Blood Coagulation- Screening assays and single factor assessment -

Jørn Dalsgaard NielsenThrombosis Centre Gentofte HospitalCopenhagen, Denmark

Platelet adhesion og activation

GP Ib-IX ¤ von Willebrand factor ¤ Collagen

Activated platelet

GP Ib-IXGP IIb-IIIa

Tromboxane A2

Serotonin

ADP

Ca++

PF3

PF4

Tissue factor

Aktivation of monocyticcells, induced by

endotoxin, cytokines, etc.

Released from TNF or IL-1-activated endothelial cells

IL-1

TNF

Activation

Endo

thel

ial d

amag

e

F XI ¤ HMWK----

F XII----

PK ¤ HMWK----

Contact activation

F XIIa

F XIa

Kallikrein

F VII F VIIaF IX

F XIa

F X

F Xa

PF3, Ca++, F VIIIa

F VIIIPCa

Prothrombin

F V

PF3, Ca++, F VaThrombin

F Vi

F VIIIi

TFPI Inactivates F VIIa ¤ F X ¤ tissue factor -kompleks

Thrombom

odulin

PC PS

Prostacyclin Nitric oxide

Inhibition

Fibrinogen

Polymerizing fibrin

Crosslinked fibrin

F XIII

F XIIIa

PlasminogenPlasmin

Fibrinogen/-Fibrin

degradation products

t-PA

PAI-1

α2-antiplasmin

Proteoglycans

Antithrombin

Heparin Cofactor II

Inhibits serine proteases

Inhibits thrombin

Activation of monocyticcells, induced by

endotoxin, cytokines, etc.

Is testing of single factors necessary in patients with suspected haemostatic dysfunction ?

• It depends on who you are addressing• A surgeon:

– Will the patient bleed ?– Can I stop bleeding with fresh-frossen plasma ?

• A haematologist:– Single factor assessment is often necessary to

establish a correct diagnosis

Indications for evaluation of haemostatic function

• Clinical problem Biochemical defect?

• Biochemical defect Clinical problem?

BleedingThromboembolism

CoagulationFibrinolysis

PlateletsEndothelium

Screening Further screening

Single factor assay

Abnormal testresult

Further screening

Single factor assay

Prophylaxis/treatment indicated?

or not?

The challenge of evaluation of clotting abnormalities

• In vitro assessment of haemostasis is difficult because the important interaction between the endothelium and blood components cannot be evaluated in a single assay.

• So-called ”global tests” can be used to test the haemostatic capacity of blood components (plasma and blood cells) but not the influence of antithrombotic properties of the endothelium.

• Thrombelastography may give a clue of a clotting defect, platelet dysfunction, or hyperfibrinolysis but will not give the final diagnosis.

Thrombelastography

Start Minutes

Amplitude

Increased in patients

with clotting defects

Decreased in patients with platelet

dysfunction or defect fibrin formation

TEG

Reaction time

Thrombelastography

• LA and HIT-2 are associated with a high risk of thrombosis

• A shortened reaction time might, therefore, be expected but is not seen because the thrombotic predisposition is provoked by endothelial dysfunction

TEG

The challenge of evaluation of clotting abnormalities

• As global tests of haemostasis neither give a consice diagnosis nor results that reliably reflect the clinical problem, more specific assays are often needed for the evaluation of thrombotic and haemorrhagic disturbances of the haemostatic system.

However, separation of the complex network of reactions may result in a number of other pitfalls and impede a comprehensive view.

The challenge of evaluation of clotting abnormalities

• Among laboratory testing, coagulation assays are the most influenced by the inaccurate standardization of the pre-analytical phase.

• Clotting times are influenced by:– time of tourniquet placement (<60 sec recommended)– needle size (19-22 gauge recommended)– citrate concentration (105-109 mM recommended)– incomplete filling of tubes (PT<80%, APTT<90%)– platelet count (<10*109/l recommended)– haemolysis and lipaemia– temperature and G-force during centrifugation– temperature and duration of storage until testing

The challenge of evaluation of clotting abnormalities

• Screening methods of coagulation should optimally be sensitive to all coagulation defects.

• This is not the case but by combination of simple procedures we can get close to the final diagnosis.

Exploring coagulation

• The present theory of the function of the coagulation system is based on numerous studies performed in the 20th century.

• The history of the discovery of clotting factors and development of assays may help understanding the use of screening assays of coagulation.

The theory of blood coagulationThe theory of blood coagulationYear 1900: the ’four factor’ theoryYear 1900: the ’four factor’ theory

Known factors:• Fibrinogen

• Prothrombin

• Thromboplastin

• Calcium

• Hammerstein. Hoppe-Seylers Zeitschrift für physiologische Chemie 1899; 28: 98.

• Morawitz, P. Ergebnisse der Physiologie biologischen Chemie und Experimental Pharmakologie 1905; 4: 307.

Cellular damage

Thromboplastin

Prothrombin + Ca

Thrombin

Fibrinogen

Fibrin

The theory of blood coagulationThe theory of blood coagulationYear 1935: the ’Quick’ testYear 1935: the ’Quick’ test

• Quick AJ. J Biol Chem 1935; 109: LXXIII

Determination of the clotting time of citrated plasma after addition of thromboplastin

and calcium chloride

Thromboplastin

Prothrombin + Ca

Thrombin

Fibrinogen

Fibrin

The theory of blood coagulationThe theory of blood coagulationYear 1947: Factor VYear 1947: Factor V

• Owren PA. Acta Med Scand 1947; Suppl: 194

Cellular damage

Thromboplastin

Prothrombin + Ca

Thrombin

Fibrinogen

Fibrin

Factor V

The theory of blood coagulationThe theory of blood coagulationYear 1947: Factor VYear 1947: Factor V

• Owren PA. Acta Med Scand 1947; Suppl: 194

Cellular damage

Thromboplastin

Prothrombin + Ca

Thrombin

Fibrinogen

Fibrin

Factor V

Factor V deficiency showed to be a rare disease,and the discovery of FV did not explain the puzzlethat the standard coagulation test: the Quick test,was normal in most patients with congenital bleeding tendency.

Mixing assays

• Whole blood clotting time and plasma clotting time are prolonged in haemophiliac patients and can be normalized by mixing patient blood/plasma with equal parts of normal blood/plasma.

• Both tests, however, have high CV%.

First description of APTT

The theory of blood coagulationThe theory of blood coagulationYear 1953: APTTYear 1953: APTT

• Langdell et al. J Lab Clin Med 1953;41:637-47.

Prothrombin + Ca

Thrombin

Fibrinogen

Fibrin

Factor VThromboplastin

Ca++

PT

”Partial thromboplastin”

Ca++

Unknown factors

Incubation

APTTKaolin

The theory of blood coagulationThe theory of blood coagulationYear 1959: the Roman numerical nomenclature Year 1959: the Roman numerical nomenclature

• suggested by an international committee under the chairmanship of Dr. IS Wright

Factor SynonymsI FibrinogenII ProthrombinIII ThromboplastinIV CalciumV Accelerator globulin; proaccelerin; labile factorVI Factor V derivative (not used now)VII Proconvertin; stable factor; autoprothrombin IVIII Antihaemophilic factor A; platelet cofactor 1IX Plasma thromboplastin component (PTC); Christmas factor; antihaemophilic

factor B; autoprothrombin II; platelet cofactor 2X Stuart-Prower factorXI Plasmathromboplastin antecedent (PTA)XII Hageman factorXIII Fibrin stabilizing factor

The theory of blood coagulationThe theory of blood coagulationYear 1964: the cascade scheme Year 1964: the cascade scheme

• Macfarlane, RG. Nature 1964; 202: 498

XII XIIa

XI XIa

IX IXa

VIII VIIIa

X Xa

V Va

II IIa

I Ia (fibrin)

Surface contact

Problems

?

?

VII ?

The theory of blood coagulationThe theory of blood coagulationYear 1975: the classic coagulation system Year 1975: the classic coagulation system

• Austen DEG & Rhymes. A laboratory manual of blood coagulation. 1975.

XII XIIa

XI XIa

IX IXa

X Xa

II IIa

I Ia (fibrin)

Surface contact

Phospholipid, Ca++, VIII

Phospholipid, Ca++, V

VIIa VII

Ca++

Tissue factorInternal pathway External pathway

X

The theory of blood coagulationThe theory of blood coagulationdiscoveries of the last decades iscoveries of the last decades

• The major in vivo importance of the external pathway

• Acceleration of coagulation by positive feed-back mechanisms

• Inibitory mechanisms of blood coagulation

Tissue factor

VII VIIa

The theory of blood coagulation todayThe theory of blood coagulation today

EXPRESSION OF TISSUE FACTOR

CONSTITUTIVE

e.g.:epithelial cellsglial cells

INDUCED

e.g.:monocytic cellsendothelial cells

PROHIBITED

e.g.:lymphocyteserythrocytes

IL-1TNF-C5a

Tissue factor

VII VIIa

XI XIa

IX IXa

X Xa

IIa II

FibrinogenFibrin

VIII VIIIa

V VaXIII

XIIIa

XL-Fibrin

Activation by a serineprotease, e.g. hepsin

The theory of blood coagulation todayThe theory of blood coagulation today

Tissue factor

VII VIIa

XI XIa

IX IXa

X Xa

IIa II

FibrinogenFibrin

VIII VIIIa

V VaXIII

XIIIa

XL-Fibrin

Activation by a serineprotease, e.g. hepsin

The theory of blood coagulation todayThe theory of blood coagulation today

XII ?

Endo

thel

ial d

amag

e

F XI ¤ HMWK----

F XII----

PK ¤ HMWK----

activationF XIIa

F XIa

Kallikrein

PlasminogenPlasmin

t-PA

PAI-1

prourokinaseurokinase

Zn2+

Activatedplatelet

Tissue factor

VII VIIa

XI XIa

IX IXa

X Xa

IIa II

FibrinogenFibrin

VIII VIIIa

V VaXIII

XIIIa

XL-Fibrin

En

do

thel

ial

cell

TM

PCa

PC

PS

TFPI

AT

HC-II

Natural inhibitors of blood coagulation Natural inhibitors of blood coagulation

The classic coagulation systemThe classic coagulation system

• Austen DEG & Rhymes. A laboratory manual of blood coagulation. 1975.

XII XIIa

XI XIa

IX IXa

X Xa

II IIa

I Ia (fibrin)

Surface contact

Phospholipid, Ca++, VIII

Phospholipid, Ca++, V

VIIa VII

Ca++

Tissue factorAPTT Prothrombin time

X

Thrombin time

Clotting defects and bleeding

• Coagulation factor deficiencies seldom cause bleeding if the level of the deficient factor is >40%.

• APTT is normal when the level of coagulation factors is >40%.

• Therefore, APTT determined in a mixture of equal parts of normal plasma and plasma from a haemophiliac patient will be normal.

• Unless an inhibitor is present.

Antibodies against coagulation factors

• Two types:– Alloantibodies:

Patients with hereditary coagulopathy may develope antibodies against the deficient factor when treated with plasma-derived or recombinant factor concentrates

– Autoantibodies: Aquired antibodies, most often against factor VIII and typically in patients with autoimmune diseases, malignancy and in women during pregnancy and post partum. In half of the cases no underlying disease can be found. Incidence: 1-5 per 1.000.000.

APTT-based inhibitor test

Patient plasma

Normal plasma

mix

Add APTT reagents

Determine APTT

Patient plasma Normal plasma

mix

Haemophilia

Factor: <1% 100% >50%

APTT: normal

Patient plasma Normal plasma

mix

Patient with inhibitor

Factor: <1% 100% <50% due to excess of antibody

APTT: prolonged

APTT-based inhibitor test

– In some patients (low responders) the neutralizing effect of the antibody can be overcome by increasing the dose of factor concentrate

– In patients with high titers of antibody recombinant factor VIIa can be used to obtain haemostasis

Treatment of bleeding in patients with antibodies against coagulation factors

AM

PL

IFIC

AT

ION

INIT

IAT

ION

FIXa

FX FXa

FVII

FVIIa

TF

Thrombin

FVa FV

FVIIIa FVIII

FXIa FXI

Prothrombin

FIX

Fibrin Fibrinogen FXIIIa FXIII

Cross-linked fibrin

FIBRIN FORMATION

The ”by-passing effect” of factor VIIa

Platelet activati

on

30-year old female with refractory bleeding

APTT Plasma

Immediate (sec)

2 h incub (sec)

Normal pool (NP) 26,5 26,7Patient (Pt) 91,3 100,8Pt:NP = 1:1 38,5 63,2

Aquired factor VIII deficiency with a progressive inhibitor to factor VIII. Bethesda titer: 5.5 BU.

Algoritm for evaluation of prolonged APTT

• Exclude preanalytical factors causing spuriously prolonged APTT– Underfilled tubes, delayed testing, venipuncture above heparin lock

etc.

• Is the patient receiving antithrombotic treatment?– E.g. heparin, thrombin inhibitors, vitamin K antagonists, fibrinolytics

• Defect fibrin formation?– Determine fibrinogen concentation thrombin time

• If not – then test for inhibitors– Lupusinhibitor (Thrombophilia)– Antibodies against a coagulation factor (Haemophilia, aquired/cong.))

• If neg. inhibitor test: Coagulation factor deficiency– Contact factor deficiency (No bleeding)– Deficiency of other clotting factors (Haemophilia)

Algorithm for evaluation of prolonged APTT

Fibrinogen < 3 M Explore hypofibrinogenaemia

Explore possible AC treatment: Heparin: Thrombin time Vitamin K antagonist: INR

No

Pt:NP 1:1 mix immediate APTT

Inhibitorpresent

Pt:NP 1:1 mix Incub 2h APTT

Corrects APTT

Fails to correct APTT

Fails to correct APTT

Factor deficiency

Corrects APTT

Pt:APTT-reagent Incub 10 minutes

Contact factor deficiency

Procoagulant factor deficiencyFails to

correct APTT

Corrects APTT

Lupusinhibitor

Specificinhibitor

Phospholipid dependent

Phospholipid independent

Yes

Prolonged preincubation with APTT reagent

Asmis et al. Thromb Res 2002;105:463-70

PK-deficient plasma

Algorithm for evaluation of prolonged APTT

Fibrinogen > 3 M Explore hypofibrinogenaemia

Explore Thrombin time: Heparin?

INR: Vitamin K antagonist?

Yes

Pt:NP 1:1 mix immediate APTT

Inhibitorpresent

Pt:NP 1:1 mix Incub 2h APTT

Corrects APTT

Fails to correct APTT

Fails to correct APTT

Factor deficiency

Corrects APTT

Pt:APTT-reagent Incub 10 minutes

Contact factor deficiency

Procoagulant factor deficiencyFails to

correct APTT

Corrects APTT

Lupusinhibitor

Specificinhibitor

Phospholipid dependent

Phospholipid independent

No

Thombosis

Symptoms

Bleeding

Bleeding

None

Evaluation of 177 consecutive cases of prolonged APTT

Results Chng et al. 2005

0%

20%

40%

60%

80%

100%

LA FXII def

FXI def FVIII def

Acq FVIII inh vWD

FXI+XII def FXII+vWD

No obvious cause

Evaluation of 177 consecutive cases of prolonged APTT

Chng et al. 2005

LA

No obvious cause

Factor deficiencies: 15 %

Factor XIII deficiency

• In FXIII deficiency the APTT, PT and thrombin time are normal.

• Moderate to severe FXIII deficiency can be diagnosed by the clot solution test.

• A fibrin clot prepared from patient plasma is placed in 8 M urea.

• Dissolution of the clot within 24 hours is suggestive of FXIII deficiency.

Blood coagulation Screening assays and single factor assessment

Jørn Dalsgaard Nielsen

Thrombosis Centre Gentofte Hospital

Copenhagen, DenmarkE-mail: jdn@dadlnet.dk