 7ndash14
91
Acknowledgements
First I thank God for every day in my life for His guide and because He was is
and will be always with me and that certainty gives me strength to take a step
forward
My sincere thanks to Prof Dr Reinhard Mischke for the opportunity to perform
my PhD under his supervision for his support and guidance for all that I learned
with him and for his friendship
I also thank to Prof Dr Manfred Kietzmann and PD Dr Mario von Depka
Prondzinski for being part of my supervision group for their time and beneficial
advices and discussion during my PhD
My gratitude to Prof Hewicker-Trautwein and Mrs Rohn for their valuable work
and availability to help me
I thank the Werlhof Institute Medical Care Center Hannover for the valuable
cooperation especially to Mrs Ober-Cassebaum Anne and Franziska for all their
help
I thank all the staff of the Small Animal Clinic of the University of Veterinary
Medicine Hannover (TiHo) My special gratitude to the internal medicine and
critical care surgery neurology ultrasound and anesthesia departments and to
the TiHo students all their cooperation made possible my research
I thank the haematology clinical chemistry and haemostasis laboratory
department of the Small Animal Clinic TiHo especially to Dirk because he was
always available for me thanks for his guide and friendship all his explanations
in the laboratory were of great value but also the conversations about different
topics of the life were of big value for me
92
I thank the Hannover Graduate School for Veterinary Pathobiology
Neuroinfectology and translational Medicine (HGNI) for the well-organized
PhD program and especially thanks to Prof Dr Beatrice Grummer Dr Tina
Selle and Mrs Tania Czeslik for his guide and help during my studies
My sincere thanks to Mrs Maritta Ledwoch for her guidance at the International
office of the TiHo ldquoI remember you since my externship in Germanyrdquo
I also thank the Student Union Hannover for all the help with the accommodation
for me and my family
I want to thank the Universidad Nacional de Costa Rica and therefore to Costa
Rica (my loved country) for the financial support to perform my PhD studies
but also for my professional formation as a veterinarian Special thanks to the
Veterinary Medicine School from Universidad Nacional de Costa Rica To all the
academic and administrative staff for their support and for believing in me
Sincerely thanks to Dr Mauricio Jimenez Soto my mentor my adviser but most
important my friend Thanks for believing in me and for always being by my side
ldquoAgradezco profundamente a mis papaacutes ldquodon Tito (Papillo) y dontildea Eida (Mami)rdquo
Todo lo bueno que hay en mi proviene de Dios y de ellos Gracias a mis hermanos
(as) cuntildeados (as) sobrinos y a toda la familia relacionada a ellos Siempre han
sido un gran apoyo y los amordquo
ldquoGracias a dontildea Alba Fran y David Gracias por toda la ayuda y por estar siempre
pendientesrdquo
I would like to thank all my friends that during a short part or during my whole
life have been with me For all the good time shared really thanks
To Willy thanks ldquogordordquo
93
I sincerely thank to Adriano and Johanna for their help and hospitality Thanks
Adriano for your guidance and support during my beginning at the TiHo for make
me feel in Costa Rica with your friendship for the tourist walks and for the bike
training I missed the coffee times
I want to thank Ina the first person to give me a smile at the start of my PhD
Thanks for your hugs and friendship
I also thank Franz for his help and for being a very nice partner Thanks for help
me always when I requested you
ldquoFinalmente quiero expresar mi gratitud y amor a Karo Faacutetima y Abigaiacutel A Karo
por tu sacrificio y soporte por las palabras de aliento por estar a mi lado en las
buenas pero en especial en las malas A Faacute por tu sacrificio por tus sonrisas por
tus abrazos y besos por bailar con migo siempre por ser mi modelo e inspiracioacuten
A Abi por ser una nueva razoacuten in mi vida para seguir adelante por todo lo que
deseo compartir con tigo por mostrarme junto con Faacutetima la felicidad en una
sonrisardquo
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