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From macrohemodynamic to the microcirculation

Donati, A.

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From Macrohemodynamic

to the Microcirculation

Abele Donati

noitalucricorciM eht ot ci

manydomehorca

M morF

– A

bele

Don

ati

General Introduction

1

From

Macrohemodynamic

to the

Microcirculation

Abele Donati


2

From Macrohemodynamic to the Microcirculation

ISBN: 978-94-6182-705-0

© Abele Donati, 2016

Printing: Off Page, www.offpage.nl


3


ACADEMISCH PROEFSCHRIFT

ter verkrijging van de graad van doctor

aan de Universiteit van Amsterdam

op gezag van de Rector Magnificus

prof. dr. ir. K.I.J. Maex

ten overstaan van een door het College voor Promoties ingestelde commissie,

in het openbaar te verdedigen in de Agnietenkapel

op dinsdag, 13 september 2016, te 10:00 uur

door

Abele Donati

geboren te Salsomaggiore Terme, Italië



4

Promotiecommissie

Promotor: Prof. dr. ir. C. Ince Universiteit van Amsterdam

Overige leden:

prof. dr. J.J. van Lieshout Universiteit van Amsterdam

prof. dr. A.J. Verhoeven Universiteit van Amsterdam

prof. dr. J.C. Preiser l'Université libre de Bruxelles

prof. dr. N.P. Juffermans Universiteit van Amsterdam

prof. dr. T.M. van Gulik Universiteit van Amsterdam

dr. E.C. Boerma Universiteit van Amsterdam

Faculteit der Geneeskunde


1

CONTENTS

General Introduction From Macrohemodynamic to the Microcirculation

3

Outline of the thesis 23

Chapter 1 Goal-Directed Intraoperative Therapy Reduces Morbidity and Length of Hospital Stay in High-Risk Surgical Patients

27

Chapter 2 Does methylene blue administration to septic shock patients affect vascular permeability and blood volume?

45

Chapter 3 Predictive value of interleukin 6 (IL-6), interleukin 8 (IL-8) and gastric intramucosal pH (pH-i) in major abdominal surgery

59

Chapter 4 A Comparison Among Portal Lactate, Intramucosal Sigmoid pH, and ∆CO2 (PaCO2 - Regional

PCO2) as Indices of Complications

in Patients Undergoing Abdominal Aortic Aneurysm Surgery

75

Chapter 5 Recombinant activated protein C treatment improves tissue perfusion and oxygenation in septic patients measured by near-infrared spectroscopy

93

Chapter 6 The aPC treatment improves microcirculation in severe sepsis/septic shock syndrome

109

Chapter 7 Levosimendan for resuscitating the microcirculation in patients with septic shock: a randomized controlled study

127


2

Chapter 8 Effects of fresh leukoreduced vs. non-leukoreduced red blood cells transfusions on microcirculation and tissue oxygenation in septic patients: a pilot study.

149

Chapter 9 Plasma Free Hemoglobin and Microcirculatory Response to Fresh or Old Blood Transfusions in Sepsis

175

Chapter 10 Towards integrative physiological monitoring of the critically ill: from cardiovascular to microcirculatory and cellular function monitoring at the bedside

199

Summary and conclusions

217

Samenvatting and conclusies

223

Reference List 229

Acknowledgments 269

Curriculum vitae and Portfolio

271

List of pubblications 275


3

GENERAL INTRODUCTION

From Macrohemodynamic to the

Microcirculation

Abele Donati,1,2,3 Roberta Domizi,1 Elisa Damiani,1 Erica Adrario,1,2 Paolo Pelaia,1,2 and Can Ince3

1Sezione di Anestesia e Rianimazione, Dipartimento di Scienze Biomediche e Sanità Pubblica, Università Politecnica delle Marche,

Ancona, Via Tronto 10, 60020 Torrette (Ancona), Italy 2AOU Ospedali Riuniti, Via Conca 71, 60020 Ancona, Italy

3Department of Translational Physiology, Academic Medical Center, University of Amsterdam, Meibergdreef 9, 1105 AZ Amsterdam, The

Netherlands

Published in: Critical Care Research and Practice 2013; Volume 2013 (2013), Article ID 892710


4

Abstract

ICU patients need a prompt normalization of macrohemodynamic

parameters. Unfortunately, this optimization sometimes does not protect

patients from organ failure development. Prevention or treatment of

organ failure needs another target to be pursued: the microcirculatory

restoration. Microcirculation is the ensemble of vessels of maximum

100 m in diameter. Nowadays the Sidestream Dark Field (SDF) imaging

technique allows its bedside investigation and a recent round-table

conference established the criteria for its evaluation. First,

microcirculatory derangements have been studied in sepsis: they are

mainly characterized by a reduction of vessel density, an alteration of

flow, and a heterogeneous distribution of perfusion. Endothelial

malfunction and glycocalyx rupture were proved to be the main reasons

for the observed microthrombi, capillary leakage, leukocyte rolling, and

rouleaux phenomenon, even if further studies are necessary for a better

explanation. Therapeutic approaches targeting microcirculation are under

investigation. Microcirculatory alterations have been recently

demonstrated in other diseases such as hypovolemia and cardiac failure

but this issue still needs to be explored. The aim of this paper is to gather

the already known information, focus the reader’s attention on the

importance of microvascular physiopathology in critical illness, and

prompt him to actively participate to achieve a more comprehensive

understanding of the issue.


5

1. Introduction

The introduction in clinical practice of pulmonary artery catheter (PAC) about 40 years ago [1] allowed clinicians to measure the cardiac output (CO) at the bedside with the thermodilution technique [2]. Moreover, with an arterial and mixed venous gas analysis, arterial (CaO2) and

mixed venous oxygen content (CvO2) could be easily calculated and

oxygen availability (DO2) and consumption (VO2) consequently

obtained by applying the following simple formulas: DO2 = CO ∗ CaO2

and VO2 = CO ∗ (CaO2 − CvO2). Old well-known physiologic data were

available at the bedside as well as clinical parameters but their interpretation and utilization as a therapeutic target was and remains controversial to date.

Shoemaker was the first clinician to try to interpret and utilize these new hemodynamic data. He was a surgeon and monitored the high risk surgical patients with PAC before, during, and after the operations [3]. He observed that patients could be divided into three groups on the basis of outcome: survived, survived with complications, and died. From the analysis of the hemodynamic data, patients with better outcome resulted to have CO, DO2, and VO2 values higher than the others and,

additionally, far higher than those considered as normal. Based on these observational data, he conceived the supernormal values of CO, DO2,

and VO2 as therapeutic goals and obtained in his trial a reduction in

mortality from 28% in the control groups to 4% in the protocol group [4]. Control groups included both patients with just a central venous catheter and patients monitored with a PAC, but with using normal values of CO, DO2, and VO2 as therapeutic targets. According to these data, physicians

began to target supernormal CO, DO2, and VO2 values in critically ill

patients, which seemed to be the best treatment. Nevertheless, the results were not so good. Gattinoni et al. did not find any difference between patients treated when targeting normal and supernormal CO, DO2, and

VO2 values or a mixed venous saturation (SvO2) higher than 70% [5].

Hayes et al. found an increased mortality in patients treated with supernormal values as target [6]. Many doubts aroused among the intensivists, especially because of the hemodynamic stress due to hypervolemia and the infusion of inotropes, such as dobutamine, with an increased risk of myocardial ischemia and arrhythmias. That is why less than twenty years ago Vincent studied the VO2/DO2 relationship and

observed that VO2 is usually independent from a wide range of DO2


6

because of compensation mechanisms [7]. At first, if CaO2 decreases,

CO increases to maintain the same DO2 levels; then, when this

compensation exhausted, another compensatory mechanism occurs due to increased oxygen extraction ratio (O2ER) which maintains normal levels

of VO2 despite the DO2 reduction. When this compensation is exhausted

too, VO2 becomes dependent on DO2 and the poorly efficient anaerobe

metabolism begins. This leads to metabolic acidosis and oxygen debt. This normally happens when O2ER is near to 60%, but in some

situations, for instance, during anaesthesia or sedation, critical O2ER

could decrease until 30%. Therapy should aim to avoid the VO2/DO2

dependency to maintain O2ER lower than 30% near to normal values. In

any case, according to the authors, only patients with high O2ER can

benefit from hemodynamic optimization, while patients with normal O2ER, even if without high CO and DO2, do not need to be subjected to

cardiovascular stress. The authors proposed the following simple dobutamine test: CO should be increased only if VO2 increases together

with CO after dobutamine infusion, otherwise this is not necessary [8].

Despite these results in critically ill patients, in high risk surgical patients, the investigators continued to observe a decreased mortality using supernormal targets, including a recent meta-analysis [9–13]. Indeed some data indicate that also for these patients the VO2/DO2 relationship

should be targeted and tested by the O2ER or the more feasible central

venous oxygen saturation (ScvO2) [14]; however, these targets also seem

to work only in high risk surgical patients and not in ICU patients. Why this discrepancy occurs?

Analyzing the Italian multicenter trial published in 1995 [5], in which ICU patients were treated following three hemodynamic goals (high values of CO, DO2, and VO2, normal values of the same ones; SvO2 >

70%) and enrolled within the first 48 hours from ICU admission, the difference between ICU and surgical patients is quite obvious: in surgical patients the moment when the “noxa patogena” begins (i.e., the operation) is exactly known and the hemodynamic treatment can be started at the same time or even before. In ICU patients it is almost always impossible to know when the pathogenic course of the illness begins, and even if that is known (i.e., trauma), ICU hemodynamic treatments are quite often started after several hours. Moreover, in the Italian study patients were enrolled even 48 hours after the ICU admission. Time is the issue. In 1995 Donati et al. published a study on a


7

cardiac index (CI)/O2ER diagram [15]. CI and O2ER values were taken

at the admission, after 12, 24, and 48 hours in any patient with a pulmonary catheter placed at the ICU admission, and values of each time point were plotted in a CI/O2ER diagram dividing survivors and

nonsurvivors. Only at 24 h after ICU admission data were significantly differentiated between survivors and nonsurvivors, with survivors in the most favourable part of the diagram (normal/high CI and normal/low O2ER). Time is the issue.

Rivers et al. more recently reported that septic shock patients who were aggressively treated, following a strict hemodynamic protocol and using the ScvO2 within the first 6 hours after hospital admission as therapeutic

goal, had a better outcome than patients treated with normal target [16]. Nevertheless, some experts argued that the use of absolute goals themselves (i.e., ScvO2, haemoglobin levels, central venous pressure,

and mean arterial pressure) may not have been so crucial, while the positive results might mainly depend on the early implementation of the protocol and the greater promptness in the therapeutic approach. Once more, time is the issue [17].

Nowadays, the use of PAC has markedly decreased since new less invasive cardiac output measuring devices are available [18], such as PiCCO system, LiDCO system [19], EV1000/VolumeView system, the pressure analytical method (PRAM), and transthoracic or esophageal Doppler devices. However, whatever the monitoring method used, macrohemodynamic has to be optimized as soon as possible within the first hours from an initial hit. We can choose as therapeutic goals high CO, DO2, and VO2 values, according to Shoemaker’s philosophy, or

more “gentle” targets such as ScvO2 or O2ER [20], or fluid optimization

following fluid-responsiveness parameters [21]. After these first hours, aiming to macrohemodynamic targets— although obviously important—is not sufficient to prevent organ failures or death, for which we need to identify some other targets. Treating the microcirculation might be the solution.

The microcirculation is the ensemble of vessels with diameter lower than 100 µm and we can distinguish between small vessels (diameter lower than 20 µm), medium vessels (diameter between 20 and 50 µm), and great vessels (50–100 µm).

Until few years ago, we were not able to observe the microcirculation at the bedside: indeed, intravital microscopy needs a back light and a circulating dye, conditions that could not be usually applied in the


8

clinical practice. The Orthogonal Polarization Spectral (OPS) [22] and, more recently, the Sidestream Dark Field (SDF) imaging have allowed clinicians to observe in vivo, at the bedside, the sublingual microcirculation: this site is not only the most accessible location to examine, but it also has to be considered as an excellent mirror for the splanchnic microcirculation, as demonstrated by Boerma few years ago [23, 24]. Figure 1(a) provides an example of the sublingual microcirculation as it appears under physiological conditions.

Figure 1: SDF images of the sublingual microcirculation. (a) Healthy

subject; (b) septic shock; (c) hypovolemia; (d) cardiogenic shock.

In 2005, a round-table conference was organized in Amsterdam in order to score the microcirculation and the following parameters have been suggested: a measure of vessel density (total or perfused vessel density), two indices of vascular perfusion (proportion of perfused vessels and microcirculatory flow index), and a flow heterogeneity index (Table 1) [25].

These indices answer the three crucial questions that we should ask: how many vessels are perfused, what is the quality of the flow, and whether there are nonperfused areas next to the well-perfused ones.


9

But now the main question is which are the alterations we can find in different pathologies, such as sepsis, hypovolemia, or cardiac failure?

2. Microcirculation in Sepsis

Sepsis is the first pathophysiologic condition in which microcirculation was studied. Microcirculatory alterations in sepsis have been found in both experimental and human studies [26, 27].

Alterations are both quantitative, such as reduced vessel density, and qualitative, such as altered blood flow (slowed, intermittent, or even stopped).

Moreover, heterogeneity of perfusion has been observed, with normally perfused areas bordering areas with altered capillary flow: the consequent increase in the distance between capillaries and cells makes hypoxia easier to quickly appear.

One of the main manifestations of heterogeneity is the appearance of areas with vascular stop flow and flow shunting from the arterial circulation to the venous, particularly in the intestinal villi, liver, diaphragm, skeletal muscle, and sublingual microcirculation. Figure 1(b) shows an example of the sublingual microcirculation during sepsis.

The PO2 gap can be used to quantify the oxygen extraction deficit that

follows such shunt, representing a marker of severity of the shunt, and it is clinically associated with blood lactate and venous PO2 increase [26].

It has been hypothesized that these phenomena (heterogeneity, stoppage and shunting of the flow, perfusion deficit) may derive from a failure in autoregulation mechanisms, first of all due to an altered expression of the inducible nitric oxide synthase (iNOS) in some areas of the vascular bed; in regions where iNOS is poorly produced, vasodilation may be impeded up to the degree that it will not be sufficient to ensure the perfusion [28, 29].

There are many reasons for these alterations: microthrombi, capillary leakage, leukocyte rolling (Figure 2), and rouleaux phenomenon, but endothelial malfunction and glycocalyx ruptures probably play a central role.


10

Table 1: Parameters for the evaluation and scoring of the microcirculation.

Microcirculation

parameter

Information

provided Measurement

Microvascular

flow index

(MFI)

Perfusion

quality (for

small, medium,

and large

vessels*)

The image is divided into four quadrants; a

number is assigned for each quadrant according

to the predominant type of flow (0 no flow; 1

intermittent; 2 sluggish; 3 continuous). The

MFI results from the averaged values.

De Backer score

(n/mm) Vessel density

The image is divided by 3 vertical and 3

horizontal lines; the De Backer score is

calculated as the number of vessels crossing the

lines divided by the total length of the lines

Total vessel

density

(mm/mm2)

Vessel density

(for small,

medium, and

large vessels*)

Total length of vessels is divided by the total

surface of the analyzed area

Perfused vessel

density

(mm/mm2)

Functional

vessel density

(for small,

medium, and

large vessels*)

Total length of perfused vessels (sluggish or

continuous) is divided by the total surface of the

analyzed area

Proportion of

perfused vessels

(%)

Perfusion

quality (for

small, medium,

and large

vessels*)

100* number of perfused vessels is divided by

the total number of vessels

Flow

heterogeneity

index (FHI)

Perfusion

heterogeneity

The difference between the highest MFI and the

lowest MFI is divided by the mean MFI. MFI is

intended as the averaged MFI of each site

*Vessel diameter classification: <20 µ small; 20–50 µ medium; 50–100 µ large. Three or five sites are evaluated. MFI of small vessels can be calculated separately.


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Figure 2: Sequence of SDF images of the sublingual microcirculation in

a septic patient, showing the passage of a rolling leukocyte in a

postcapillary venule.

It is well known that the endothelial cell is the crucial component of this auto-regulated system. Two endothelial functions among the others are the shear stress transduction and the activation of the response to catecholamines, prostaglandins, endothelin, bradykinin, thromboxane, and adenosine. It also participates to cell-to-cell communication in order to attend at local signs integration [30, 31].

Endotoxemia damages the endothelial cell, thereby breaking this chain and potentially impeding a sufficient tissue perfusion to be assured.

The septic status is also associated with the reduction of the arteriolar muscular tone (with a lower response to adrenergic stimuli), impairment of red blood cells deformability (even more for the old ones), and increase in platelet aggregation tendency [30–33].

However, the exact reaction of platelets, red blood cells, and leucocytes is poorly understood.

Red blood cells become rigid and unshrinkable, haematic viscosity increases, and fibrin deposition rises. When, finally, platelets aggregate, microthrombi appear and drastically occlude smaller vessels [32, 33].

Bateman et al. observed that the leukocyte rolling, adhesion, and activation is an early step in septic evolution and it has to be attributed to an upregulation of adhesion molecules and inflammatory cascade [34].

The following radical oxygen species production may be considered as the main factor responsible for the glycocalyx rupture.


12

The glycocalyx represents a blood-tissue interface deriving from the endothelial cells and consisting of a proteoglycan, hyaluronan, and glycosaminoglycan-made layer, combined with plasmatic proteins. It participates to vascular tone regulation and mechanic impulses transduction and is also responsible for RBCs velocity variation. A rupture in its structure impairs all these mechanisms [34–36].

As already said, restoring a normal microcirculation is essential for a good outcome; therefore, this target has to be included in any therapeutic approach in septic shock resuscitation.

A therapeutic strategy combining volume resuscitation, use of vasopressors, inotropic agents, vasodilators, and RBC transfusions (aimed to obtain an adequate global oxygen delivery) will not succeed in improving the outcome if it cannot recruit the microcirculation nor restore the microvascular flow [37].

Dubin et al. demonstrated in twenty septic shock patients that reaching a good MAP (>65 mmHg) with increasing doses of norepinephrine can improve cardiac index, pulmonary pressures, systemic vascular resistance, and left and right ventricular stroke work indexes, but not the microvascular perfusion. It might be harmful in some patients [38].

Sakr et al., as well as many other authors, demonstrated that the recovery of macrohemodynamic stability does not necessarily match with microhemodynamic improvement, organ function restoration, and improved survival; experimental models of resuscitated septic shock show that microvascular perfusion is often altered despite the normalization of systemic and regional hemodynamic [39].

Bateman and Walley showed that microhemodynamic restoration leads to organ function improvement and evident decrease in mortality [40].

Furthermore, according to Top et al., persistent microcirculatory alteration can be prognostic of mortality [41].

Therefore, blood pressure, cardiac index, and other macrohaemodynamical variables have not to be considered as reliable markers of septic shock recovery.

De Backer et al. showed no correlation between arterial blood pressure and microvascular perfusion during sepsis, while they demonstrated the relation between the proportion of perfused capillaries and mortality [42]. Similar results were then reported on a larger sample by Sakr et al. [43].


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In the last years, many researchers focused their attention on the microvascular response to pharmacological interventions, in an attempt to find therapies able to restore the microcirculatory flow.

The activated-protein C (aPC) is among the most interesting and studied drugs [44]; a nonrandomized study, conducted by de Backer et al., have demonstrated that an infusion of aPC in septic patients can improve the microcirculatory flow. An increase in the proportion of perfused capillaries and a more rapid resolution of hyperlactatemia have been also found in the aPC treated patients, unlike the control group [45].

In a prospective observational open study, Donati et al. measured the tissue oxygen saturation (StO2) using the near infrared spectroscopy

during a vascular occlusion test and demonstrated an improvement in both the StO2 downslope and upslope in patients treated with aPC, unlike

the controls, reflecting an improved microvascular reactivity [46].

An aPC administration during experimental endotoxaemia can improve intestinal microcirculation by protecting functional capillary density and exerts an anti-inflammatory effect by reducing leukocyte rolling and adherence to the endothelium in each submucosal venule; protection from leukocytic inflammation is probably mediated by a modulation of adhesion molecules expression on the surface of leukocytes and endothelial cells [47].

Unfortunately, aPC was removed from clinical use by the company after the PROWESS-Shock trial in septic shock patients because it failed to reduce mortality, compared to placebo [48].

Morelli et al. obtained good results using terlipressin (a vasopressin analogue, relatively selective for V1 receptors) as adjunctive vasopressor agent in experimental models of vasodilatatori hyperdynamic septic shock unresponsive to catecholamine infusion; both bolus and continuous infusion of terlipressin seem to improve the microcirculation [49–51].

It is not clearly understood whether a continuous or intermittent infusion is to be preferred; recent studies demonstrated that a continuous therapy is associated with less organ dysfunction in endotoxemic sheep, but the relationship with septic shock outcome is not clear [52].

Finally it was recently demonstrated that levosimendan is better than dobutamine in improving the microcirculation in stabilized septic shock patients [53].


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3. Microcirculation in Cardiac Failure

Few years after these studies many authors turn their attention to the microvascular reactions to acute heath failure (AHF) and cardiogenic shock.

De Backer et al. evaluated the sublingual microcirculation in 40 patients within the first 48 hours after an AHF and found alterations similar to those of sepsis; while the capillary density and the perfusion of large vessels were preserved, the proportion of perfused small vessels (PPVs) was acutely reduced and the extent of such reduction strictly related to survival [54]. An example of sublingual microcirculation derangement during cardiogenic shock can be seen in Figure 1(d).

The reliability of these results is so strong that the sublingual SDF was used by Lam et al. to evaluate the effective myocardial recovery and the optimization of organ perfusion in STEMI patients treated with PCI and a percutaneous left ventricular support [55].

The main difference between cardiogenic shock and septic microvascular derangement is that microvascular alterations in AHF are not completely independent from changes in macrocirculation; indeed, a relationship between cardiac output and microcirculatory status can be seen.

Therefore, a good therapeutic strategy should target restoring both macro- and microcirculation. Many clinical approaches have been considered, aiming to evaluate which one fulfills both objectives. For example, Erol-Yilmaz et al. proved that the cardiac resynchronization therapy (CRT) used to improve the systemic pressure can stabilize also the microcirculation [56]. Additionally, Munsterman et al. demonstrated that the intra-aortic balloon pump used to mechanically support the hearth often impairs the microcirculatory flow and its withdrawal can paradoxically improve the microcirculatory flow of small vessels [57].

Besides, den Uil et al. used the SDF imaging in patients with AHF during and after a low dose administration on nitroglycerin , which is sufficient to decrease central venous pressure and pulmonary capillary wedge pressure. They found a significant increase of PCD in patients responding to NTG; therefore, NTG does affect not only uniquely the cardiac muscle but also any peripheral tissue [58].

Future studies might examine the response to higher doses of NTG, nitroprusside (which releases NO by non-enzymatic means), and hydralazine (a nonNO donor vasodilator) in order to better understand the situation of nonrespondant patients.


15

4. Microcirculation in Hypovolemia

Functional capillary density deteriorates in hypovolemia when the mean blood pressure drastically decreases.

The ultimate goal of volume replacement therapy is to improve organ perfusion thereby sustaining an adequate oxygenation. Too few studies have been conducted to evaluate the effects of this strategy on microcirculation, but colloids generally tend to be considered superior to crystalloids in improving tissue perfusion [59]. However, whether they can also improve the outcomes still needs to be proved.

Microcirculatory changes are smaller in hypovolemia than sepsis; for similar blood pressure levels, hypovolemic rats showed a lower percentage of nonperfused capillaries than septic shock rats, and the red blood cell velocity was nearly always preserved. Even if the observed alterations are fewer than those in sepsis, they are though related to mortality in animal models [60]. A typical example of sublingual microcirculation during hypovolemia is provided in Figure 1(c).

Hemorrhagic shock leads to intestinal microvascular endothelium damage; the endothelial cells become oedematous and cell membrane and mitochondria are quickly injured; SOD activity is enhanced and the activity of CAT and GSH-PX decreases. Korzonek and Gwóźdź proved that an I.V. administration of endothelin-1 can restore normal blood pressure, prevent it to fall, and restrict the ischemic injury on microcirculation, thereby prolonging the survival in animals with hemorrhagic shock [61].

In addition, the results presented by Fang et al. suggest that in hypovolemia, as well as cardiac failure, microvascular alterations are not completely independent from global haemodynamic parameters [62].

An experimental study by Legrand et al. showed that kidneys are particularly prone to hypoxia even in a high MAP-directed fluid resuscitation (>80 mmHg); the renal microvascular PO2 does not

improve compared with fluid resuscitation targeting to a . Moreover, a decreased renal oxygenation persists after blood transfusion [63].

These findings need to be confirmed in human studies and resuscitation strategy for hemorrhagic shock remains controversial.

5. Conclusions

Further research is required to improve microcirculatory flow knowledge. A recent multicenter prevalence study [64] is aimed to assess the


16

relationship between microcirculatory dysfunction and severity of illness and to investigate the prevalence of sublingual microcirculatory alterations in intensive care unit (ICU) patients, regardless of their underlying disease, monitored at a single time point in all the different participating centers. This is the first step towards a more comprehensively understanding of what happens at the microcirculatory level during life-threatening illness, to identify the relationship with macrohemodynamics and to evaluate whether drugs used in ICU to improve hemodynamic status and organ functions can also improve the microcircuvascular flow. The biggest step forward will be made when treatments selectively targeted to resuscitate the microcirculation will be found.

For the moment, according to our knowledge, we can state: treat the macrohemodynamic as soon as possible, but if the patient does not get better, look at the microcirculation and try to resuscitate it!

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18

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37. M. Y. Rady, E. P. Rivers, and R. M. Nowak, “Resuscitation of the critically ill in the ED: responses of blood pressure, heart rate, shock index, central venous oxygen saturation, and lactate,” American Journal of Emergency Medicine, vol. 14, no. 2, pp. 218–225, 1996.

38. A. Dubin, M. O. Pozo, C. A. Casabella et al., “Increasing arterial blood pressure with norepinephrine does not improve microcirculatory blood flow: a prospective study,” Critical Care, vol. 13, no. 3, article no. R92, 2009.

39. Y. Sakr, M. J. Dubois, D. de Backer, J. Creteur, and J. L. Vincent, “Persistent-microcirculatory alterations are associated


20

with organ failure and death in patients with septic shock,” Critical Care Medicine, vol. 32, no. 9, pp. 1825–1831, 2004.

40. R. M. Bateman and K. R. Walley, “Microvascular resuscitation as a therapeutic goal in severe sepsis,” Critical Care, vol. 9, no. 4, pp. S27–S32, 2005.

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42. D. de Backer, J. Creteur, J. C. Preiser, M. J. Dubois, and J. L. Vincent, “Microvascular blood flow is altered in patients with sepsis,” American Journal of Respiratory and Critical Care Medicine, vol. 166, no. 1, pp. 98–104, 2002.

43. Y. Sakr, M. J. Dubois, D. de Backer, J. Creteur, and J. L. Vincent, “Persistent-microcirculatory alterations are associated with organ failure and death in patients with septic shock,” Critical Care Medicine, vol. 32, no. 9, pp. 1825–1831, 2004.

44. J. L. Vincent, “Drotrecogin alfa (activated): the treatment for severe sepsis?” Expert Opinion on Biological Therapy, vol. 7, no. 11, pp. 1763–1777, 2007.

45. D. de Backer, C. Verdant, M. Chierego, M. Koch, A. Gullo, and J. L. Vincent, “Effects of drotrecogin alfa activated on microcirculatory alterations in patients with severe sepsis,” Critical Care Medicine, vol. 34, no. 7, pp. 1918–1924, 2006.

46. A. Donati, M. Romanelli, L. Botticelli et al., “Recombinant activated protein C treatment improves tissue perfusion and oxygenation in septic patients measured by near-infrared spectroscopy,” Critical Care, vol. 13, supplement 5, p. S12, 2009.

47. J. N. Hoffmann, B. Vollmar, M. W. Laschke, J. M. Fertmann, K. W. Jauch, and M. D. Menger, “Microcirculatory alterations in ischemia-reperfusion injury and sepsis: effects of activated protein C and thrombin inhibition,” Critical Care, vol. 9, supplement 4, pp. S33–S37, 2005.

48. V. M. Ranieri, B. T. Thompson, P. S. Barie et al., “Drotrecogin alfa (activated) in adults with septic shock,” The New England Journal of Medicine, vol. 366, no. 22, pp. 2055–2064, 2012.

49. A. Morelli, C. Ertmer, P. Pietropaoli, and M. Westphal, “Terlipressin: a promising vasoactive agent in hemodynamic support of septic shock,” Expert Opinion on Pharmacotherapy, vol. 10, no. 15, pp. 2569–2575, 2009.

50. A. Morelli, A. Donati, C. Ertmer et al., “Short-term effects of terlipressin bolus infusion on sublingual microcirculatory blood


21

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51. A. Morelli, A. Donati, C. Ertmer et al., “Effects of vasopressinergic receptor agonists on sublingual microcirculation in norepinephrine-dependent septic shock,” Critical Care, vol. 15, no. 5, p. R217, 2011.

52. M. Lange, C. Ertmer, S. Rehberg et al., “Effects of two different dosing regimens of terlipressin on organ functions in ovine endotoxemia,” Inflammation Research, vol. 60, no. 5, pp. 429–437, 2011.

53. A. Morelli, A. Donati, C. Ertmer et al., “Levosimendan for resuscitating the microcirculation in patients with septic shock: a randomized controlled study,” Critical Care, vol. 14, no. 6, article R232, 2010.

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55. K. Lam, K. D. Sjauw, J. P. S. Henriques, C. Ince, and B. A. de Mol, “Improved microcirculation in patients with an acute ST-elevation myocardial infarction treated with the Impella LP2.5 percutaneous left ventricular assist device,” Clinical Research in Cardiology, vol. 98, no. 5, pp. 311–318, 2009.

56. A. Erol-Yilmaz, B. Atasever, K. Mathura et al., “Cardiac resynchronization improves microcirculation,” Journal of Cardiac Failure, vol. 13, no. 2, pp. 95–99, 2007.

57. L. D. H. Munsterman, P. W. G. Elbers, A. Ozdemir, E. P. A. van Dongen, M. van Iterson, and C. Ince, “Withdrawing intra-aortic balloon pump support paradoxically improves microvascular flow,” Critical Care, vol. 14, no. 4, article R161, 2010.

58. C. A. den Uil, W. K. Lagrand, P. E. Spronk et al., “Low-dose nitroglycerin improves microcirculation in hospitalized patients with acute heart failure,” European Journal of Heart Failure, vol. 11, no. 4, pp. 386–390, 2009.

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62. X. Fang, W. Tang, S. Sun et al., “Comparison of buccal microcirculation between septic and hemorrhagic shock,” Critical Care Medicine, vol. 34, no. 12, pp. S447–S453, 2006.

63. M. Legrand, E. G. Mik, G. M. Balestra et al., “Fluid resuscitation does not improve renal oxygenation during hemorrhagic shock in rats,” Anesthesiology, vol. 112, no. 1, pp. 119–127, 2010.

64. N. A. Vellinga, E. C. Boerma, M. Koopmans et al., “Study design of the Microcirculatory Shock Occurrence in Acutely Ill Patients (microSOAP): an international multicenter observational study of sublingual microcirculatory alterations in intensive care patients,” Critical Care Research and Practice, vol. 2012, Article ID 121752, 7 pages, 2012.


23

Outline of the thesis

Outline of the Thesis

24

Outline of the thesis

The current clinical studies presented in this thesis have been conducted

in the University Surgical-Medical ICU of AOU Ospedali Riuniti of

Ancona, Italy.

Hemodynamic monitoring has allowed the physicians to know the human

blood flow, that is the cardiac output, at the bedside since 1970 with the

pulmonary catheter. Moreover with arterial and mixed venous blood

gases arterial and mixed venous oxygen content, global oxygen

availability and global oxygen consumption, oxygen extraction ratio

could easily be calculated.

After almost 20 years Shoemaker demonstrated for the high risk surgical

patients that using as therapetucal goal supranormal hemodynamic values

patients’ outcome can be improved. But not everything can be explained

from global hemodynamic. In this septic shock cardiac output is very

high, oxygen avaibility very high but oxygen consumption very low and

patients die with normal/high global hemodynamic parameters.

Hemodynamic monitoring allows clinicians to manage cardiac output and

macrocirculation, but some patients, despite optimal macrohemodynamic

management, remain still critical.

The problem of tissue dysoxia raised and macrohemodynamic could not

explain everything. Tonometry was created by Fiddian-Green:

monitoring regional tissue perfusion of the gut was available. And for the

first time great differences between global hemodynamic and regional

perfusion were found: sometimes the last one was alterated with normal

or even high cardiac output and oxygen availability. This expecially

happened in septic shock. But this was not enough: other techniques were

made not only to monitor tissue perfusion but to visualize the

microcirculation at the bedside and these techniques have been applied

not only in septic shock but also in other critically conditions. Important

microcirculatory alterations have been found in septic shock patients and

thank to these techniques nowadays we know much more of the

pathophysiology and we are ready to explore new therapies for the

critically ill patient.


25

In critically ill patients we need to have a balance between oxygen

delivery and consumption: the oxygen extraction ratio represents this

balance. In Chapter 1 we aimed to compare the number of patients with

postoperative organ failure and length of hospital stay between those

randomized to conventional vs a protocolized strategy designed to

maintain O2ER < 27%..

In Chapter 2 we aimed to assess the effects of the inhibition of guanylate

cyclase, an enzyme involved in sepsis-related vascular and myocardial

dysfunctions, on hemodynamic variables including blood volume and

pulmonary vascular permeability during septic shock. Fifteen patients

with septic shock associated with persisting hypotension despite

conventional treatment including fluid loading, vasopressors, and

inotropes received a bolus dose of methylene blue (3 mg/kg)

intravenously over 10 mins. All the patients were monitored with

pulmonary catheter and a femoral artery catheter to calculate not only

CO, VO2 and DO2, but also hemodynamic volumes, such as

intrathoracic blood volume and extra vascular lung water.Hemodynamic

variables were recorded before methylene blue and 20 mins, 1 hr, and 2

hrs after the end of methylene blue infusion.

In the past years macrocirculation monitoring appeared to be not enough

to improve patient outcome and splanchnic perfusion was monitored with

gastric tonometer. In Chapter 3 we aimed to demonstrate that in

abdominal surgery, intraoperative splanchnic ischemia is directly

correlated to the increase of IL-6 and IL-8 plasma levels and to the

incidence of postoperative complications. In Chapter 4 we aimed to

demonstrate that PHi and ∆CO2 are sensitive prognostic indices during

abdominal aortic aneurysm surgery and that tonometry may identify

patients at higher risk of organ failure in the postoperative period.

Muscular perfusion during sepsis can be monitored with near-infrared

spectroscopy and vascular occlusion test can be applied to test tissue

metabolism and vascular reactivity in septic patients. In Chapter 5 we

aimend to demonstrate that treatment with activated protein C (rh-aPC)

may improve muscle oxygenation (StO2 baseline) and reperfusion (StO2

upslope) and that rh-aPC treatment may increase tissue metabolism (StO2


26

downslope). Sepsis causes important microcirculatory alterations.

Sublingual microcirculation can be monitored at the bedside with the

side-dark field (SDF) imaging and microcirculatory parameters can be

calculated off-line. In Chapter 6 we aimed to demonstrate that activated

protein C may improve also the microcirculation in patients with severe

sepsis/septic shock.

In chapter 7 the objective of the randomized controlled, double-blinded

clinical study was to elucidate the effects of levosimendan on systemic

and microvascular hemodynamics. On this basis, we aimed at rejecting

the null hypothesis that there is no difference in sublingual microvascular

blood flow - as measured by sidestream dark-field (SDF) imaging - in

patients with fluid-resuscitated septic shock treated with levosimendan as

compared with an active comparator drug (that is, dobutamine).

In a pilot study (Chapter 8) the primary aim was to compare the effects of

non-leukodepleted or leukodepleted RBC transfusions on microvascular

flow in septic patients. In addition, it was determined whether transfusion

of either non-leukodepleted or leukodepleted packed RBC units could

increase microcirculatory density and reactivity to improve tissue

oxygenation during sepsis. In Chapter 9 we evaluated whether old red

blood cell transfusion increases plasma fHb in sepsis and how the

microvascular response may be affected.

In Chapter 10 we discuss how current hemodynamic monitoring of

critically ill patients is mainly focused on monitoring of pressure-derived

hemodynamic variables related to systemic circulation. Increasingly,

oxygen transport pathways and indicators of the presence of tissue

dysoxia are now being considered. In addition to the microcirculatory

parameters related to oxygen transport to the tissues, it is becoming

increasingly clear that it is also important to gather information regarding

the functional activity of cellular and even subcellular structures to gain

an integrative evaluation of the severity of disease and the response to

therapy.

Chapter 1

27

Chapter 1

Goal-Directed Intraoperative Therapy

Reduces Morbidity and Length of

Hospital Stay in High-Risk Surgical

Patients

Abele Donati, MD; Silvia Loggi, MD; Jean-Charles Preiser, MD, PhD; Giovanni Orsetti, MD; Cristopher Mu¨ nch, MD; Vincenzo Gabbanelli,

MD; Paolo Pelaia, MD; and Paolo Pietropaoli, MD†

Published in: CHEST 2007; 132:1817–1824

Chapter 1

28

Abstract Background: Postoperative organ failures commonly occur after major abdominal surgery, increasing the utilization of resources and costs of care. Tissue hypoxia is a key trigger of organ dysfunction. A therapeutic strategy designed to detect and reverse tissue hypoxia, as diagnosed by an increase of oxygen extraction (O2ER) over a predefined threshold, could

decrease the incidence of organ failures. The primary aim of this study was to compare the number of patients with postoperative organ failure and length of hospital stay between those randomized to conventional vs a protocolized strategy designed to maintain O2ER < 27%.

Methods: A prospective, randomized, controlled trial was performed in nine hospitals in Italy. One hundred thirty-five high-risk patients scheduled for major abdominal surgery were randomized in two groups. All patients were managed to achieve standard goals: mean arterial pressure> 80 mm Hg and urinary output > 0.5 mL/kg/h. The patients of the “protocol group” (group A) were also managed to keep O2ER < 27%.

Measurements and main results: In group A, fewer patients had at least one organ failure (n 8, 11.8%) than in group B (n 20, 29.8%) [p < 0.05], and the total number of organ failures was lower in group A than in group B (27 failures vs 9 failures, p < 0.001). Length of hospital stay was significantly lower in the protocol group than in the control group (11.3 ± 3.8 days vs 13.4 ± 6.1 days, p < 0.05). Hospital mortality was similar in both groups. Conclusions: Early treatment directed to maintain O2ER at < 27%

reduces organ failures and hospital stay of high-risk surgical patients. Clinical trials.gov reference No. NCT00254150

Key words: central venous saturation; goal-directed therapy; high-risk surgical patient; oxygen extraction ratio Abbreviations: ASA American Society of Anesthesiologists; CVP central venous pressure; HR heart rate; MAP mean arterial pressure; NS not significant; O2ER oxygen extraction ratio; O2ERe oxygen

extraction ratio estimated; PRBC packed RBC; SaO2 arterial oxygen

saturation; ScvO2 central venous saturation

Chapter 1

29

The development of postoperative organ failures severely affects the prognosis of surgical patients and substantially increases the utilization of resources and cost of care. The prevalence of organ failures ranges from 27 to 77%. Length of stays in the ICU and in the hospital as well as postoperative mortality are largely increased in “high-risk” patients, for whom preoperative risk factors are unavoidable.1,2 Therefore, the use of early and efficient therapeutic strategies able to detect and to treat potential triggers of organ failures, such as tissue hypoperfusion, is particularly important in this highrisk population. If hypoperfusion is not adequately managed, tissue hypoxia could occur, resulting from an impairment of the adaptive mechanisms of myocardial contractile function, under the influence of inflammatory mediators, and the peripheral tissues will then increase their oxygen extraction (O2ER).1–4

When O2ER increases over a threshold value, venous oxygen saturation

will decrease and lactic acidosis can ultimately occur.5 Hence, the use of O2ER calculated from arterial and mixed venous oxygen saturation as a

therapeutic goal is appropriate to monitor goal-directed hemodynamic strategies because it reflects the balance between oxygen delivery and consumption.6,7 The ensuing therapeutic approach will then imply the application of a standardized algorithm as soon as O2ER reaches a

predefined threshold. This concept, which differs from the “preoperative optimization of oxygen delivery”8,9 or the strategies aiming at the maintenance of stroke volume,10 was already assessed in mixed populations of critically ill patients11,12 and patients with early sepsis.13

The interpretation of venous oxygen saturation is eventually similar when mixed venous blood drawn from a pulmonary artery catheter is replaced by venous blood drawn from a central venous line.14 Indeed, evidence suggests that a multifaceted goaldirected strategy, including fluid challenge, blood transfusion, and inotropes titrated to keep central venous oxygen saturation higher than a predetermined threshold of 70%, was associated with decreased mortality and rate of organ failures when applied from the early phase of septic shock or severe sepsis.13 The aim of the present multicenter, prospective, randomized study was to compare the outcomes of patients randomized to a conventional management or to a therapeutic strategy guided by O2ER estimate (O2ERe) calculated from

the arterial oxygen saturation (SaO2) and central venous saturation

(ScvO2), Specifically, we hypothesized that the use of a goal-directed

protocol aimed at maintaining the O2ERe below a previously defined

“critical” (able to discriminate survivors from nonsurvivors) value of 27%15 during surgical interventions in high-risk patients will reduce the

Chapter 1

30

rate of postoperative organ failures, hospital length of stay, and mortality, as compared with the standard management based on the monitoring of mean arterial pressure (MAP), central venous pressure (CVP), and urinary output.16,17

Materials and Methods

This prospective, randomized, controlled, multicenter study was approved by the Hospital Ethical Committee of Ancona for all the institutions involved in the trial. Written informed consent was obtained preoperatively from the patients. The study was performed in nine Italian hospitals during 48 months. In these hospitals, major abdominal surgery and abdominal aortic surgery were routinely performed, and these patients were usually admitted after surgery in the ICUs (5 to 12 beds).

Inclusion and Exclusion Criteria

Patients scheduled for elective abdominal extensive surgery or abdominal aortic surgery were eligible. After enrollment, the patients were randomized to one of the two groups of treatment (group A or group B) by a telephone system on a 24 h/d, 7 d/wk basis. Randomization was based on a permuted-block algorithm, allowing stratification for each center. The exclusion criteria from the study were age < 16 years and preexistent neurologic or malignant hematologic diseases.

Study Protocol

In preparation for surgery, the patients were equipped with central and peripheral venous and arterial catheters, respectively. Standard monitoring included continuous recording of ECG, body temperature, heart rate (HR), pulse oximetry, and arterial BP. CVP, ScvO2, arterial

blood gas levels, lactate concentration, body temperature, and urinary output were recorded hourly. Hemoglobin concentration was measured when deemed necessary by the anesthesiologist. For the purpose of the study, blood gas levels measured on arterial and central venous samples, arterial lactate, and O2ERe (SaO2 - ScvO2/SaO2) were recorded after

induction of anesthesia, hourly after cutaneous incision, throughout surgery, half an hour after the end of anesthesia, hourly during the first 6 h of the postoperative period, and on postoperative day 1 (Fig 1).

Chapter 1

31

Figure 1

Therapeutic protocol. In addition to the standard management (group B),

a standardized therapeutic protocol designed to restore and/or keep

O2ERe < 27% was applied to patients randomized to group A. Intra-op

intraoperative; Preop preoperative; Postop postoperative.

In both groups, the patients were managed to achieve predefined standard goals: MAP > 80 mm Hg, urinary output > 0.5 mL/kg/h, and CVP from 8 to 12 cm H2O until the first postoperative day. The patients of the “protocol group” (group A) were managed to keep O2ERe < 27%,

following algorithms detailed in Figure 1. In brief, a fluid challenge (colloids, 250 to 1,000 mL infused over 30 min to restore CVP to at least 10 mm Hg), dobutamine (incremental doses of 3 mcg/kg/min up to 15 mcg/kg/min), and/or packed RBCs (PRBCs) [in cases of hemoglobin concentration < 10 g/dL or intraoperative hemorrhage > 1,000 mL] could be administered. Colloids were preferred to crystalloids because this is consistent with standard practice at our institutions.

There was no specific requirement regarding the type of anesthesia in any of the groups.

Chapter 1

32

Outcome Measures

The primary end point of this study was the number of patients who had at least one new postoperative organ failure described using the sequential organ failure assessment score recorded daily during the stay in the ICU (Table 1), with the expectation of a 50% reduction with the use of the tested therapeutic protocol. Secondary end points included the number of organ failures during the ICU stay, length of hospital stay, and hospital mortality.

Table 1.Definition of Organ Failures*

Cardiocirculatory: MAP < 80 mm Hg and CVP > 18 mm Hg and urinary output < 0.5 mL/kg/h; acute myocardial infarction† ; myocardial ischemia defined as an ST-segment depression or elevation > 1 mm‡

Respiratory: mechanical ventilation or requirement for continuous positive airway pressure for > 24 h

Renal: serum creatinine concentration > 2 mg/dL or need for renal replacement therapy

Hepatic: ALT and AST > 80 UI and total bilirubin > 2 mg/dL or AST and ALT > 200 or total bilirubin > 3 mg/dL

Hematology: platelets < 50,000 × 103/µL; leukocytosis < 2,500 or > 30,000 × 103/µL; disseminated intravascular coagulation, defined as fall of platelet count > 50% with increase of prothrombin time ≥ 50% or increase of partial thromboplastin time > 20% and increase d-dimer > 500 ng/mL

CNS: Glasgow coma scale score < 7

* Modified from Gattinoni et al.11 ALT alanine aminotranferase; AST aspartate aminotransferase. † Myocardial infarction was defined when ECG (ST-segment elevation, new bundle-branch block, 20% have other changes, eg, ST-segment depression or T-wave inversion), and an increase of troponin levels > 0.2 ng/mL were both present. ‡ ECG was performed every day for the first 3 postoperative days, then after 3 days and when the clinician judged necessary.

Statistical Analysis

A total of 130 patients was the calculated sample size needed to detect, in a one-sided test performed with a 0.05 type I error, an absolute difference between the two groups on the number of patients who had at least one new postoperative organ failure of 20% with a 80% power, assuming a 40% of patients with complications in the control group (based on an

Chapter 1

33

historical database). A one-sided formulation was chosen to compute the sample size because the trial was designed to test whether therapy in the protocol group (group A) was more effective than therapy in the control group (group B), and we had no interest in formally demonstrating the opposite alternative hypothesis. At each time point, means and SDs for continuous variables were calculated for both groups of patients and were compared using two-way analysis of variance, with Bonferroni posttest for multiple comparisons to assess differences at each time between group A and group B. Fisher exact test was used to test differences in therapeutic interventions and in outcome, measured as death and organ failures. Student t test was used to test differences between groups and differences in the length of hospital stay; p values were considered significant if < 0.05 (GraphPad 2.0; GraphPad; San Diego, CA).

Results

Three hundred twenty-four patients were assessed for eligibility, but 189 patients were excluded: 153 patients because they did not met inclusion criteria, and 36 patients because the refused to participate the study. One hundred thirty-five patients were eventually enrolled in the study: 68 patients in group A, and 67 patients in group B. All the patients enrolled concluded the study and were included in the analysis.

The patients of both groups did not differ in terms of demographic variables: age, male/female ratio, American Society of Anesthesiologists (ASA) class,18 type and duration of surgical procedures, and blood loss (Table 2).

The assigned intervention could be performed and follow-up was complete for each randomized patient. There was no patient excluded from the analysis.

Therapeutic Interventions

Fluid challenge with colloids was administered to 27 patients in each group (1,940 ± 673 mL vs 1,805 ± 611 mL and 2,191 ± 377 mL vs 2,209 ± 381 mL, during and after surgery, for groups A and B, respectively; p not significant [NS]) [Fig 2]. Remarkably, patients in group A received the fluid challenge earlier than the patients in group B (during operation, 10 patients vs 8 patients; during and after operation, 9 patients vs 6 patients; only after surgery, 8 patients vs 13 patients of groups A and B, respectively). Similarly, 10 patients in each group received

Chapter 1

34

Table 2.Patient Characteristics*

Characteristics Group

A

Group

B

p

Value

Patients, No. 68 67

Age, yr 66.0 ±

7.7 66.1 ±

7.1 NS

Male/female gender, No. 45/23 43/24 NS

Patients subclassified according to ASA class

ASA class II 9 11 NS

ASA class III 49 45 NS

ASA class IV 10 11 NS

Type of surgery

Abdominal aortic aneurysm 21 19 NS

Intestinal resection for cancer 32 38 NS

Duodenopancreatectomy 7 5 NS

Aortoiliac bypass 8 5 NS

Total 68 67 NS

Operative time, h 3.4 ± 1.1 3.3 ± 1.0 NS

Blood loss, mL 340 ± 178

354 ± 196

NS

* Data are presented as mean ± SD unless otherwise indicated.

PRBCs (260 ± 130 mL per patient vs 271 ± 173 mL per patient for groups A and B, respectively; p NS), but earlier in group A (more

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35

transfusions during operation) than in group B (more transfusions after operation). In contrast, dobutamine was administered much more often in group A (30 patients, 44.1%) than in group B (3 patients, 4.5%) [p < 0.01], during (10 patients vs 1 patient), during and after (11 patients vs 1 patient), or only after surgery (9 patients vs 1 patient). The mean dose of dobutamine was also higher in group A (2.6 ± 4.0 mcg/kg/min vs 0.4 ± 2.2 mcg/kg/min and 2.1 ± 3.7 vs 0.3 ± 1.7 mcg/kg/min during and after surgery for groups A vs B, respectively; p < 0.001). Both fluid challenge and dobutamine infusion were used in more patients of group A than group B (9 patients vs 0 patients and 6 patients vs 1 patient during and after surgery, respectively; p < 0.001).

Hemodynamic and Oxygen-Derived Variables

Importantly, most of the standard variables used to monitor the hemodynamic status (MAP, urinary output [Fig 3, top left, A, and bottom

left, C]), HR, PaO2/fraction of inspired oxygen, and body temperature

were similar in both groups. Importantly, CVP was higher in group B than in group A during the late postoperative time (Fig 3, center left, B). O2ERe and lactate were higher in group B than group A, and conversely

ScvO2 was higher in group A than group B (Fig 3, top right, D, to bottom

right, F).

Rate of Organ Failures and Outcome

Dramatic differences in the rate of organ failures and in the length of hospital stay were seen (Fig 4). In group A, fewer patients had at least one organ failure (n 8, 11.8%) than in group B (n 20, 29.8%) [p < 0.05], and the total number of organ failures was lower in group A than in group B (27 failures vs 9 failures, p < 0.001). The incidence of each type of organ failure was decreased, with the exception of respiratory failure. There was no dysfunction of the CNS noted in any group. These impairments in organ function were mostly transient.

However, the length of hospital stay was significantly lower in group A than in group B (11.3 ± 3.8 days vs 13.4 ± 6.1 days, p < 0.05), but hospital mortality was similar in both groups (2.9% and 3.0% for groups A and B, respectively), and this mortality rate was actually expected from the preoperative status of the patients.

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Figure 2

Therapeutic interventions. Total fluids, PRBC, and dobutamine (Dobu)

administered to the patients randomized to standard care (group B [B])

and to patients assigned to a standardized therapeutic protocol designed

to restore and/or keep O2ERe < 27% (group A [A]). The number of

patients receiving fluid challenges (left panel), PRBCs (middle panel),

and dobutamine (right panel) are shown. For each set of data, the

number of patients recorded during the total period of observation (left

bars), the intraoperative period (middle bars), and the postoperative

period (right bars) are shown.

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Figure 3

Time course of MAP (mm Hg), CVP (mm Hg), urinary output (mL/h),

blood lactate (mmol/L), ScvO2 (%), and O2ERe recorded after induction

of anesthesia (T0), hourly after cutaneous incision (T1a–f), throughout

surgery, during the first 6 h of the postoperative period (T2a–f), and on

postoperative day 1. Group A (O2ERe group) is represented by solid line,

and group B (standard management group) is represented by dotted line.

Data are shown as mean ± SD. *p < 0.05 and **p < 0.01 between

groups.

Discussion

This study clearly confirms that a goal-directed therapy titrated to keep O2ER ratio calculated from central venous sample (O2ERe) value lower

than a predefined threshold of 27% reduces the incidence of postoperative organ failures and length of hospital stay. The critical value of 27% for O2ERe, as representative of the hypoxic threshold, was

already reported by previous investigators15 as a predictor of survival in high-risk surgical patients. These encouraging results have been simply achieved by an earlier and more aggressive hemodynamic management, which does not require any additional invasive or expensive equipment or procedures and is operator independent. Importantly, the feasibility of the

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38

tested protocol was confirmed in the present trial because each patient randomized to the “aggressive” therapeutic strategy was treated as initially assigned, and because O2ERe was always maintained below the

critical value in patients randomized to the group A, as recommended. Due to the close monitoring, there was less concern for the incidence of adverse events such as pulmonary edema, arrhythmia, and increase of HR in spite of dobutamine infusion.

Figure 4

Number and type of organ failures observed in group A (O2ERe group,

dotted column) and in group B (standard management group, black

column). The total number of organ failures was lower in group A than in

group B (p < 0.01). The incidence of each type of organ failure was

decreased, with the exception of respiratory failure.

The preoperative and postoperative characteristics of patients studied here were very usual in the setting of scheduled major abdominal surgery in terms of age, gender, duration of surgical procedure, ASA class, and incidence of postoperative organ failures with conventional management.2,8,9,19–21 The issue of whether the therapeutic approach tested here may decrease postoperative mortality would require a much larger sample of patients. However, as the organ failures are usually transient and as the crude mortality is low, we would not expect a major impact of this therapeutic strategy on vital outcome. In contrast, the cost-effectiveness ratio of this therapeutic strategy, although not assessed, is

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39

probably very advantageous. Pearse and colleagues22 found that early-goal directed therapy was effective to reduce complications after major surgery. In any case, they used hemodynamic monitoring to assess oxygen availability, but this is not always available in all hospitals.

We speculate that the prevention of tissue hypoxia as soon as a warning signal is detected (ie, increase in O2ERe) in patients for whom oxygen

utilization cannot be adapted explains these results and may also help to prevent postoperative organ failures in high-risk patients surgical patients.1,3,23 Although not investigated in the present trial, the underlying mechanisms of tissue hypoxia could involve an impairment in myocardial contractility, a loss of vasogenic peripherical control leading to a large heterogeneity in perfusion, coagulation abnormalities, vascular permeability, endothelial dysfunction,24 and a reduction of the capacity of tissues to adapt the oxygen utilization to the supply due to anesthetic drugs and hypothermia.24,25 In any case, increasing oxygen availability by correcting hypovolemia and/or an inadequately low cardiac output is the only possibility to reverse ongoing tissue hypoxia. The timing of therapeutic intervention is definitely a key issue, as shown by the data of the present trial, when the same amount of fluids and PRBC was administered earlier in group A than in group B. Only the dose and the frequency of use of dobutamine were higher in group A than in group B. However, the dose was much lower than in some previous studies,12,15,19,26 where the hemodynamic target could not be achieved with 20 to 25 mcg/kg/min of dobutamine. As cardiac function was often compromised in the patients studied here, the frequent use of dobutamine was actually expected. As the preoperative use of betablockers was similar in both groups (data not shown), the absence of difference in HR rate in spite of a more frequent use of higher doses of dobutamine in group A than group B is somewhat surprising. A partial explanation could be related to the earlier fluid load in group A, thereby preventing the need for a compensatory increase in HR. The doses of dobutamine required were lower than in other studies, 2,12 in which the therapeutic goal was cardiac output and oxygen delivery, that we did not record. The present data suggest that the optimization of O2ER could be achieved

with low doses of dobutamine in conjunction with appropriate fluid loading. Dobutamine was preferred over other tested agents such as adrenaline or dopexamine8 because we hypothesized that a transient myocardial dysfunction was a significant and correctable causative factor of tissue hypoxia unresponsive to fluid loading. However, we cannot anticipate the effects of other agents with positive inotropic effects.

The efficacy of therapies guided to reach a hemodynamic goal was usually confirmed in conditions of tissue hypoperfusion and possible

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early and reversible tissue hypoxia such as the initial phases of trauma, severe sepsis, and surgery,2,8 –10,13,15,21–23,27 but was no longer confirmed in protracted conditions, likely associated with irreversible organ dysfunctions perhaps related to cell death.11,12,26 We might explain the discrepancy between these latter findings and the success of the approach tested here by the control and rapid prevention of tissue hypoxia as soon as a warning signal was believed, in contrast to the indiscriminate use of a standard therapy with its potential side effects regardless of the presence or the stage of tissue hypoxia. Therefore, the data presented here cannot be extrapolated to conditions where more complex impairments of oxygen utilization and other mechanisms of cell injury can occur. Indeed, the interpretation of O2ERe can then become much more complex than

during surgery. In any case, also a meta-analysis28 showed that interventions aimed to hemodynamic optimization of high risk surgical patients reduce mortality, with an odd ratio of 0.61 (95% confidence interval, 0.46 to 0.81). In this study, we compared two potential indexes of tissue level oxygenation: ScvO2/O2ERe and arterial lactate.

Consistently, lactate rose later than ScvO2 and O2ERe and only when

these were not corrected aggressively (in group B). Interestingly, organ failures were observed much more often in patients with at least one elevated lactate value (24 organ failures in 53 patients) than in patients without any elevation of lactate (6 organ failures in 82 patients) [p < 0.001]. The changes in ScvO2 and O2ERe are transient, however, while

the later increase in lactate lasts longer. Taken together, these findings are consistent with the basic assumption of tissue hypoperfusion that leads to hypoxia, decreased oxygen consumption, and eventual production of lactate, cell injury, and organ failure.

Conclusions

In conclusion, during major abdominal surgery, the findings presented here argue for a close monitoring of O2ER calculated from central

venous blood sample and for the routine use of a therapeutic algorithm designed to correct an increase in O2ER < 27%.

Appendix

Hospitals Participating the Study

Ancona University Hospital; Fano Hospital; Perugia University Hospital; Varese University Hospital; “Porta Roma” Hospital, Verona; San Salvatore Hospital, Pesaro; “Galliera” Hospital, Genova; Jesi Hospital; and Senigallia Hospital.

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References 1. Shoemaker WC, Appel PL, Kram HB. Role of oxygen debt in the

development of organ failure sepsis, and death in high risk surgical patients. Chest 1992; 102:208–215

2. Lobo SM, Salgado PF, Castillo VG, et al. Effects of maximizing oxygen delivery on morbidity and mortality in high-risk surgical patients. Crit Care Med 2000; 28:3396–3404

3. Shoemaker WC, Appel PL, Kram HB. Tissue oxygen debt as determinant of lethal and nonlethal postoperative organ failure. Crit Care Med 1988; 16:1117–1120

4. Donati A, Battisti D, Recchioni A, et al. Predictive value of interleukin 6 (IL-6), interleukin 8 (IL-8) and gastric intramucosal pH (pH-i) in major abdominal surgery. Intensive Care Med 1998; 24:329–335

5. Cain SM. Appearance of excess lactate in anesthetized dogs during anemic and hypoxic hypoxia. Am J Physiol 1965; 209:604 – 610

6. Vincent JL. The relationship between oxygen demand, oxygen uptake, and oxygen supply. Intensive Care Med 1990; 16(suppl 2):S145–S148

7. Elliott DC. An evaluation of the end points of resuscitation. J Am Coll Surg 1998; 187:536–547

8. Boyd O, Grounds RM, Bennet ED. A randomized clinical trial of the effect of deliberate perioperative increase of oxygen delivery on mortality in high-risk surgical patients.

9. Wilson J, Woods I, Fawcett J, et al. Reducing the risk of major surgery: randomized controlled trial of preoptimization of oxygen delivery. BMJ 1999; 318:1099–1103

10. Gan TJ, Soppitt A, Maroof M, et al. Goal-directed intraoperative fluid administration reduces length of hospital stay after major surgery. Anesthesiology 2002; 97:820–826

11. Gattinoni L, Brazzi L, Pelosi P, et al. A trial of goal-oriented hemodynamic therapy in critically ill patients. N Engl J Med 1995; 333:1025–1036

12. Hayes MA, Timmins AC, Yau EHS, et al. Elevation of systemic oxygen delivery in the treatment of critically ill patients. N Engl J Med 1994; 330:1717–1722

13. Rivers E, Nguyen B, Havstad S, et al. Early goal-directed therapy in the treatment of severe sepsis and septic shock. N Engl J Med 2001; 345:1368–1377

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14. Reinhart K, Rudolph T, Bredle DL, et al. Comparison of central-venous to mixed-venous oxygen saturation during changes in oxygen supply/demand. Chest 1989; 95:1216–1221

15. Bland RD, Shoemaker WC, Abraham E, et al. Hemodynamic and oxygen transport patterns in surviving and nonsurviving postoperative patients. Crit Care Med 1985; 13:85–90

16. Rady MY, Rivers EP, Nowak RM. Resuscitation of the critically ill in the ED: responsiveness of blood pressure, heart rate, shock index, central venous oxygen saturation, and lactate. Am J Emerg Med 1996; 14:218–225

17. Cortez A, Zito J, Lucas CE, et al. Mechanism of inappropriate polyuria in septic patients. Arch Surg 1977; 112:471–476

18. Owens WD, Felts JA, Spitznagel EL. AMA physical status classifications. Anesthesiology 1978; 49:239–243

19. Yu M, Levy MM, Smith P, et al. Effect of maximizing oxygen delivery on morbidity and mortality rates in critically ill patients: a prospective, randomized, controlled study. Crit Care Med 1993; 2:830–837

20. Fleming A, Bishop M, Shoemaker WC. Prospective trial of supranormal values as goals of resuscitation in severe trauma. Arch Surg 1992; 127:1175–1181

21. Bishop MH, Shoemaker WC, Appel PL et al. Prospective randomized trial of survivor values of cardiac index, oxygen delivery, and oxygen consumption as resuscitation end-points in severe trauma. J Trauma 1995; 38:780–787

22. Pearse R, Dawson D, Fawcett J, et al. Early goal-directed therapy after major surgery reduces complications and duration of hospital stay: a randomized, controlled trial (ISRCTN38797445). Crit Care 2005; 9:R687–R693

23. Bilkovski RN, Rivers EP, Horst HM. Targeted resuscitation strategies after injury. Curr Opin Crit Care 2004; 10:529–538

24. Karimova A, Pinsky DJ. The endothelial response to oxygen deprivation: biology and clinical implications. Intensive Care Med 2001; 27:19–31

25. Lugo G, Arizpe D, Dominguez G, et al. Relationship between oxygen consumption and oxygen delivery during anesthesia in high-risk surgical patients. Crit Care Med 1993; 21:64–69

26. Tuchschmidt J, Fried J, Astiz M, et al. Elevation of cardiac output and oxygen delivery improves outcome in septic shock. Chest 1992; 102:216–220

27. Cain SM, Bradley WE. Critical oxygen transport values at lowered body temperature in rats. J Appl Physiol 1983; 55:1713–1717

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28. Poeze M, Grave JWM, Ramsay G. Meta-analysis of hemodynamic optimization: relationship to methodological quality. Crit Care 2005; 9:R771–R779

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Chapter 2

45

Chapter 2

Does methylene blue administration to

septic shock patients affect vascular

permeability and blood volume?

Abele Donati, MD; Giovanna Conti, MD; Silvia Loggi, MD; Cristopher Münch, MD; Rosanna Coltrinari, MD; Paolo Pelaia, MD; Paolo

Pietropaoli, MD; Jean-Charles Preiser, MD, PhD

From the Department of Medicosurgical Emergency (AD, GC, SL, MC, RC, P. Pelaia), University of Ancona, Italy; the Department of

Anesthesiology and Intensive Care (P. Pietropaoli), University La Sapienza, Roma, Italy; and the Department of Intensive Care

(JC-P), Centre Hospitalier Notre-Dame and Reine Fabiola, Charleroi and Erasme University Hospital, Brussels, Belgium.

Published in: Critical Care Medicine 2002, 30:2271-2277

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46

Abstract

Objective: To assess the effects of the inhibition of guanylate cyclase, an enzyme involved in sepsis-related vascular and myocardial dysfunctions, on hemodynamic variables including blood volume and pulmonary vascular permeability during septic shock. Design: Prospective, open study with repeated measurements. Setting: A medicosurgical intensive care unit of a university hospital. Patients: Fifteen patients with septic shock associated with persisting hypotension despite conventional treatment including fluid loading, vasopressors, and inotropes. Interventions: A fiberoptic catheter was inserted for the determination of blood and extravascular volumes by the thermal-dye double indicator technique, using indocyanine green (COLD system). A bolus dose of methylene blue (3 mg/kg) was infused intravenously over 10 mins. COLD-derived variables were recorded before methylene blue and 20 mins, 1 hr, and 2 hrs after the end of methylene blue infusion. Measurements and Main Results: Standard hemodynamic and oxygen-derived variables; total, intrathoracic, systolic, and diastolic cardiac blood volumes; extravascular lung water; plasma osmolarity; and lactate and protein concentrations were recorded. Mean arterial and pulmonary artery pressures, systemic and pulmonary vascular resistances, and left ventricular stroke work index increased, and blood lactate transiently decreased after methylene blue (p < .05). The other variables recorded were unchanged during the 2-hr period following methylene blue infusion. Conclusions: This study confirmed the acute vasoconstrictive and positive inotropic effects of methylene blue during septic shock. These effects were not associated with changes in blood volume, myocardial diastolic function, or pulmonary vascular permeability assessed by extravascular lung water. KEY WORDS: double indicator technique; nitric oxide; guanylate cyclase; myocardial depression; vascular permeabilità

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Despite some recent advances in diagnosis, monitoring, and therapy, the mortality rate of patients with septic shock remains very high. Septic shock is typically associated with arterial hypotension, related to arteriolar vasodilation and myocardial depression (1). In addition, a reduction in blood volume related to increases in vascular permeability and extravasation of plasma commonly occurs and explains the requirement for large amounts of fluids during the initial phase of septic shock. The preservation of ventricular diastolic function is an important adaptive mechanism to maintain stroke volume, which has been correlated with survival of patients with septic shock (2). Therefore, the study of vascular permeability and blood volume, in addition to standard hemodynamic variables, can refine the assessment of new therapeutic modalities for septic shock.

The soluble intracellular enzyme guanylate cyclase (GC) is activated during septic shock to produce cyclic guanosine monophosphate (cGMP) (3, 4), presumably under the influence of several mediators, including nitric oxide (NO), carbon monoxide, or the hydroxyl radical (1, 4, 5). Increases in the intracellular concentration of cGMP are followed by relaxation of myocardial and vascular smooth muscle and by an increase in vascular permeability (6–8). The plasma concentrations of cGMP are correlated with the severity of myocardial depression during septic shock (9). Among the potential activators of GC, NO probably plays a major role, because its production is increased (10 –12) following activation of the inducible NO synthase enzyme (13). The activation of GC by other mediators than NO also can contribute to the cardiovascular alterations of septic shock, as the production of carbon monoxide and the hydroxyl radical can be increased by bacterial endotoxin (14, 15). Interestingly, a NO independent regulatory site recently has been described on GC (16).

Inhibition of NO generation has been suggested as a promising therapy for septic shock but was unfortunately associated with an increase in the overall mortality rate in a recent phase III trial (17), presumably related to the inhibition of some beneficial effects of NO. Because some of the beneficial effects of NO are mediated via other pathways than GC (1), administration of a GC inhibitor such as methylene blue (MB) could be a safer therapeutic option than the inhibition of NO production. During experimental endotoxin shock in large animals, infusion of MB was followed by the reversal of endotoxin-induced cardiovascular alterations (18) and the attenuation of lung injury by decreasing lung fluid filtration, pulmonary microvascular pressure, and permeability (19). Similarly, selective inhibition of GC during endotoxin shock was followed by the restoration to preendotoxin values of cardiovascular variables in two different models (20, 21). In patients, MB was used by seven different

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48

teams in adults during severe septic shock already treated with vasopressors (22–28). The administration of 2– 4 mg/kg MB was consistently associated with increases in blood pressure, systemic vascular resistance, and left ventricular stroke index. There was no major side effect associated with MB administration, although a slight and transient increase in pulmonary vascular resistance (22) and a transient decrease in the PaO2/FIO2 ratio (28) were reported.

These effects of MB could be related to vasoconstriction and positive inotropic effects as well to an increase in blood volume, itself related to a decrease in vascular permeability (19). The actual contributions of the effects of MB on cardiovascular contractility and blood volume and vascular permeability during human septic shock are not known. In the present study we attempted to further document the hemodynamic effects of MB by measuring total blood volume; intrathoracic, systolic, and diastolic intraventricular blood volumes; and pulmonary vascular permeability assessed by the extravascular lung water index, by using the double indicator technique.

PATIENTS AND METHODS

This prospective, open study was approved by the institutional review board of the Universityof Ancona Medical School. Before enrollment,a signed informed consent was obtained from the patients’ relatives. The study was performed in the medicosurgical intensivecare unit of a university regional hospital.

The inclusion criteria were the presence of septic shock unresponsive to conventional therapy (29), that is, when the patient remained hypotensive (systolic blood pressure <90 mm Hg) despite fluid loading titrated to achieve a pulmonary artery occlusion pressure(PAOP) of 14–16 mm Hg, and despite vasopressor agent infusion, including norepinephrineand/or dopamine infusion (1.5–3 mcg/kg-1·min-1, started when urine output was <0.5 mL· kg-1·hr-1 during the 4 hrs before study inclusion). Dobutamine was infused when cardiac index was < 3 L·min-1·m-2. When a patient was found eligible, the demographic data, source of infection, microorganisms involved, Acute Physiologic and Chronic Health Evaluation II (30), and sepsis-related organ failure assessment (31) scores were recorded. A fiberoptic catheter was introduced in the femoral artery (COLD system; Pulsion,Munich, Germany).

A baseline (T0) set of measurements including heart rate, intravascular pressures,and cardiac output was recorded, and blood was sampled to determine arterial and mixed venous blood gases as well as plasma

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49

concentrations of indocyanine green, lactate, osmolarity,and protein. There after, intravenous MB infusion was started (3 mg/kg over 10mins). The measurements and blood sampling were repeated 20 mins (T1), 1 hr (T2), and 2hrs (T3) after the end of MB infusion. The rate of administration of catecholamines was unchanged throughout the study protocol.

Analytical Techniques, Calculations, and Statistical Analysis.

Hemoglobin was measured by the OSM3 hemoglobinometer (Radiometer, Copenhagen, Denmark). Lactate concentration was detected in arterial blood by colorimetric assay (Johnson & Johnson, NY).

Protein concentration was detected with a colorimetric test. Plasma osmolarity was detectedby measuring the plasma frozen time with anosmometer (Menarini, Firenze, Italy).

Derived variables including systemic and pulmonary vascular resistance, left and right ventricular stroke work indexes, oxygen delivery, oxygen consumption, oxygen extraction, and venous admixture were calculated by standard formulas. Plasmatic disappearance rate of indocyanine; total blood volume (TBV); global,right, and left heart end-diastolic volumes; right ventricular end-systolic volume and ejection fraction; intrathoracic TBV (ITBV);and extravascular lung water were determined from the thermodilution curve and the indocyanine green concentration, determined by spectrophotometry (wavelength 805 nm, with 930 nm as reference), after a bolus infusion ofa 2.5-mg/mL solution of cold indocyaninegreen (0.4 mg/kg) infused via a central vein.

The results are expressed as median and25th–75th percentile values. The values recorded at each time were compared by using analysis of variance for repeated measures (Friedman test) and were corrected for multiplecomparisons (Dunn’s test; GraphPadPrism 2.00). A p < .05 was considered statistically significant.

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RESULTS

Fifteen patients were included. Demographic data, admission diagnosis, site of infection, microorganisms involved, and vasoactive/inotropic therapy at the time of MB infusion are shown in the Table 1. Median age was 67 (43.5–70.5), and theAcute Physiology and Chronic Health Evaluation II at admission and sepsis related organ failure assessment score at inclusion had medians of 22 (21th–27thpercentile) and 14 (11.5th–15th percentile), respectively. Intensive care unit mortality rate was 60%.

Baseline Values. The patients were hypotensive and had a hyperdynamic circulatory status despite fluid loading and vasoactive treatment including norepinephrine (1.2±1.8 mcg·kg-1·min-1, 15 patients) and dopamine(3.3±1.3 mcg·kg-1·min-1, 12 patients). Most patients had an elevated cardiac output, although inotropic support with dobutamine(9.2 ± 4.4 mcg·kg-1·min-1) was required in 12 patients. Accordingly, systemic vascular resistance and left stroke work index were below the

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nonpathologic range. Oxygen derived variables were within the norma lrange, but plasma lactate concentration was elevated. TBVI was in the normal range, while end-diastolic volume index and ITBVI were elevated. The patients were mechanically ventilated for hypoxemia requiring high FIO2(0.61 ± 0.17) and positive end-expiratory pressure

(6.4 ± 3.2 cm H2O). The PAOP was<18 mm Hg, and extravascular lung water was increased. Pulmonary vascular resistance was slightly elevated, where as right ventricularfunction variables (i.e., right ventricular ejection fraction and right ventricular stroke work index) were below the normal range. Plasma osmolarity and protein concentration were within the normal range.

Effects of MB (Table 2). In each patient of this study, mean arterial pressure had significantly increased (p < .001) already at T1 and stayed higher than T0 up to 1 hr post infusion (Figs. 1 and 2). Pulmonary artery pressure significantly increased at T1 and T2 (p < <01). Because cardiac output and filling pressures were unchanged, systemic vascular resistance and left ventricular stroke work index increased (p < .001 and p < .05, respectively). Pulmonary vascular resistance transiently increased at T1 (p < .01), and right ventricular stroke work index slightly increased after MB administration. PaO2/FIO2, venous admixture, oxygen consumption,

oxygen extraction, and oxygen delivery were unaffected by MB. Plasma lactate transiently decreased 20 mins after the infusion of MB (p < .05), while 1 hr and 2 hrs after the infusion of MB, no significant differences compared with baseline were found.

The COLD-derived variables including plasmatic disappearance rate of indocyanine, end-diastolic volume index, ITBV index, left and right-sided blood volumes, and extra vascular lung water index remained unchanged during the 2-hr observation period. Similarly, right ventricular function variables remained unchanged during the study. TBVI was unchanged duringthe study period.

Plasmatic osmolarity slightly increased, while protein concentration remained constant throughout the study period.

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52

DISCUSSION

The findings of this preliminary, uncontrolled study confirm previous reports of a long-lasting increase of arterial pressure following MB infusion. The actual mortality rate of the patients treated withMB (60%) was not significantly different than the expected mortality rate predictedby the Acute Physiology and Chronic Health Evaluation II (43% ± 20%; p = .71). The effect of MB on blood pressure appears to be related to increases in vascular tone, as indicated by the increase in systemic vascular resistance and left ventricular performance.The increase in vascular tone following exposure to MB is consistent with in

vitro observations (32) and with clinical reports (22–28). The data of the present study indicate that the diastolic function was not affected by MB, because TBVI and end-diastolic ventricular volumes were stable. The improvement of left ventricular performance seems to be related to an increase in systolic function, consistent with previous reports (23, 24, 26). In contrast, there was no significant change in right-sided pressures or functional variables despite transient increases in pulmonary artery pressures and resistance.

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Figure 1. Effects of methylene blue intravenous administration (3 mg/kg over 10

mins, started after T0) on hemodynamic variables recorded 20 mins (T1), 1 hr (T2),

and 2 hrs (T3) after T0. Values are expressed as median (25th–75th percentile). *p <

.05 vs. T0 value; **p < .01 vs. T0 value. CI, cardiac index; MAP, mean arterial

pressure; LVSWI, left ventricular stroke work index; SVRI, systemic vascular

resistance index; PVR, pulmonary vascular resistance; Qs/Qt, venous admixture;

CVP, conventional vasopressors; WP, pulmonary artery occlusion pressure.

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54

Figure 2. Effects of methylene blue intravenous administration (3 mg/kg

over 10 mins, started immediately after T0) on the variables determined

by the double indicator technique, recorded 20 mins (T1), 1 hr (T2), and

2 hrs (T3) after T0. Values are expressed as median (25th–75th

percentile). *p < .05 vs. T0 value; **p < .01 vs. T0 value. ITBVI,

intrathoracic total blood volume index; EVLWI, extravascular lung water

index; TBVI, total blood volume index; CPldy, plasma disappearance

rate of indocyanine.

Importantly, in a canine model of endotoxic shock, the positive inotropic effectof MB was markedly increased during endotoxemia, compared with non septic conditions (18). In vitro, incubation of myocardial fibers in the presence of asynthetic analog of cGMP, 8-bromocGMP,is followed by a decrease in the contractile response to catecholamines (33), while MB increased the contractility of isolated myocardial fibers previously exposed to endotoxin (34) or plasma from patients with septic shock (3). Plasma and intracellular cGMP concentrations were not monitored in the previous clinical studies (22–28), which would be mandatory to confirm the inhibition of GC by MB. The responsiveness to catecholamines was been assessed in this study, since the rate of infusion of drugs was not changed during and after MB administration.

The values of blood volumes were not below the normal range, indicating that fluid resuscitation was adequate and that the patients were not hypovolemic. Importantly, the hypoxemia (mean PaO2/FIO2, <200 mm

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55

Hg) was probably not related to left ventricular dysfunction, because the PAOP value was <18 mm Hg.

The data of the present study suggest that the acute hemodynamic changes observed during the 2 hrs following MB administration were unrelated to changes in blood volume and vascular permeability, although an alteration in extravascular lung water was unlikely in such a short period. Later changes of the COLD derived variables cannot be ruled out, of course, but would be unlikely to contribute to the acute cardiovascular effects of MB. The thermal-dye double indicator technique allows a reliable, although indirect, bedside assessment of the blood volumes and extravascular lung water, an index of pulmonary vascular permeability, provided that hydrostatic pressuresare unchanged. This latter requirement was satisfied, because PAOP remained stable throughout the study period. The double indicator technique has some limitations including the possible interference of changes in hepatic perfusion in the clearance of indocyanine green and the underestimation of extravascular lung water. Evgenov et al. (19) found in sheep a decrease in lung lymph flow andprotein clearance during the early phase of endotoxemia after administration of 10mg/kg MB. The species, the dose of MB used, and the technique used to determine pulmonary permeability can probably explain the differences between the results of the two studies.

The slight increase in plasma osmolarity was an unexpected finding that could be related to the chemical properties of MB itself or to the solutions administered before MB as fluid loading. Alternatively,the free water clearance may have been increased, but this was not measured. Potential concerns include an interference of MB in the spectrophotometric reading of indocyanine green concentration. However, this is unlikely, since MB has a longer elimination curve than indocyanine green, while the time needed for a determination is only 3 mins; moreover,the COLD system was recalibrated and the indocyanine concentration zeroed before each measurement.

REFERENCES

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2. Parrillo JE, Parker MM, Natanson C, et al: Septic shock in humans. Advances in the understanding of pathogenesis, cardiovascular dysfunction, and therapy. Ann Intern Med 1990; 113:227–242

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3. Kumar A, Brar R, Wang P, et al: Role of nitric oxide and cGMP in human septic seruminduced depression of cardiac myocyte contractility. Am J Physiol 1999; 276:R265–R276

4. Beasley D, McGuiggin M: Interleukin 1 activates soluble guanylate cyclase in human vascular smooth muscle cells through a novel nitric oxide-independent pathway. J Exp Med

1994; 179:71–80 5. Wu CC, Szabo C, Chen SJ, et al: Activation of soluble guanylyl

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9. Hartemink KJ, Groeneveld AB, de Groot MC, et al. Alpha-atrial natriuretic peptide, cyclic guanosine monophosphate, and endothelin in plasma as markers of myocardial depression in human septic shock. Crit Care Med 2001; 29:80–87

10. Evans T, Carpenter A, Kinderman H, et al: Evidence of increased nitric oxide production in patients with the sepsis syndrome. Circ

Shock 1993; 41:77–81 11. Gomez-Jimenez J, Salgado A, Mourelle M, et al: L-arginine:

Nitric oxide pathway in endotoxemia and human septic shock. Crit Care Med 1995; 23:253–258

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oxidative stress in the rat small intestine: Role of nitric oxide. Shock 1996; 5:217–222

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16. Stasch JP, Becker EM, Alonso-Alija C, et al: NO-independent regulatory site on soluble guanylate cyclase. Nature 2001; 410:212–215

17. Grover R, Zaccardelli D, Colice G, et al: An open-label dose escalation study of the nitric oxide synthase inhibitor, N(G)-methyl-Larginine hydrochloride (546C88), in patients with septic shock. Glaxo Wellcome International Septic Shock Study Group. Crit Care Med 1999; 27:913–922

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function after a short infusion of methylene blue. Braz J Med

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Chapter 4

A Comparison Among Portal Lactate,

Intramucosal Sigmoid pH, and ∆CO2

(PaCO2 - Regional PCO2) as indices of

Complications in Patients Undergoing

Abdominal Aortic Aneurysm Surgery

Abele Donati, MD*, Oriana Cornacchini, MD*, Silvia Loggi, MD*, Sandro Caporelli, MD*, Giovanna Conti, MD*, Stefano Falcetta, MD*, Francesco Alo` , MD†, Gabriele Pagliariccio, MD†, Elisabetta Bruni,

MD*, Jean-Charles Preiser, MD, PhD‡, and Paolo Pelaia, MD*

*Department of Neuroscience, Anesthesia and Intensive Care Unit, and †Department of Vascular Surgery, Marche

Polytechnique University, Ancona, Italy; and ‡Department of Intensive

Care, University Hospital of Liege, Liege, Belgium

Published in: Anesth Analg 2004;99:1024–31

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Abstract

Our aim in this observational, prospective, noncontrolled study was to detect, in 29 patients who underwent abdominal aortic aneurysm (AAA) surgery, correlations between the incidence of postoperative organ failure and intraoperative changes in arterial and portal blood lactate; changes in intramucosal sigmoid pH (pHi); differences between sigmoid PCO2 and

arterial PCO2 ( ∆CO2); and hemoglobin (Hb). Hb, arterial blood lactate

concentrations, pHi, and ∆CO2 (air tonometry) were recorded at the start

of anesthesia (T0), before aorta clamping (T1), 30 minutes after clamping (T2), and at the end of surgery (T3). Portal venous lactate concentrations were recorded at T1 and T2. Patients were stratified into two groups: group A patients had no postoperative organ failure, and group B patients had one or more organ failures. As compared with group A(n=16),group B patients (n=13)had a lower pHi value at T2 and T3 and a higher ∆CO2

at T3. A pHi value of <7.15 was a predictor of organ failure, with a sensitivity of 92.3%, a specificity of 68.8%, and positive and negative predictive values of 70.6% and 91.7%, respectively, whereas a ∆CO2

value of>28mmHg predicted later organ failure with a sensitivity of 92.3%, a specificity of 62.5%, and positive and negative predictive values of 66.6% and 90.9%, respectively. Portal venous lactate concentrations werelarger in group B at T2 (P<0.001),and an increase>5g/dLpredicted later postoperative organ failure with a sensitivity of 92.3%, a specificity of 100%, and positive and negative predictive values of 100% and 94.1%, respectively. The comparison of the receiving operator characteristic curves to test the discrimination of each variable and the logistic regression analysis revealed that the increase in portal lactate was the best predictor for the development of postoperative organ failure. Hb concentration was significantly smaller in group B at T0 (13.8 ± 1.0 g/dL versus 12.2 ± 2.2 g/dL) and T2 (10.9 ± 1.2 g/dL versus 9.1± 1.9 g/dL). In conclusion, both pHi and ∆CO2 are

reasonably sensitive prognostic indices of organ failures after AAA surgery, but they are less specific and accurate than portal venous lactate. Gut hypoxia is a phenomenon that occurs during major abdominal operations (1,2). During surgical repair of abdominal aortic aneurysms (AAA) (3), gut hypoxia often occurs as a consequence of clamping of the aorta, reduced intraoperative arterial blood pressure, activation of the inflammatory cascade, or effects of anesthetics. Importantly, the presence of gut hypoxia is associated with an increased incidence of postoperative complications (4), including multiple organ dysfunction syndrome. Therefore, early detection of gut hypoxia is important to optimize therapeutic management.

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However, diagnosis and assessment of the severity of gut hypoxia are challenging. Inadequate splanchnic perfusion can be indirectly detected by several approaches, including increased concentrations of portal venous lactate and a widened portal venous/arterial lactate gradient (5– 8). Intestinal tonometry is another technique of clinical monitoring of intestinal perfusion: it can detect early mucosal ischemia, reflected by a decrease in intramucosal intestinal pH (pHi) or a widening of the gradient between regional and arterial PCO2 ( ∆CO2) (9). Both pHi and ∆CO2

were validated as reliable prognostic indices for the development of organ failure after major surgery (10). The aim of this study was to compare the values of portal venous lactate, sigmoidal pHi, and ∆CO2 as

early indicators of postoperative organ failure after AAA surgery.

Methods

This observational, prospective, open-labeled noncontrolled study was approved by the Hospital Ethical Committee, and informed consent was obtained from the patients before surgery. A cohort of 29 consecutive patients undergoing elective transperitoneal surgery for AAA was enrolled in the study. Twenty-six patients were male and 3 were female, with a mean age of 73.0 ± 6.9 yr.

A post hoc stratification of the patients in both groups was planned according to the absence (group A) or the presence (group B) of one or more postoperative organ failures, defined as shown in Table 1 (11). The observation period lasted until patient discharge from the hospital. Clinicians managing the patients postoperatively were not aware of the tonometric and lactate values.

All patients were treated before surgery with H2- blockers (ranitidine 300 mg per os) and premedicated with diazepam (0.1 mg/kg). No patient had an epidural catheter placed. Anesthesia was induced with fentanyl (0.01 mg/kg) and sodium thiopental (4 mg/ kg), and muscle relaxation was induced with vecuronium (0.08 mg/kg). After orotracheal intubation, anesthesia was maintained with sevoflurane, nitrous oxide, vecuronium, and fentanyl; fraction of inspired oxygen was maintained at 0.40. Intraoperative monitoring included 1) on-line electrocardiogram, 2) pulse oximetry, 3) continuous arterial blood pressure, 4) monitoring of blood gas variables, 5) sigmoid tonometry, 6) urinary output, and 7) hemoglobin (Hb) concentration. Transfusion of packed red cells was considered for Hb levels less than 8 g/dL, and no patient received any red cell transfusion before surgery. Dobutamine infusion was started when cardiac failure was suspected (hypotension with mean arterial blood pressure less than 70 mm Hg not responsive to fluid challenge; oliguria

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with urine output less than 0.5 mL · kg-1 · h-1). All the patients were tracheally extubated during the first postoperative hour.

Blood gas analysis was performed with the automatic analyzer RapidLab 865 Chiron Diagnostic (Bayer, Leverkusen, Germany), and values were corrected for blood temperature. Hb concentration was measured by using the same instrument.

Measurement of lactate levels in systemic arterial and portal circulation was performed with a Vitros 950 Chemistri System analyzer. This system is based on an enzymatic technique that determines by spectroscopy the color changes due to the presence of a lactate-activated chromogenic substrate. Systemic lactate determinations were performed at the time of anesthesia induction (T0), before clamping (T1), 30 min after clamping (T2), and at the end of surgery (T3). Portal venous lactate determinations were performed at T1 and T2 through a sample of portal venous blood taken by the surgeon, and the difference in portal venous lactate concentration (T2 - T1; ∆ portal venous lactates) was calculated.

A TRIP tonometer (Tonometrics Division, Instrumental Corp., Helsinki, Finland) was used to measure regional CO2 (PrCO2) after automatic

calibration of the instrument. This tonometer is based on principles and techniques described elsewhere (12,13). The tonometer is a silicon 8F catheter with a balloon tip and a semipermeable membrane that allows diffusion of gas, but not fluids. A tonometer is introduced intrarectally, positioned in the sigmoid colon, and connected to the Tonocap (Datex, Helsinki, Finland) device to perform air tonometry. The surgeon manually checked the positioning of the tonometer during the intervention. The tonometer was maintained in situ throughout the operation and was removed after tracheal extubation. PaCO2 was

measured at T0, T1, T2, and T3. At the same time, pH was recorded and an arterial blood sample was taken to measure PaCO2 and [H+], used for

the calculation of ∆CO2 (PrCO2 - PaCO2).

Outcome was defined as the absence or presence of one or more postoperative organ failures. At each time point, the means and standard deviations of heart rate, mean arterial blood pressure, PaO2/fraction of

inspired oxygen ratio, PaCO2, pHi, ∆CO2, systemic and portal venous

lactates, and Hb were calculated for both groups of patients. Gaussian distribution was also verified with the Kolmogorov-Smirnov test. Continuous variable measurements expressed as means were compared by using two-way analysis of variance, with Student’s t-test or Student’s t-test with Welch’s correction if variances were significantly different and with Bonferroni’s correction for multiple comparisons when

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appropriate. Sensitivity, specificity, and positive and negative predictive values to predict postoperative complications were calculated for pHi, ∆CO2, and ∆portal venous lactates. Fisher’s exact test was used to test

significance, and power was calculated for each analysis.

All P values are two sided, and a threshold < of 0.05 was used to assign significance (GraphPad 2.0; Graph- Pad Inc., San Diego, CA). The area under the receiving operator characteristic curve (ROC) was calculated to detect the discrimination ability of the variables for complications (SPSS 10.1; SPSS Inc., Chicago, IL). Finally, logistic regression was performed to detect which variables had an independent significant value to predict outcome (SPSS 10.1).

Results

Of the 29 patients enrolled in this study, 16 were classified in group A, and 13 qualified for group B, because these experienced at least 1 postoperative new organ failure (Table 1). Six patients developed acute renal failure, three developed heart failure, three had hepatic failure, and one had a multiorgan failure and eventually died. Infrarenal aortic clamping was used in all patients.

There was no difference between groups in age, number of preoperative morbidities, aortic clamping time, and type of bypass performed (aortoaortic or aortoiliac). Patients without complications were discharged from the hospital significantly earlier than patients with complications (Table 2).

Table 1. Definition of Organ Failure, Modified (11)

Renal: serum creatinine concentration >2 mg/dL or the need for renal support*

Hematology: platelets <50,000 x 103/mL; white blood cells <500 or >30,000 x 103/mL; disseminated intravascular coagulation, defined as decrease of platelet count <50% with increase of prothrombin time (PT) ≥50% or increase of PT ≥20% and increase of fibrin. Degradation products >1:40 (or d-dimer >500 ng/mL)*

Respiratory: mechanical ventilation or continuous positive airway pressure for more than 24 h

Cardiocirculatory: mean arterial blood pressure <80 mm Hg, central venous pressure >18 mm Hg, and urine output <0.5 mL kg-1 h-1; acute myocardial infarction†; myocardial ischemia defined at

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electrocardiogram (ECG) as ST segment depression or elevation >1 mm not associated with branch blocks‡

Hepatic: alanine amino transferase (ALT) and aspartate amino transferase (AST) >80 IU/L and total bilirubin >2 mg/dL or AST and ALT >200 or total bilirubin >3 mg/dL*

Central nervous system: Glasgow Coma Scale score <7*

One altered value in one laboratory result was sufficient to define organ failure.

* Daily measurements were performed for at least 1 wk.

† Myocardial infarction was defined both clinically and with ECG criteria (ST segment elevation, some with bundle branch block, 20% with other changes,

e.g., ST depression or T-wave inversion), with an increase of troponin levels >0.2 ng/mL.

‡ ECG was performed every day for the first 3 postoperative days, then after 3 days and when the clinician judged necessary.

Table 2. Clinical Features, Surgical Notes, and Length of Postoperative Stay in the Hospital Within Each Group

Variable Group A Group B

No. patients 16 13

Age,yr, mean(sd) 73.6 (6.7) 72.3 (7.2)

Patients with preexisting comorbidities

7a 7

Hypertensive and/or ischemic cardiomyopathy

5 4

Chronic obstructive pulmonary disease

2 3

Head stroke 1 0

Duration of aortic clamping, min, mean (sd)

44.7 (11.2) 47.3 (7.2)

Aortoaortic/aortobisiliac bypass

7/9 5/8

Postoperative length of stay, d, mean (sd)

9.6 (2.0) 15.0 (9.4)*

a One patient had two preexisting comorbidities.

* P < 0.05 between group A and group B.

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Arterial lactate concentrations increased significantly from T0 to T3 in group A (P < 0.01) and from T0 to T2 and T3 (P < 0.01 and P < 0.001, respectively) in group B (Table 3). The trend of systemic arterial lactate levels was similar in the two groups.

The ∆ portal venous lactates significantly increased in group B (P < 0.001) but not in group A (Fig. 1). The difference between groups was significant at T2 (P < 0.001). With a cutoff value for ∆ portal venous lactates at 5 mg/dL, 12 of the 13 patients in group B and none of the patients in group A had values equal to or more than 5 mg/dL. The sensitivity to predict postoperative organ failure was 92.3%, and the specificity was 100% (P < 0.001; power, 99%). The positive and negative predictive values were 100% and 94.1%, respectively. The area under the ROC was 0.971 (se, 0.031; 95% confidence interval [CI], 0.911– 1.031; P < 0.001 versus the null hypothesis area = 0.5) (Fig. 2).

The pHi decreased in both groups during surgery (Table 3). Overall, the decrease was larger in group B than in group A (P < 0.01). As compared with the value recorded at the same time point, the decrease from T0 was larger in group B than in group A for T2 (P < 0.05) and T3 (P < 0.01). The mean of the lowest values (the lowest pHi value at any of the observation points of each patient) was significantly lower in group B than in group A (6.99 ± 0.15 versus 7.15 ± 0.17; P < 0.05). A lowest pHi value less than 7.15 predicted the occurrence of organ failure with a sensitivity of 92.3% and a specificity of 68.8% (P < 0.01; power, 91%) (Fig. 3, top). The positive and negative predictive values were 70.6% and 91.7%, respectively. The area under the ROC was 0.755 (se, 0.096; 95% CI, 0.567–0.943; P < 0.020) (Fig. 2).

There was a significant increase of ∆CO2 over time within each group

(Table 3), although it was larger in group B than in group A (P < 0.01). ∆CO2 was significantly larger in group B than in group A at T2 and T3

(P < 0.05). The mean peak ∆CO2 (the highest CO2 value at any of the

observation points of each patient) was significantly lower in group A (32.9 ± 24.2 mm Hg) than in group B (53.9 ± 28.4 mm Hg; P < 0.05). With a cutoff level of 28 mm Hg ∆CO2, sensitivity was 92.3% and

specificity was 62.5% (P < 0.01; power, 82%) (Fig. 3, bottom). Positive and negative predictive values were 66.6% and 90.9%, respectively. The area under the ROC was 0.740 (se, 0.094; 95% CI, 0.556– 0.925; P = 0.028) (Fig. 2).

The other differences between the patients from group A and those from group B included Hb values, intraoperative blood loss, and number of

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transfusions. Blood loss was larger in group B than in group A (1119 ± 645 mL versus 647 ± 462 mL; P < 0.05). The area under the ROC was 0.692 (se, 0.104; 95% CI, 0.489– 0.895; P = 0.079). Accordingly, Hb progressively decreased from T0 to T1 in group B (P < 0.05), from T0 to T2 in both groups (P < 0.001), and from T0 to T3 in both groups (P < 0.01 in group A and P < 0.05 in group B). The average values of Hb at T0 and T2 were significantly less in group B than in group A (Table 3).

The number of packed red cells transfused was still more in group B than in group A (1.5 U [range, 0–3 U] versus 0.4 U [range, 0–3 U]; P < 0.05). The area under the ROC was 0.702 (se, 0.103; 95% CI, 0.501–0.903; P = 0.066). No other blood products were transfused during the operation. The area under the ROC for the Hb values at T0 as a predictor of complications was 0.755 (SE 0.091; 95% CI, 0.577–0.932; P=0.020). No other significant difference was found in the other variables (Table 3).

Comparing the differences between areas of the 3 ROC curves (Fig. 2), ∆ portal venous lactates was significantly more powerful than pHi (difference between areas, 0.216; se, 0.086; 95% CI, 0.047–0.386; P = 0.012) and ∆CO2 (difference between areas, 0.231; se, 0.092; 95% CI,

0.051–0.410; P = 0.012). No differences were found between pHi and ∆CO2 ROC curves (difference between areas, 0.015; se, 0.049; 95% CI,

0.082 to 0.111; P = 0.770) to predict postoperative organ failure.

The ∆ portal venous lactates ROC curve was also more powerful than Hb at T0 (difference between areas, 0.216; se, 0.089; 95% CI, 0.042–0.391; P = 0.015), the number of transfused packed red cells (difference between areas, 0.269; se, 0.097; 95% CI, 0.079–0.460; P = 0.006), and blood loss (difference between areas, 0.279; se, 0.098; 95% CI, 0.087–0.470; P = 0.004). The ROC curves for pHi, ∆CO2, Hb at T0, number of

transfused packed red cells, and blood loss did not differ. The logistic regression revealed that the independent predictors of postoperative organ failure included ∆ portal venous lactates (odds ratio, 3.513; P = 0.029), Hb at T0 (odds ratio, 0.498; P = 0.045), and ∆CO2 (odds ratio,

1.490; P = 0.049).

Discussion

In this study, we compared three indices of gut hypoxia recorded during surgery and found that the changes in each of these three variables were sensitive predictors for the development of later organ failures. However, the portal venous lactate gradient was a more accurate and more specific predictor of postoperative organ failure than pHi or ∆CO2, as indicated

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by the sensitivity and specificity, the positive and negative predictive values, the comparison of the ROC curves, and the logistic regression.

The findings of this study strongly support the role of gut hypoxia in the pathogenesis of multiple organ dysfunction after AAA surgical repair. The association between low Hb and an increased incidence of organ failure suggests that gut hypoxia is amplified by preoperative and postoperative anemia. The balance between the benefit of blood transfusions on gut perfusion and the potential deleterious effects (14) can hardly be deducted from the data recorded in this study, although

Table 3. Trends of Each Variable in the Two Groups (Mean ± sd)

Time 0 Time 1

Variable Group A Group B Group A Group B

Intramucosal pH 7.41 ± 0.14 7.37 ± 0.23 7.27 ± 0.17* 7.15 ± 0.13

Difference between sigmoid PCO2 and

arterial PCO2

(mm Hg)

7 ±6 11 ± 10 20 ± 16 31 ± 21*

Hemoglobin concentration (g/dL)

13.8 ± 1.0† 12.2 ± 2.2 11.7 ± 1.5 11.3 ± 1.4*

Arterial lactate concentration (mg/dL)

7.6 ± 1.7 6.6 ± 1.0 8.3 ± 4.0 7.9 ± 3.1

Portal venous lactates (mg/dL)

9.8 ± 3.1 8.1 ± 2.3

Heart rate (bpm) 73 ± 11 77 ± 12 70 ± 11 66 ± 12

Mean arterial blood pressure (mm Hg)

99 ± 20 102 ± 18 83 ± 22 91 ± 19

PaO2/FiO2 326 ± 48 317 ± 41 337 ± 41 346 ± 38

PaCO2 (mm Hg) 38.5 ± 4.2 38.0 ± 4.1 39.3 ± 3.8 39.1 ± 4.5

FiO2 = fraction of inspired oxygen; NS = not significant.

* P < 0.05 in each group between T1 or T2 or T3 and T0; † P < 0.05 between group A and B; ‡ P < 0.001 in each group between T1 or T2 or T3 and T0; § P < 0.01 between group A and B; װP < 0.015 in each group between T1 or T2 or T3 and T0; ¶ P < 0.001 between group A and B; # P

< 0.001 in each group between T2 and T1.

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Table 3. Continued

Time 2 Time 3 P value

(ANOVA between groups)

Group A Group B Group A Group B

7.17 ± 0.18†‡

7.01 ± 0.16‡

7.24 ± 0.14‡§

7.06 ± 0.16‡

<0.01

31 ± 25†‡ 51 ± 29‡ 20 ± 16† 38 ± 26± <0.01

10.9 ± 1.2‡§

9.1 ± 1.9‡ 11.4 ± 1.6± 10.8 ± 1.6* <0.01

NS ‡7.8 ± 14.5 װ4.9 ± 11.6 װ8.3 ± 12.7 2.5 ± 9.4

10.7 ± 2.4¶ 19.4 ± 5.8# ±0.001

70 ± 11 71 ± 15 72 ± 10 74 ± 11 NS

82 ± 17 83 ± 13 98 ± 23 87 ± 15 NS

313 ± 62 311 ± 55 327 ± 60 331 ± 54 NS

39.5 ± 4.7 39.4 ± 7.3 39.8 ± 4.9 40.1 ± 6.6 NS

Figure 2. Area under the receiver operating characteristic curves of

intramucosal sigmoid pH (pHi), gradient between regional and arterial

PCO2 (∆CO2), and ∆ portal venous lactates.

the differences in the amount of blood transfusions could represent a risk factor per se for the development of organ failure and could therefore be

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a confounding factor for the interpretation of the results. However, there are other studies that suggest that patients with a lower level of hematocrit are affected by more complications, such as myocardial ischemia (15,16). In any case, patients with complications had lower Hb levels before surgery, and intraoperative transfusion did not reverse this association.

Figure 1. Portal venous lactate concentrations at different times in the

two groups. Time T1 is before clamping; T2 is 30 min after clamping.

Columns indicate mean and sd at each time of each group. #P < 0.001

versus T1 in group B; *P < 0.001, group A versus group B at T2.

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Figure 3. (Upper panel) The lowest intramucosal sigmoid pH (pHi) value

at any of the observation points of each patient of the two groups. With

pHi 7.15 as the cutoff, there was 92.3% sensitivity and 68.8% specificity

to predict complications and a positive and negative predictive value of

70.6% and 91.7%, respectively (power = 91%). Columns indicate mean and sd at each time of each group. (Lower panel) The highest gradient

between regional and arterial PCO2 (∆CO2) value at any of the

observation points of each patient of the two groups. With a ∆CO2 of 28

mm Hg as the cutoff, there was 92.3% sensitivity and 62.5% specificity to

predict complications and a positive and negative predictive value of

66.6% and 90.0%, respectively (power = 82%). Columns indicate mean

and sd at each time of each group.

The ischemia/reperfusion phenomenon can be advocated to explain our findings. Indeed, ischemia/ reperfusion occurs during clamping and declamping of the abdominal aorta (17,18) and can trigger the absorption

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of bacteria or bacterial endotoxins across the ischemic intestinal wall and the release of cytokines from the intestine. These cytokines, also through vascular endothelial damage, may play a significant role in the pathogenesis of organ dysfunction (17,19). In this study, we did not record the microbiological or immunological data that could confirm such a mechanism. However, we (2) and others (1) previously observed increased levels of proinflammatory cytokines during abdominal and AAA surgery in patients who developed postoperative complications.

The reasons for the better specificity of the lactate gradient than the tonometric variables may be related to the fact that portal lactate is released by the full thickness of the intestinal wall in case of hypoxia, whereas the variables recorded by tonometry reflect only mucosal perfusion. In addition, the absence of severe hepatic failure at the time of surgery suggests that the portal lactate is appropriately cleared by the liver, as illustrated by the poor sensitivity of arterial lactate concentrations as an index of gut hypoxia and predictor of postoperative organ failure (20,21). Very importantly, there was no organ failure in patients with a portal venous lactate gradient less than 5 mg/ dL. In some patients without an increase in portal lactate gradient or any organ failure, a preexisting thrombosis of the inferior mesenteric artery could be present (22), and therefore gut perfusion is not affected by infrarenal aortic clamping.

The positive predictive values of pHi and ∆CO2 recorded by tonometry

were reasonably high and consistent with those in previous reports. There was a tendency for lower pHi and higher ∆CO2 from the beginning of

anesthesia, and this was consistent with Poeze et al. (23), who demonstrated that patients with lower preoperative pHi have a worse outcome than patients with normal pHi. In both groups, there was a significant reduction of pHi and increase of ∆CO2. During anesthesia, a

decrease in global hemodynamic indices is normal (24), and oxygen debt is common during anesthesia (25). The degree of this debt determines postoperative complications and death (25,26). The major advantage of this noninvasive technique of monitoring the gut mucosal perfusion is the early detection of an ischemic intestinal lesion before the occurrence of clinically detectable symptoms and signs (27). Another major advantage over portal lactate determination is the possibility of postoperative monitoring that was assessed and validated by others (13). Indeed, previous studies have shown that a sigmoid pHi less than 7.1 for more than two hours is associated with large concentrations of endotoxins and cytokines (28) and is predictive of major complications and death. In a study by Bjo¨rck and Lindberg (29), the severity of the mucosal ischemic

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lesions classified by colonoscopy was correlated with the values of sigmoid pHi monitoring with a sensitivity of 90% and a specificity of 100%.

Other groups have tested the diagnostic accuracy of sigmoid pHi in the evaluation of colon ischemia in patients undergoing AAA surgery (30,31). Sigmoid pHi was related to the severity of colonic damage andthe duration of ischemia (30,31). In this study, the level of sigmoid pHi that appears able to discriminate patients with complications is 7.15, and most (12 of 13) patients with complications showed sigmoid pHi levels less than the cutoff. Most levels (11 of 16) were more than the cutoff in patients without complications. In our study, we showed that pHi decreases before aortic clamping, but the lowest values were shown 30 minutes from clamping and at the end of surgery. This is consistent with published results by other groups indicating that the lowest levels of sigmoid pHi were found between four and six hours after aortic clamping (12). In this study, sigmoid pHi monitoring was performed only at the end of surgery and not during postoperative evaluation. A good correlation between direct and tonometer-obtained measurement of pHi has been reported by other groups as well (22).

Work by Heino and Hartikainen (32) confirmed the sensitivity of ∆CO2

in identifying regional splanchnic hypoperfusion, as previously suggested by Schlichtig and Boweles (33). In our study, calculation of the difference between PrCO2 and PaCO2 was not only a sensitive marker,

but also an independent predictor of organ failure. Differences in surgical techniques and the presence of collateral circulation between the superior and inferior mesenteric arteries were not accounted for in this study. The possible presence of collateral circulation supports our hypothesis that patients with significant anastomosis between splanchnic and systemic circulation may display lower degrees of colon ischemia with higher sigmoid pHi levels and lower portal venous lactates. In all patients with complications, the difference between PrCO2 and PaCO2 was increased

before, during, and after clamping, as shown previously by Schlichtig and Boweles (33).

In conclusion, although the pathogenesis of the reduced pHi is still controversial, the results of this pilot study indicate that pHi and ∆CO2

are sensitive prognostic indices during AAA surgery and therefore suggest that tonometry may identify patients at higher risk of organ failure in the postoperative period. The ∆ portal venous lactates are more specific then tonometric variables, and this study confirms experimental studies that indicate portal venous lactate as an important index of gut hypoperfusion. Further studies with a large number of patients need to

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prospectively validate our cutoff points for pHi and ∆CO2. New

therapeutic strategies, such as fenoldopam infusion (a dopamine D1- like receptor agonist) during surgery, could be tested in a prospective study to treat splanchnic ischemia by applying the cutoff points (pHi <7.15 and ∆CO2 >28 mm Hg) that were used in our study.

References 1. Mythen MG, Wehb AR. The role of gut mucosal hypoperfusion

in the pathogenesis of post-operative organ dysfunction. Intensive Care Med 1994;20:203–9.

2. Donati A, Battisti D, Recchioni A, et al. Predictive value of interleukin 6 (IL-6), interleukin 8 (IL-8) and gastric intramucosal pH (pH-i) in major abdominal surgery. Intensive Care Med 1998;24:329–35.

3. Deitch EA, Berg R, Specian R. Endotoxin promotes the translocation of bacteria from the gut. Arch Surg 1987;122:185–90.

4. Dantzker DR. The gastrointestinal tract: the canary of the body? JAMA 1993;270:1247–8.

5. Schlichting E, Lyberg T. Monitoring of tissue oxygenation in shock: an experimental study in pigs. Crit Care Med 1995;23: 1703–10.

6. Roding B, Schenk WG. Mesenteric blood flow after hemorrhage in anaesthetized and unanaesthetized dogs. Surgery 1980;68:857–61.

7. Jakob SM, Merasto-Minkkinen M, Tenhunen JJ, et al. Prevention of systemic hyperlactatemia during splanchnic ischemia. Shock 2000;14:123–7.

8. Heino A, Hartikainen J, Merasto ME, et al. Systemic and regional effects of experimental gradual splanchnic ischemia. J Crit Care 1997;12:98–2.

9. Brinkmann A, Calzia E, Tra¨ger K, Radermaker P. Monitoring the hepatosplanchnic region in the critically ill patient: measurement techniques and clinical relevance. Intensive Care Med 1998;24:542–56.

10. Doglio GR, Pusajo JF, Egunola MA. Gastric mucosal pH as a prognostic index of mortality in critically ill patients. Crit Care Med 1991;19:1037–40.

11. Gattinoni L, Brazzi L, Pelosi P, et al. A trial of goal-oriented hemodynamic therapy in critically ill patients. N Engl J Med 1995;333:1025–32.

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12. Dawson AM. Small bowel tonometry: assessment of small gut mucosal oxygen tension in dog and man. Nature 1965;206:943–4.

13. Fiddian-Green RG. Gastric intramucosal pH, tissue oxygenation and acid-base balance. Br J Anaesth 1995;74:591–606.

14. Hebert PC, Wells G, Blajchmann MA. Multicenter, randomized, controlled clinical trial of transfusion requirements in critical

3. care. N Engl J Med 1999;340:409–17. 15. Hogue CW Jr, Goodnough LT, Monk TG. Perioperative ischemic

episodes are related to hematocrit level in patients undergoing radical prostatectomy. Transfusion 1998;38:924–31.

16. Nelson AH, Fleisher LA, Rosenbaum SH. Relationship between postoperative anemia and cardiac morbidity in high-risk vascular patients in the intensive care unit. Crit Care Med 1993;21:860–6.

17. Soong CV, Blair PHB, Halliday MI. Bowel ischemia and organ impairment in elective abdominal aortic aneurysm repair. Br J Surg 1994;81:965–8.

18. Hess W, Frank C, Hornburg B. Prolonged oxygen debt after abdominal aortic surgery. J Cardiothorac Vasc Anesth 1997;11:149–54.

19. Ueno H, Hirasawa H, Oda S, et al. Coagulation/fibrinolysisabnormality and vascular endothelial damage in the pathogenesis of thrombocytopenic multiple organ failure. Crit Care Med 2002;30:2242–8.

20. Tollefson DJF, Ernst CB. Colon ischemia following aortic reconstruction. Ann Vasc Surg 1991;5:485–9.

21. Mythen MG, Purdy G, Mackie IJ. Postoperative multiple organ dysfunction syndrome associated with gut mucosal hypoperfusion, increased neutrophil degranulation and C1-esterase inhibitor depletion. Br J Anaesth 1993;71:858–63.

22. Velazquez OC, Baum RA, Carpenter JP, et al. Relationship between preoperative patency of the inferior mesenteric artery and subsequent occurrence of type II endoleak in patients undergoing endovascular repair of abdominal aortic aneurysms. J Vasc Surg 2000;32:777–88.

23. Poeze M, Takala J, Greve JWM, Ramsay G. Pre-operative tonometry is predictive for mortality and morbidity in high-risk surgical patients. Intensive Care Med 2000;26:1272–81.

24. Bacher A, Mayer N, Mittlboeck M, Zadrobilek E. Anaesthesia and systemic oxygenation. Acta Anaesthesiol Scand 1996;40:869–75.

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25. Lugo G, Arizpe D, Dominguez G, et al. Relationship between oxygen consumption and oxygen delivery during anesthesia in high-risk surgical patients. Crit Care Med 1993;21:64–9.

26. Shoemaker WC, Appel PL, Kram HB. Role of oxygen debt in the development of organ failure sepsis, and death in high-risk surgical patients. Chest 1992;102:208–15.

27. Hamilton-Davies C, Mythen MG, Salmon JB. Comparison of commonly used clinical indicators of hypovolemia with gastrointestinal tonometry. Intensive Care Med 1997;23:276–81.

28. Bjorck M, Edberg B. Early detection of major complications after abdominal aortic surgery: predictive value of sigmoid colon and gastric intramucosal pH monitoring. Br J Surg 1994;81:25–30.

29. Bjo¨rck M, Lindberg F. pHi monitoring of the sigmoid colon after aortoiliac surgery: a five-year prospective study. Eur J Vasc Endovasc Surg 2000;20:273–80.

30. Syk I, Brunkwall J, Ivanov K. Postoperative fever, bowel ischemia and cytokine response to abdominal aortic aneurysm repair. Eur J Vasc Endovasc Surg 1998;15:398–405.

31. Klok T, Moll FL, Leusink JA. The relationship between sigmoid intramucosal pH and intestinal arterial occlusion during aortic reconstructive surgery. Eur J Vasc Endovasc Surg 1996;11:304–7.

32. Heino A, Hartikainen J. Systemic and regional pCO2 gradients as

markers of intestinal ischaemia. Intensive Care Med 1998;24:599–604.

33. Schlichtig R, Boweles SA. Distinguishing between aerobic and anaerobic appearance dissolved CO2 in intestine during low

flow. J Appl Physiol 1994;76:2443–51.

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Chapter 5

Recombinant activated protein C

treatment improves tissue perfusion and

oxygenation in septic patients measured

by near-infrared spectroscopy Abele Donati1, Michela Romanelli1, Laura Botticelli1, Agnese Valentini1,

Vincenzo Gabbanelli1, Simonetta Nataloni1, Tiziana Principi1, Paolo Pelaia1, Rick Bezemer2 and Can Ince2

1Department of Neuroscience, Intensive Care Unit, Marche Polytechnical University, Via Tronto 10/A, 60020 Torrette di Ancona, Italy

2Department of Intensive Care, Erasmus MC University Hospital Rotterdam, 3000 Rotterdam, The Netherlands

Published in: Critical Care 2009, 13(Suppl 5):S12 (doi:10.1186/cc8010)

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Abstract

Introduction: The purpose was to test the hypothesis that muscle perfusion, oxygenation, and microvascular reactivity would improve in patients with severe sepsis or septic shock during treatment with recombinant activated protein C (rh-aPC) (n 11) and to explore whether these parameters are related to macrohemodynamic indices, metabolic status or Sequential Organ Failure Assessment (SOFA) score. Patients with contraindications to rhaPC were used as a control group (n 5). Materials and methods: Patients were sedated, intubated, mechanically ventilated, and hemodynamically monitored with the PiCCO system. Tissue oxygen saturation (StO2) was measured using near-infrared

spectroscopy (NIRS) during the vascular occlusion test (VOT). Baseline StO2 (StO2 baseline), rate of decrease in StO2 during VOT (StO2

downslope), and rate of increase in StO2 during the reperfusion phase

(StO2 upslope) were determined. Data were collected before (T0), during

(24 hours (T1a), 48 hours (T1b), 72 hours (T1c) and 96 hours (T1d)) and 6 hours after stopping rh-aPC treatment (T2) and at the same times in the controls. At every assessment, hemodynamic and metabolic parameters were registered and the SOFA score calculated. Results: The mean ± standard deviation Acute Physiology and Chronic Health Evaluation II score was 26.3 ± 6.6 and 28.6 ± 5.3 in rh-aPC and control groups, respectively. There were no significant differences in macrohemodynamic parameters between the groups at all the time points. In the rh-aPC group, base excess was corrected (P <0.01) from T1a until T2, and blood lactate was significantly decreased at T1d and T2 (2.8 ± 1.3 vs. 1.9 ± 0.7 mmol/l; P <0.05). In the control group, base excess was significantly corrected at T1a, T1b, T1c, and T2 (P <0.05). The SOFA score was significantly lower in the rh-aPC group compared with the controls at T2 (7.9 ± 2.2 vs. 12.2 ± 3.2; P <0.05). There were no differences between groups in StO2 baseline. StO2 downslope in the rh-

aPC group decreased significantly at all the time points, and at T1b and T2 (–16.5 ± 11.8 vs. –8.1 ± 2.4%/minute) was significantly steeper than in the control group. StO2 upslope increased and was higher than in the

control group at T1c, T1d and T2 (101.1 ± 62.1 vs. 54.5 ± 23.8%/minute) (P <0.05). Conclusions: Treatment with rh-aPC may improve muscle oxygenation (StO2 baseline) and reperfusion (StO2 upslope) and, furthermore, rh-aPC

treatment may increase tissue metabolism (StO2 downslope). NIRS is a

simple, real-time, non-invasive technique that could be used to monitor the effects of rh-aPC therapy at microcirculatory level in septic patients.

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Introduction

During sepsis, the microcirculatory and mitochondrial dysfunction plays a key role in the development of severe sepsis, septic shock, and multiple organ failure [1]. This condition is characterized by microcirculatory perfusion heterogeneity, arteriovenous shunting, and impaired autoregulation mainly due to disturbed coagulation, inflammation, and leukocyte-endothelium interaction [1-3]. It is now well established in septic patients that restoring basic macrohemodynamic parameters, such as blood pressure, in itself does not lead to improved patient outcome, and that normalization of microcirculatory and mitochondrial function may be necessary as an endpoint [2,4].

To this end, recombinant activated protein C (rh-aPC) has been used to restore the coagulative cascade, the inflammatory response, leukocyte adhesion and migration, and endothelial function. In previous studies, rh-aPC has been shown to decrease end-organ dysfunction and mortality if administered in the early stages of sepsis [5-7].

Near-infrared spectroscopy (NIRS) is a rapid, continuous, and non-invasive monitoring system of hemoglobin oxygen saturation in muscle and the brain, and has been used to assess the presence and extent of both circulatory and metabolic disorders in intensive care patients and trauma patients [8,9]. The monitoring system uses near-infrared light (680 to 800 nm) to illuminate tissue, which is mainly absorbed by hemoglobin and myoglobin [10]. Due to the selected wavelength range and the high corresponding spectral absorbance by (de)oxyhemoglobin, the NIRS measurements are confined to vessels with a diameter <500 µm.

Using NIRS, oxyhemoglobin can be distinguished from deoxyhemoglobin because of their differing optical absorption spectra. The ratio of oxyhemoglobin concentration to deoxyhemoglobin concentration is used to calculate a parameter called tissue oxygen saturation (StO2), describing the oxygenation of the microvasculature in

a certain volume of (muscular) tissue. In addition to steady-state StO2

values, NIRS can be used in combination with a vascular occlusion test (VOT), which consists of a baseline phase, an ischemia phase, a reperfusion phase, and a reactive hyperemia phase. Using this methodology in many studies of sepsis, it has been demonstrated in a variety of ways that, following a brief period of ischemia, there is an anomalous tissue reperfusion profile due to disturbed microcirculatory functioning [11,12].

The purpose of the present study was to test the hypothesis that rh-aPC treatment corrects tissue perfusion and microcirculatory reperfusion in

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septic patients, evaluated with NIRS in combination with a VOT, and to explore whether the NIRS parameters are related to macrohemodynamic indices, metabolic status, and Sequential Organ Failure Assessment (SOFA) score.

Materials and methods

Patients

The study was designed as a prospective observational investigation. For the experimental group (rh-aPC group), we enrolled all patients admitted to the 12-bed polyvalent intensive care unit of the University Hospital of Ospedali Riuniti, Ancona, Italy with a diagnosis of severe sepsis or septic shock - based on the criteria of the International Sepsis Definitions Conference ACCP/SCCM [13] - that could receive rh-aPC treatment (continued infusion of 24 µg/kg/hour for 96 hours). We included patients with two or more sepsis-related organ failures (that is, cardiovascular, pulmonary or renal dysfunction, thrombocytopenia, metabolic acidosis with high lactates) or sepsis-correlated acute respiratory distress syndrome. Patients with absolute or relative contraindications to rh-aPC therapy were enrolled into the control group. At the onset of severe sepsis or septic shock, the Acute Physiology and Chronic Health Evaluation II score was calculated.

All patients were sedated, intubated, and mechanically ventilated. They were hemodynamically monitored by arterial femoral catheter with the PiCCO system (Pulsion, Munich, Germany). All patients received fluid challenge, and, if necessary, continuous infusion of inotropic (dobutamine) and vasopressor (norepinephrine) agents to maintain a normal cardiac index and intrathoracic blood volume index and to maintain the mean arterial pressure between 70 and 100 mmHg.

Near-infrared spectroscopy

StO2 was measured by a tissue spectrometer (InSpectra™ Model 325;

Hutchinson Technology Inc., Hutchinson, MN, USA). The spectrometer consists of light detection circuitry and an optical cable that transmits light to tissues and receives scattered light from tissues. The maximum depth of the tissue volume sampled is estimated to be equal to the distance between the sending and receiving fibers of the probe (probe spacing). A probe spacing of 15 mm was used, with the probe placed on an adhesive surface on the skin of the volar surface of the forearm at the level of the brachio-radial muscle. The VOT was applied using a sphygmomanometer cuff around the same arm that was inflated to 60 mmHg above the systolic arterial pressure to obtain an arterial occlusion (stagnant ischemia) until StO2 decreased to 40%. StO2 was monitored

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continuously before (baseline) and during (ischemia) pneumatic compression and after cuff release (reperfusion).

Data were analyzed using InSpectra™ software to plot and measure the StO2 curve characteristics; that is, baseline StO2 (StO2 baseline), rate of

decrease in StO2 during the VOT during the (StO2 downslope), and rate

of increase in StO2 reperfusion phase (StO2 upslope). Data were

collected before (T0), during (24 hours (T1a), 48 hours (T1b), 72 hours (T1c), and 96 hours (T1d)), and 6 hours (T2) after rh-aPC treatment (that is, 102 hours from T0), and at the same times in the controls. At all time points (except at 96 hours) the following measurements were obtained: mean arterial pressure, dose of norepinephrine, arterial blood lactate and base excess, cardiac index and intrathoracic blood volume index, and SOFA score.

Statistical analysis

Results are expressed as the mean ± standard deviation, and as the median (first to third interquartile range) for catecholamines. Parametric statistics were applied for all parameters-except for catecholamines, for which nonparametric statistics were utilized. A two-way analysis of variance test was used to assess differences between groups; a paired t test was applied to test differences between times within each group, while an unpaired t test with Welch correction when indicated was applied to test differences at each time between groups. The Friedman test was used to test significant differences during time within each group, the Wilcoxon test was used to test differences between each time point and T0, and the Mann-Whitney U test was used to test for differences between groups. P < 0.05 was considered statistically significant.

Results

We studied 11 patients (four female and seven male) with severe sepsis or septic shock who received rh-aPC therapy (continued infusion of 24 µg/kg/hour for 96 hours) and a control group of five patients (two female and three male) who could not receive rh-aPC because of contraindications. The patient characteristics are presented in Table 1. On admission to the intensive care unit, the mean Acute Physiology and Chronic Health Evaluation II score was 26.3 ± 6.6 for the rh-aPC group and 28.6 ± 5.3 for the control group, with a risk of death of 41.0 ± 22.9% and 57.6 ± 25.7%, respectively. The mortality rate was 36.4% in the rh-aPC group and 60% in the control group.

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Four patients in the rh-aPC group (Table 1, Patients 2, 4, 9, and 11) and one patient in the control group (Table 1, Patient 4) presented severe sepsis/septic shock on intensive care unit admission, while the other patients developed sepsis after admission to the intensive care unit.

From the two-way analysis of variance test, significant differences between groups were found for StO2 downslope (P < 0.01), StO2

upslope, the SOFA score (P < 0.05) and the mean arterial pressure (P < 0.001).

The Friedman test showed that the norepinephrine and dobutamine rates significantly decreased only in the rh-aPC group (P < 0.01), and not in the control group (Table 2).

The SOFA score, compared with T0, was significantly lower at T1c and T1d (10.1 ± 2.3 vs. 8.8 ± 2.0 and 8.0 ± 2.3; P < 0.05) and at T2 (7.9 ± 2.2; P < 0.01) (Figure 1). At T2 the SOFA score was significantly reduced compared with the control group (7.9 ± 2.2 vs. 12.2 ± 3.2; P < 0.05). In the control group, no differences were found with respect to baseline values.

There were no significant differences in the macrohemodynamic parameters (cardiac index and intrathoracic blood volume index) at T0, during therapy, and at T2 (Table 2). The mean arterial pressure was no different at T0 between groups, while it was significantly increased during treatment only in the rh-aPC group (T2 93.8 ± 12.8 vs. T0 81 ± 10.9 mmHg) (Figure 2).

With regard to metabolic acidosis in the rh-aPC group, base excess was significantly corrected (P < 0.01) after 24 hours from T0 and remained corrected until T2 (Figure 3a). In the control group, base excess was significantly corrected at T1a, T1b, T1c, and T2 (P < 0.05) (Figure 3a). Blood lactate was significantly decreased in the rh-aPC group at T1d and T2 (2.8 ± 1.3 vs. 1.9 ± 0.7 mmol/l; P < 0.05) (Figure 3b).

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Table 1. Patient characteristics

Patient characteristics

Patient Diagnosis at admission Contraindication

rh-aPC group

1 Postoperative triple aortocoronary bypass

2 Mediastinitis after odontogenic abscess

3 Postoperative liver trauma

4 Severe acute respiratory distress syndrome

5 Severe trauma

6 Severe trauma

7 Postoperative urgent aortocoronary bypass

8 Postoperative esophagectomy

9 Postoperative intestinal perforation

10 Necrotic-hemorrhagic pancreatitis

11 Pulmonary aspergillosis

Control group

1 Intracerebral hematoma Craniotomy

2 Postoperative cerebral aneurysm Craniotomy

3 Postoperative abdominal aortic aneurysm Risk of bleeding

4 Septic shock for cholecystitis Risk of intracranial bleeding

5 Pancreatitis Risk of bleeding

rh-aPC, recombinant activated protein C.

Donati et al. Critical Care 2009 13(Suppl 5):S12 doi:10.1186/cc8010

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Table 2. Cardiac index, ITBVI, norepinephrine dose, and dobutamine dose before, during, and after rh-aPC treatment

Cardiac index, ITBVI, norepinephrine dose, and dobutamine dose before, during,

and after rh-aPC treatment

T0 T1a T1b T1c T1d T2

rh-aPC group

Cardiac index 4.6 ± 1.3 4.6 ± 1.3 3.8 ± 0.7 4.1 ± 1.7 4.0 ± 1.0 4.1 ± 0.7

ITBVI 883 ± 207

874 ± 189

884 ± 203

922 ± 223

876 ± 213

872 ± 176

Norepinephrine*

0.22

(0.16 to 0.35)

0.22

(0.15 to 0.38)

0.22

(0.14 to 0.34)

0.22

(0.11 to 0.24)

0.17

(0.02 to 0.23)

0.14

(0.01 to 0.21)

Dobutamine* 3.4 (1 to

4.8) 2.8 (1 to

3.25) 2.1 (0 to

3.4) 2.1 (0 to

3.1) 2 (0 to

3.1) 2 (0 to

3.1)

Control group

Cardiac index 4.2 ± 0.6 4.1 ± 1.1 4.3 ± 0.7 4.2 ± 0.5 4.1 ± 0.8 4.1 ± 0.7

ITBVI 892 ± 249

894 ± 211

926 ± 208

897 ± 265

913 ± 280

891 ± 260

Norepinephrine

0.17

(0.06 to 0.37)

0.37

(0.11 to 0.5)

0.27

(0.11 to 0.37)

0.27

(0.11 to 0.37)

0.37

(0.11 to 0 to 4)

0.37

(0.11 to 0.4)

Dobutamine 4.2 (0 to

5.3) 0 (0 to

3.7) 0 (0 to

3.7) 2.3 (0 to

3.7) 2.3 (0 to

3.7) 2.3 (0 to

3.7)

Data are presented as the mean ± standard deviation or as the median (first to third interquartile range). Data were collected before (T0), during (24 hours (T1a), 48 hours

(T1b), 72 hours (T1c), and 96 hours (T1d)), and 6 hours (T2) after recombinant activated protein C (rh-aPC) treatment (that is, 102 hours from T0). ITBVI, intrathoracic blood

volume index. *P < 0.01, Friedman test in the rh-aPC group.

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Figure 1.

Sequential Organ Failure Assessment score before, during, and after recombinant activated

protein C treatment. The Sequential Organ Failure Assessment (SOFA) score in the

recombinant activated protein C (rh-aPC) group before, during, and after rh-aPC treatment,

and in the control group at the same times. Data were collected before (T0), during (24 hours

(T1a), 48 hours (T1b), 72 hours (T1c), and 96 hours (T1d)), and 6 hours (T2) after rh-aPC

treatment (that is, 102 hours from T0). ANOVA, analysis of variance. Figure 2.

Mean arterial pressure before, during, and after recombinant activated protein C treatment.

Mean arterial pressure (MAP) in the recombinant activated protein C (rh-aPC) group before,

during, and after rh-aPC treatment, and in the control group at the same times. Data were

collected before (T0), during (24 hours (T1a), 48 hours (T1b), 72 hours (T1c), and 96 hours

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(T1d)), and 6 hours (T2) after rh-aPC treatment (that is, 102 hours from T0). ANOVA,

analysis of variance.

Figure 3.

Base excess and blood lactate before, during, and after recombinant

activated protein C treatment. (a) Arterial base excess (BE) and (b)

blood lactate in the recombinant activated protein C (rh-aPC) group

before, during, and after rh-aPC treatment, and in the control group at

the same times. Data were collected before (T0), during (24 hours (T1a),

48 hours (T1b), 72 hours (T1c), and 96 hours (T1d)), and 6 hours (T2)

after rh-aPC treatment (that is, 102 hours from T0). ANOVA, analysis of

variance; n.s., not significant.

StO2 baseline was significantly higher at T1a, T1c and T2 (Figure 4),

while in the control group it was significantly higher at T2. StO2

downslope decreased significantly at all the time points (Figure 5a) only in the rh-aPC group, and it was significantly steeper in the rh-aPC-treated

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patients than in the control patients at T1b and T2 (-16.5 ± 11.8 vs. -8.1 ± 2.4%/minute). StO2 upslope increased significantly in the rh-aPC group

at T1b, T1c, T1d and T2 (Figure 5b), and was significantly higher than in the controls at T1c, T1d and T2 (101.1 ± 62.1 vs. 54.5 ± 23.8%/minute). Figure 4.

Baseline tissue oxygen saturation before, during, and after recombinant activated protein C

treatment. Baseline tissue oxygen saturation (StO2 baseline) in the recombinant activated

protein C (rh-aPC) group before, during, and after rh-aPC treatment, and in the control

group at the same times. Data were collected before (T0), during (24 hours (T1a), 48 hours

(T1b), 72 hours (T1c), and 96 hours (T1d)), and 6 hours (T2) after rh-aPC treatment (that is,

102 hours from T0). ANOVA, analysis of variance; n.s., not significant.

Figure 5.

Tissue oxygen saturation decrease and increase with the vascular occlusion test before,

during, and after recombinant activated protein C treatment. (a) Rate of decrease in tissue

oxygen saturation (StO2 downslope) and (b) rate of increase in tissue oxygen saturation (StO2

upslope) in the recombinant activated protein C (rh-aPC) group before, during, and after rh-

aPC treatment, and in the control group at the same times. Data were collected before (T0),

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during (24 hours (T1a), 48 hours (T1b), 72 hours (T1c), and 96 hours (T1d)), and 6 hours (T2)

after rh-aPC treatment (that is, 102 hours from T0). ANOVA, analysis of variance.

Discussion The present prospective observational study investigated the effects of rh-aPC treatment on the SOFA score, macrohemo-dynamic parameters, and metabolic acidosis in severe sepsis and septic shock. Additionally, and more importantly in the context of sepsis, the tissue oxygenation, metabolism, and microvascular reperfusion dynamics were assessed using NIRS in combination with a VOT to study any beneficial effects of rh-aPC therapy at the microcirculatory level. It was shown that rh-aPC treatment significantly lowered the SOFA score, increased the mean arterial pressure, and reduced the blood lactate concentration. Furthermore, rh-aPC had positive effects on the VOT-derived StO2

parameters; both StO2 downslope and StO2 upslope increased

significantly, indicating raised oxygen consumption/metabolism and indicating improved microvascular reperfusion following ischemia. Early goal-directed therapy focused on restoring macrohemodynamics has been shown to be insufficient in preventing cellular hypoxia and organ failure due to the heterogeneous nature of sepsis-related microcirculatory dysfunction. It has been shown that improvement in hemodynamic parameters with vasoconstrictors, such as norepinephrine, could make tissue perfusion worse [14], and several studies have demonstrated the positive effects of vasodilators on microcirculatory recruitment even in hemodynamically resuscitated septic patients [15]. rh-aPC treatment has been shown to improve end-organ function and to decrease mortality if started in the early stages of sepsis [5-7] by restoring the coagulative cascade, the inflammatory response, leukocyte adhesion and migration, and endothelial function. In addition to the anticoagulant, profibrinolytic [16], and anti-inflammatory effects and the antioxidant properties [17,18], rh-aPC acts at the microcirculation level to enhance the proportion of perfused capillaries and improve local autoregulation [1,19-21]. These studies were mostly carried out in animals. De Backer and co-workers [22] were the first to show the beneficial effects of rh-aPC on microcirculatory perfusion by direct observation of the sublingual microcirculation of septic patients using OPS imaging. To our knowledge, however, the direct effects of rh-aPC treatment on tissue oxygenation have not been studied before. Many studies have shown the relevance of StO2 in the assessment of the

metabolic and microcirculatory state in septic patients. Doerschug and colleagues [23], De Blasi and colleagues [10], Skarda and colleagues [24], and Pareznik and colleagues [25] all showed that tissue oxygen

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consumption was lower in septic patients than in nonseptic patients or healthy volunteers and that septic patients have slower tissue reoxygenation following ischemia. In addition, Creteur and colleagues [12] demonstrated that persistent alteration of StO2 resaturation

correlated with worse outcome and multiorgan failure. The steady-state tissue oxygenation (StO2 baseline) did not change as a

result of rh-aPC treatment, which indicates that the balance between tissue oxygen delivery and consumption is unaltered by rh-aPC infusion. StO2 downslope, in contrast, increased significantly after starting the rh-

aPC therapy, indicating increased cellular oxygen consumption. Additionally, StO2 upslope increased significantly due to rh-aPC

treatment, which indicates the improved ability of the microcirculation to be reperfused after a brief period of ischemia. Microvascular function is therefore improved by rh-aPC treatment. This finding is also supported by the reduced SOFA score and lactate levels. The present study has some limitations: firstly, the small number of patients - in particular in the control group, where patients affected by head trauma and intracranial hypertension could have an altered systemic hemodynamic; and secondly, because the NIRS technique itself has some limitations. StO2 downslope has been asserted to indicate the muscle

oxygen consumption, but oxygen consumption cannot be directly measured as the amount of hemoglobin in the respective muscle blood volume is not known. The parameter being measured is the oxygen consumption rate extrapolated from the decrease in saturation of hemoglobin (StO2 decrease rate, %/minute), which is an index of the

basic metabolism of the thenar muscle. Moreover, whether the concentration of hemoglobin affects the oxygen consumption rate is not known. One argument regarding the reperfusion rate is that NIRS does not measure blood flow and it must be assumed that an increase in StO2

reflects endothelium-dependent vasodilation. The extent to which comorbidites such as atherosclerosis, age, gender or mental stress may influence this parameter is not known. Regardless of these limitations, the ability to provide a non-invasive, reproducible estimate of the oxygen consumption rate of skeletal muscle at the bedside renders this technique potentially useful in clinical practice.

Conclusion Treatment of septic patients with continuous infusion of rh-aPC may improve tissue oxygenation, cellular metabolism, and microvascular

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reactivity, and may significantly reduce the SOFA score and lactate levels. NIRS in combination with a VOT was able to detect microcirculatory and metabolic changes associated with sepsis and rh-aPC treatment. Abbreviations NIRS: near-infrared spectroscopy; rh-aPC: recombinant activated protein C; SOFA: Sequential Organ Failure Assessment; StO2: tissue oxygen

saturation; StO2 downslope: rate of decrease in tissue oxygen saturation;

StO2 upslope: rate of increase in tissue oxygen saturation; VOT: vascular

occlusion test.

References 1. Ince C: The microcirculation is the motor of sepsis. Crit

Care 2005, 9(Suppl 4):13-19. 2. De Backer D, Creteur J, Preiser J, Dubois MJ, Vincent JL:

Microvascular blood flow is altered in patients with sepsis. Am J Respir Crit Care 2002, 166:98-104.

3. Astiz ME, DeGent GE, Lin RY, Rackow EC: Microvascular function and rheologic changes in hyperdynamic sepsis. Crit Care Med 1995, 23:265-271.

4. Trzeciak S, McCoy JV, Dellinger RP, Arnold RC, Rizzuto M, Abate NL, Shapiro NI, Parrillo JE, Hollenberg SM, Microcirculatory Alterations in Resuscitation and Shock (MARS) investigators: Early increases in microcirculatory perfusion during protocol-directed resuscitation are associated with reduced multi-organ failure at 24 h in patients with sepsis. Intensive Care Med 2008, 34:2210-2217.

5. Bernard GR, Vincent JL, Laterre PF, LaRosa SP, Dhainaut JF, Lopez-Rodriguez A, Steingrub JS, Garber GE, Helterbrand JD, Ely EW, Fisher CJ Jr, Recombinant Human Protein C Worldwide Evaluation in Severe Sepsis (PROWESS) study group: Efficacy and safety of recombinant human activated protein C for severe sepsis. N Engl J Med 2001, 344:699-709.

6. Vincent JL, Bernard GR, Beale R, Doig C, Putensen C, Dhainaut JF, Artigas A, Fumagalli R, Macias W, Wright T, Wong K, Sundin DP, Turlo MA, Janes J: Drotrecogin alfa (activated) treatment in severe sepsis from the global open-label trial ENHANCE: further evidence for survival and safety and implications for early treatment. Crit Care Med 2005, 33:2266-2277.

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7. Dellinger RP, Carlet JM, Masur H, Gerlach H, Calandra T, Cohen J, Gea-Banocloche J, Keh D, Marshall JC, Parker MM, Ramsay G, Zimmerman JL, Vincent JL, Levy MM: Surviving Sepsis Campaign Management Guidelines Committee: Surviving sepsis campaign guidelines for management of severe sepsis and septic shock. Crit Care Med 2004, 32:858-873.

8. Boushel R, Langberg H, Olesen J, Gonzales-Alonzo J, Bulow J, Kjaer M: Monitoring tissue oxygen availability with near infrared spectroscopy (NIRS) in health and disease. Scand J Med Sci Sports 2001, 11:213-222.

9. Ward KR, Ivatury RR, Barbee RW, Terner J, Pittman R, Filho IP, Spiess B: Near infrared spectroscopy for evaluation of the trauma patient: a technology review. Resuscitation 2006, 68:27-44.

10. De Blasi RA, Palmisani S, Alampi D, Mercieri M, Romano R, Collini S, Pinto G: Microvascular dysfunction and skeletal muscle oxygenation assessed by phase-modulation near-infrared spectroscopy in patients with septic shock. Intensive Care Med 2005, 31:1661-1668.

11. Knotzer H, Pajk W, Dunser MW, Majer S, Mayr AJ, Ritsch N, Friesenecker B, Hasibeder WR: Regional microvascular reactivity in patients with different degree of multiple organ dysfynction syndrome. Anesth Analg 2006, 102:1187-1193.

12. Creteur J, Carollo T, Soldati G, Buchele G, De Backer D, Vincent JL: The prognostic value of muscle StO2 in septic

patients. Intensive Care Med 2007, 33:1549-1556. 13. Levy MM, Fink MP, Marshall JC, Abraham E, Angus D,

Cook D, Cohen J, Opal SM, Vincent JL, Ramsay G: 2001 SCCM/ESICM/ACCP/ATS/SIS International Sepsis Definitions Conference. Intensive Care Med 2003, 29:530-538. Jhanji S, Stirling S, Patel N, Hinds CJ, Pearse RM: The effect of increasing doses of norepinephrine on tissue oxygenation and microvascular flow in patients with septic shock. Crit Care Med 2009, 37:1961-1966.

14. Spronk PE, Ince C, Gardien MJ, Mathura KR, Oudemans-van Straaten HM, Zandstra DF: Nitroglycerin in septic shock after intravascular volume resuscitation. The Lancet 2002, 360:1395-1396.

15. Macias WL, Yan SB, Williams MD, Um SL, Sandusky GE, Ballard DW, Planquois JM: New insights into the protein C pathway: potential implications for the biological activities

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of drotrecogin alfa (activated). Crit Care 2005, 9(Suppl 4):38-45.

16. Yamaji K, Wang Y, Liu Y, Abeyama K, Hashiguchi T, Uchimura T, Krishna Biswas K, Iwamoto H, Maruyama I: Activated protein C, a natural anticoagulant protein, has antioxidant properties and inhibits lipid peroxidation and advanced glycation end products formation. Thromb Res 2005, 115:319-325.

17. Gierer P, Hoffmann JN, Mahr F, Menger MD, Mittlmeier T, Gradl G, Vollmar B: Activated protein C reduces tissue hypoxia, inflammation, and apoptosis in traumatized skeletal muscle during endotoxiemia. Crit Care Med 2007, 35:1966-1971.

18. Hoffmann JN, Vollmar B, Laschke MW, Inthorn D, Fertmann J, Schildberg FW, Menger MD: Microhemodynamic and cellular mechanisms of activated protein action during endotoxemia. Crit Care Med 2004, 32:1011-1017.

19. Isobe H, Okajima K, Uchiba M, Mizutani A, Harada N, Nagasaki A, Okabe K: Activated protein C prevents endotoxin-induced hypotension in rats by inhibiting excessive production of nitric oxide. Circulation 2001, 104:1171-1175.

20. Marechal X, Favory R, Joulin O, Montaigne D, Hassoun S, Decoster B, Zerimech F, Neviere R: Endothelial glycocalyx damage during endotoxemia coincides with microcirculatory dysfunction and vascular oxidative stress. Shock 2008, 29:572-576.

21. De Backer D, Verdant C, Chierego M, Koch M, Gullo A, Vincent JL: Effects of drotrecogin alfa activated protein C on microcirculatory alterations in patients with severe sepsis. Crit Care Med 2006, 34:1-7.

22. Doerschug KC, Delsing AS, Schmidt GA, Haynes WG: Impairments in microvascular reactivity are related to organ failure in human sepsis. Am J Physiol Heart Circ Physiol 2007, 293:1065-1071.

23. Skarda DE, Mulier KE, Myers DE, Taylor JH, Beilman GJ: Dynamic near-infrared spectroscopy measurements in patients with severe sepsis. Shock 2007, 27:345-353.

24. Pareznik R, Knezevic R, Voga G, Podbregar M: Changes in muscle tissue oxygenation during stagnant ischemia in septic patients. Intensive Care Med 2006, 32:87-92.

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Chapter 6

The aPC treatment improves

microcirculation in severe sepsis/septic

shock syndrome

Abele Donati1,2*, Elisa Damiani1, Laura Botticelli1, Erica Adrario1, Maria

Rita Lombrano1, Roberta Domizi1, Benedetto Marini1, Jurgen WGE Van Teeffelen3, Paola Carletti1,

Massimo Girardis4, Paolo Pelaia1 and Can Ince2

1Anesthesia and Intensive Care Unit, Department of Biomedical Science and Public Health, Università Politecnica delle Marche, via Tronto 10,

60126 Torrette di Ancona, Italy. 2Department of Translational Physiology, Academic Medical Center, University of Amsterdam, Meibergdreef 9, Amsterdam 1105 AZ, The

Netherlands. 3Department of Physiology, Cardiovascular Research Institute

Maastricht, Maastricht University, PO Box 616, 6200 MD Maastricht, The Netherlands.

4Surgical ICU, Anesthesia and Intensive Care Department, University Hospital of Modena, Modena, Italy.

Published in: BMC Anesthesiology 2013, 13:25 (doi:10.1186/1471-2253-

13-25)

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Abstract

Background: The role of recombinant activated protein C (aPC) during sepsis is still controversial. It showed antiinflammatory effect and improved the microvascular perfusion in experimental models of septic shock. The present study was aimed at testing the hypothesis that recombinant aPC therapy improves the microcirculation during severe sepsis.

Methods: Prospective observational study on patients admitted in a 12-beds intensive care unit of a university hospital from July 2010 to December 2011, with severe sepsis and at least two sepsis-induced organ failures occurring within 48 hours from the onset of sepsis, who received an infusion of aPC (24 mcg/kg/h for 96 hours) (aPC group). Patients with contraindications to aPC administration were also monitored (no-aPC group). At baseline (before starting aPC infusion, T0), after 24 hours (T1a), 48 hours (T1b), 72 hours (T1c) and 6 hours after the end of aPC infusion (T2), general clinical and hemodynamic parameters were collected and the sublingual microcirculation was evaluated with sidestream dark-field imaging. Total vessel density (TVD), perfused vessel density (PVD), De Backer score, microvascular flow index (MFIs), the proportion of perfused vessels (PPV) and the flow heterogeneity index (HI) were calculated for small vessels. The perfused boundary region (PBR) was measured as an index of glycocalyx damage. Variables were compared between time points and groups using non parametric or parametric statistical tests, as appropriate.

Results: In the 13 aPC patients mean arterial pressure (MAP), base excess, lactate, PaO2/FiO2 and the Sequential Organ Failure Assessment

(SOFA) score significantly improved over time, while CI and ITBVI did not change. MFIs, TVD, PVD, PPV significantly increased over time and the HI decreased (p < 0.05 in all cases), while the PBR did not change. No-aPC patients (n = 9) did not show any change in the microcirculation over time. A positive correlation was found between MFIs and MAP. TVD, PVD and De Backer score negatively correlated with norepinephrine dose, and the SOFA score negatively correlated with MFIs, TVD and PVD.

Conclusions: aPC significantly improves the microcirculation in patients with severe sepsis/septic shock. Trial registration: NCT01806428

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Sepsis is a major problem in intensive care units (ICU), with high

incidence and mortality rate [1]. Although many studies have been

conducted in order to find an effective therapy, the treatment for such a

complex syndrome is still a source of investigation, mainly due to the

uncertainty about its pathophysiology. According to the current

hypothesis, microcirculatory alterations may contribute to the defect in

oxygen extraction during sepsis and play a major role in the progression

to multiorgan dysfunction [2]. For this reason the microcirculation may

represent the best target for therapy aimed to improve organ dysfunction

and outcome. Recombinant activated protein C (aPC) had been approved

for the treatment of patients with severe sepsis in 2001, after the

PROWESS Study showed that it was able to significantly reduce

mortality [3]. Unfortunately, these results were not confirmed [4] and the

drug was withdrawn from the market ten years later. Nevertheless, the

role of aPC in sepsis therapy is still a controversial issue and papers

stressing its benefits continue to be published [5-7]. APC is an endogen

protein with anti-inflammatory, anticoagulant and profibrinolitic

properties; it showed to inhibit the generation of thrombin by inactivating

factor Va and factor VIIIa [8] and this was thought to be the most

important mechanism for its therapeutic action in sepsis. Then this theory

was abandoned as its anti-inflammatory action seemed to be of major

relevance: preclinical studies demonstrated a host of beneficial effects

targeting NF-kB pathway [9] together with the ability to reduce the

apoptosis and decrease citonecrosis during sepsis [10]. Moreover aPC

can influence the endothelial cell function, by inhibiting white blood cells

rolling and adhesion [11], preventing the activation of inducible nitric

oxide synthase (iNOS) [12] and exerting a protective role towards the

endothelial glycocalyx [13]. Several experimental studies showed that

aPC may integratively improve the microvascular perfusion during sepsis

[14-17]. Only one study was conducted so far on severely septic patients

in order to translate these experimental evidences into a clinical setting

[18].

The aim of the present study was to evaluate the effect of aPC infusion

on the microcirculation in patients with severe sepsis/septic shock.

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Methods

The study was approved by the Ethics Committee of AOU “Ospedali

Riuniti Umberto I–Lancisi–Salesi” of Ancona (Italy) and informed

consent was obtained from the patients or their relatives.

Patient population

This prospective observational study was conducted in a 12-beds ICU of

“Azienda Ospedaliera-Universitaria–Ospedali Riuniti: Umberto I–

Lancisi–Salesi” of Ancona (Italy) from July 2010 to December 2011 and

included patients who received an aPC infusion (24 mcg/kg/hr for 96 hrs)

for the presence of severe sepsis/ septic shock and at least two sepsis-

induced organ failures within 48 hours of the onset of sepsis, with no

contraindications to aPC treatment (recent head trauma or intracranial

bleeding). Exclusion criteria were: hematologic or advanced

malignancies, liver cirrhosis, severely impaired consciousness (Glasgow

Coma Scale score <7), and therapeutic limitations (do-not-resuscitate

orders).

The sample size was calculated on the Microvascular Flow Index (MFI):

13 patients proved to be sufficient to demonstrate a change in MFI of 0.5

(standard deviation = 0.5) with a power of 90% and an alpha error of

0.05.

During the same study period, the microcirculation was monitored also in

those patients who met the inclusion criteria but did not receive aPC

because of contraindications (recent head trauma or intracranial bleeding)

(no-aPC group).

General management

All patients were sedated (propofol 2–4 mg/kg/h or midazolam 0.1–0.3

mg/kg/h and sufentanil 0.15–2 µg/kg/h or remifentanil 1.2–4.8 µg/kg/h),

intubated and mechanically ventilated. All patients were equipped with a

central venous catheter and a femoral artery catheter and hemodynamic

parameters were monitored using the PiCCO system (Pulsion, Munich,

Germany). Fluids (crystalloids and colloids), vasopressors

(norepinephrine) and inotropic agents (dobutamine) were provided

according to individual needs, in order to maintain the Intrathoracic

Blood Volume Index (ITBVI) within the range of normality (800–1000

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ml/m2), a normal cardiac index value, and mean arterial pressure

(MAP) > 65 mmHg.

Measurements

The Acute Physiology and Chronic Health Evaluation (APACHE) II

score [19] was obtained on the day of the inclusion. Temperature (T),

heart rate (HR), MAP and complete hemodynamic assessment were

obtained in all patients before starting aPC infusion (T0), at 24 hours (±3

hrs) (T1a), 48 hours (±3 hrs) (T1b), 72 hours (±3 hrs) (T1c) and finally 6

hours (±3 hrs) after the end of aPC infusion (T2). Arterial blood samples

were withdrawn simultaneously in order to measure blood gases,

hemoglobin, and lactate levels (Omni Roche Diagnostic, Monza, Italy).

Results of routine biological blood samples were collected and The

Sequential Organ Failure Assessment (SOFA) score [20] was calculated

daily up to day 5. Patients who did not receive aPC infusion were

monitored at the same time points.

Microcirculatory evaluation and analysis

The sublingual microcirculation was evaluated using sidestream dark

field (SDF) imaging (Microscan, Microvision Medical, Amsterdam, the

Netherlands) by an investigator blinded to the patient’s group at the five

time points previously described (T0, T1a, T1b, T1c and T2). SDF

technique is described in detail elsewhere [21]. After removal of saliva

and other secretions with a gauze, the device was gently applied without

pressure to the lateral side of the tongue, in an area approximately 1.5–4

cm from the tip of the tongue. Five different microcirculatory sites (at

least 10 sec/site) were recorded at each time point with adequate focus

and contrast and every effort was made to avoid movement and pressure

artifacts. A random number was assigned to each sequence; poor-quality

images were discarded and three images for each time point were

selected and analyzed using a computer software package (Automated

Vascular Analysis Software, Microvision Medical BV, Amsterdam, the

Netherlands). According to the consensus report on the performance and

evaluation of microcirculation using SDF imaging [22], Total Vessel

Density (TVD), Perfused Vessel Density (PVD) and De Backer score

were calculated, providing index of microvascular vessel density; the

Proportion of Perfused Vessels (PPV) and the Microvascular Flow Index

(MFI), reflecting microcirculatory blood flow velocity, were analyzed

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semiquantitatively in small- (diameter < 20 µm) (MFIs) and medium-

sized vessels (20–100 µm), as described previously [23]. The Flow

Heterogeneity Index (HI) was also calculated, providing an index of

heterogeneous microcirculatory perfusion, which is common during

sepsis [24]. Each sequence was analyzed by two investigators, both

blinded to the origin of the clip: one investigator from AOU Ospedali

Riuniti (Ancona, Italy) and the other from Academic Medical Center

(Amsterdam, the Netherlands). Inter-observer variability was calculated

for all the sequences analyzed: the coefficient of variability ranged from

4.5% to 8.7% for MFI, from 3.5% to 5.7% for the TVD and from 4.3% to

7.9% for the PPV.

Microvascular glycocalyx assessment

SDF videos of at least 40 consecutive frames of approximately 950 µm

by 700 µm sublingual tissue surface area were analyzed using the

software GlycoCheck ICU (Maastricht University Medical Center,

Maastricht, The Netherlands) in order to measure the Perfused Boundary

Region (PBR). The PBR is the dimension of the permeable part of the

endothelial glycocalyx which allows the penetration of flowing red blood

cells. Erythrocytes usually have a limited access into an intact

glycocalyx: when this is compromised and starts losing its protective

capacity, its permeability increases, allowing circulating cells to approach

the luminal endothelial membrane more closely. As a result, the

dimension of the erythrocyte PBR will increase [25].


Data were analyzed using GraphPad 5.0 program (GraphPad Software,

Inc, La Jolla, CA, USA). Data are presented as median [25th–75th

percentiles]. Descriptive statistics were computed for all study variables.

A Kolmogorov-Smirnov test was used, and stratified distribution plots

were examined to verify the normality of distribution of continuous

variables. Nonparametric measures of comparison were used when

variables evaluated were not normally distributed. Differences between

aPC and no-aPC group were assessed using a chi-square, Fisher’s exact

test, and unpaired t-test or Mann–Whitney U test as appropriate. One-

way analysis of variance for repeated measures with Bonferroni post test

or Friedman test with Dunn’s post-test were used to assess the evolution

Chapter 6

115

of microvascular perfusion in each group. Relationships between

variables were assessed by Spearman’s correlation.

Differences were considered significant at (two-sided) p value < 0.05.

Results

Thirteen patients receiving aPC were included during the study period.

All the patients survived until the end of aPC infusion. General

characteristics, hemodynamic and general management data are reported

in Tables 1 and 2, respectively.

As shown in Table 2, MAP, PaO2/FiO2 and BE were significantly

higher at T2 compared to T0, while lactate levels, norepinephrine

infusion rate and SOFA score were significantly decreased.

Nine patients with contraindications to aPC infusion were monitored. All

of them survived until the end of the study protocol. No-aPC patients did

not significantly differ from aPC patients for age, gender, APACHE II

score (Table 1), SOFA score, hemodynamic parameters and blood gas

values (Table 2). Baseline microcirculatory variables were similar in no-

aPC and aPC patients, except for PVD which was significantly lower in

the aPC-group (p < 0.05, Table 3). Hemodynamic, blood gases and the

microvascular flow did not show any significant change during the whole

study period. A significant increase in the SOFA score was seen at T2

compared to T0 (Tables 2 and 3, Figure 1).

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Microcirculatory variables are shown in Table 3. All the microvascular

parameters improved over time, with significant increases in MFI, MFIs

(Figure 1A), TVD (Figure 1C), PVD (Figure 1D) and PPV (Figure 1E)

and a decrease in the HI. A slight not significant decrease in PBR could

be noted over time (Figure 1F).

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Table 2. Hemodynamic and general management data

Table 3. Microcirculatory data

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118

Figure 1 Microcirculatory variables: aPC-group vs. no-aPC group. (a) Microvascular Flow

Index for small vessels; (b) De Backer score; (c) Total ll Vessel Density, (d) Perfused Vessel

Density; (e) Proportion of Perfused Vessels; (f) Perfused Boundary Region. # p < 0.05, vs. T0

in the aPC-group (## p < 0.01; ### p < 0.0001). * p < 0.05, aPC-group vs. aPC-group at the

same time point.

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As shown in Table 2 and Figure 1, in aPC patients compared to no-aPC

the MFI was higher at T1c (p = 0.02) and T2 (p = 0.001), MFIs, PVD,

PPV and De Backer score were higher (p = 0.03, p = 0.04, p = 0.04,

p = 0.03 respectively) and HI was lower (p = 0.01) at T2.

A significant correlation was found between MFIs and MAP (r = 0.3,

p < 0.01) but their changes over time were not correlated. TVD, De

Backer score and PVD were negatively correlated with norepinephrine

dose (r = −0.4, p < 0.01; r = −0.3, p < 0.05; r = −0.2, p < 0.05,

respectively). No relationship was seen between changes in CI or in

ITBVI and changes in microcirculatory variables. The SOFA score was

negatively correlated with MFIs (r = −0.3, p < 0.01), TVD (r = −0.5,

p < 0.01) and PVD (r = −0.4, p < 0.01). ì

Discussion

The present study confirms that aPC treatment improves the

microcirculation in severe septic/septic shock patients. This is in

accordance with the conclusions of De Backer et al. [18], who studied the

effects of aPC on the microcirculation using orthogonal polarization

spectral imaging and evaluated changes in the De Backer score and PPV.

We tried to perform a more comprehensive analysis of the

microcirculation by calculating further parameters for vessel density

(TVD, PVD), microvascular perfusion (MFI, PPV) and flow distribution

(HI). According to De Backer’s findings, we found an improvement in

the microvascular density following aPC infusion, suggesting a

recruitment of capillaries which were not perfused before, and an

increase in the amount of continuously perfused vessels. Additionally, the

flow in the smaller vessels was improved and more homogeneously

distributed–as results from the increase in MFIs and the decrease in the

HI, respectively–suggesting a benefit of aPC on the rheology of the

microcirculation during sepsis.

Moreover, whereas we found a stable improvement in the

microcirculation, De Backer et al. had reported a transient early

worsening in microcirculatory variables after aPC cessation, although

they had neither indicated significant differences towards previous days,

nor found a persistence of such a deterioration in the following 3 days.

This discrepancy is not immediately clear to explain: indeed, aPC was

infused at the same dose in both studies; differences in patient baseline

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characteristics between the two studied cannot be excluded, although

baseline SOFA scores were similar in the two studies. Differences in time

point measurements may have played a role: contrarily to De Backer, we

only evaluated the microcirculation every 24 hours and might have

missed a temporary slight alteration occurring during the end of aPC

infusion. Still, if such a transient deterioration did occur, this might do

nothing but confirm the role of aPC in influencing the microvascular

function: indeed, it is logical to expect the end of the administration of a

drug to be reflected by a transient instability at the level where it should

act. Actually, looking at our raw data, the PPV seemed to slightly

decrease after aPC cessation, even if the difference towards previous time

points was not significant.

Data for this study had been collected before the publication of the

PROWESS SHOCK trial and the withdrawal of aPC from the market.

However, discrepancies between PROWESS and PROWESS SHOCK

trials are still a matter of concern [26]. Several experimental evidences

support the beneficial effect of aPC on the microcirculation [14-17]. In an

experimental model of sepsis, Marechal et al. showed that aPC

administration was able to preserve either microvascular perfusion and

glycocalyx integrity [13]. Nevertheless, in our study the microvascular

recovery seems not to be related to a glycocalyx restoration, although a

slight even if not significant PBR decrease was found during aPC

infusion.

A significant decrease in blood lactate and an increase in BE were seen

during aPC treatment, which may reflect an improved tissue oxygen

uptake. This is consistent with our previous findings: using near infrared

spectroscopy, we demonstrated that aPC infusion may increase tissue

oxygenation and microvascular reactivity in severely septic patients,

leading to an improved cellular metabolism [27]. MAP also increased

after aPC administration. Nevertheless, this does not seem to be the

reason for the microvascular improvement, since either we and De

Backer found no relationship between changes in MAP and the

improvement in MFIs. Moreover, CI and ITBVI did not change in aPC-

nor in no-aPC patients and were not correlated to microvascular

variables, according to previous evidences [28,29]. We agree with De

Backer in rather relating the increase in MAP to an effect of a better

microvascular tone. Indeed, many experimental studies demonstrated that

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aPC can protect the microcirculation from endotoxic shock thanks to its

anti-inflammatory properties [15,30-32] and the rise in MAP can be

reasonably explained by its ability to inhibit the iNOS, thereby

preventing the arteriolar vasodilation [12].

The case-crossover analysis, in which each patient served as his/her own

control, is a major limitation of our study. We cannot be sure that the

microvascular improvement depended on different treatments or the

independent evolution in the patient’s condition rather than aPC infusion.

We monitored 9 severe sepsis/septic shock patients who did not receive

aPC because of contraindications. The fact that these patients did not

show any improvement in the microcirculation over time would suggest

that aPC infusion can really exert a beneficial effect on microvascular

perfusion. However, beyond the lack of differences in general

characteristics and the collected baseline clinical data, these patients were

different from aPC-patients by definition and the comparison between

them cannot provide any conclusion. As a major limitation, our analysis

is lacking of a real control group with patients adequately matched to

those receiving aPC, which would allow to reliably discriminate the

effects of aPC on the microcirculation by controlling for possible

confounding factors. However, the case crossover design was required

for ethical reasons.

A further limitation of our study is that many factors were not considered

which might have independently affected the microcirculation. Moreover,

the sample was too small and heterogeneous to adjust for potential

confounders.

The ability to improve the microcirculation would be of major

importance since the persistence of microvascular alterations during

septic shock proved to be associated with the occurrence of organ failures

and mortality [28]. In our cohort, microvascular flow and density

negatively correlated with norepinephrine requirement and SOFA score.

Unfortunately, our study was not designed to look at mortality.

Therefore, the relation between aPC treatment and outcome could not be

investigated, nor was it possible to reliably test an association between

the microvascular improvement and survival. As a limitation, our data do

not allow to support that an aPC-induced improvement in microvascular

perfusion may eventually result in lower risk of mortality.

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Conclusions

The present study supports a role of aPC treatment in improving the

microvascular perfusion in severe sepsis/septic shock patients. The

relationship between the aPC-induced microvascular improvement and

the outcome is still to be demonstrated.

Abbreviations

ICU: Intensive care unit; aPC: Activated protein C; MAP: Mean arterial

pressure; CI: Cardiac index; ITBVI: Intrathoracic blood volume index;

TVD: Total vessel density; PVD: Perfused vessel density; MFI:

Microvascular flow index; MFIs: Microvascular flow index for small

vessels; PPV: Proportion of perfused vessels; HI: Flow heterogeneity

index; PBR: Perfused boundary region; APACHE II: Acute physiology

and chronic health evaluation II; SOFA: Sequential organ failure

assessment; T: Body temperature; HR: Hearth rate; BE: Base excess;

SDF: Sidestream dark field.

Acknowledgements

We thank all the patients who kindly gave their consent to the

participation in this study and all the nurse and medical staff for the

support and contribution to the realization of this work.

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Chapter 7

Levosimendan for resuscitating the

microcirculation in patients with septic

shock: a randomized controlled study

Andrea Morelli1*, Abele Donati2, Christian Ertmer3, Sebastian Rehberg3,

Matthias Lange3, Alessandra Orecchioni1, Valeria Cecchini1, Giovanni Landoni4, Paolo Pelaia2, Paolo Pietropaoli1, Hugo Van Aken3, Jean-Louis

Teboul5, Can Ince6,7 and Martin Westphal3

1 Department of Anesthesiology and Intensive Care, University of Rome, 'La Sapienza', Viale del Policlinico 155, Rome 00161, Italy 2 Department of Neuroscience-Anesthesia and Intensive Care Unit,

Università Politecnica delle Marche, Via Tronto 10, Torrette di Ancona 60020, Italy

3 Department of Anesthesiology and Intensive Care, University Hospital of Muenster, Albert-Schweitzer-Str. 33, Muenster 48149,

Germany 4 Department of Anesthesia and Intensive Care, Università Vita-

Salute San Raffaele, Via Olgettina 60, Milan 20132, Italy 5 Hôpital de Bicêtre, Service of Medical Intensive Care, Centre Hospitalier de Bicêtre, rue du Général Leclerc 78, Le Kremlin-

Bicêtre 94270, France 6 Department of Translational Physiology, Academic Medical

Center, University of Amsterdam, Meibergdreef 9, Amsterdam 1105 AZ, The Netherlands

7 Department of Intensive Care, Erasmus MC, University Medical Center Rotterdam, 's-Gravendijkwal 230, Rotterdam 3015 CE, The

Netherlands

Published in: Critical Care 2010, 14:R232 (doi:10.1186/cc9387)

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Abstract

Introduction The purpose of the present study was to investigate microcirculatory blood flow in patients with septic shock treated with levosimendan as compared to an active comparator drug (i.e. dobutamine). The primary end point was a difference of ≥ 20% in the microvascular flow index of small vessels (MFIs) among groups. Methods

The study was designed as a prospective, randomized, double-blind clinical trial and performed in a multidisciplinary intensive care unit. After achieving normovolemia and a mean arterial pressure of at least 65 mmHg, 40 septic shock patients were randomized to receive either levosimendan 0.2 µg·kg-1·min-1 (n 20) or an active comparator (dobutamine 5 µg·kg-1·min-1; control; n 20) for 24 hours. Sublingual microcirculatory blood flow of small and medium vessels was assessed by sidestream dark-field imaging. Microcirculatory variables and data from right heart catheterization were obtained at baseline and 24 hours after randomization. Baseline and demographic data were compared by means of Mann-Whitney rank sum test or chi-square test, as appropriate. Microvascular and hemodynamic variables were analyzed using the Mann-Whitney rank sum test. Results Microcirculatory flow indices of small and medium vessels increased over time and were significantly higher in the levosimendan group as compared to the control group (24 hrs: MFIm 3.0 (3.0; 3.0) vs. 2.9 (2.8; 3.0); P .02; MFIs 2.9 (2.9; 3.0) vs. 2.7 (2.3; 2.8); P < .001). The relative increase of perfused vessel density vs. baseline was significantly higher in the levosimendan group than in the control group (dMFIm 10 (3; 23)% vs. 0 (-1; 9)%; P .007; dMFIs 47 (26; 83)% vs. 10 (-3; 27); P < .001). In addition, the heterogeneity index decreased only in the levosimendan group (dHI -93 (-100; -84)% vs. 0 (-78; 57)%; P < .001). There was no statistically significant correlation between systemic and microcirculatory flow variables within each group (each P > .05). Conclusions Compared to a standard dose of 5 µg·kg-1·min-1 of dobutamine, levosimendan at 0.2 µg·kg-1·min-1 improved sublingual microcirculatory blood flow in patients with septic shock, as reflected by changes in microcirculatory flow indices of small and medium vessels. Trial registration NCT00800306.

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Introduction

Microvascular dysfunction plays a pivotal role in the pathophysiology of septic shock and may occur even in the presence of normal systemic oxygen supply and mean arterial pressure [1]. In this regard, several vasoactive agents, including inotropes, vasodilators, and inodilators, have been investigated in the attempt to preserve or improve microcirculatory blood flow in patients with severe sepsis or septic shock [1-5].

In recent years, much attention has been paid to the use of the calcium sensitizer levosimendan in the treatment of septic myocardial dysfunction [6-10]. Levosimendan increases myocardial contractility while simultaneously exerting vasodilatory properties via activation of ATP-dependent potassium channels (KATP) [11]. In addition, levosimendan exerts anti-ischemic, anti-inflammatory, and anti-apoptotic properties, thereby affecting important pathways in the pathophysiology of septic shock [12-14]. It has been speculated that, owing to these beneficial effects, levosimendan may positively affect myocardial performance and regional hemodynamics, thereby improving microcirculatory perfusion [6-10,12,15,16].

The objective of the present randomized controlled, double-blinded clinical study was, therefore, to elucidate the effects of levosimendan on systemic and microvascular hemodynamics. On this basis, we aimed at rejecting the null hypothesis that there is no difference in sublingual microvascular blood flow - as measured by sidestream dark-field (SDF) imaging [17] - in patients with fluid-resuscitated septic shock treated with levosimendan as compared with an active comparator drug (that is, dobutamine).

Materials and methods

Patients

After approval by the local institutional ethics committee, the study was performed in an 18-bed multidisciplinary intensive care unit (ICU) at the Department of Anesthesiology and Intensive Care of the University of Rome 'La Sapienza'. Informed consent was obtained from the patients' next of kin. Enrolment of patients started in January 2008 and ended in April 2009. We enrolled patients who fulfilled the criteria of septic shock that required norepinephrine (NE) to maintain a mean arterial pressure (MAP) of at least 65 mm Hg despite appropriate volume resuscitation (pulmonary arterial occlusion pressure [PAOP] 12 to 18 mm Hg and central venous pressure [CVP] 8 to 12 mm Hg) [18]. Exclusion criteria of the study were age of less than 18 years, pregnancy, significant valvular heart disease, present or suspected acute coronary syndrome, and

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limitations to the use of inotropes (that is, ventricular outflow tract obstruction and mitral valve systolic anterior motion). All patients were sedated with sufentanil and midazolam and received mechanical ventilation using a volume-controlled mode.

Hemodynamics, global oxygen transport, and acid-base balance

Systemic hemodynamic monitoring of the patients included a pulmonary artery catheter (7.5-F; Edwards Lifesciences, Irvine, CA, USA) and a radial artery catheter. MAP, right atrial pressure, mean pulmonary arterial pressure, and PAOP were measured at end-expiration. Heart rate was analyzed from a continuous recording of electrocardiogram with ST segments monitored. Cardiac index (CI) was measured using the continuous thermodilution technique (Vigilance II; Edwards Lifesciences). Systemic vascular resistance index, pulmonary vascular resistance index, and left and right ventricular stroke work indices were calculated by means of standard equations. Arterial and mixed-venous blood samples were withdrawn to determine oxygen tensions and saturations as well as carbon dioxide tensions, standard bicarbonate, base excess, pH, and lactate concentrations. SvO2 was measured

discontinuously by intermittent mixed-venous blood gas analyses (Gem 4000 Premier; Instrumentation Laboratory Company, Bedford, MA, USA). Systemic oxygen delivery index (DO2I), oxygen consumption

index, and oxygen extraction ratio were calculated by means of standard formulae.

Microvascular network

Microvascular blood flow was visualized by means of an SDF imaging device (MicroScan®; MicroVision Medical, Amsterdam, The Netherlands) with a 5× magnification lens [17]. The optical probe was applied to the sublingual mucosa after gentle removal of saliva with a gauze swab. Three discrete fields were captured with precaution to minimize motion artifacts. Individual sequences of approximately 15 seconds were analyzed off-line with the aid of dedicated software (Automated Vascular Analysis 3.0; Academic Medical Center, University of Amsterdam, The Netherlands) in a randomized fashion by a single investigator who was unaware of the study protocol. Vessel density was automatically calculated from the software as the total vessel lengths of the small, medium, and large vessels, divided by the total area of the image [17]. The 'De Backer score' was calculated as described previously [17] and is based on the principle that density of the vessels is proportional to the number of vessels crossing arbitrary lines. In this score, three equidistant horizontal lines and three equidistant vertical lines are drawn on the screen, and then the De Backer score can be

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calculated as the number of small, medium, and large vessels crossing the lines, divided by the total length of the lines [17]. Vessel density was also calculated as the total vessel lengths divided by the total area of the image [17]. Both indices were automatically calculated by means of dedicated software (Automated Vascular Analysis 3.0). Perfusion was then categorized by eye as present (normal continuous flow for at least 15 seconds), sluggish (decreased but continuous flow for at least 15 seconds), absent (no flow for at least 50% of the time), or intermittent (no flow for less than 50% of the time) [17]. The proportion of perfused vessels (PPV) was calculated as follows: 100 × [(total number of vessels - [no flow + intermittent flow])/total number of vessels]. Perfused vessel density (PVD) was calculated by multiplying vessel density by the proportion of perfused vessels [17]. Microvascular flow index [17] was used to quantify microvascular blood flow. In this score, flow is characterized as absent (0), intermittent (1), sluggish (2), or normal (3) [17]. Since our investigation was focused on small and medium vessels, calculations were performed separately for vessels with diameters of smaller than 20 µm (MFIs) and of larger than 20 µm but smaller than 50 µm (MFIm). Vessel size was determined with the aid of a micrometer scale. For each patient, values obtained from the three mucosa fields were averaged [17]. To assess flow heterogeneity between the different areas investigated, we used the heterogeneity index. The latter was calculated as the highest site flow velocity minus the lowest site flow velocity, divided by the mean flow velocity of all sublingual sites [17]. Percentage changes from baseline for all variables were determined as dVariable 100 × [(Value24 hours /ValueBL) - 1] [19].

Study design

Patients were enrolled within the first 24 hours from the onset of septic shock after having established normovolemia (PAOP 12 to 18 mm Hg and CVP 8 to 12 mm Hg) [18] and an MAP of at least 65 mm Hg using norepinephrine, if needed. Packed red blood cells were transfused when hemoglobin concentrations decreased to below 7 g/dL [18] or if the patient exhibited clinical signs of inadequate systemic oxygen supply. Forty patients were randomly allocated to the treatment with either (a) intravenous levosimendan 0.2 µg/kg per minute (without a loading bolus dose) for 24 hours or (b) intravenous dobutamine 5 µg/kg per minute as active comparator ( control) in a double-blinded manner (each n 20). The consort diagram is presented in Figure 1. Systemic and pulmonary hemodynamic variables, microcirculatory flow variables, blood gases, and norepinephrine requirements were determined at baseline and 24 hours after randomization. After the 24-hour intervention period, study

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drugs were discontinued and open-label dobutamine was started if judged as appropriate by the attending ICU physician.

Figure 1. Consort diagram. MAP, mean arterial pressure.


An a priori analysis of sample size revealed that at least 17 patients per group were required to demonstrate a minimum difference of 20% between groups in the primary endpoint with an estimated standard deviation of 20%, a test power of 80%, and an alpha error of 5%. Data are expressed as median (25th; 75th percentile) if not otherwise specified. Sigma Stat 3.10 software (Systat Software, Inc., Chicago, IL, USA) was used for statistical analysis. Baseline and demographic data were compared with a Mann-Whitney rank sum test or chi-square test, as appropriate. Microvascular and hemodynamic variables were analyzed with a Mann-Whitney rank sum test. The correlation between systemic and microcirculatory flow variables within each group was tested by means of Spearman rank order correlation. A P value of less than 0.05 was considered statistically significant for all tests.

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Results

Demographic data

Baseline characteristics, including age, gender, body weight, and origin, as well as onset time of septic shock, Simplified Acute Physiology Score II (SAPS II), and mortality were not different among groups (Table 1). In addition, there was no significant difference between groups at baseline in any of the investigated hemodynamic or microcirculatory variables.

Hemodynamic and oxygen transport variables

Systemic and pulmonary hemodynamic variables were comparable between groups. SvO2 and arterial pH tended to be higher whereas NE

requirements tended to be lower in the levosimendan group (Table 2). However, these differences did not reach statistical significance.

Concomitant therapies

Activated protein C was administered in five patients in the control group and in four patients in the levosimendan group. Three patients in each group required continuous renal replacement therapy during the study period. These treatments were equally distributed among groups (each P value of greater than 0.05).

Microcirculatory variables

Microcirculatory data are presented in Figures 2, 3 and 4. MFIm and MFIs were significantly higher (MFIm 3.0 [3.0; 3.0] versus 2.9 [2.8; 3.0]; P 0.02; MFIs 2.9 [2.9; 3.0] versus 2.7 [2.3; 2.8]; P < 0.001) and heterogenity index was lower after 24 hours of treatment with levosimendan versus dobutamine (heterogenity index 0.63 [0.44; 0.87] versus 0.26 [0.12; 0.51]; P 0.001). Since baseline data varied (non-significantly) among groups, relative changes from baseline were calculated and compared between groups. Relative increases from baseline of MFIs, MFIm, PPV, and PVD (that is, dMFIs, dMFIm, dPPV, and dPVD) were significantly higher in the levosimendan group (Figure 3 and 4). In addition, the heterogeneity index decreased relative to baseline only in the levosimendan group. Correlation analyses (that is, DO2I and CI versus MFIm and MFIs in

each group)revealed no statistically significant results. (each P > 0.05; Figure 5).

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Table 1

Characteristics of the study patients

Levosimendan (n 20) Control (n 20) P value

Age, years 68 (55; 74) 66 (54; 78) 0.98

Gender, male 70% 65% 1.00

SAPS II 55 (45; 61) 57 (46; 64) 0.90

Cause of septic shock

Endocarditis (n 1) Peritonitis (n 8)

Pneumonia (n 11)

Peritonitis (n 4) Pneumonia (n

16) 0.10

Onset of septic shock, hoursa

20 (18; 24) 18 (13; 22) 0.13

ICU mortality 13/20 15/20 0.50

ICU length of stay, days

14 (11; 19) 27 (9; 47) 0.32

Data are presented as median (25th; 75th percentile). Control, dobutamine 5 µg/kg per minute. aOnset of septic shock defines the time elapsed from the onset of septic shock until administration of study drug. ICU, intensivecare unit; SAPS II, Simplified Acute Physiology Score II.

Table 2. Hemodynamic and metabolic data of the study patients

Levosimendan (n 20) Control (n 20) P value

CI, L/min per m2 BL 3.6 (2.9; 4.3) 3.9 (2.9; 4.6) 0.70

24 hours 4.1 (3.5; 5.1)a 4.1 (3.3; 5.0) 0.66

HR, beats per min BL 96 (87; 107) 95 (90; 106) 0.75

24 hours 94 (86; 104) 98 (87; 114) 0.36

MAP, mm Hg BL 70 (67; 72) 72 (70; 74) 0.11

24 hours 72 (69; 73) 73 (70; 75) 0.13

PAOP, mm Hg BL 18 (15; 18) 19 (15; 21) 0.25

24 hours 16 (16; 18) 17 (14; 21) 0.52

RAP, mm Hg BL 14 (11; 16) 14 (11; 16) 0.81

24 hours 13 (11; 14) 14 (10; 18) 0.27

LVSWI, g·m/m2 BL 26 (21; 32) 30 (25; 36) 0.13

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24 hours 34 (29; 38)a 32 (29; 38) 0.56

DO2I, mL/min per m2 BL 431 (363; 531) 492 (393; 550) 0.27

24 hours 512 (438; 612) 519 (436; 593) 0.93

VO2I, mL/min per m2 BL 111 (93; 151) 126 (112; 153) 0.18

24 hours 127 (107; 144) 149 (110; 178) 0.24

O2-ER, percentage BL 28 (24; 32) 29 (22; 34) 0.99

24 hours 25 (20; 27)a 27 (21; 36) 0.17

SaO2, percentage BL 98 (96; 99) 98 (95; 99) 0.99

24 hours 99 (99; 99)a 99 (94; 99) 0.02

PaCO2, mm Hg BL 45 (41; 50) 41 (37; 51) 0.35

24 hours 41 (37; 44) 41 (36; 49) 0.42

SvO2, percentage BL 72 (66; 75) 70 (66; 78) 0.95

24 hours 77 (74; 81)a 71 (62; 78) 0.06

Hba, g/dL BL 8.6 (8.0; 8.9) 9.0 (8.0; 9.6) 0.96

24 hours 8.5 (8.0; 8.9) 8.8 (8.0; 9.3)

0.42

pHa, -log10c(H+) BL 7.29 (7.25; 7.34) 7.28 (7.25;7.38) 0.87

24 hours 7.38 (7.29; 7.40)a 7.32 (7.23; 7.37) 0.06

aBE, mmol/L BL -4.9 (-6.9; -2.5) -3.8 (-9.0; 0.0) 0.72

24 hours -2.9 (-5.0; -0.6) -3.8 (-8.9; 1.8) 0.74

Lactate, mmol/L BL 2.3 (1.3; 2.9) 1.9 (1.3; 2.9) 0.72

24 hours 1.9 (1.2; 2.5) 1.6 (1.3; 3.6) 0.61

Fluid input, mL/24h BL NA NA NA

24 hours 5,700 (4,700; 6,050)

4,850 (4,150; 5,200)

0.01

NE dosage, µg/kg per min

BL 0.4 (0.2; 0.9) 0.4 (0.3; 0.7) 0.72

24 hours 0.3 (0.1; 0.9) 0.4 (0.3; 1.1) 0.10

Data are presented as median (25th; 75th percentile). Control, dobutamine 5 µg/kg per minute. aP < 0.05 versus baseline (BL) within groups. aBE, arterial base excess; CI, cardiac index; DO2I, systemic oxygen delivery index; Hba,

arterial hemoglobin concentration; HR, heart rate; LVSWI, left ventricular stroke work index; MAP, mean arterial pressure; NA, not applicable; NE, norepinephrine; O2-ER, oxygen extraction ratio; PaCO2, arterial partial pressure of

carbon dioxide; PAOP, pulmonary arterial occlusion pressure; pHa, arterial potentia hydrogenii; RAP, right atrial

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pressure; SaO2, arterial oxygen saturation; SvO2, mixed-venous oxygen saturation; VO2I, oxygen consumption

index.

Discussion

The major finding of the present study is that levosimendan improved microvascular perfusion in patients with septic shock, as indicated by increases in MFIs, MFIm, and PVD. Notably, this improvement was related to enhanced convection rather than changes in diffusion distance.

The role of levosimendan in severe sepsis or septic shock is still not fully elucidated and remains controversial [12,14-16,20-26]. However, there is increasing evidence that under normovolemic conditions, continuous infusion with levosimendan attenuates septic myocardial dysfunction [6-10,27,28] without aggravating hemodynamic instability. In harmony with previous reports [6-10,27,28], levosimendan did not influence arterial blood pressure or NE requirements in the present study. Furthermore, we noticed neither an increase in heart rate nor new onsets of

tachyarrhythmias following levosimendan infusion in our fluid-resuscitated septic shock patients.

These findings strengthen the assumption that under normovolemic conditions, the decrease in vascular resistance (owing to the opening of KATP channels) following levosimendan infusion may be compensated by a simultaneous increase in myocardial contractility.

The hypothesis that constituted the basis of our study was that (besides the effects on myocardial contractility) levosimendan - by its vasodilatory effects - improves microcirculatory blood flow by increasing the driving pressure of blood flow at the entrance of the microcirculation [3].

In fact, we noticed that levosimendan improved sublingual microcirculation, as indicated by significant increases in MFIs, MFIm, dMFIs, and dMFIm. In addition, we observed an increase in dPVD following levosimendan infusion, further indicating an improvement of the microcirculation. We focused our investigation on the effects of the study drug on MFI of the small and medium vessels since alterations in such microvessels are typically associated with organ dysfunction and - if persisting - poor outcome [1-5].

Whereas the increases in MFI suggest that levosimendan ameliorated blood flow within the perfused vessels, the increase in PPV with a concomitant decrease in heterogeneity index indicates a recruitment of non-perfused vessels and hence a reduction of the diffusion distance

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between capillaries. In light of these findings, it is most likely that levosimendan enhanced both convection and diffusion, thereby improving oxygen delivery at the level of the microcirculation.

Figure 2.

Absolute changes in microcirculatory variables. BL, baseline; DBS, De

Backer score; HI, heterogenity index; MFIm, microvascular flow index of

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medium vessels (∅ 20 to 50 µm); MFIs, microvascular flow index of small

vessels (∅ <20 µm); PVD, perfused vessel density; VD, vessel density.

Figure 3.

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Absolute and relative changes in microcirculatory variables. BL,

baseline; dPPV, relative changes in proportion of perfused vessels; PPV,

proportion of perfused vessels.

Figure 4.

Relative changes in microcirculatory variables. Data represent relative

changes from baseline at 24 hours. dDBS, relative changes in De Backer

score; dHI, relative changes in heterogeneity index; dMFIm, relative

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changes in microvascular flow index of medium vessels (∅ 20 to 50 µm);

dMFIs, relative changes in microvascular flow index of small vessels (∅

<20 µm); dPVD, relative changes in perfused vessel density; dVD,

relative changes in vessel density.

Figure 5.

Correlation analyses of systemic and microcirculatory flow variables.

Data represent percentage changes in cardiac index (dCI) and systemic

oxygen delivery index (dDO2I) plotted against percentage changes in

microvascular flow indices of medium (dMFIm) and small (dMFIs)

vessels within each group. Solid and dashed lines represent regression

lines for levosimendan and control, respectively. CI, cardiac index;

DO2I, systemic oxygen delivery index; MFIm, microvascular flow index

of medium vessels (∅ 20 to 50 µm); MFIs, microvascular flow index of

small vessels (∅ <20 µm).

Although the increases in SvO2 and pH noticed in the levosimendan

group may further indicate an improvement in microcirculatory blood flow, it has to be considered that an improvement in pulmonary function (increase in PaO2 [arterial oxygen partial pressure] and SaO2 [arterial

oxygen saturation] with a concomitant decrease in PaCO2 [arterial partial

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pressure of carbon dioxide]) following levosimendan administration might have contributed to these changes. This assumption is supported by recent experimental and clinical studies showing that levosimendan in fact improves pulmonary function and gas exchange [8,12,14,20,25,26]. However, it may well be that levosimendan (secondary to its vasodilatatory properties) has promoted microvascular shunting and thereby increased venous oxygen saturation.

Our results are in line with those of an experimental study by Schwarte and colleagues [29], who reported that levosimendan selectively increases gastric microvascular mucosal oxygenation in dogs. Whereas a previous experimental study [30] showed that levosimendan improved microvascular oxygenation in experimental sepsis, our study demonstrates for the first time that levosimendan selectively increases microvascular blood flow in the clinical setting. However, the present study design does not allow us to exclude whether non-hemodynamic effects of levosimendan, such as the ability to decrease cytokine synthesis, plasma levels of endothelin-1, ICAM-1 (intercellular adhesion molecule-1), and VCAM-1 (vascular cell adhesion molecule-1) [12,13,26], might have contributed to the improvement of microcirculation.

Notably, the lack of modifications in the proportion of perfused vessels observed in the control group (in which the patients were treated with dobutamine as an active comparator at a dose of 5 µg/kg per minute) varies from the study of De Backer and colleagues [2], who reported that the same dose of dobutamine increased microvascular density and the proportion of perfused vessels, a finding that clearly indicated an improved microcirculation in a series of septic shock patients. However, despite the use of an equivalent dobutamine dose [2], there is a marked difference in the study designs in terms of time frame. In this regard, the previously reported short-term response to dobutamine after 2 hours [2] was outside the scope of our investigation. A likely explanation might be related to the fact that we performed microcirculatory evaluation at the end of 24 hours of drug infusion in progressed septic shock. It is well recognized that, owing to adrenergic receptor and signaling abnormalities, the efficacy of catecholamines often gradually decreases over time [31]. This may account for the attenuated hemodynamic effects of 5 µg/kg per minute dobutamine infusion in patients with severe septic shock [7,32,33] in comparison with patients with less severe sepsis [34]. On this basis, it is conceivable that microvessels may reach a near maximal vasodilation in the early phase of dobutamine administration lasting for a brief period [2,32,35], whereas after 24 hours, the effects of 5 µg/kg per minute of dobutamine on the microcirculation are attenuated.

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In this light, our findings support the hypothesis formulated by De Backer and colleagues [2] that stronger vasodilatory compounds, such as levosimendan, may be more effective than dobutamine for improving microcirculatory blood flow. However, these postulated advantages of levosimendan remain to be further elucidated in larger clinical trials.

The present study has some limitations that we would like to acknowledge. First, we administered a fixed dose of 5 µg/kg per minute of dobutamine and cannot exclude the possibility that a higher dose would have resulted in different findings. However, it is important to note that our intention was not to perform a direct comparison between dobutamine and levosimendan but to use the selected dobutamine dose as an 'active comparator' to facilitate blinding of the study drugs. Indeed, randomization of levosimendan versus placebo would have unmasked group allocation because of the strong hemodynamic effects of levosimendan. Second, in the present study, the improvement in microvascular perfusion was independent from changes in CI. However, it is also possible that these variables might correlate in a way that is more complex than the linear correlation of percentage changes in CI and oxygen delivery. Therefore, a possible correlation should be clarified in future larger studies. Third, owing to the lack of investigation of specific variables, we cannot conclude whether anti-ischemic and anti-inflammatory effects, as well as effects at the cellular level [13], have contributed to the improved microcirculatory blood flow with levosimendan. In addition, we investigated the changes in microvascular perfusion of the sublingual mucosa which might not be representative of alterations in other tissues [1]. Furthermore, owing to the pharmacokinetic characteristics of the study drug, the present study protocol required a relatively long time interval (24 hours of drug infusion) that does not allow the exclusion of a direct time-dependent effect unrelated to the specific agent. Finally, we have chosen changes in MFIs as the primary endpoint of this study. Since we investigated only a small number of septic shock patients treated over a relative brief period, the risk of positive results in a study with numerous secondary variables has to be taken into account. Thus, caution should be exercised in interpreting the results of the secondary outcome variables.

Conclusions

This is the first prospective, randomized clinical study investigating the effects of levosimendan on sublingual microcirculation in patients with septic shock. Our results demonstrate that levosimendan at 0.2 µg/kg per minute (when compared with a standard dose of 5 µg/kg per minute of dobutamine) improves sublingual microcirculatory blood flow in volume-

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resuscitated septic shock patients and that this effect was not correlated with changes in systemic flow variables.

Abbreviations

CI: cardiac index; CVP: central venous pressure; dMFIm: relative increases of microvascular flow index of medium vessels; dMFIs: relative increases of microvascular flow index of small vessels; DO2I:

systemic oxygen delivery index; dPVD: relative increase in perfused vessel density; ICU: intensive care unit; KATP: ATP-dependent potassium; MAP: mean arterial pressure; MFIm: microvascular flow index of medium vessels; MFIs: microvascular flow index of small vessels; NE: norepinephrine; PAOP: pulmonary arterial occlusion pressure; PPV: proportion of perfused vessels; PVD: perfused vessel density; SDF: sidestream dark-field; SvO2: mixed-venous oxygen

saturation.

References

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8. Morelli A, Teboul JL, Maggiore SM, Vieillard-Baron A, Rocco M, Conti G, De Gaetano A, Picchini U, Orecchioni A, Carbone I, Tritapepe L, Pietropaoli P, Westphal M: Effects of levosimendan on right ventricular afterload in patients with acute respiratory distress syndrome: a pilot study. Crit Care Med 2006, 34:2287-2293.

9. Powell BP, De Keulenaer BL: Levosimendan in septic shock: a case series. Br J Anaesth 2007, 99:447-448.

10. Pinto BB, Rheberg S, Ertmer C, Westphal M: Role of levosimendan in sepsis and septic shock. Curr Opin Anaesthesiol 2008, 21:168-177.

11. Toller WG, Stranz C: Levosimendan, a new inotropic and vasodilator agent. Anesthesiology 2006, 104:556-569.

12. Scheiermann P, Ahluwalia D, Hoegl S, Dolfen A, Revermann M, Zwissler B, Muhl H, Boost KA, Hofstetter C: Effects of intravenous and inhaled levosimendan in severe rodent sepsis. Intensive Care Med 2009, 35:1412-1419.

13. Antoniades C, Tousoulis D, Koumallos N, Marinou K, Stefanadis C: Levosimendan: beyond its simple inotropic effect in heart failure. Pharmacol Ther 2007, 114:184-197.

14. Rehberg S, Ertmer C, Vincent JL, Spiegel HU, Köhler G, Erren M, Lange M, Morelli A, Seisel J, Su F, Van Aken H, Traber DL, Westphal M: Effects of combined arginine vasopressin and levosimendan on organ function in ovine septic shock. Crit Care Med 2010, 38:2016-2023.

15. Dubin A, Murias G, Sottile JP, Pozo MO, Barán M, Edul VS, Canales HS, Etcheverry G, Maskin B, Estenssoro E: Effects of levosimendan and dobutamine in experimental acute endotoxemia: a preliminary controlled study. Intensive Care Med 2007, 33:485-494.

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20. Erbüyün K, Vatansever S, Tok D, Ok G, Türköz E, Aydede H, Erhan Y, Tekin I: Effects of levosimendan and dobutamine on experimental acute lung injury in rats. Acta Histochem 2009, 111:404-414.

21. Barraud D, Faivre V, Damy T, Welschbillig S, Gayat E, Heymes C, Payen D, Shah AM, Mebazaa A: Levosimendan restores both systolic and diastolic cardiac performance in lipopolysaccharide-treated rabbits: comparison with dobutamine and milrinone. Crit Care Med 2007, 35:1376-1382.

22. Cunha-Goncalves D, Perez-de-Sa V, Dahm P, Grins E, Thörne J, Blomquist S: Cardiovascular effects of levosimendan in the early stages of endotoxemia. Shock 2007, 28:71-77.

23. Dubin A, Maskin B, Murias G, Pozo MO, Sottile JP, Barán M, Edul VS, Canales HS, Estenssoro E: Effects of levosimendan in normodynamic endotoxaemia: a controlled experimental study.

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Bonnin P, Payen D, Mebazaa A: Cardiac and renal effects of levosimendan, arginine vasopressin, and norepinephrine in lipopolysaccharide-treated rabbits. Anesthesiology 2005, 103:514-521.

25. Oldner A, Konrad D, Weitzberg E, Rudehill A, Rossi P, Wanecek M: Effect of levosimendan a novel inotropic calcium-sensitizing drug, in experimental septic shock. Crit Care Med 2001, 29:2185-2193.

26. Scheiermann P, Ahluwalia D, Hoegl S, Dolfen A, Revermann M, Zwissler B, Muhl H, Boost KA, Hofstetter C: Inhaled levosimendan reduces mortality and release of proinflammatory mediators in a rat model of experimental ventilator-induced lung injury. Crit Care Med 2008, 36:1979-1981.

27. Ramaswamykanive H, Bihari D, Solano TR: Myocardial depression associated with pneumococcal septic shock reversed by levosimendan. Anaesth Intensive Care 2007, 35:409-413.

28. Matejovic M, Krouzecky A, Radej J, Novak I: Successful reversal of resistent hypodynamic septic shock with levosimendan. Acta Anaesthesiol Scand 2005, 49:127-128.

29. Schwarte LA, Picker O, Bornstein SR, Fournell A, Scheeren TW: Levosimendan is superior to milrinone and dobutamine in selectively increasing microvascular gastric mucosal oxygenation in dogs. Crit Care Med 2005, 33:135-142.

30. Fries M, Ince C, Rossaint R, Bleilevens C, Bickenbach J, Rex S, Mik EG: Levosimendan but not norepinephrine improves microvascular oxygenation during experimental septic shock. Crit Care Med 2008, 36:1886-1891.

31. Landry DW, Oliver JA: The pathogenesis of vasodilatory shock. N Engl J Med 2001, 345:588-595.

32. Duranteau J, Sitbon P, Teboul JL, Vicaut E, Anguel N, Richard C, Samii K: Effects of epinephrine, norepinephrine or combination of norepinephrine and dobutamine on gastric mucosa in septic shock. Crit Care Med 1999, 27:893-900.

33. Levy B, Nace L, Bollaert PE, Dousset B, Mallie JP, Larcan A: Comparison of systemic and regional effects of dobutamine and dopexamine in norepinephrine-treated septic shock. Intensive Care Med 1999, 25:942-948.

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34. Creteur J, De Backer D, Vincent JL: A dobutamine test can disclose hepatosplanchnic hypoperfusion in septic patients. Am J Respir Crit Care Med 1999, 160:839-845.

35. Lebuffe G, Levy B, Nevière R, Chagnon JL, Perrigault PF, Duranteau J, Edouard A, Teboul JL, Vallet B: Dobutamine and gastric-to-arterial carbon dioxide gap in severe sepsis without shock. Intensive Care Med 2002, 28:265-271.

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Chapter 8

Effects of fresh leukoreduced vs. non-

leukoreduced red blood cells transfusions

on microcirculation and tissue

oxygenation in septic patients: a pilot

study.

Abele Donati1,4, Elisa Damiani1, Michele Maria Luchetti2, Roberta

Domizi1, Claudia Scorcella1, Andrea Carsetti1, Vincenzo Gabbanelli1, Paola Carletti1, Rosella Bencivenga3, Hans Vink4, Erica Adrario1,

Armando Gabrielli2, Paolo Pelaia1, Can Ince5

1 Anaesthesia and Intensive Care Unit, Department of Biosciences and Public Health, Università Politecnica delle Marche, Ancona, Italy. 2 Department of Clinical and Molecular Sciences, Clinica Medica,

Università Politecnica delle Marche, Ancona, Italy. 3 Immunohematology and Transfusional Medicine, AOU Ospedali

Riuniti, Ancona, Italy. 4 Department of Physiology, Cardiovascular Research Institute

Maastricht, Maastricht University, Maastricht, The Netherlands

5 Department of Translational Physiology, Academic Medical Center, Amsterdam, The Netherlands.

Published in: Critical Care 2014, 18:R33 (doi:10.1186/cc13730)

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ABSTRACT

INTRODUCTION: Microvascular alterations impair tissue oxygenation during sepsis. A red blood cell (RBC) transfusion increases oxygen (O2)-delivery but rarely improves tissue O2

uptake in septic patients. Possible causes include RBC alterations due to prolonged storage or residual leukocyte-derived inflammatory mediators. The aim of this study was to compare the effects of two types of transfused-RBCs on microcirculation in septic patients. METHODS: In a prospective randomized trial, 20 septic patients were divided into two separate groups and received either non-leukodepleted (n = 10) or leukodepleted (n = 10) RBC transfusions. Microvascular density and perfusion were assessed with sidestream dark-field (SDF) imaging sublingually, before and 1 hour after transfusions. Thenar tissue O2-saturation (StO2) and tissue haemoglobin index (THI) were

determined with near-infrared spectroscopy (NIRS), and a vascular occlusion test was performed. The microcirculatory perfused boundary region was assessed in SDF images as an index of glycocalyx damage and glycocalyx compounds (syndecan-1, hyaluronan, heparan sulfate) were measured in the serum. RESULTS: No differences were observed in microvascular parameters at baseline and after transfusion between the groups, except for the proportion of perfused vessels (PPV) and blood flow velocity, which were higher after transfusion in the leukodepleted group. Microvascular flow index in small vessels (MFI) and blood flow velocity exhibited different responses to transfusion between the two groups (P = 0.03 and P = 0.04, respectively), with a positive effect of leukodepleted RBCs. When looking at within-group changes, microcirculatory improvement was only observed in patients that received leukodepleted RBC transfusion as suggested by the increase in De Backer score (P = 0.02), perfused vessel density (P = 0.04), PPV (P = 0.01) and MFI (P = 0.04). Blood flow velocity decreased in the non-leukodepleted group (P = 0.03). THI and StO2-upslope increased in both groups. StO2 and StO2-

downslope increased in patients who received non-leukodepleted RBC transfusions. Syndecan-1 increased after the transfusion of non-leukodepleted RBCs (P = 0.03). CONCLUSIONS: This study does not show a clear superiority of leukodepleted over non-leukodepleted RBC transfusions on microvascular perfusion in septic patients, although it suggests a more favourable effect of leukodepleted RBCs on microcirculatory convective flow. Further studies are needed to confirm these findings.

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TRIAL REGISTRATION: NCT01584999 Keywords: blood transfusion, leukoreduction, microcirculation, sepsis, glycocalyx, sidestream dark field imaging, near infrared spectroscopy.

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INTRODUCTION

Anaemia is a common problem in Intensive Care Units (ICU) [1]. Red blood cell (RBC) transfusion aimed at increasing O2 delivery is generally

considered a life-saving treatment [2]. Nonetheless, a restrictive transfusion strategy may result in equivalent or improved clinical outcomes in comparison to liberal transfusion strategies [3]. This raises concern whether transfusion practice is beneficial in general.

Altered properties of stored blood products might explain this paradoxical effect. Biochemical and haemorrheological alterations of packed RBC units due to prolonged storage (depletion of ATP and 2,3-diphosphoglycerate, membrane phospholipid vesiculation and loss, protein and lipid peroxidation, loss of deformability) [4-6] may affect their O2 delivery capacity [7-9]. Residual leukocytes in RBC-units may

also compromise the efficacy of blood transfusion by producing cytokines such as Interleukin (IL)-1b, IL6, IL8, TNFα, which may interfere with immune function [10], alter circulating lymphocytes and enhance neutrophil activity in recipients [11,12]. These effects, collectively referred to as “transfusion-related immunomodulation”, may be responsible for higher incidences of infections among transfused patients [13]. Moreover, cytokines may contribute to RBC membrane alterations during storage [14] and impair RBC rheology. These effects should be in theory prevented by leukocyte reduction, however, clinical data remain controversial [15].

The effects of blood transfusion in septic patients are poorly understood, and in particular how it influences tissue microcirculation. Sepsis-induced microvascular dysfunction [16], endothelial and glycocalyx damage [17], pathological shunting and heterogeneous perfusion [18] may hamper blood transfusion-based restoration of tissue RBC delivery and oxygenation [19]. Moreover, altered RBCs and/or inflammatory mediators in blood bags may further compromise microvascular perfusion and tissue O2-uptake. Only a few studies have previously

investigated the response of the microcirculation to blood transfusion in septic populations [20-22] and found no global effect on microvascular perfusion; unfortunately, however, the characteristics of the transfused RBCs in these studies were not examined. The primary aim of the present study was to compare the effects of non-leukodepleted or leukodepleted RBC transfusions on microvascular flow in septic patients. In addition, it was determined whether transfusion of either non-leukodepleted or leukodepleted packed RBC units could increase microcirculatory density and reactivity to improve tissue oxygenation during sepsis.

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MATERIALS AND METHODS

The study protocol was approved by the local medical ethics committee of the Azienda Ospedaliero Universitaria (AOU) Ospedali Riuniti of Ancona in Italy (NCT01584999, www.clinicaltrials.gov). Written informed consent was obtained from the enrolled patients or their next of kin.

Patients

Between February 2011 and 2012, adult patients admitted to the 12-bed Intensive Care Unit of the AOU Ospedali Riuniti of Ancona with sepsis, severe sepsis, or septic shock as diagnosed according to standard criteria [23] and requiring blood transfusion for Hb levels <8 g/dL or as indicated by the attending physician (in accordance with the local hospital guidelines) were eligible to participate in this prospective randomized study. Exclusion criteria for this study were: age <18 years, previous blood transfusions during ICU stay, previous history of coagulation disorders, cardiogenic or haemorrhagic shock, pregnancy, factors impeding the sublingual microcirculation evaluation (oral surgery, maxillofacial trauma). All patients were monitored with an arterial catheter. Sedation and analgesia was provided according to individual needs, as well as the type of fluids infused (crystalloids and colloids) and adrenergic agents (norepinephrine, dobutamine). The goal was to maintain a mean blood pressure of 65 mmHg as recommended by the international guidelines of the Surviving Sepsis Campaign (2008) [24]. Fluid infusion and furosemide treatment were titrated according to individual needs, in order to maintain adequate urine output (>0.5 mL/kg/h) [24].

Interventions

The original study protocol included 3 separate groups of septic patients receiving transfusion of fresh (<10 days storage) non-leukodepleted, fresh leukodepleted or old (>15 days storage) non-leukodepleted RBC units, respectively. The analysis presented herein was focused on the role of leukocyte reduction; therefore, only the data from the first two groups (hereafter referred to as non-leukodepleted and leukodepleted groups) are reported. A parallel analysis focused on the role of prolonged storage will be reported separately. Blood product randomisation was performed through sealed envelopes by a physician at the blood bank, who blindly provided the blood bags to the ICU; neither the attending physician nor the investigators nor the patients were aware of the type of RBCs transfused. Post-storage leukoreduction was performed by a physician at the blood bank using the filter Sepacell RZ-200 (Fenwal, Inc., Lake

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Zurich, IL, USA) within a maximum of 5 days after donor blood withdrawal.

Basic haemodynamic and blood gas parameters

All measurements were performed immediately before and 1 hour after RBC transfusions. These time points were chosen on the basis of those reported in previous studies [20-22]. We recorded temperature (T), heart rate (HR) and mean arterial pressure (MAP). Arterial blood samples were withdrawn in order to assess haemoglobin (Hb) level, whole blood cell counts, blood gases (pH, paO2, paCO2, SaO2, paO2/FiO2, HCO3-, base

excess [BE]), lactate (Lac), creatinine and glucose levels. Arterial blood samples were immediately centrifuged and plasma and serum were stored at −70°C for subsequent analysis. For each participant, the Simplified Acute Physiology Score (SAPS) II was obtained at admission and the Sequential Organ Failure Assessment (SOFA) score [25] on the study day.

Microcirculation measurements with sidestream dark-field (SDF)

imaging

Sublingual microcirculatory density and flow were monitored using sidestream dark-field (SDF) imaging (Microscan, Microvision Medical, Amsterdam, the Netherlands); details on the SDF imaging technique have been described elsewhere [26]. Briefly, the Microscan is a hand-held video microscope system that epi-illuminates a tissue of interest with stroboscopic green (530 nm) light emitting diodes (LEDs). Hb absorbs the 530 nm wavelength light, which in turn is captured via the imaging probe’s light guide and a charge coupled device camera. Clear images of flowing RBCs are depicted as dark moving globules in the lumen of blood vessels against a white/grayish background. After the removal of saliva and other secretions with a gauze, the SDF probe, covered by a sterile disposable cap, was gently applied on the sublingual mucosa of the floor of the mouth at the base of the tongue. Videos from 5 different sites (at least 10 sec/site) were recorded at both time points with adequate focus and contrast and every effort was made to avoid movement and pressure artefacts. Poor-quality images were discarded and 3 images for each time point were selected and analyzed using a computer software package (Automated Vascular Analysis Software, Microvision Medical BV, Amsterdam, The Netherlands). According to the consensus report on the performance and evaluation of microcirculation using SDF imaging [27], total vessel density (TVD) and perfused vessel density (PVD) were calculated for small vessels (diameter <20 µm). The De Backer score was calculated as described previously [27]. In brief, the SDF image was divided by three equidistant horizontal and three equidistant vertical

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lines; the De Backer Score was calculated as the number of the small (diameter <20 µm) and medium (diameter 20–100 µm) vessels crossing the lines divided by the total length of the lines. The proportion of perfused vessels (PPV) and the microvascular flow index (MFI), reflecting microcirculatory blood flow velocity, were analyzed semi-quantitatively in small vessels, as described elsewhere [28]. The flow heterogeneity index (HI) was also calculated as the highest MFI minus the lowest MFI divided by the mean MFI, providing an index of heterogeneous microcirculatory perfusion. Quantitative blood flow velocity was measured through the use of space-time diagrams [29]. Three lines were manually traced in the spacetime diagram and the average orientation was used to calculate the blood flow velocity [30].

Peripheral O2 and Hb measurements with near-infrared spectroscopy

(NIRS)

Near-infrared reflectance spectrophotometry (InSpectra™ Model 650; Hutchinson Technology Inc., Hutchinson, MN, USA) was used to measure peripheral tissue oxygen saturation (StO2) and tissue Hb index

(THI) [31,32] at baseline and during a vascular occlusion test (VOT). A 15 mm-sized probe was placed on the skin of the thenar eminence and a sphygmomanometer cuff was placed around the (upper) arm to occlude the brachial artery. After a 3-minute period of StO2 signal stabilization,

arterial inflow was arrested by inflation of the cuff to 50 mmHg above the systolic arterial pressure. The cuff was kept inflated until the StO2

decreased to 40% and then released [33]. StO2 was continuously

recorded during the reperfusion phase until stabilization [33]. The StO2

downslope (StO2down, %/minute) was calculated from the regression

line of the first minute of StO2 decay after occlusion, providing an index

of O2 consumption rate. The StO2 upslope (StO2up, %/minute) was

obtained from the regression line of StO2 increase in the reperfusion

phase. The area under the curve (AUC) of the hyperemic response was also calculated. StO2 upslope and the area under the curve (AUC) StO2

reflect microvascular reactivity [33]. All the parameters were calculated using a computer software package (Version 3.03 InSpectra Analysis Program; Hutchinson Technology Inc.).

Microvascular glycocalyx assessment

A series of 10 Microscan video fragments of at least 40 consecutive frames were automatically analysed using the GlycoCheck ICU software package (Maastricht University Medical Center, Maastricht, The

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Netherlands) in order to measure vascular lumen perfused boundary region (PBR). The PBR includes the dimension of the permeable part of the endothelial glycocalyx which allows the penetration of flowing RBCs. Erythrocytes usually have limited access into an intact glycocalyx, when this is compromised and starts losing its protective capacity, its permeability increases, allowing circulating cells to approximate the luminal endothelial membrane. As a result, the dimension of the erythrocyte PBR will increase [34]. This methodology has been extensively described elsewhere [35]. Briefly, measurement lines perpendicular to the vessel direction are arranged automatically every 10 µm along each visible vessel with a diameter <50 µm. Each line represents a measurement site, at each measurement site a total of 21 parallel (every 0.5 µm) intensity profiles is plotted and RBC column width (RBCW, full width half maximum) is determined at each line for all 40 consecutive frames in a movie, revealing a total of 840 RBC column width measurements at a measurement site (21 profiles × 40 frames). The associated (cumulative) distribution of the RBC column widths for these 840 measurements was used to determine median RBC column width (P50), as well as lower and upper percentiles of the RBC column width

distribution. The RBC perfused diameter (position of the outer edge of the RBC perfused lumen) is derived from the RBC column width distribution by linear extrapolation of all RBC column width percentiles between P25 and P75. The PBR is defined as the distance of median (P50) RBC column width to the outer edge of the extrapolated RBC perfuseddiameter.

Serum measurements of glycocalyx damage markers

Concentrations of syndecan-1 (Human sCD138/Syndecan-1 ELISA Gen-Probe Diaclone SAS), heparan sulfate (Human heparan sulfate HS ELISA Kit, Cusabio Biotech Co., LTD.) and hyaluronan (Hyaluronic Acid Quantitative Test kit, Corgenix, Inc. ©), three main components of the endothelial glycocalyx [36], were measured in serum using the corresponding enzyme-linked immunosorbent assay (ELISA) kits according to manufacturer’s instructions.

Sample size calculation

Sample size calculation was computed on the basis of MFI data. A total of 9 patients per group showed to be sufficient to detect a statistically significant change in MFI of 0.4 (standard deviation = 0.3) after blood transfusion with a power of 80% and an alpha error of 0.05.

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Statistical analysis was performed using GraphPad Prism version 5 (GraphPad Software, La Jolla, CA). A Mann Whitney U test was used to evaluate differences between the two groups at baseline and after blood transfusion. Wilcoxon matched-pairs signed rank test was used for comparative analysis of data sets obtained before and 1 hour after RBC transfusion. A Spearman coefficient was evaluated to study the correlation between variables. All data are presented as median (25th-75th percentiles). Differences were considered significant at p values <0.05.

RESULTS

Twenty patients were enrolled in the study (10 patients per group). Patient characteristics are presented in Table 1. All patients were mechanically ventilated. All patients received 2 [2,3] packed RBC units and all transfused RBCs were fresh: median age was 4 [3.5-5] days for non-leukodepleted and 3 [1.5-3] days for leukodepleted RBCs.

SOFA score, hematologic, haemodynamic and gas exchange variables

SOFA score, hematologic, haemodynamic and gas exchange variables before and 1 hour after blood transfusion are presented in Table 2. Hb and Hct increased after blood transfusion in both groups (p < 0.01). After blood transfusion a decrease in BE was only found in nonleukodepleted group (p < 0.05). Baseline MAP and PaO2/FiO2 were lower in the

nonleukodepleted group compared with the leukodepleted group. MAP increased after transfusion only in the non-leukodepleted group (p = 0.04). No other significant differences between groups or time points were found.

SDF- and NIRS-derived variables

Microcirculatory and NIRS-derived variables before and 1 hour after blood transfusion are presented in Table 3. When comparing groups, at baseline there were no statistically significant differences, but after transfusion higher PPV and blood flow velocity were observed in the leukodepleted group (Figure 1). Concerning the changes between before and after transfusion, MFI and blood flow velocity exhibited different responses (MFI: -0.02 [−0.1 -0.04] in non-leukodepleted group and 0.17 [0–0.54] in leukodepleted group, p = 0.03; blood flow velocity: -56 [−183 -16] in the non-leukodepleted group and 68 [11–170] in leukodepleted group, p = 0.04). When looking at within group changes, compared to baseline, MFI, PVDs, PPV and De Backer score increased in patients that received leukodepleted RBCs, but not in those who received non-leukodepleted RBCs (Figure 1).

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Table 1 - Patient characteristics for the two groups.

Non-leukodepleted group

Leukodepleted group

(n=10) (n=10)

Age, years 69.5 (65.25-72) 74 (63.5-79)

Sex (male; female) 5; 5 7; 3

SAPS II (on admission) 37 (28.25-74) 41 (34.75-47.25)

Sepsis (n) 2 5

Severe sepsis (n) 3 2

Septic shock (n) 5 3

Source of infection (n)

lung 4 3

abdomen 1 3

urinary tract 2 1

miscellaneous 3 3

Adrenergic doseA

norepinephrineA 5; 0.047 (0.015-0.37) 3; 0.155 (0.05-0.26)

dobutamineA 1; 2.074 (2.074-2.074) 1; 2.31 (2.31-2.31)

SAPS II = Simplified Acute Physiology Score II; ICU = Intensive Care Unit. A number of patients; dose in µg/kg*min [median (interquartile range)].

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Blood flow velocity decreased only in the non-leukodepleted group (Figure 1). No correlation was found between changes in microvascular parameters and MAP after blood transfusion. The change in PPV after blood transfusion was negatively correlated with baseline PPV in the leukodepleted group (r = −0.72, p = 0.02); this relationship was lacking

in non-leukodepleted group (r = −0.36, p = 0.3).

No significant differences were observed in NIRS-derived parameters at baseline and after transfusion. As regards within-group changes, StO2

upslope and THI were elevated in both groups (Figure 2C). Baseline StO2 (Figure 2A) and StO2 downslope (Figure 2B) increased in the non-

leukodepleted group. No difference in the AUC for StO2 was found

either in the between-group and the within-group analysis.

Glycocalyx measurements

Baseline heparan sulfate was higher in the leukodepleted group, no significant differences were observed for baseline syndecan-1, hyaluronan and PBR between the groups. After blood transfusion PBR, heparan sulfate and hyaluronan did not change between the two groups (Figure 3). Syndecan-1 increased after the transfusion of non-leukodepleted RBCs (Figure 3B). A minor correlation was found between PBR values and heparan sulfate levels (r = 0.35, p = 0.03), as well as between PBR changes and heparan sulfate changes after blood transfusion in the whole sample (r = 0.46, p = 0.04) (Figure 4). No correlation was found between PBR values and syndecan-1 or hyaluronan levels.

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Figure 1 Individual changes in microcirculatory parameters

after blood transfusion in non-leukodepleted and leukodepleted groups. (A) Microcirculatory Flow Index (in small vessels); (B) Total small

Vessel Density; (C) Perfused small Vessel Density; (D) Proportion of

Perfused small Vessels; (E) De Backer score; (F) Blood Flow Velocity. *

p < 0.05, Wilcoxon matched-pairs signed rank test; # p < 0.05, Mann–

Whitney U test.

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Figure 2 Individual changes in NIRS-derived variables after blood transfusion in non-leukodepleted and leukodepleted groups. (A) StO2

baseline; (B) StO2 downslope (ischemic phase during the vascular

occlusion test); (C) StO2 upslope (reperfusion phase during the vascular

ocllusion test). * p < 0.05, Wilcoxon matched-pairs signed rank test.

Figure 3 Effects of the transfusion of non-leukodepleted and

leukodepleted RBCs on the endothelial glycocalyx. (A) Perfused

Boundary Region; (B) Syndecan-1; (C) Heparan Sulfate; (D)

Hyaluronan. * = p < 0.05, Wilcoxon matched-pairs signed rank test; # p

< 0.05, Mann–Whitney U test.

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Figure 4 Correlation between PBR and serum Heparan Sulfate. (A)

Correlation between all PBR values and serum Heparan Sulfate values;

(B) correlation between changes in PBR and serum Heparan Sulfate

after blood transfusion. PBR = Perfused Boundary Region; HS =

Heparan Sulfate

DISCUSSION

The present study does not show a clear superiority of leukodepleted over non-leukodepleted RBC transfusion on microvascular perfusion in septic patients, but the more favourable changes observed in MFI and blood flow velocity suggest a positive effect of leukodepleted blood transfusion on microcirculatory convective flow. The within group analysis indicates that patients who received leukodepleted RBCs exhibited a more consistent overall improvement in the microvascular parameters. The lack of further differences in the between group comparisons may just reflect that the study was underpowered.

The sublingual microcirculation is used as a model to study and extrapolate information representing splanchnic blood flow. Persistent microvascular alterations are associated with the occurrence of organ failures and death in patients with septic shock [37]. Previous studies showed that blood transfusions were not able to reverse microcirculatory hypoperfusion in patients with severe sepsis [20,22]. However, the characteristics of the transfused RBCs, in terms of storage and leukocyte reduction, were not examined in previous investigations. The present study did not demonstrate a clear advantage of leukodepleted over non-leukodepleted RBC transfusion on the microcirculation. Indeed, the sublingual microvascular parameters were mostly not significantly different after transfusion between the two groups and the difference in PPV might totally depend on the basal disparity. Nevertheless, the MFI, which was the primary endpoint of the study, showed significantly different changes after the transfusion of leukodepleted versus non-leukodepleted RBC units. Similarly, different effects were observed

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through the quantification of blood flow velocity, which was higher after the transfusion of leukodepleted RBCs. These results would suggest a beneficial effect of leukodepleted blood transfusion on the convective flow in the microcirculation. Lower adhesiveness to vascular endothelial cells has been reported for leukodepleted compared to non-leukodepleted and buffy-coat-poor blood [38,39], and may account for a better haemorrheological impact of leukodepleted RBCs. In addition, the within-group analysis indicated more consistent improvements in several microvascular parameters in patients who received leukodepleted RBCs, as represented by increased microvascular density and percentage of perfused vessels. The transfusion of non-leukodepleted RBCs did not yield any notable improvement in the sublingual microcirculation; a reduction in blood flow velocity was observed. Nevertheless, several points should be considered. First, the responses observed may depend not only on the type of transfused RBCs but also on the underlying clinical and microvascular status. We studied a heterogeneous population by including patients with sepsis, severe sepsis or septic shock. Second, the two groups were not adequately balanced, in fact the patients in the non-leukodepleted group appeared to be more severely ill in general as indicated by the lower MAP and PaO2/FiO2, higher SOFA score (even if

not significant) and higher proportion of patients with septic shock. Therefore, definite conclusions cannot be extrapolated from the results of the present study, as the meaning of any comparison between the groups remains uncertain. Notably however, one would have expected the more severely ill patients in the non-leukodepleted group to show bigger microvascular improvements: indeed, the microcirculatory response to blood transfusion demonstrated a negative correlation with the baseline status in patients with severe sepsis [20]. In our study the increase in PPV was inversely related to baseline values in patients that received leukodepleted RBCs; interestingly, this correlation was lacking in non-leukodepleted group. Finally, most patients were studied several days after their ICU admittance and were already haemodynamically stable, as reflected by their low SOFA score, normal heart rate, low arterial lactate levels and absence of metabolic acidosis. Conversely, metabolic alkalosis was seen in both groups; the most plausible reason was that 17 patients out of 20 had been treated with furosemide for some days before their inclusion in the study. Moreover, most patients did not show big microcirculatory alterations at baseline, with PPV above 75% in all patients and the median MFI was above 2.6 in both groups [30]. This may have conditioned the response observed. The transfusion of leukodepleted RBCs was able to improve microvascular perfusion and tissue oxygenation in patients with a relatively healthy microcirculation [40,41] but did not show any significant effect in severely septic patients

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with dysfunctional microcirculation [20]. Therefore, our findings could not be extended to septic patients with severe microvascular derangement. Moreover, it remains a matter of debate whether most of our patients with an already resuscitated microcirculation really required an increase in microvascular perfusion and if such an increase could have any beneficial impact on outcome.

We found an increase in serum syndecan-1 levels after the transfusion of non-leukodepleted RBCs, suggesting fragmentation of the endothelial glycocalyx. The glycocalyx is believed to fulfil an important role in maintaining microvascular haemorrheological homeostasis. Interestingly however, it can be easily damaged by oxidative stress and inflammatory mediators [36,42,43]. We did not find any change in hyaluronan, heparan sulfate and PBR in either group, nor did we find differences in these parameters after blood transfusion between the groups. Therefore, our results do not allow any conclusion on the effect of the transfusion of the two types of RBCs on the glycocalyx. Large variability was seen in the baseline glycocalyx status in the studied patients, perhaps in relation to the severity of sepsis, this heterogeneity may have produced different responses to blood transfusion.

The transfusion of banked leukodepleted RBCs, stored up to 42 days, showed no substantial effect on muscle StO2 and microvascular

reactivity in critically ill patients [21]; similar results were found in severely septic patients with the transfusion of non-leukodepleted RBCs stored up to 42 days [22]. Blood transfusions increase blood viscosity [41], thus leading to shear stress-induced vasodilation by nitric oxide production [44]. During hypoxia RBCs can release nitric oxide and ATP, thereby exerting a direct vasodilator effect [21]; these properties may be impaired during prolonged storage [4,5]. In the present study microvascular reactivity improved after blood transfusion in both groups, as reflected by the increase in NIRS-derived StO2 upslope during the

reperfusion phase of the VOT. We could speculate that the transfusion of fresh RBCs with preserved haemorrheological properties was responsible for the observed effect. Nevertheless, the discrepancy between our results and previous findings might reasonably be explained also by differences in the studied patient populations.

Despite the increase in THI in both groups, StO2 and StO2 downslope

increased only in the non-leukodepleted group. It should be noted that similar absolute change, although not significant, was observed in the leukodepleted group. We acknowledge that this study may have been underpowered to detect changes in these parameters. An increase in muscle and sublingual tissue oxygenation was reported after the

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transfusion of leukodepleted RBCs in haematology outpatients [41]. On the contrary, previous studies in critically ill and severely septic patients did not show any effect of blood transfusion on tissue oxygenation measured by NIRS technique [21,22]. Substantial differences in the baseline microvascular status may again explain these discrepant results.

The transfusion of leukodepleted RBCs has been associated with reduced hospital length of stay [45], incidence of infections [46], transfusion-related acute lung injury [47] and acute kidney injury [48] in various populations of patients. It has been reported that transfusion of leukodepleted RBCs is not associated with increased mortality in septic shock patients [49]. It has been demonstrated that pre-storage leukoreduction can prevent the accumulation of cytokines and other inflammatory mediators from residual leukocytes, thereby avoiding potentially detrimental effects in the recipient [50-52]. All these findings derived from studies using pre-storage leukoreduction. Notably, in our study RBC units underwent a post-storage leukoreduction before transfusion. This procedure does not prevent the accumulation of leukocyte-derived cytokines during the first days of storage. This might be one of the reasons why we found only slight differences in the microcirculation after blood transfusion between the two groups. Unfortunately, we could not investigate the potential advantages of pre-storage leukoreduction, since this is not usually performed in our blood bank. Nevertheless, microvascular improvement observed in the leukodepleted group might also suggest that post-storage leukoreduction can still prevent deleterious effects of the transfusion of RBC units containing allogenic leukocytes. Further studies comparing pre- and post-storage leukocyte reduction would be needed to clarify this point.

Our study has several limitations that should be considered when extrapolating the data reported in this clinical investigation. First, the low number of patients enrolled may have resulted in underpowered statistical analysis and differences in some variables may not have been detected. Second, the inclusion of patients with different severity of sepsis could have influenced the microvascular responses to blood transfusion. Unfortunately, the small sample size did not allow a post hoc analysis in order to distinguish between sepsis, severe sepsis and septic shock subgroups. Our study was designed to include a heterogeneous population of patients with different severity of sepsis. Future studies should be focused on more homogeneous subgroups of septic patients. Third, the baseline differences observed between non-leukodepleted and leukodepleted groups prevented a proper between-group comparison; in fact, we acknowledge that our results are mostly based on a within-group analysis which cannot reliably support the benefit of leukodepleted over

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non-leukodepleted RBCs in septic patients. Finally, the inclusion of stable septic patients with an already restored microvascular perfusion; patients with a dysfunctional microcirculation may have revealed a different response to the same blood transfusions. This point should be addressed in future studies. Despite these limitations, our study remains interesting as it provides a comprehensive evaluation of microcirculatory responses to blood transfusion in a heterogeneous population of septic patients. As a pilot study on a small number of patients, our investigation cannot provide conclusive answers, nor was it aimed at this goal. Our objective was to explore whether the transfusion of leukodepleted RBCs could provide any advantage on the microcirculation in septic patients. Adequately powered studies should be performed in order to better define the potential benefits observed.

CONCLUSIONS

In this pilot study we were not able to demonstrate a clear benefit of leukodepleted over nonleukodepleted RBC transfusion on the microcirculation in septic patients. However, our results suggest a more favourable effect of leukodepleted RBCs on microcirculatory convective flow. In addition, the within-group analysis showed more consistent improvements in several microvascular parameters in patients that received leukodepleted RBCs. The lack of further differences in the between-group comparisons may just reflect that the study was underpowered. Further studies are needed to confirm these findings.

List of abbreviations:

AUC StO2, Srea under the curve of StO2; BE, Base excess; HI,

Heterogeneity index; ICU, Intensive care unit; Lac, Lactate; MFI, Microvascular flow index; NIRS, Near infrared spectroscopy; PBR, Perfused boundary region; PPV, Proportion of perfused vessels; PVD, Perfused vessel density; RBCs, Red blood cells; SAPS, Simplified acute physiology score; SDF, Sidestream dark field; SOFA, Sequential organ failure assessment; StO2, Tissue oxygen saturation; StO2down, StO2

downslope; StO2up, StO2 upslope; THI, Tissue hemoglobin index; TVD,

Total vessel density; VOT, Vascular occlusion test.

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Chapter 9

Plasma Free Hemoglobin and

Microcirculatory Response to Fresh or

Old Blood Transfusions in Sepsis

Elisa Damiani1, Erica Adrario1, Michele Maria Luchetti2, Claudia Scorcella1, Andrea Carsetti1, Nicoletta Mininno1, Silvia Pierantozzi1, Tiziana Prinicipi1, Daniele Strovegli1, Rosella Bencivenga3, Armando Gabrielli2, Rocco Romano1, Paolo Pelaia1, Can Ince4, Abele Donati1,4

1 Anaesthesia and Intensive Care Unit, Department of Biosciences and Public Health, Università Politecnica delle Marche, Ancona, Italy. 2 Department of Clinical and Molecular Sciences, Clinica Medica,

Università Politecnica delle Marche, Ancona, Italy. 3 Immunohematology and Transfusional Medicine, AOU Ospedali

Riuniti, Ancona, Italy. 4 Department of Translational Physiology, Academic Medical Center,

Amsterdam, The Netherlands.

Published in: PLoS ONE 10(5): e0122655.

doi:10.1371/journal.pone.0122655

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Abstract

Background

Free hemoglobin (fHb) may induce vasoconstriction by scavenging nitric

oxide. It may increase in older blood units due to storage lesions. This

study evaluated whether old red blood cell transfusion increases plasma

fHb in sepsis and how the microvascular response may be affected.

Methods

This is a secondary analysis of a randomized study. Twenty adult septic

patients received either fresh or old (<10 or >15 days storage,

respectively) RBC transfusions. fHb was measured in RBC units and in

the plasma before and 1 hour after transfusion. Simultaneously, the

sublingual microcirculation was assessed with sidestream-dark field

imaging. The perfused boundary region was calculated as an index of

glycocalyx damage. Tissue oxygen saturation (StO2) and Hb index (THI)

were measured with near-infrared spectroscopy and a vascular occlusion

test was performed.

Results

Similar fHb levels were found in the supernatant of fresh and old RBC

units. Despite this, plasma fHb increased in the old RBC group after

transfusion (from 0.125 [0.098–0.219] mg/mL to 0.238 [0.163–0.369]

mg/mL, p = 0.006). The sublingual microcirculation was unaltered in

both groups, while THI increased. The change in plasma fHb was

inversely correlated with the changes in total vessel density (r = -0.57

[95% confidence interval -0.82, -0.16], p = 0.008), De Backer score (r = -

0.63 [95% confidence interval -0.84, -0.25], p = 0.003) and THI (r = -

0.72 [95% confidence interval -0.88, -0.39], p = 0.0003).

Conclusions

Old RBC transfusion was associated with an increase in plasma fHb in

septic patients. Increasing plasma fHb levels were associated with

decreased microvascular density.

Trial Registration ClinicalTrials.gov NCT01584999

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Introduction

Anaemia is common in the Intensive Care Units (ICUs) [1].

Approximately 40% of patients receive packed red blood cell (RBC)

transfusions during their ICU stay [2]. The goal of blood transfusion is to

increase blood oxygen (O2)-carrying capacity, thus restoring tissue

oxygenation [3]. Although potentially life-saving in individual patients,

transfusion practice was associated with increased morbidity and/or

mortality in different patient populations [4, 5].

Stored packed RBCs may develop alterations over time, collectively

referred to as “storage lesions”, which compromise their

hemorrheological properties and O2-delivery capacity [6]. These include

depletion of adenosine triphosphate and 2,3-diphosphoglycerate,

membrane phospholipid peroxidation and vesiculation, protein oxidation,

loss of deformability and increased osmotic fragility [7]. Increasing

hemolysis and release of cell-free hemoglobin (fHb) were documented as

a function of time during prolonged storage [8]. fHb is a potent scavenger

of nitric oxide (NO), the most important endogenous vasodilator [9], and

may therefore be responsible for microvascular perfusion disturbances

[10].

Endothelial dysfunction and impaired microcirculatory blood flow are

leading aspects in the pathophysiology of sepsis [11, 12]. Persistent

microvascular alterations are associated with organ failure and death in

patients with septic shock [13]. Severe deregulation in the NO system is a

major cause of sepsis-induced microvascular perfusion failure [11].

Interestingly, increased plasma fHb levels are associated with higher

mortality in patients with sepsis [14, 15]. A reduction in NO availability

induced by the transfusion of stored RBCs may synergize with the

underlying endothelial dysfunction and be responsible for tissue

hypoperfusion. In the present study, we aimed to evaluate whether the

transfusion of old RBCs increases plasma fHb in septic patients and how

this may affect the microvascular response to blood transfusion.

Materials and Methods

This study is a secondary analysis of a prospective randomized pilot trial

whose primary aim was to evaluate the effects of fresh (<10 days storage)

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non-leukodepleted, fresh leukodepleted or old (>15 days storage) non-

leukodepleted RBCs transfusion on the microcirculation in septic

patients. A comparison between the first two groups (fresh non-

leukodepleted and fresh leukodepleted) was focused on the potential role

of leukocyte reduction and reported previously [16]. Herein, we focus our

attention on the role of storage and report the comparison between fresh

non-leukodepleted and old non-leukodepleted groups. Data related to the

fresh RBC group in this report have been already presented in [16] as

“fresh non-leukodepleted” group.

The study protocol was approved by the local Ethics Committee of

“Azienda Ospedaliera Universitaria (AOU) Ospedali Riuniti” of Ancona

in Italy (NCT01584999, www.clinicaltrials.gov). Written informed

consent was obtained from the enrolled patients or their next of kin.

Patients

Between February 2011 and 2012, adult patients admitted to the 12-bed

Intensive Care Unit of the AOU Ospedali Riuniti of Ancona with sepsis,

severe sepsis, or septic shock as diagnosed according to standard criteria

[17] and requiring blood transfusion for Hb levels <8 g/dL or as indicated

by the attending physician (in accordance with the local hospital

guidelines) were eligible to participate. Exclusion criteria were: age <18

years, previous blood transfusions during their ICU stay, previous history

of coagulation disorders, cardiogenic or hemorrhagic shock, pregnancy,

factors impeding the sublingual microcirculation evaluation (oral surgery,

maxillofacial trauma). Sedation and analgesia were provided according to

individual needs, as well as the type of fluids infused (crystalloids and

colloids) and adrenergic agents (norepinephrine, dobutamine). The goal

was to maintain a mean arterial pressure of 65 mmHg as recommended

by the international guidelines of the Surviving Sepsis Campaign (2008)

[18]. Fluid infusion and furosemide treatment were titrated according to

individual needs, in order to maintain an adequate urine output (>0.5

mL/kg/h) [18].

Interventions

The enrolled patients from the primary study randomly received either

fresh non-leukodepleted RBCs (<10 day storage), fresh leukodepleted

RBCs (<10 day storage) or old non-leukodepleted RBCs (>15 days

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storage). Blood product randomisation was performed through sealed

envelopes by a physician at the blood bank, who blindly provided the

blood bags to the ICU; neither the attending physician nor the

investigators nor the patients were aware of the type of RBCs transfused.

Herein, we present data from the fresh non-leukodepleted and old non-

leukodepleted groups, hereafter referred to as fresh RBC and old RBC

groups.

Basic haemodynamic and blood gas parameters

All measurements were performed in each patient immediately before

and 1 hour after the end of all RBC transfusions; these time points were

chosen on the basis of those reported in previous studies [19–21]. We

recorded temperature (T), heart rate (HR) and mean arterial pressure

(MAP). Arterial blood samples were withdrawn in order to assess Hb

level, whole blood cell counts, blood gases (pH, paO₂, paCO₂, SaO₂,

paO₂/FiO₂, HCO₃-, base excess [BE]) and lactate (Lac) levels. For each

participant, the Simplified Acute Physiology Score (SAPS) II was

obtained at admission and the Sequential Organ Failure Assessment

(SOFA) score [22] on the study day.

Free haemoglobin measurement

Arterial blood samples were withdrawn before and 1 hour after

transfusion and immediately centrifuged; plasma samples were stored at -

70°C for subsequent analysis. In addition, samples were withdrawn from

each transfused RBC-unit; the supernatant was obtained by centrifugation

and stored at -70°C for subsequent analysis. fHb was quantified in each

sample through colorimetric assay using the Drabkin’s reagent (Sigma-

Aldrich, Saint Louis, Missouri, USA).

Microcirculation measurements with sidestream dark-field (SDF)

imaging

Sublingual microcirculatory density and flow were monitored using

sidestream dark-field (SDF) videomicroscopy (Microscan, Microvision

Medical, Amsterdam, the Netherlands) before and 1 hour after

transfusion. Details on the SDF imaging technique have been described

elsewhere [16, 23]. Videos from 5 different sites (at least 10 sec/site)

were recorded at both time points with adequate focus and contrast and

every effort was made to avoid movement and pressure artefacts. Poor-

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quality images were discarded and 3 images for each time point were

selected and analysed using a computer software package (Automated

Vascular Analysis Software, Microvision Medical BV, Amsterdam, The

Netherlands). According to the consensus report on the performance and

evaluation of microcirculation using SDF imaging [24], total vessel

density (TVD) and perfused vessel density (PVD) were calculated for

small vessels (diameter <20 µm). The De Backer score was calculated as

described previously [24]. The proportion of perfused vessels (PPV) and

the microvascular flow index (MFI), reflecting microcirculatory blood

flow velocity, were analysed semi-quantitatively in small vessels, as

described elsewhere [25]. The flow heterogeneity index (HI) was also

calculated as the highest MFI minus the lowest MFI divided by the mean

MFI, providing an index of heterogeneous microcirculatory perfusion.

In addition, SDF videos were automatically analysed using the

GlycoCheck ICU software package (Maastricht University Medical

Center, Maastricht, The Netherlands) in order to measure vascular lumen

perfused boundary region (PBR). The PBR is considered an index of the

dimension of the permeable part of the endothelial glycocalyx which

allows the penetration of flowing RBCs [16, 26, 27]. Erythrocytes usually

have limited access into an intact glycocalyx, when this is compromised

and starts losing its protective capacity, its permeability increases,

allowing circulating cells to approximate the luminal endothelial

membrane. As a result, the dimension of the erythrocyte PBR will

increase. A detailed description of this methodology can be found

elsewhere [28].

Peripheral O2 and Hb measurements with near-infrared spectroscopy

(NIRS)

Before and 1 hour after transfusion, near-infrared reflectance

spectrophotometry (InSpectra™ Model 650; Hutchinson Technology

Inc., Hutchinson, MN, USA) was used on the thenar emincence to

measure peripheral tissue oxygen saturation (StO2) and tissue Hb index

(THI) [29, 30] at baseline and during a vascular occlusion test (VOT),

using a 40% StO2 target for the ischemic phase [16, 31]. StO₂ was

continuously recorded during the reperfusion phase until stabilization

[32]. The StO₂ downslope (%/minute) was calculated from the regression

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line of the first minute of StO₂ decay after occlusion, providing an index

of O₂ consumption rate. The StO₂ upslope (%/minute) was obtained from

the regression line of StO₂ increase in the reperfusion phase. The area

under the curve (AUC) of the hyperaemic response was also calculated.

StO₂ upslope and the area under the curve (AUC) StO₂ reflect

microvascular reactivity [32]. All the parameters were calculated using a

computer software package (Version 3.03 InSpectra Analysis Program;

Hutchinson Technology Inc.).

Sample size calculation and statistical analysis

The sample size had been originally calculated on the basis of MFI data

(primary endpoint) [16]. The secondary analysis presented herein focused

on a different objective (changes in plasma free Hb levels). The power of

this analysis was assessed a posteriori.

Statistical analysis was performed using GraphPad Prism version 5

(GraphPad Software, La Jolla, CA). A Mann Whitney U test was used to

evaluate differences between the two groups at baseline and after blood

transfusion. Wilcoxon matched-pairs signed rank test was used for

comparative analysis of data sets obtained before and 1 hour after RBC

transfusion. A Spearman coefficient was evaluated to study the

correlation between variables. In a supplementary analysis, non-normally

distributed data were normalized whenever possible through logarithmic

or reciprocal transformation and a two-way analysis of variance

(ANOVA) for repeated measures was performed with Bonferroni post-

hoc test in order to compare changes in the parameters of interest

between the two groups. Data are presented as median (25th-75th

percentiles), unless otherwise indicated. Differences were considered

significant at p values <0.05.

Results

Twenty patients were studied in total (10 patients per group). Patient

characteristics are shown in Table 1. Sixteen patients out of 20 received 2

blood units, 3 patients received only 1 RBC-unit (1 patient in the fresh

RBC group, 2 in the old RBC group) and 1 patients in the fresh RBC

group received 3 blood units. No other blood components were given

during the study period. Storage was 4 [3.5–5] days in the fresh RBC

group and 30 [22–30] days in the old RBC group. None of the included

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patients had a medical history of hemoglobinopathies, erythrocyte

membrane defects, enzymatic defects of microangiopathies or any other

disease that could induce hemolysis thus influencing plasma fHb levels.

Hematologic, hemodynamic and gas exchange variables

Hematologic, hemodynamic and gas exchange variables before and 1

hour after transfusion in the two groups are presented in Table 2.

Baseline differences were found between the two groups: MAP was

higher and lactate levels lower in the old RBC group (p<0.001 and

p<0.01, respectively). Hb and Hct were elevated after transfusion in both

groups (p<0.01 in all cases). MAP increased after transfusion in the fresh

RBCs group (p = 0.04). BE decreased in both groups. Lactate levels

differed significantly after transfusion between the two groups (p<0.01).

Results of two-way ANOVA are reported in S1 Table.

Free hemoglobin

fHb levels in the supernatant of blood units did not differ between fresh

and old RBCs (0.103 [0.073–0.149] mg/mL in fresh RBC-units, 0.111

[0.070–0.187] mg/mL in old RBC-units, p = 0.4). No correlation was

found between the age of the transfused RBC-units and fHb levels in the

supernatant (r = 0.03 [95% CI -0.46, 0.49], p = 0.9; data not shown).

Baseline plasma fHb levels did not significantly differ between the two

groups (p = 0.07). Plasma fHb was elevated after transfusion only in old

RBCs group (Table 3, Fig 1A). No difference was found in plasma fHb

after transfusion between the two groups (p = 0.28). Changes in plasma

fHb (delta fHb [before-after transfusion]) differed between the two

groups (-0.041 [-0.237, -0.057] mg/mL in the fresh RBCs group, 0.065

[0.024–0.161] mg/mL in the old RBCs group, p = 0.04, Fig 1B). Two-

way ANOVA showed a significant interaction between time and type of

RBCs transfused, without revealing however any significant difference

between the two groups at each time point (S1 Table and S1 Fig).

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Table 1. Patient characteristics for the two groups.

A post-hoc analysis showed that our study with a sample of 10 patients

per group was able to demonstrate the observed change in fHb after

blood transfusion with a power >90% (type II error of 0.06).

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Table 2. Hematologic, hemodynamic and gas exchange variables in the

two groups (baseline and 1 hour after transfusion).

Microvascular response to fresh or old RBC transfusion

Sublingual microvascular parameters and NIRS-derived variables before

and 1 hour after transfusion in the two groups are presented in Table 3.

No difference was found in baseline values between the groups. We

could not find any significant change in MFI, PPV, TVD, PVD, De

Backer score, HI and PBR after the transfusion of either fresh or old

RBCs. The change in PPV after transfusion was inversely related to the

baseline PPV value in the whole sample (r = -0.53 [95% CI -0.79–0.10],

p = 0.02; data not shown). Changes in TVD, PVD and De Backer score

were not correlated to their baseline values (r = 0.18 [95% CI -0.30, 0.59]

p = 0.4, r = 0.03 [95% CI -0.43, 0.48] p = 0.9, r = -0.21 [95% CI -0.61,

0.26] p = 0.4, respectively).

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Fig 1. Changes in plasma free haemoglobin after blood transfusion in the

two groups. (A) Individual changes in plasma free haemoglobin after

blood transfusion in the two groups; **p<0.01, Wilcoxon matched-pair

signed rank test. (B) Delta values (after-before transfusion) of plasma

free haemoglobin in the two groups. *p<0.05, Mann-Whitney U test.

Open circles indicate patients in the fresh RBC group, full circles

patients in the old RBC group.

StO2, StO2 downslope and StO2 upslope increased in the fresh RBCs

group. THI was elevated after transfusion in both groups. The AUC StO2

remained unaltered in both groups. Changes in NIRS-derived variables

were not correlated to their baseline values.

All SDF- and NIRS-derived parameters did not differ after transfusion

between the groups. Two-way ANOVA did not show any significant

effect of the type of transfused RBCs on the changes in SDF- or NIRS-

derived parameters after blood transfusion (S1 Table).

No correlation was found between baseline SOFA score and

microvascular changes after blood transfusions. The change in MFIs

tended to correlate with the baseline MAP (r = 0.47 [95% CI, 0.01–0.77],

p = 0.04).

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Table 3. Plasma free haemoglobin, sublingual microvascular parameters

and NIRS-derived variables in the two groups (baseline and 1 hour after blood transfusion).

Free haemoglobin and microcirculation

The change in fHb (delta fHb [after-before transfusion]) was negatively

correlated with changes in TVD (r = -0.57 [95% CI -0.82, -0.16], p =

0.008), De Backer score (r = -0.63 [95% CI -0.84, -0.25], p = 0.003) and

THI (r = -0.71 [95% CI -0.88, -0.39], p = 0.0003) (Fig 2 and S2 Fig). The

change in PVD and StO2 tended to be inversely correlated with the

change in fHb (r = -0.40 [95% CI -0.72, 0.07], p = 0.08 and r = -0.40

[95% CI -0.72, 0.06], p = 0.08, respectively) (Fig 2B–2E and S2 Fig).

Changes in MFI, PPV, StO2 upslope, StO2 downslope and AUC StO2

were not correlated with the change in fHb.

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Fig 2. Correlation analysis between the change in plasma fHb (X axis)

and changes in: (A) total small vessel density, (B) perfused vessel density,

(C) De Backer score, (D) tissue haemoglobin index, (E) tissue oxygen

saturation (Y axis).

Open circles indicate patients in the fresh RBC group, full circles patients

in the old RBC group.

Discussion

In the present study, the transfusion of old RBCs was associated with an

increase in plasma fHb in septic patients. We were not able to

demonstrate any improvement in the microcirculation after transfusion in

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either group, nor did we find any clear difference in the microcirculatory

response to the transfusion of fresh versus old RBCs. The change in

plasma fHb after transfusion was negatively correlated with changes in

sublingual microvascular density and peripheral tissue Hb content.

Increasing fHb concentration in the supernatant of packed RBCs has been

described as a function of time during blood storage [8, 33], and the

transfusion of 2 packed RBC units increased circulating fHb levels in

patients with hematologic malignancies [33]. In the present study, septic

patients who received old blood transfusions showed an increase in

plasma fHb, despite the absence of significantly higher fHb levels in the

supernatant of old blood units. It is possible that the transfusion of older

fragile erythrocytes led to premature intravascular RBC rupture in the

recipients. We cannot exclude that the underlying critical illness had

influenced our results: spontaneous changes unrelated to blood

transfusion might reasonably explain the decrease in fHb observed in 6

patients out of 20.

RBC storage lesions can be responsible for the association between the

transfusion of older blood and adverse outcomes [34]. Old RBCs

decreased microvascular oxygenation and flow in rat isovolemic

exchange models [35, 36]. Nonetheless, clinical data remain controversial

[20, 37–39]. Marik et al. were the first to report a harmful effect of

duration of RBC storage on systemic and tissue oxygenation in septic

patients [40], but this association was not confirmed [41]. RBC storage

time showed no influence on the sublingual microvascular response to

the transfusion of leukodepleted blood in patients with severe sepsis [19].

In the present study, the transfusion of non-leukodepleted blood did not

apparently affect the sublingual microcirculation and no difference could

be seen in the microvascular response to the transfusion of fresh versus

old RBCs. Whereas both fresh and old RBC transfusions were able to

increase the peripheral tissue Hb content, StO2, StO2 downslope and

StO2 upslope increased significantly only in the fresh RBC group. Of

note however, the old RBC group showed similar absolute changes: the

study may have been underpowered to detect significant differences in

these parameters.

We found an inverse relationship between the changes in the

microcirculation and the change in plasma fHb. Independently of the age

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of the transfused blood or the baseline microvascular status, an increase

in circulating fHb after transfusion was associated with a decrease in the

sublingual microvascular density and a lower increase in peripheral tissue

Hb content. We can speculate that these effects were mediated by

disturbances in NO metabolism: cell-fHb can react with NO much faster

than RBC-encapsulated Hb [42] and inhibit the NO-mediated

vasodilation. Experimental studies showed that the infusion of the

supernatant from stored RBCs produces potent vasoconstriction that is

correlated with the amount of fHb in the storage medium [8]. Blood

transfusion increased NO consumption in patients with hematologic

malignancies [33]. Our results are consistent with these findings. Sepsis-

induced deregulation in NO production is associated with impaired

microvascular perfusion and reduced O2 consumption [11]. Although

inhibiting NO during sepsis increases blood pressure, it also reduces

microvascular blood flow and exacerbates abnormal oxygen transport

[43]. We found an increase in MAP in the fresh RBC group but not in the

old RBC group: this would contradict the previous findings. However,

the baseline between-group discrepancy may have confounded the results

and prevents to draw any conclusion on this point. Increasing NO-

scavenging by fHb due to old RBC transfusion may synergize with the

underlying endothelial dysfunction, thus reducing NO bioavailability and

producing relative vasoconstriction [44]. Notably, we studied stable

patients with low baseline microvascular alterations, as indicated by PPV

above 70% in all patients and median MFI above 2.6 in both groups [45]:

the observed interaction might be more pronounced and deleterious in

presence of severe underlying microcirculatory dysfunction. Although

our results would suggest that there may be a relationship between the

changes in plasma fHb and the microvascular response after transfusion,

several points remain to be clarified, namely the role of the patient’s

underlying clinical condition and microcirculatory perfusion, and the

potential advantages of pre-storage leukodepletion.

The microcirculatory response to blood transfusion is likely to be

influenced by the underlying clinical and microvascular condition of the

recipient. In our study, the heterogeneity of the studied population, which

included patients with different severity of sepsis, may have been a

source of variability in the response observed. More importantly, baseline

disparities between the two groups (lower MAP and higher lactate levels

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in the fresh RBC-group) reasonably influenced the results and add to the

uncertainty of our data. In fact, a relationship seemed to exist between

baseline MAP and the increase in MFI. However, patients who received

old RBC transfusions did not show any significant improvement in

microvascular convective flow despite higher baseline MAP. Other

factors could have played a role. In previous studies, the microvascular

response to blood transfusion was negatively correlated with the baseline

microcirculatory status rather than the age of the transfused blood units

[19, 20]. Accordingly, in the present study the change in PPV was

inversely related to its baseline value.

Our analysis was focused on a single aspect of packed-RBC storage

lesions; other potentially important factors, such as loss of RBC

deformability and accumulation of residual leukocyte-derived cytokines

within the storage medium, were not considered. Some studies suggest

that pre-storage leukodepletion may abrogate the detrimental effects of

packed RBC aging [46]. The real role of inflammatory mediators from

residual leukocytes in the development of storage lesions remains to be

clarified. Future studies should investigate whether pre-storage

leukodepletion may really preserve the integrity of stored RBCs and

prevent the release of fHb.

The first limitation of the present study is that it is a secondary analysis

of a randomized pilot study with a different primary endpoint. Moreover,

we enrolled a small number of patients. However, a post-hoc analysis

showed that our study had a power >90% to detect the observed variation

in plasma fHb, which was the main objective of this investigation. The

heterogeneity of the studied patient population may have been a source of

variability, thus preventing to detect different effects of fresh versus old

RBC transfusions. In addition, baseline differences between the two

groups reasonably influenced the microvascular response observed and

impeded a proper between-group comparison. Another limitation of our

analysis is the fact that a large number of statistical tests was performed

on a small sample size; it is possible that some of the observed

associations were due to chance. Our investigation was designed as a

pilot study aimed to detect any possible relationship between the type of

transfused blood and changes in plasma fHb and/or microcirculation:

therefore, a higher type I error rate was deemed acceptable and a

correction for multiple comparisons was not applied as it would have

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substantially reduced the probability of finding any statistically

significant associations [47]. A two-way ANOVA with Bonferroni post-

hoc test performed after normalization of the data revealed a significant

interaction between the type of transfused RBCs and the changes in

plasma fHb after transfusion. However, our results are not conclusive and

require validation from larger studies.

In our previous report [16], we explored the impact of blood transfusion

on the endothelial glycocalyx. Unfortunately, it was not possible to

evaluate the relationship between variations in plasma fHb and markers

of glycocalyx disruption (hyaluronan, syndecan-1, heparan sulphate) as

these were not measured for the old RBC group due to cost reasons.

Since the glycocalyx plays a major role in the shear stress-induced

release of NO [48, 49], future studies should be addressed to investigate

its impact on NO bioavailability after the transfusion of stored blood.

Finally, only non-leukodepleted RBCs were used: this may limit the

direct applicability of our results, as most developed countries currently

use leukodepleted blood units. Nevertheless, the use of non-

leukodepleted RBCs is still the standard practice in Italy, and universal

leukoreduction has not been implemented yet in several countries

including USA.

Conclusions

In the present study, the transfusion of old RBCs was associated with an

increase in plasma fHb in a small and heterogeneous population of septic

patients. The sublingual microcirculation appeared globally unaffected by

the transfusion of either fresh or old RBCs. Independently of the type of

blood received and the baseline microvascular status, increasing plasma

fHb levels after transfusion were associated with decreasing sublingual

microcirculatory density and lower increase in peripheral tissue Hb

content after transfusion. Further studies are needed to confirm these

findings.

Acknowledgments

We thank all the patients for their participation in the study and the

medical and nurse staff for their collaboration to the realization of this

work. We are grateful to Dr. Hans Vink for his contribution in the

analysis of SDF videos.

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Author Contributions

Conceived and designed the experiments: ED CI AD. Performed the

experiments: ED EA CS AC NM SP TP DS RB. Analyzed the data: ED

MML AG. Contributed reagents/materials/analysis tools: EA RR PP AD.

Wrote the paper: ED CI AD.

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Chapter 10

Towards integrative physiological

monitoring of the critically ill: from

cardiovascular to microcirculatory and

cellular function monitoring at the

bedside

Abele Donati1,2, Dick Tibboel3 and Can Ince1,3

1 Department of Intensive Care, Erasmus MC, University Medical Center Rotterdam, 's-Gravendijkwal 230, 3015 CE Rotterdam, the

Netherlands

2 Department of Biomedical Science and Public Health Anesthesia and ICU, Università Politecnica delle Marche, via Tronto 10, 60126

Torrette di Ancona, Italy

3 Intensive Care and Department of Pediatric Surgery, Erasmus Medical Center - Sophia Children's Hospital, Postbox 2040, 3000

CA, Rotterdam, the Netherlands

Published in: Critical Care 2013, 17(Suppl 1):S5 (doi:10.1186/cc11503)

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Abstract

Current hemodynamic monitoring of critically ill patients is mainly

focused on monitoring of pressure-derived hemodynamic variables

related to systemic circulation. Increasingly, oxygen transport pathways

and indicators of the presence of tissue dysoxia are now being

considered. In addition to the microcirculatory parameters related to

oxygen transport to the tissues, it is becoming increasingly clear that it is

also important to gather information regarding the functional activity of

cellular and even subcellular structures to gain an integrative evaluation

of the severity of disease and the response to therapy. Crucial to these

developments is the need to provide continuous measurements of the

physiological and pathophysiological state of the patient, in contrast to

the intermittent sampling of biomarkers. As technological research and

clinical investigations into the monitoring of critically ill patients have

progressed, an increasing amount of information is being made available

to the clinician at the bedside. This complexity of information requires

integration of the variables being monitored, which requires

mathematical models based on physiology to reduce the complexity of

the information and provide the clinician with a road map to guide

therapy and assess the course of recovery. In this paper, we review the

state of the art of these developments and speculate on the future, in

which we predict a physiological monitoring environment that is able to

integrate systemic hemodynamic and oxygen-derived variables with

variables that assess the peripheral circulation and microcirculation,

extending this real-time monitoring to the functional activity of cells and

their constituents. Such a monitoring environment will ideally relate

these variables to the functional state of various organ systems because

organ function represents the true endpoint for therapeutic support of the

critically ill patient.

The rise of cardiovascular monitoring

In the early 1960s, Weil realized the importance of continuous

monitoring of physiological parameters coupled with calculations to

provide real-time information on the hemodynamic status of patients at

the bedside. One can argue that this characteristic - namely, the

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continuous monitoring and support of physiological variables - defines

the health of critically ill patients. The introduction by Weil and Safar of

monitors attached to digital computers to continuously monitor

respiratory and hemodynamic measurements was a defining moment in

the development of critical care medicine [1]. This technology, used in

conjunction with the pulmonary artery catheter introduced by Swan [2],

provided the intensivist with a powerful platform to semicontinuously

monitor the functional state of the heart as the main motor driving

systemic circulation. By including measurements of arterial and mixed

venous gas analysis, the arterial oxygen content and the mixed venous

oxygen content could easily be calculated. Consequently, oxygen

delivery and oxygen consumption could be calculated from known

formulae, and a target for titration therapy was formulated.

Shoemaker was the main proponent of driving systemic circulation by

targeting high values of cardiac output, oxygen delivery and oxygen

consumption. The basic idea behind this approach was that maximizing

the oxygen delivery of the systemic circulation would ensure ample

oxygen for the organ beds at risk. In initial studies, Shoemaker utilized

the hemodynamic data obtained from the pulmonary artery catheter in

high-risk adult surgical patients before, during and after surgical

procedures. From these observational data, he established a protocol

using supernormal values for cardiac output, oxygen delivery and oxygen

consumption as the therapeutic goals. Indeed, this approach seemed to be

favorable in surgical patients because it resulted in improved outcomes

[3]. Donati and colleagues demonstrated that this approach was also

successful in reducing morbidity and the length of hospital stay in high-

risk surgical patients [4]. However, the effectiveness of this strategy in

other critically ill patients remains controversial. Gattinoni and

colleagues, for example, found no difference between patients who were

treated with a protocol that targeted normal values for cardiac output,

oxygen delivery and oxygen consumption, supernormal values or mixed

venous saturation >70% [5]. Hayes and colleagues found an increased

mortality in patients who were treated using the supranormal values

protocol [6]. The condition of normal or reduced oxygen extraction

seemed to be a key hemodynamic component that contraindicated

targeting supranormal values of oxygen delivery. Vincent suggested that

the application of a dobutamine challenge to identify the effect of

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increasing systemic oxygen delivery was an effective strategy to achieve

hemodynamic optimization for the critically ill patient [7].

Adding the peripheral circulation to the equation

Simply targeting the parameters related to the systemic circulation was

ineffective in resuscitating the various organ systems because the

microvasculature was unable to effectively regulate the flow of oxygen-

carrying blood to match regional needs. This inability is presumably

caused by the pathogenic action of the inflammatory mediators, reactive

oxygen species and hypoxemia on vascular regulatory mechanisms, such

as autoregulation. This dysfunction is augmented by certain therapies

such as intravascular fluids and vasoactive mediators that override the

endogenous physiological mechanisms regulating homeostasis. The

consequent mismatch has its impact not only between different organ

systems but also at the level of the microcirculation. This defect

manifests itself as a reduced oxygen extraction deficit that is

characterized by shunting within vulnerable, weak micro-circulatory

units and organ beds. Reduced extraction results in elevated venous

oxygen levels in the presence of signs of regional dysoxia, such as

elevated levels of lactate and elevated tissue carbon dioxide (CO2) [8].

Weil, in advance of experimental evidence, first understood the origin of

the sequence of events relating to oxygen transport dysfunction during

circulatory failure [9,10]. He classified four states of shock:

hypovolemic, obstructive, cardiogenic and distributive. All of these states

indicate that the ultimate target of shock is the cellular starvation of

oxygen availability. The first three states of shock, however, are

associated with the reduction in cardiac output that is the primary cause

of the ensuing tissue dysoxia. Distributive shock can occur in the

presence of a normal or even elevated cardiac output and describes a

defect in the vascular trafficking of cardiac output between and within the

various organ beds, resulting in local tissue dysoxia in the presence of

otherwise normal systemic hemodynamics. When such a distributive

defect occurs, increased cardiac output is ineffective in resolving the

regional tissue dysoxia. In their editorial 'Expanding from the Macro to

the Microcirculation', Weil and Tang identified the need to monitor the

microcirculation to monitor and treat distributive shock [11]. This

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realization, in combination with the idea that microcirculatory

dysfunction leads to organ dysfunction, formed the basis for the

appreciation of the microcirculation as a central focus in the pathogenesis

of multiorgan dysfunction [12].

The need to monitor hemodynamic and oxygen-derived variables of the

peripheral circulation was demonstrated by the introduction of CO2

gastric tonometry by Fiddian-Green and Baker to identify splanchnic

dysoxia during states of shock [13]. The significance of this monitoring

for the critically ill was revealed by the landmark study of Guteriez and

coworkers, who found that septic shock patients whose gastric CO2 did

not normalize following resuscitation had a higher chance of dying than

did those whose gastric CO2 normalized [14]. The physiological

mechanisms underlying tissue CO2 production was controversial at that

time, however, with one school of thought indicating that its origin was

due to mitochondrial dysfunction and another school supporting the idea

that abnormal CO2 reflected an abnormal perfusion of the tissues [15].

However, a number of experimental studies performed by Dubin and

coworkers [16] as well as clinical investigations by Creteur and

colleagues [17] have now firmly established that elevated tissue CO2

reflects a perfusion deficit in the microcirculation. The importance of

monitoring the peripheral circulation in the context of distributive shock

was further expanded upon by the work of Lima and Bakker, who

investigated the clinical significance of assessing peripheral perfusion by

physical examination. In so doing, Lima and colleagues identified

abnormalities in peripheral perfusion as being associated with high

Sequential Organ Failure Assessment scores [18].

A closer look at the microcirculation

Clinical monitoring of the microcirculation has previously been limited to

indirect measures such as lactate, tissue CO2 and subjective assessment

of peripheral perfusion. Hand-held intravital microscopes offer a different

approach [19,20], which incorporates specialized optics such as crossed

polarized green light and/or dark-field illumination to filter out the

surface reflections as developed much earlier by Slaaf and colleagues and

Sherman and colleagues [21,22]. This technology allows for observation

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of the flowing red blood cells in the microcirculation of the mucosal

surfaces of organ beds [19,23]. These hand-held intravital microscopes

were subsequently used to directly observe the microcirculation on organ

surfaces at the bedside in various clinical scenarios [24-33]. Sublingual

microcirculatory observations identified microcirculatory obstructions to

be characteristic of septic patients who are resistant to therapy despite

corrected systemic hemodynamics [25,32,33]. De Backer and coworkers

first demonstrated a correlation between the severity of microcirculatory

alterations and morbidity and outcome in septic patients, whereas no such

relationship existed for conventional systemic hemodynamic variables

[26]. We recently further demonstrated this phenomenon in septic

pediatric patients [34]. These findings were reproduced using an early

goal-directed therapy treatment by Tryziack and coworkers, who found

that an early effective recruitment of the microcirculation predicted

Sequential Organ Failure Assessment improvement 24 hours following

early goal-directed therapy [25].

Based on the idea that active recruitment of the microcirculation is

needed for resuscitation, vasodilatory therapy (for example,

nitroglycerin) was shown to be especially effective in recruiting

obstructed sublingual microcirculation in pressure-resuscitated septic

patients [24]. Similar improvement was not found in fluid-optimized

septic patients [35]. In septic shock patients, levosimendan was

demonstrated superior to dobutamine for recruiting microcirculation [36],

while the addition to norepinephrine of continuously infused low-dose of

terlipressin or vasopressin did not affect sublingual microcirculatory

blood flow [37].

In cardiac surgery patients, blood transfusions are effective in improving

microcirculatory oxygen availability by recruiting previously unfilled

microcirculatory capillaries, thereby reducing the diffusion distances

between capillaries and tissue cells; this result emphasizes the importance

of viscosity in recruiting the microcirculation during resuscitation [38].

The importance of viscosity was further demonstrated in a study of septic

patients by Dubin and colleagues, who showed that highly viscous starch

solutions can recruit the micro-circulation more effectively than less

viscous crystalloids [39]. These studies highlight the unique ability of

microcirculatory monitoring to measure not only flow (convection) but

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also the diffusive capacity of the circulation to transport oxygen by

measuring the functional capillary density [31,39,40].

In studies of septic patients, fluid responsiveness has been evaluated at

the level of the microcirculation. The type and timing of fluid

administration have been found to be an important aspect of fluid

efficacy in recruiting microcirculation [39,41]. Vasopressor therapy,

although effective in increasing blood pressure, can have limited or even

deleterious effects on improving perfusion of the microcirculation

[30,42]. One consistent finding from various investigators has been that

microcirculatory alterations often manifest themselves at the capillary

level by normalized or even elevated flow in the larger venules

[24,26,33]. These observations describe the nature of the distributive

defect that occurs during shock (especially during the resuscitation phase,

as obstructions in the capillary vessels affect the persistence of flow in

the larger microvessels) and, furthermore, directly illustrate the nature of

the functional shunting that is associated with sepsis and other forms of

distributive shock [8]. In particular, the heterogeneity of capillary

function has been found by many to be a key characteristic feature of this

type of distributive shock [33]. This observation led Tryziack and co-

workers to analyze microcirculation images to develop a heterogeneity

index to quantify this type of microcirculatory alteration [25].

An additional level of heterogeneity can be attributed to the physiological

diversity of the patients themselves. In particular, differences in age

influence the response of the patients, with each age group having its own

characteristic phenotype and response to critical illness. In this respect,

critically ill pediatric and neonatal patients form a special group because

they present a completely separate level of (patho)physiological diversity

relative to adult patients [43]. For example, as an infant grows during the

first years of life, systolic and diastolic pressures are low and heart rates

are high. The cardiac output and stroke volume continue to rise until the

age of 5 years. Changing cardiovascular physiology is also reflected in

the development of the microcirculation, which exists during the initial

days and months following birth as a rich network of microcirculatory

capillaries that diminishes in density as the infant grows [44].

The response to critical illness is also largely divergent between pediatric

and adult patients. A diminished systemic vascular resistance is a

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hallmark of adult sepsis but is not observed in the pediatric patient.

Furthermore, septic shock in pediatric patients, in contrast to adult

patients, is often characterized by a hypodynamic response with low

cardiac output and high systemic vascular resistance, although a rapid

switch can be made. The septic pediatric patient also has a diminished

contractile reserve and a poor response to volume loading and inotropic

support [43]. Hemodynamic monitoring in these very small patients is

indeed a challenging task because the possibilities for invasive

hemodynamic monitoring are limited. For instance, Swan-Ganz

monitoring has never become common practice in the pediatric age

group. Hemodynamic monitoring using hand-held intravital microscopes

could offer advantages in these patients; besides targeting an important

physiological compartment, this method offers the additional advantage

of being largely non-invasive.

The potential application of monitoring the microcirculation in pediatric

patients using hand-held intravital microscopic techniques has been

exemplified in the work of Top and Tibboel. Using orthogonal

polarization spectral imaging, Top and colleagues demonstrated in septic

children that persistent microcirculatory alterations were the single most

sensitive and specific indicator of outcome [34]. In a recent study, Paize

and coworkers further supported the importance of monitoring

microcirculatory alterations in pediatric patients by observing that certain

microcirculatory alterations in patients with severe meningococcal

disease are associated with clinical recovery [45]. Others have shown that

some therapies, such as hypothermia and blood transfusions, positively

impact the microcirculation of critically ill pediatric and neonatal patients

[46,47].

The cardiovascular response of neonatal patients is a largely unexplored

area and presents a further level of complexity, not only owing to their

small size but also owing to their complex response to hypoxia [48]. This

issue is truly a physiological challenge. Whereas hypoxemia is

considered a pathological condition in older patients, hypoxia may be

viewed as a physiological condition for the neonate, to which the neonate

is continuously adapting. Only when these adaptive mechanisms fail does

the neonate present as critically ill. These adaptive mechanisms require

support that is essential for promoting development [49]. Monitoring the

success of the microcirculation at providing blood flow in combination

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with an assessment of tissue oxygenation is anticipated to form an

important platform to realize this support.

Cell function monitoring

The microcirculation is an integrative physiological compartment in

which red blood cells, leucocytes, blood constituents, endothelial cells,

smooth muscle cells, parenchymal cells and the intracellular components

of these cellular systems integratively and symbiotically function

together to ensure optimal oxygen and nutrient transport for the

utilization of the parenchymal cells. Indeed, adequate function in terms of

perfusion and oxygen transport can be regarded as an indication of

success for all of these cellular systems. Microcirculatory dysfunction of

this system caused by pathogenic factors, such as inflammation, oxidative

stress and hypoxemia, however, can lead to organ dysfunction [12]. Fully

understanding the nature of the insult and the indication for appropriate

therapy requires insight into the function of the individual subcellular

building blocks of the microcirculation. The future of monitoring will

need to integrate the functional state of the various cellular constituents

into microcirculatory monitoring.

The ability of red blood cells to carry hemoglobin-bound oxygen to the

microcirculation is, of course, one of the main functions of the

cardiovascular system. The oxygenation state of hemoglobin in red blood

cells can be measured quite effectively at the bedside using

spectrophotometry, and we used this method to demonstrate the efficacy

of blood cell transfusion to improve oxygen availability in the

microcirculation in adult anemic hematological patients [50]. Leukocytes

form an important source of pathogenic activation, resulting in tissue

damage that contributes to organ dysfunction. The ability to monitor

leukocyte activation at the bedside using direct observation of their

rolling and sticking to the endothelium could therefore provide an

important indication of the state of inflammation and possibly the

response to therapy. Hand-held intravital microscopy of the sublingual

bed was first used for this purpose by Baur and coworkers in patients

following the release of the clamp after cardiac surgery [51].

The endothelial cell forms the central regulatory player in the

orchestration of the physiology of microcirculation. The cell plays an

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important signaling role in the regulation of vessel tone in addition to

inflammation and hemostasis. Assessment of its function can be

accomplished by administering compounds that target endothelial cell

function and observing the microcirculatory response. De Backer and

coworkers used this approach by topically administering acetylcholine

sublingually in septic patients who demonstrated enhanced

microcirculatory perfusion [52].

A critical subcellular component that has come to prominence recently

due to its relevance to critical illness is the endothelial glycocalyx [53].

This gel-like layer lining the endothelial cells forms the barrier between

the intravascular lumen and the endothelial cells. Shedding and

disruption of the glycocalyx has been associated with many states of

endothelial dysfunction, including the loss of autoregulation and the

development of tissue edema and organ dysfunction. Vink and coworkers

developed a method to measure the integrity of the glycocalyx by

analyzing images obtained from sublingual intravital microscopy [54].

They further developed a software platform to assess the functional state

of the glycocalyx directly at the bedside [55].

The routine clinical application of such measurements using the current

orthogonal polarization spectral/sidestream dark-field hand-held intravital

microscopes and analog video cameras [19,23] has been criticized

[56,57]. This criticism is based on the fact that these devices have poor

reproducibility and image quality [58,59] and require time-consuming

off-line analysis of the acquired images. These devices also suffer from

pressure artifacts imposed by the weight of the devices [56,57] and from

an inability to implement automatic analysis software to process the

generated images [60]. In addition, higher resolution optics and image

sensors are required to allow for software analysis and the identification

of the subcellular structures associated with microcirculatory function.

For these devices to enter the clinical arena, technological advances are

therefore mandatory [56,57].

A new hand-held intravital microscope has been developed recently that

is based on incident dark-field imaging [22], containing a computer-

controlled high-resolution imaging sensor [61]. Such technological

advances might possibly address the earlier critiques of the conventional

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devices but will need to be validated with regard to these critiques before

such hand-held vital microscopes can truly enter the clinical arena.

Towards an integrated physiological monitoring system

The above summary has highlighted the need to extend monitoring of the

physiological determinants of organ function from the macro to the micro

and down to the cellular level. However, a crucial component of this

monitoring is the need to include functional indicators of organ function

because it is the successful restoration of organ function that determines

the success of intensive care. These indicators of organ function need to

be continuous, specific and quantitative. These initiatives are important

because they describe a road map for the new developments that are

needed to provide complete physiological monitoring of critically ill

patients. The information from new sensors and physiological variables,

as well as measures of organ function, will require a much higher level of

integration than is currently available, and mathematical models of

physiology and pathophysiology are expected to play an important role in

this integration. From this perspective, these innovations represent a

challenge for industry.

By integrating information on all of the characteristics of the patient -

including disease, co-morbidities and age - into the evolution of this

integrated physiological monitoring system, we anticipate the

development of an environment in which the complete continuum of

human development, as well as diseases and their response to therapy,

can be monitored. Intensive care medicine offers a unique environment

for this development, which ultimately may be relevant to other areas of

medicine.

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217


Hemodynamic monitoring allows clinicians to manage cardiac output and

macrocirculation, but some patients, despite optimal macrohemodynamic

management, remain still critical. In this thesis we show that the

introduction in clinical practice of pulmonary artery catheter (PAC) with

the thermodilution technique about 40 years ago allowed clinicians to

measure bedside cardiac output (CO) and to calculate other physiological

parameters, such as oxygen delivery (DO2) and oxygen consumption

(VO2), but their interpretation and utilization as a therapeutic target was

and remains controversial to date. Shoemaker was the first clinician who

tried to interpret and utilize these new hemodynamic data in high risk

surgical patients. He conceived the supernormal values of CO, DO2, and

VO2 as therapeutic goals and obtained a reduction in mortality from 28%

in the control groups to 4% in the protocol group.

Postoperative organ failures commonly occur after major abdominal

surgery, increasing the utilization of resources and costs of care. Tissue

hypoxia is a key trigger of organ dysfunction. A prospective, randomized,

controlled trial was performed in nine hospitals in Italy. One hundred

thirty-five high-risk patients scheduled for major abdominal surgery were

randomized in two groups. All patients were managed to achieve

standard goals: mean arterial pressure> 80 mm Hg and urinary output >

0.5 mL/kg/h. The patients of the “protocol group” (group A) were also

managed to keep O2ER < 27%, the other group received “standard”

therapy (Group B). In group A, fewer patients had at least one organ

failure (n 8, 11.8%) than in group B (n 20, 29.8%) [p < 0.05], and the

total number of organ failures was lower in group A than in group B (27

failures vs 9 failures, p < 0.001). Length of hospital stay was significantly

lower in the protocol group than in the control group (11.3 ± 3.8 days vs

13.4 ± 6.1 days, p < 0.05). Hospital mortality was similar in both groups.

Early treatment directed to maintain O2ER at < 27% reduces organ

failures and hospital stay of high-risk surgical patients. (Chapter 1).

A prospective, open study with repeated measurements on fifteen patients

with septic shock was performed in a medical-surgical ICU. A bolus

dose of methylene blue (3 mg/kg) was infused intravenously over 10

mins. Hemodynamic variables were recorded before methylene blue and

20 mins, 1 hr, and 2 hrs after the end of methylene blue infusion.

Standard hemodynamic and oxygen-derived variables; total,

intrathoracic, systolic, and diastolic cardiac blood volumes; extravascular

lung water; plasma osmolarity; and lactate and protein concentrations


218

were recorded. Mean arterial and pulmonary artery pressures, systemic

and pulmonary vascular resistances, and left ventricular stroke work

index increased, and blood lactate transiently decreased after methylene

blue (p < .05). The other variables recorded were unchanged during the

2-hr period following methylene blue infusion. This study confirmed the

acute vasoconstrictive and positive inotropic effects of methylene blue

during septic shock. These effects were not associated with changes in

blood volume, myocardial diastolic function, or pulmonary vascular

permeability assessed by extravascular lung water. (Chapter 2).

In the past years macrocirculation monitoring appeared to be not enough

to improve patient outcome and splanchnic perfusion was monitored with

gastric tonometer. In 12 patients who underwent abdominal surgery,

intraoperative splanchnic ischemia, as documented by gastric

intramucosal pH-i, is directly correlated to the increase of IL-6 plasma

levels and to the incidence of postoperative complications, while IL-8

levels shows no correlation with surgical complications (Chapter 3). PHi

and ∆CO2 have been demonstrated to be sensitive prognostic indices

during abdominal aortic aneurysm surgery in 29 patients. Tonometry may

identify patients at higher risk of organ failure in the postoperative

period. The ∆ portal venous lactates are more specific then tonometric

variables, and are an important index of gut hypoperfusion (Chapter 4).

Muscular perfusion during sepsis can be monitored with near-infrared

spectroscopy and vascular occlusion test can be applied to test tissue

metabolism and vascular reactivity in septic patients. We tested the

hypothesis that muscle perfusion, oxygenation, and microvascular

reactivity would improve in patients with severe sepsis or septic shock

during treatment with recombinant activated protein C (rh-aPC) (n. 11)

and to explore whether these parameters are related to

macrohemodynamic indices, metabolic status or Sequential Organ

Failure Assessment (SOFA) score. Patients were sedated, intubated,

mechanically ventilated, and hemodynamically monitored with the

PiCCO system. Tissue oxygen saturation (StO2) was measured using

near-infrared spectroscopy (NIRS) during the vascular occlusion test

(VOT). Baseline StO2 (StO2 baseline), rate of decrease in StO2 during

VOT (StO2 downslope), and rate of its increase (StO2 upslope). Patients

with contraindications to rhaPC were used as a control group. Treatment

with activated protein C (rh-aPC) has improved muscle oxygenation

(StO2 baseline) and reperfusion (StO2 upslope) and, furthermore, rh-aPC

treatment has increased tissue metabolism (StO2 downslope) (Chapter 5).


219

Sepsis causes important microcirculatory alterations that can be observed

at the bedside at sublingual level and can be monitored with the side-dark

field (SDF) imaging and microcirculatory parameters can be calculated

off-line. We demonstrated that activated protein C significantly improves

the microcirculation in 13 patients with severe sepsis/septic shock

compared to 9 patients who did not received aPC because controindicated

(Chapter 6).

We investigated microcirculatory blood flow in patients with septic shock

treated with levosimendan (20 patients) as compared to an active

comparator drug (dobutamine, 20 patients) in a prospective, randomized,

double-blind clinical trial. The primary end point was a difference of ≥

20% in the microvascular flow index of small vessels (MFIs) among

groups. Microcirculatory flow indices of small and medium vessels

increased over time and were significantly higher in the levosimendan

group as compared to the control group (24 hrs: MFIm 3.0 (3.0; 3.0) vs.

2.9 (2.8; 3.0); P .02; MFIs 2.9 (2.9; 3.0) vs. 2.7 (2.3; 2.8); P < .001). The

relative increase of perfused vessel density vs. baseline was significantly

higher in the levosimendan group than in the control group (dMFIm 10

(3; 23)% vs. 0 (-1; 9)%; P .007; dMFIs 47 (26; 83)% vs. 10 (-3; 27); P <

.001). In addition, the heterogeneity index decreased only in the

levosimendan group (dHI -93 (-100; -84)% vs. 0 (-78; 57)%; P < .001).

There was no statistically significant correlation between systemic and

microcirculatory flow variables within each group (each P > .05).

Compared to a standard dose of 5 µg·kg-1·min-1 of dobutamine,

levosimendan at 0.2 µg·kg-1·min-1 improved sublingual

microcirculatory blood flow in patients with septic shock, (Chapter 7).

In Chapter 8 a prospective randomized trial was performed, 20 septic

patients were divided into two separate groups and received either non-

leukodepleted (n = 10) or leukodepleted (n = 10) RBC transfusions.

Microvascular density and perfusion were assessed with sidestream dark-

field (SDF) imaging sublingually, before and 1 hour after transfusions.

Thenar tissue O2-saturation (StO2) and tissue haemoglobin index (THI)

were determined with near-infrared spectroscopy (NIRS), and a vascular

occlusion test was performed. The microcirculatory perfused boundary

region was assessed in SDF images as an index of glycocalyx damage

and glycocalyx compounds (syndecan-1, hyaluronan, heparan sulfate)

were measured in the serum. No differences were observed in

microvascular parameters at baseline and after transfusion between the

groups, except for the proportion of perfused vessels (PPV) and blood

flow velocity, which were higher after transfusion in the leukodepleted


220

group. Microvascular flow index in small vessels (MFI) and blood flow

velocity exhibited different responses to transfusion between the two

groups (P = 0.03 and P = 0.04, respectively), with a positive effect of

leukodepleted RBCs. When looking at within-group changes,

microcirculatory improvement was only observed in patients that

received leukodepleted RBC transfusion as suggested by the increase in

De Backer score (P = 0.02), perfused vessel density (P = 0.04), PPV (P =

0.01) and MFI (P = 0.04). Blood flow velocity decreased in the non-

leukodepleted group (P = 0.03). THI and StO2-upslope increased in both

groups. StO2 and StO2-downslope increased in patients who received

non-leukodepleted RBC transfusions. Syndecan-1 increased after the

transfusion of non-leukodepleted RBCs (P = 0.03). In conclusion this

study does not show a clear superiority of leukodepleted over non-

leukodepleted RBC transfusions on microvascular perfusion in septic

patients, although it suggests a more favourable effect of leukodepleted

RBCs on microcirculatory convective flow. In a secondary analysis of the

previous randomized study, twenty adult septic patients received either

fresh or old (<10 or >15 days storage, respectively) RBC transfusions.

fHb was measured in RBC units and in the plasma before and 1 hour

after transfusion. Simultaneously, the sublingual microcirculation was

assessed with sidestream-dark field imaging. The perfused boundary

region was calculated as an index of glycocalyx damage. Tissue oxygen

saturation (StO2) and Hb index (THI) were measured with near-infrared

spectroscopy and a vascular occlusion test was performed. Similar fHb

levels were found in the supernatant of fresh and old RBC units. Despite

this, plasma fHb increased in the old RBC group after transfusion (from

0.125 [0.098–0.219] mg/mL to 0.238 [0.163–0.369] mg/mL, p = 0.006).

The sublingual microcirculation was unaltered in both groups, while THI

increased. The change in plasma fHb was inversely correlated with the

changes in total vessel density (r = -0.57 [95% confidence interval -0.82,

-0.16], p = 0.008), De Backer score (r = -0.63 [95% confidence interval -

0.84, -0.25], p = 0.003) and THI (r = -0.72 [95% confidence interval -

0.88, -0.39], p = 0.0003). In conlusion old RBC transfusion was

associated with an increase in plasma fHb in septic patients. Increasing

plasma fHb levels were associated with decreased microvascular density.

(Chapter 9).

It is clear the need to extend monitoring of the physiological determinants

of organ function from the macro to the micro and down to the cellular

level. However, a crucial component of this monitoring is the need to

include functional indicators of organ function because it is the


221

successful restoration of organ function that determines the success of

intensive care. These indicators of organ function need to be continuous,

specific and quantitative. The information from new sensors and

physiological variables, as well as measures of organ function, will

require a much higher level of integration than is currently available, and

mathematical models of physiology and pathophysiology are expected to

play an important role in this integration. From this perspective, these

innovations represent a challenge for industry. By integrating information

on all of the characteristics of the patient - including disease, co-

morbidities and age - into the evolution of this integrated physiological

monitoring system, we anticipate the development of an environment in

which the complete continuum of human development, as well as

diseases and their response to therapy, can be monitored. Intensive care

medicine offers a unique environment for this development, which

ultimately may be relevant to other areas of medicine (Chapter 10).

In conclusion, with this thesis we have demonstrated that monitoring

microcirculation could help physician to treat critically ill patients. With

these new techniques, we can observe in vivo the physiopathological

alterations and study the effects of therapies aiming to fix these

alterations.


222


223

Samenvatting en conclusies

Hemodynamische monitoring stelt clinici in staat om het

hartminuutvolume en de macrocirculatie aan te sturen. Desalniettemin

blijven sommige patiënten ondanks optimale macrohemodynamische

behandeling kritisch ziek. In dit proefschrift laten wij zien dat de

introductie in de klinische praktijk van de arteria pulmonaliskatheter

(PAC) met de thermodilutiemethode ongeveer 40 jaar geleden clinici in

staat stelde om het hartminuutvolume aan het bed te meten, en om andere

fysiologische parameters - zoals zuurstofaanbod (DO2) en

zuurstofverbruik (VO2)- te berekenen. De interpretatie en het gebruik

van deze parameters als therapeutische eindpunten is tot op heden

controversieel. Shoemaker was de eerste clinicus die een poging deed

om deze nieuwe hemodynamische data te interpreteren en te gebruiken

bij hoog risico chirurgische patiënten. Hij gebruikte supranormale

waarden van hartminuutvolume, DO2 en VO2 als therapeutische

eindpunten en bewerkstelligde daarmee een mortaliteitsreductie van 28%

in de controlegroep naar 4 % in de protocolgroep.

Postoperatief orgaanfalen treedt vaak op na grote buikchirurgie. Dit leidt

tot toegenomen zorgconsumptie en daarmee ook tot toegenomen kosten.

Hypoxie van de weefsels is een belangrijke uitlokkende factor van

orgaandysfunctie. Een prospectief, gerandomiseerd onderzoek met een

controlegroep werd uitgevoerd in negen Italiaanse ziekenhuizen.

Honderdvijfendertig hoog-risico patiënten die grote buikchirurgie

ondergingen werden gerandomiseerd over twee groepen. Bij alle

patiënten werden gestandaardiseerde eindpunten nagestreefd: een mean

arterial pressure > 80 mmHg en een diurese > 0.5 ml/kg/uur. Bij de

patiënten in de “controlegroep” (groep A) werd ook een O2ER <27%

nagestreefd, de andere groep (groep B) werd middels de

“standaardtherapie” behandeld. Falen van één orgaan trad in groep A

(n=8, 11.8%) minder vaak op dan in groep B (n=20, 29.8%) [p<0.05], en

het totale aantal orgaanfalen was lager in groep A dan in groep B (27

orgaanfalen versus 9 orgaanfalen, p<0.001). De opnameduur in het

ziekenhuis was significant lager in de protocolgroep dan in de

controlegroep (11.3±3.8 dagen versus 13.4±6.1 dagen, p < 0.05).

Ziekenhuismortaliteit was in beide groepen gelijk.

Vroege behandeling gericht op het handhaven van O2ER<27% reduceert

orgaanfalen en opnameduur bij hoog risico chirurgische patiënten

(Hoofdstuk 1).


224

Een prospectieve, open studie met herhaalde metingen in vijftien

patiënten met septische shock werd uitgevoerd in een medische-

chirurgische ICU. Een bolus methyleenblauw (3 mg/kg) werd intraveneus

toegediend in een periode van 10 minuten. Hemodynamische variabelen

werden gemeten voor toediening van methyleenblauw en 20 minuten, 1

uur en 2 uur na het eind van de methyleenblauwinfusie. Standaard

hemodynamische variabelen en variabelen betreffende zuurstoftransport-

en verbruik; totale, intrathoracale, systolische en diastolische cardiale

bloedvolumina; extravasculair longwater; plasmaosmolariteit en lactaat-

en eiwitconcentraties werden gemeten. Mean arterial pressure en a.

pulmonalisdruk, systemische en pulmonale vaatweerstanden, en left

ventricular stroke work index namen toe na methyleenblauw (P<0.05).

Het lactaatgehalte liet een passagère daling zien (p<0.05). De overige

gemeten variabelen veranderden niet gedurende de 2 uur na toediening

van methyleenblauw. Deze studieresultaten bevestigen de acute

vasoconstrictieve en positief inotrope effecten van methyleenblauw bij

septische shock. Deze effecten waren niet geassocieerd met

veranderingen in bloedvolume, diastolische functie van het myocard of

permeabiliteit van de longvaten (zoals uitgedrukt in extravasculair

longwater). (Hoofdstuk 2).

In de voorbije jaren bleek dat monitoren van de macrocirculatie niet

voldoende was om de prognose van patiënten te verbeteren en perfusie

van het splanchnicusgebied werd gemeten met tonometrie van de maag.

Bij 12 patiënt die buikchirurgie ondergingen, is intraoperatieve ischemie

van het splanchnicusgebied (zoals gemeten met de intramucosale pH-i in

de maag) direct gecorreleerd met de toename van IL-6 plasmaniveaus en

met de incidentie van postoperatieve complicaties, terwijl IL-8 waarden

geen correlatie met chirurgische complicaties laten zien (Hoofdstuk 3). Er

is aangetoond dat pH-i en ∆CO2 gevoelige prognostische indicatoren zijn

gedurende chirurgie vanwege een aneurysma van de abdominale aorta bij

29 patiënten. Tonometrie zou patiënten kunnen identificeren die een

verhoogde kans op postoperatief orgaanfalen hebben. De ∆ portaal

veneus lactaat is meer specifiek dan tonometrische variabelen en is een

belangrijke indicator van hypoperfusie van de darm. (Hoofdstuk 4)

Perfusie van de spieren tijdens sepsis kan in kaart worden gebracht met

near infrared spectroscopie en een vasculaire occlusietest kan worden

aangewend om weefselmetabolisme en vasculaire reactiviteit te testen in

septische patiënten. Wij testten de hypothese dat perfusie van de spieren,

oxygenatie en microvasculaire reactiviteit zouden verbeteren in patiënten

met ernstige sepsis of septische shock gedurende de behandeling met


225

geactiveerd proteïne C (rh-aPC) (n=11) en wij exploreerden of deze

parameters gerelateerd waren aan macrohemodynamische indicatoren,

metabole status of Sequential Organ Failure Assessment (SOFA) score.

Patiënten waren gesedeerd, geïntubeerd en werden mechanisch beademd.

Hemodynamische monitoring vond plaats met het PiCCO-systeem.

Zuurstofsaturatie van de weefsels (StO2) werd gemeten met near infrared

spectroscopie (NIRS) gedurende de vasculaire occlusietest (VOT). De

uitgangswaarde van de StO2 (StO2 baseline), de mate van afname in

StO2 gedurende de VOT (StO2 downslope) en de mate van toename

(StO2 upslope). Patiënten met contra-indicaties voor rh-aPC werden

gebruikt als controlegroep. Behandeling met rh-aPC heeft oxygenatie

(StO2 baseline) en reperfusie (StO2 upslope) van de spieren verbeterd.

Ook heeft rh-aPC het weefselmetabolisme verhoogd (StO2 downslope).

(Hoofdstuk 5)

Sepsis veroorzaakt belangrijke afwijkingen in de microcirculatie die aan

het bed kunnen worden waargenomen op het sublinguale niveau. Deze

afwijkingen kunnen in beeld worden gebracht met Sidestream Dark Field

(SDF) imaging en microcirculatoire parameters kunnen offline worden

berekend. Wij lieten zien dat geactiveerd proteïne C de microcirculatie in

13 patiënten met ernstige sepsis/septische shock significant verbetert in

vergelijking met 9 patiënten waarbij geen aPC werd toegediend vanwege

contra-indicaties. (Hoofdstuk 6)

Wij onderzochten microcirculatoire doorbloeding bij patiënten met

septische shock die werden behandeld met levosimendan (20 patiënten)

in vergelijking met een actief vergelijkbaar medicijn (dobutamine, 20

patiënten) in een prospectieve, gerandomiseerde, dubbelblinde klinische

studie. Het primaire eindpunt was een verschil van minstens 20 % in de

microvascular flow index van de kleine vaten (MFIs) tussen beide

groepen. De MFI van kleine en middelgrote vaten nam toe over de tijd en

was significant hoger in de levosimendangroep in vergelijking met de

controlegroep (24 uur: MFIm 3.0 (3.0; 3.0) versus 2.9 (2.8; 3.0); P .02;

MFIs 2.9 (2.9;3.0) versus 2.7 (2.3;2.8); P<.001). De relatieve toename

van de ‘perfused vessel density’(dichtheid van de geperfundeerde vaten)

in vergelijking met de uitgangswaarde was significant hoger in de

levosimendangroep dan in de controlegroep (dMFIm 10 (3; 23)% versus

0 (-1; 9)%; P .007; dMFIs 47 (26;83)% versus 10 (-3; 27); P<.001).

Daarnaast nam de heterogeniciteitsindex alleen af in de

levosimendangroep (dHI -93 (-100;-84)% versus 0 (-78; 57)%; P<.001).

Er was geen statistisch significante correlatie tussen systemische en


226

microcirculatoire perfusievariabelen in elke groep. In vergelijking met

dobutamine in een standaarddosis van 5 µg·kg-1·min-1, verbeterde

levosimendan in een dosering van 2 µg·kg-1·min-1 de doorbloeding van

de sublinguale microcirculatie bij patiënten met septische shock.

(Hoofdstuk 7)

In hoofdstuk 8 werd een prospectieve gerandomiseerde studie uitgevoerd.

Twintig septische patiënten werden verdeeld over twee verschillende

groepen en kregen respectievelijk non-leukodeplete (n = 10) of

leukodeplete (n = 10) erytrocytentransfusies toegediend. Microvasculaire

dichtheid en perfusie werden beoordeeld met sublinguale Sidestream

Dark Field (SDF) imaging voor en 1 uur na de transfusies. Weefsel O2-

saturatie (StO2) van de duimmuis en weefsel hemoglobine index (THI)

werden bepaald met near-infrared spectroscopie (NIRS), en een

vasculaire occlusietest werd uitgevoerd. De microcirculatoire ‘perfused

boundary region’ werd op de SDF beelden beoordeeld als een indicator

voor beschadiging van de glycocalyx en bestanddelen van de glycocalyx

(syndecan-1, hyaluronan, heparansulfaat) werden gemeten in het serum.

Er werden geen verschillen gezien in microvasculaire parameters voor en

na transfusie in beide groepen, met uitzondering van de ‘proportion of

perfused vessels’ (PPV) en de perfusiesnelheid, welke hoger waren na

transfusie in de leukodeplete groep. Microvascular flow infex (MFI) van

de kleine vaten en de perfusiesnelheid vertoonden verschillende reacties

op transfusie in de twee groepen (respectievelijk P = 0.03 en P= 0.04),

met een positief effect van leukodeplete erytrocytentransfusies. Wanneer

gekeken wordt naar veranderingen binnen beide groepen, dan wordt

gezien dat verbetering van de microcirculatie alleen optreedt in patiënten

die leukodeplete erythrocytentransfusies toegediend kregen zoals werd

gesuggereerd door de toename in de De Backer score (P = 0.02),

‘perfused vessel density’(P=0.04), PPV (P=0.01) en MFI (P=0.04). De

perfusiesnelheid nam af in de non-leukodeplete groep (P=0.03). THI en

StO2-upslope nam toe in beide groepen. StO2 en StO2-downslope namen

toe in patiënten die non-leukodeplete erytrocytentransfusies toegediend

kregen. Syndecan-1 nam toe na de transfusie van non-leukodeplete

erytrocytentransfusies (P=0.03). Concluderend toont deze studie geen

duidelijke superioriteit aan van leukodeplete erytrocytentransfusies in

vergelijking met non-leukodeplete erytrocytentransfusies op

microvasculaire perfusie in septische patiënten, hoewel deze studie wel

suggereert dat er een gunstiger effect is van leukodeplete

erytrocytentransfusies op convectieve doorbloeding van de

microcirculatie. In een secundaire analyse van voornoemde


227

gerandomiseerde studie, kregen twintig volwassen septische patiënten

ofwel verse ofwel oude (respectievelijk <10 of > 15 dagen bewaard)

erytrocytentransfusies. fHb werd gemeten in het getransfundeerde bloed

en in het plasma voor en 1 uur na transfusie. De sublinguale

microcirculatie werd gelijktijdig in kaart gebracht met sidestream dark

field imaging. De ‘perfused boundary region’ werd berekend als een maat

voor beschadiging van de glycocalyx. Zuurstofsaturatie van de weefsels

(StO2) en Hb index (THI) werden gemeten met near infrared

spectroscopie en een vasculaire occlusietest werd uitgevoerd. Gelijke

fHb-waarden werden gemeten in het supernatant van verse en oude

erytrocytentransfusies. Desondanks werd een toename in fHb in het

plasma gemeten bij patiënten die oude erytrocytentransfusies toegediend

kregen (van 0.125 [0.098-0.219] mg/ml tot 0.238 [0.163-0.369] mg/ml, p

= 0.006). De sublinguale microcirculatie was onveranderd in beide

groepen, terwijl THI toenam. De verandering in plasma fHb was

omgekeerd evenredig gecorreleerd aan de veranderingen in de totale

vaatdichtheid ( r = -0.57 [95% betrouwbaarheidsinterval -0.82, -0.16, p =

0.008), De Backer (r = -0.63 [95% betrouwbaarheidsinterval -0.88, -

0.39], p = 0.003). Concluderend waren oude erytrocytentransfusies

geassocieerd met een toename in plasma fHb bij septische patiënten.

Gestegen plasma fHb-waarden waren geassocieerd met een afgenomen

microvasculaire dichtheid. (Hoofdstuk 9)

Het is duidelijk dat er een noodzaak is om de monitoring van

fysiologische determinanten van orgaanfunctie uit te breiden van macro

naar micro tot op celniveau. Een cruciale component van deze

monitoring is de noodzaak om functionele indicatoren van orgaanfunctie

hierin mee te nemen omdat het succesvolle herstel van orgaanfunctie de

goede afloop van intensive care bepaalt. Deze indicatoren van

orgaanfunctie dienen continu, specifiek en kwantitatief te zijn. De

informatie van nieuwe sensoren en fysiologische variabelen, evenals

maten van orgaanfunctie, zullen een veel hoger niveau van integratie

vereisen dan dat momenteel gebruikelijk is. Het valt te verwachten dat

mathematische modellen van fysiologie en pathofysiologie een

belangrijke rol zullen spelen bij deze integratie. Vanuit dit perspectief

bekeken zijn deze innovaties een uitdaging voor de industrie. Door

integratie van informatie over alle patiëntkarakteristieken - onder meer

ziekte, co-morbiditeit en leeftijd- in de ontwikkeling van dit model van

geïntegreerde fysiologische monitoring, verwachten wij een omgeving te

ontwikkelen waarin het complete continuüm van de humane

ontwikkeling, evenals ziekten en de respons op behandeling, kan worden


228

gemonitord. Intensive care geneeskunde is een unieke omgeving voor

deze ontwikkeling, die uiteindelijk ook relevant zou kunnen zijn voor

andere gebieden binnen de geneeskunde. (Hoofdstuk 10)

Concluderend hebben wij met dit proefschrift laten zien dat monitoring

van de microcirculatie artsen behulpzaam kan zijn bij het behandelen van

kritisch zieke patiënten. Met deze nieuwe technieken kunnen wij in vivo

fysiopathologische veranderingen observeren en kunnen wij de effecten

bestuderen van behandelingen gericht op het verbeteren van deze

afwijkingen.

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• Ince C, van Kuijen A, Milstein D, Yuruk K, Folkow L, Fokkens W, Blix A: Why Rudolph's nose is red. Br Med J 2012, 345:e8311.

• Boerma EC, van der Voort PHJ, Spronk PE, Ince C: Relationship between sublingual and intestinal microcirculatory perfusion in patients with abdominal sepsis. Crit Care Med 2007, 35:1055-1060.

• Dubin A, Pozo MO, Casabella CA, Pálizas F Jr, Murias G, Moseinco MC, Kanoore Edul VS, Pálizas F, Estenssoro E, Ince C: Increasing arterial blood pressure with norepinephrine does not improve microcirculatory blood flow: a prospective study. Crit Care 2009, 13:R92.

• Pottecher J, Deruddre S, Teboul JL, Georger JF, Laplace C, Benhamou D, Vicaut E, Duranteau J: Both passive leg raising and intravascular volume expansion improve sublingual microcirculatory perfusion in severe sepsis and septic shock patients. Intensive Care Med 2010, 36:1867-1874.

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• Sakr Y, Dubois MJ, De Backer D, Creteur J, Vincent JL: Persistent microcirculatory alterations are associated with organ failure and death in patients with septic shock. Crit Care Med 2004, 32:1825-1831.

• Edul VS, Enrico C, Laviolle B, Vazquez AR, Ince C, Dubin A: Quantitative assessment of the microcirculation in healthy volunteers and in patients with septic shock. Crit Care Med 2012, 40:1443-1448.

• Top AP, Ince C, de Meij N, van Dijk M, Tibboel D: Persistent low microcirculatory vessel density in non survivors of sepsis in the pediatric intensive care. Crit Care Med 2011, 39:8-13.

• Boerma EC, Koopmans M, Konijn A, Kaiferova K, Bakker AJ, van Roon EN, Buter H, Bruins N, Egbers PH, Gerritsen RT, Koetsier PM, Kingma P, Kuiper MA, Ince C: Effects of nitroglycerin on sublingual microcirculatory blood flow in patients with severe sepsis/septic shock after a strict resuscitation protocol: a double-blind randomised placebo controlled trial. Crit Care Med 2009, 38:93-100.

• Morelli A, Donati A, Ertmer C, Rehberg S, Lange M, Orecchioni A, Cecchini V, Landoni G, Pelaia P, Pietropaoli P, Van Aken H, Teboul JL, Ince C, Westphal M: Levosimendan for resuscitating the microcirculation in patients with septic shock: a randomized controlled study. Crit Care 2010, 14:R232.

• Morelli A, Donati A, Ertmer C, Rehberg S, Kampmeier T, Orecchioni A, Di Russo A, D'Egidio A, Landoni G, Lombrano MR, Botticelli L, Valentini A, Zangrillo A, Pietropaoli P, Westphal M: Effects of vasopressinergic receptor agonists on sublingual microcirculation in norepinephrine-dependent septic shock. Crit Care 2011, 15:R217.

• Yuruk K, Almac E, Bezemer R, Goedhart P, de Mol B, Ince C: Blood transfusions recruit the microcirculation during cardiac surgery. Transfusion 2010, 51:961-967.

• Dubin A, Pozo MO, Casabella CA, Murias G, Pálizas F Jr, Moseinco MC, Kanoore-Edul VS, Pálizas F, Estenssoro E, Ince C: Comparison of 6% hydroxyethyl starch 130/0.4 and saline solution for resuscitation of the microcirculation during the early goal-directed therapy of septic patients. J Crit Care 2010, 25:659.e1-e8.

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• De Backer D, Hollenberg S, Boerma C, Goedhart P, Büchele G, Ospina-Tascon G, Dobbe I, Ince C: How to evaluate the microcirculation? Report of a round table conference. Crit Care 2007, 11:R101-R111.

• Ospina-Tascon G, Neves AP, Occhipinti G, Donadello K, Büchele G, Simion D, Chierego ML, Silva TO, Fonseca A, Vincent JL, De Backer D: Effects of fluids on microvascular perfusion in patients with severe sepsis. Intensive Care Med 2010, 36:949-955.

• Boerma EC, van der Voort PHJ, Ince C: Sublingual microcirculatory flow is impaired by the vasopressin-analogue terlipressin in a patient with catecholamine-resistant septic shock. Acta Anaesth Scand 2005, 49:1387-1390.

• Top AP, Tasker RC, Ince C: The microcirculation of the critically ill pediatric patient. Crit Care 2011, 15:213.

• Top AP, van Dijk M, van Velzen JE, Ince C, Tibboel D: Functional capillary density decreases after the first week of life in term neonates. Neonatology 2011, 99:73-77.

• Paize F, Sarginson R, Makwana N, Baines PB, Thomson APJ, Sinha I, Hart CA, Riordan A, Hawkins KC, Carrol ED, Parry CM: Changes in the sublingual microcirculation and endothelial adhesion molecules during the course of severe meningococcal disease treated in the paediatric intensive care unit. Intensive Care Med 2012, 38:863-871.

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• Yuruk K, Bartels SA, Milstein DM, Bezemer R, Biemond BJ, Ince C: Red blood cell transfusions and tissue oxygenation in anemic hematology outpatients. Transfusion 2011, 52:641-646.

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269

Acknowledgment

To my wife Donatella, for her love and for every kind of supports she has

given to me during these years

To Martina, Nicolò and Gioele

To my father and my mother

To Prof. Paolo Pietropaoli because he instilled in me the seed of the

research

To Prof. Paolo Pelaia for his support, for having allowed me to do

clinical research and having introduced in the university team

To all the young doctors and residents that have helped me to do

research, in particular Andrea, Claudia, Elisa and Roberta.

Finally, last but not least, to Prof. Can Ince, for having introduced me in

the “microcirculatory world”, for his help and support and for this Thesis

Curriculum Vitae

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Curriculum Vitae

271

Curriculum vitae

Master Degree in Medicine and Surgery on the 30th of October 1987 with honors at the University of Ancona.

Specialization in Anesthesia and Intensive Care July 1990 at the University of Ancona.

Registered at Italian Medical Association since January 1988.

Job

Assistant and then consultant at the University Anesthesiology and Intensive Care Unit of the Regional Hospital Torrette of Ancona from 01/02/1990 to 31/10/2008.

From November the 1st, 2008 Researcher at the Polytechnic University of Marche, Faculty of Medicine, (MED/41, Anesthesiology) agreement with the Azienda Ospedaliera-Universitaria Ospedali Riuniti, Ancona, at the SOD of Anaesthesia and Intensive Care.

From November the 1st, 2011 to present, Associate Professor of Anesthesiology at the Polytechnic University of Marche, Faculty of Medicine.

Teaching

1996 to present Professor at the School of Anesthesia and Intensive Care and Professor of Anaesthesia and Intensive Care for the Master Degree in Nursing since 2009 to present.

Research

His fields of interest have been since from the beginning hemodynamic monitoring and sepsis. He published 63 papers on these arguments and in the last years he started to monitor microcirculation in septic patients with side-dark field technique and after with incident-dark field technique. He has been part of the steering committee in the microSOAP study.

Portfolio

Name PhD student: Abele Donati

PhD period: December 2008-December 2015

Name PhD supervisor: Prof.Dr. Can Ince

Curriculum Vitae

272

PhD Training

• Training in the analysis of microcirculation at The Laboratory of Translational Physiology at the Academic Medical Center, Amsterdam, in December 2008.

• Project management of clinical trials including analysis and interpretation of microcirculation in different clinical setting, under the supervision of Prof. Can Ince.

Scientific Director of the following Congresses and Courses.

• Basic hemodynamic monitoring, Ancona April 13, 2012

• Sepsis Highlights 2012, Ancona, November 27, 2012

• Hemodynamic monitoring, Ancona May 13, 2013



• Hemodynamic monitoring, Ancona May 24, 2015


Invited Oral Presentations (from December 2008 at present)

• SMART 2009 Congress held in Milan on 6-8 May 2009, with a presentation entitled: Xigris: clinical effects on microcirculation

• Meeting "The recombinant activated protein C: what have we learned in 10 years", held in Milan September 22, 2010, with a presentation entitled: Resuscitation macrocircolatoria and microcirculatory the patient in septic shock: How-monitoring and therapy.

• SIAARTI Congress held in Parma 13 to 16 October 2010 with the presentation entitled: Tissue Oxygen Saturation

• Lectures "Microcirculation in Sepsis", held in Valencia, Spain, Hospital Clinico de Valencia, 17 December 2010

• New Star Congress 2011, Rome 16 to 18 March 2011, presentation entitled: Sepsis and microcirculation

• Congress: Fluid Challenge: lights and shadows, held in Ancona September 29, 2012, with a presentation entitled: Semiotics and microcirculation in shock.

• Symposium of Microcirculation and Glycocalix, Lisbon October 16, 2012, with a presentation entitled: Transfusion of different types of red blood cells in septic patients: effects on microcirculation and Glycocalyx

• 67° SIAARTI Congress, Roma 16-19 October 2013. Normobaric oxygen paradox and microcirculation.

Curriculum Vitae

273

• Pisa 23th of May 2014: Glycocalix alteration in critically ill patients.

• SMART Educational: “Gestione delle Infezioni in Terapia Intensiva: dalla Prevenzione al Buon Uso degli Antibiotici.” in SMART Congress, Milan, 27 May 2014. Presentation: Terapie aggiuntive: cosa ci rimane.

• Forlì, 26th of September 2014. Inquadramento patogenetico ed early goal therapy.

• 69° SIAARTI Congress, Venezia 22-25 October 2014. Measuring and manage the microcirculatin in sepsis.

• SMART Educational: “Gestione delle Infezioni in Terapia Intensiva: dalla Prevenzione al Buon Uso degli Antibiotici.” in SMART Congress, Milan, 27 May 2014. Presentation: Il microcircolo: come cambia il mio approccio?

• Modena 7th of March 2015: Blood purifications for sepsis.

• SIAARED Congress, Riva del Garda, 13th of May 2015: La MAP, qual’è il numero magico?

• 70° SIAARTI congress, Bologna 14-17 October 2015, Presentation: il microcircolo è ancora il motore della disfunzione d’organo nella sepsi?

List of Publications

274


275


1. Pietropaoli P, Valente M, De Pace F, G Sambo, Donati A, Adrario E, Giovannini C: Validity and limits of SvO2 in

intensive care. Minerva Anestesiol 1991, vol. 57 suppl. 1 to No. 12, 127-32

2. Donati A, Teddy G, Bini G, Luzi A, Valente M, Giovannini C, De Ritis GC, Pietropaoli P: Validity of 'index V / Q in the monitoring of critically ill patients Minerva Anestesiol 1992, vol. 58, No. 1-2, 707-713.

3. Pietropaoli P, Valente M, Martorano P, Mancinelli G, Donati A, Tanara L: Incidence and significance of lesions associated to outcome of TBI. Minerva Anestesiol 1995, 61, Suppl 1 to No. 9, 167-169.

4. A Donati et al.: The COLD in septic shock. Minerva Anestesiol 1999, 65 suppl. 2.

5. Pietropaoli P Caporelli S, De Pace F, Donati A, Adrario E, Luzi A, Munch C, Giovannini C, Frezzotti AR Intraoperative lactic acidosis, can it be treated? Clinico-experimental, prospective, sequential study. Minerva Anestesiol 1994 Dec; 60 (12) :707-13.

6. Donati A, Cola L, Danieli R, Munch C, Mancinelli G, Achilli A, Pietropaoli P: Prediction of outcome in critically ill patients using oxyphoretic and haemodynamic parameters. Minerva Anestesiol 1996; 62 (7-8) :243-8.

7. Donati A, Cola L, Danieli R, Adrario And Givoannini C, Pietropaoli P: Predictability associated with hemodynamic parameters ossiforetici: relationship between cardiac index and oxygen extraction. Minerva Anestesiol 1995, vol.61: 241-7.

8. Donati A, Battisti D, Recchioni A, Paoletti P, Conti G, S Caporelli, Adrario And Pelaia P, Pietropaoli P: Predictive value of interleukin 6 (IL-6), interleukin 8 (IL-8) and gastric intramucosal pH ( pH-i) in major abdominal surgery. Intensive Care Med 1998, 24:329-335.

9. Donati A, Valente M, Münch C, Gabbanelli V, Montozzi A, Pietropaoli P: Mathematical Model for predicting outcome in patients in ICU patients articulated dul trend dell'Apache II score and the disease causes of hospitalization. Minerva Anestesiol 1998, vol.64: 271-9.

10. A Donati et al.: Hemodynamic modifications after anesthesia subaracnoid Evaluated with Transthoracic echocardiography. APEX 1998.

11. Donati A, Münch C, Marini B, Teddy G, Coltrinari R, Pietropaoli P: Transesophageal Doppler ultrasonography evaluation of


276

hemodynamic changes videolaparoscopy During cholecystectomy. Minerva Anestesiol 2002, 68, 6: 549-554

12. Donati A, Conti G, Loggi S, Münch C, Coltrinari R, Pelaia P, Pietropaoli P, Preiser JC.: Does methylene blue administration to septic shock patients affect vascular permeability and blood volume? Crit Care Med 2002, 30:2271-7.

13. Donati A, S Loggi, Pelaia P, Pietropaoli P, Preiser JC: Author reply to: Methylene blue, a renaissance of an old drug?. Crit Care Med 2003, 31:1602.

14. Donati A, Gabbanelli V, Nataloni S, S Pantanetti, Principles P, R Natalini, Pelaia P: Improving the quality of care in an intensive care unit using a clinical information system. Minerva Anestesiol 2003, 69 (Suppl 1-9) :103-105.

15. Donati A, S Loggi, Coltrinari R, G Sambo, Carletti P, Marini B, Pelaia P: Swan-Ganz catheter. Minerva Anestesiol 2003 (Suppl 1-9): 309-313.

16. Donati A, Gabbanelli V, Pantanetti S, Scala C, Carbini C, Valentini, Antognini M, Pelaia P, P Pietropaoli: To verify four 5-year-old mathematical models to predict the outcome of ICU patients. Minerva Anestesiol 2003,69:897-905.

17. Donati A, S Loggi, Coltrinari R, P Pelaia: intrathoracic blood volume as index of cardiac output variations. Acta Anaesthesiol Scand. 2004, 48:386-7.

18. Pietropaoli P, Donati A. Clinical information systems and quality of care in intensive care medicine: state of the art. Minerva Anestesiol 2004; 70:521-3.

19. Gabbanelli V, Pantanetti S, Donati A, Montozzi A, C Carbini, Pelaia P. Initial distribution volume of glucose as noninvasive indicator of cardiac preload: comparison with intrathoracic blood volume.Intensive Care Med 2004 Nov; 30:2067-73.

20. Donati A, Cornacchini O, S Loggi, Caporelli S, Conti G, Falcetta S, Alo F, Pagliariccio G, Brown E, Preiser JC, P. Pelaia A comparison among portal lactate, intramucosal sigmoid Ph, and deltaCO2 (PaCO2 - regional PCO2) as indices of complications

in patients undergoing abdominal aortic aneurysm surgery. Anesth Analg. 2004, 99:1024-31.

21. Donati A, Ruzzi M, Adrario And Pelaia P, Coluzzi F, V Gabbanelli, P. Pietropaoli A new and feasible model for predicting operative risk. Br J Anaesth. 2004 93:393-9.

22. Gabbanelli V; Pantanetti S, Donati A, et al. Initial distribution volume of glucose as noninvasive indicator of cardiac preload: comparison with intrathoracic blood volume. Intensive Care Med 2004; 30:2067-2073


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23. Donati A, Mercuri G, S Iuorio, Sinkovetz L, M Scarcella, C Trabucchi, Pelaia P, Pietropaoli P. Haemodynamic modifications after unilateral subarachnoid anesthesia Evaluated with Transthoracic echocardiography. Minerva Anestesiol 2005; 71:75-81.

24. Gabbanelli V; Pantanetti S, Donati A, Principi T; Pelaia P. Correlation between hyperglycemia and mortality in a medical and surgical Intensive Care Unit (ICU). Minerva Anestesiol. 2005, 71 (11) :717-25.

25. Donati A. About global volume-related hemodynamic variables and outcome.Crit Care Med 2006.34 (5): 1585.

26. Donati A, Preiser JC. Methylene blue: an old-timer or a compound ready for revival? Crit Care Med 2006, 34 (11) :2862-3.

27. Nataloni S, Gabbanelli V, Rossi R, Donati A, Pantanetti S, P. Pelaia Successful voriconazole treatment of Aspergillus infection early in two immunocompromised patients not in Intensive Care Unit. Minerva Anestesiol. 2007, 73 (6) :371-5.

28. Donati A, Romanelli M, Romagnoli L, L Botticelli, Beato V, Pelaia P. The monitoring of peripheral perfusion during sepsis. Minerva Anestesiol 2007, 73 (Suppl 1 to 10) :203-5.

29. Donati A, Scarcella M, Nardella R, Zompanti V, Sinkovetz L, S Iuorio, Pelaia P. Fluid challenge in patients Submitted to spinal block. Minerva Anestesiol. 2007, 73 (4) :213-8.

30. Donati A, Loggi S, Preiser JC, Teddy G, Münch C, Gabbanelli V, Pelaia P, Pietropaoli P. Goal-directed intraoperative therapy Reduces morbidity and length of hospital stay in high-risk surgical patients. Chest. 2007, 132 (6) :1817-24.

31. Donati A, Gabbanelli V, Pantanetti S, Carletti P, Principi T, Marini B, Nataloni S, G Sambo, P. Pelaia The Impact of a Clinical Information System in an Intensive Care Unit. J Clin Monit Comput, 2008; 22:31.

32. Donati A, Preiser JC. Early Goal-Directed Therapy is a therapeutic strategy to reduce morbidity and length of hospital stay in high-risk surgical patients. Chest 2008, 134:215-216.

33. Donati A, Preiser JC. Corticosteroids and septic shock: a new episode of a never-ceasing story? Crit Care Med 2008 May; 36:1658-9

34. Donati A, Nardella R, Gabbanelli V, Scarcella M, Romanelli M, Romagnoli L, Botticelli L, S Pantanetti, Pelaia P. The ability of PiCCO versus Lidco variables to detect changes in cardiac index: a prospective clinical study. Minerva Anestesiol 2008; 74:367-74.


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35. Principi T, G Falzetti, Elisei D, Donati A, Pelaia P. Behavior of B-type natriuretic peptide During mechanical ventilation and spontaneous breathing after extubation. Minerva Anestesiol. 2009, 75:179-83.

36. Rossi A, Falzetti G, Donati A, Pelaia P. Desflurane versus Sevoflurane to reduce blood loss in maxillofacial surgery. J Oral Maxillofac Surg 2010;1007-1012. 68

37. Cruz DN, Antonelli M, Fumagalli R, Foltran F, Brienza N, Donati A, Malcangi V, Petrini F, Volta G, Bobbio Pallavicini FM, Rottoli F, Giunta F, Ronco C. Early use of polymyxin B hemoperfusion in abdominal septic shock: the EUPHAS randomized controlled trial. JAMA. 2009 Jun 17;301(23):2445-52.

38. A. Morelli,A. Donati,C. Ertmer,S. Rehberg,M. Lange,A. Orecchioni,V. Cecchini,G. Landoni,P. Pelaia,P. Pietropaoli,H. V. Aken,J. Teboul,C. Ince,M. Westphal (2010). Levosimendan for resuscitating the microcirculation in patients with septic shock: a randomized controlled study.. CRITICAL CARE (ISSN:1466-609X). R232- 14

39. A. Donati,E. Adrario,P. Pelaia,J. Preiser (2010). Methylene blue as the future protecting agent for ischemic brain injury?. CRITICAL CARE MEDICINE (ISSN:0090-3493). 2265- 2266. 38

40. A. Donati,E. Adrario,P. Pelaia,J. Preiser (2010). Disorder of osmoregulation as a new pathogenetic mechanism of septic shock?. CRITICAL CARE MEDICINE (ISSN:0090-3493). 2068- 2069. 38

41. Morelli,A. Donati,C. Ertmer,S. Rehberg,A. Orecchioni,A. D. Russo,P. Pelaia,P. Pietropaoli,M. Westphal (2011). Short-term effects of terlipressin bolus infusion on sublingual microcirculatory blood flow during septic shock.. INTENSIVE CARE MEDICINE (ISSN:1432-1238). 963- 969. 2011 Jun;37(6)

42. Donati, P. Pelaia, P. Pietropaoli, J.C. Preiser (2011). Do use ScvO2 and O2ERe as therapeutical goals.. MINERVA

ANESTESIOLOGICA (ISSN:1827-1596). 483- 484. 77/2011 43. Morelli,A. Donati,C. Ertmer,S. Rehberg,T. Kampmeier,A.

Orecchioni,A. D. Russo,A. D'Egidio,G. Landoni,M. R. Lombrano,L. Botticelli,A. Valentini,A. Zangrillo,P. Pietropaoli,M. Westphal (2011). Effects of vasopressinergic receptor agonists on sublingual microcirculation in norepinephrine-dependent septic shock.. CRITICAL CARE (ISSN:1364-8535). R217- 15


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44. S. Falcetta,L. Pecora,G. Orsetti,P. Gentili,A. Rossi,V. Gabbanelli,E. Adrario,A. Donati,P. Pelaia (2012). The Bonfils fiberscope: a clinical evaluation of its learning curve and efficacy in difficult airway management.. MINERVA ANESTESIOLOGICA (ISSN:0375-9393). 176- 184. 78(2) 2012

45. N. A. R,E. C. Boerma,M. Koopmans,A. Donati,A. Dubin,N. I. Shapiro,R. M. Pearse,J. Bakker,C. Ince (2012). Study Design of the Microcirculatory Shock Occurrence in Acutely Ill Patients (microSOAP): an International Multicenter Observational Study of Sublingual Microcirculatory Alterations in Intensive Care Patients.. CRITICAL CARE RESEARCH AND PRACTICE (ISSN:2090-1305) p. 121752 - Vol. 2012

46. Nataloni,A. Carsetti,V. Gabbanelli,A. Donati,E. Adrario,P. Pelaia (2013). A rare case of central venous catheter malpositioning in polytraumatic patient not recognized by chest x-ray.. JOURNAL OF VASCULAR ACCESS (ISSN:1129-7298) p. 97 - 98 Vol. 14

47. A. Donati, E. Damiani, L. Botticelli,E. Adrario,M. R. Lombrano,R. Domizi,B. Marini,J. W. Van,P. Carletti,M. Girardis,P. Pelaia,C. Ince (2013). The aPC treatment improves microcirculation in severe sepsis/septic shock syndrome.. BMC ANESTHESIOLOGY (ISSN:1471-2253) p. 25 - Vol. 13

48. A. Morelli, A. Donati, C. Ertmer,S. Rehberg,T. Kampmeier,A. Orecchioni,A. D'Egidio,V. Cecchini,G. Landoni,P. Pietropaoli,M. Westphal,M. Venditti,A. Mebazaa,M. Singer (2013). Microvascular Effects of Heart Rate Control With Esmolol in Patients With Septic Shock: A Pilot Study*. CRITICAL CARE MEDICINE (ISSN:0090-3493) p. 2162 - 2168 Vol. 41

49. Biagioni Emanuela; Venturelli Claudia; Klein David J; Buoncristiano Marta; Rumpianesi Fabio; Busani Stefano; Rinaldi Laura ; Donati Abele, Girardis Massimo (2013). ENDOTOXIN ACTIVITY LEVELS AS A PREDICTION TOOL FOR RISK OF DETERIORATION IN PATIENTS WITH SEPSIS NOT ADMITTED TO THE ICU: A PILOT OBSERVATIONAL STUDY . JOURNAL OF CRITICAL CARE (ISSN:0883-9441) p. 612 - 617 Vol. 28

50. Abele Donati, Roberta Domizi, Elisa Damiani,Erica Adrario, Paolo Pelaia, Can Ince (2013). From macrohemodynamic to the microcirculation . CRITICAL CARE RESEARCH AND PRACTICE (ISSN:2090-1313) Vol. 2013

51. Abele Donati, Dick Tibboel and Can Ince (2013). Towards integrative physiological monitoring of the critically ill: from cardiovascular to microcirculatory and cellular function


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monitoring at the bedside. CRITICAL CARE (ISSN:1466-609X) Vol. 17

52. S. Falcetta,L. Pecora,G. Orsetti,P. Gentili,A. Rossi,V. Gabbanelli,E. Adrario,A. Donati,P. Pelaia (2013). Is Trachlight really better than the Bonfils fibrescope?. ACTA ANAESTHESIOLOGICA SCANDINAVICA (ISSN:1399-6576) p. 529 - Vol. 57

53. A. Donati,E. Damiani,R. Domizi,R. Romano,E. Adrario,P. Pelaia,C. Ince,M. Singer (2013). Alteration of the sublingual microvascular glycocalyx in critically ill patients.. MICROVASCULAR RESEARCH Nov;90:86-9. (ISSN:0026-2862)

54. Donati A, Damiani E, Domizi R, Botticelli L, Castagnani R, Gabbanelli V, Nataloni S, Carsetti A, Scorcella C, Adrario E, Pelaia P, Preiser JC. (2014) Glycaemic variability, infections and mortality in a medical-surgical intensive care unit. Crit Care Resusc. Mar;16(1):13-23.

55. Donati A, Carsetti A, Tondi S, Scorcella C, Domizi R, Damiani E, Gabbanelli V, Münch C, Adrario E, Pelaia P, Cecconi M (2014). Thermodilution vs pressure recording analytical method in hemodynamic stabilized patients. J Crit Care. Apr;29(2):260-4. doi: 10.1016/j.jcrc.2013.11.003.

56. Damiani E, Adrario E, Girardis M, Romano R, Pelaia P, Singer M, Donati A (2014). Arterial hyperoxia and mortality in critically ill patients: a systematic review and meta- nalysis. Crit Care. Dec 23;18(6):711. doi: 10.1186/s13054-014-0711-x.

57. Donati A, Damiani E, Adrario E, Romano R, Pelaia P. (2014). Pain and discomfort management during central venous catheter insertion. Indian J Crit Care Med. Jul;18(7):417-8. doi: 10.4103/0972-5229.136066. No abstract available.

58. Donati A, Damiani E, Domizi R, Botticelli L, Castagnani R, Gabbanelli V, Nataloni S, Carsetti A, Scorcella C, Adrario E, Pelaia P, Preiser JC. (2014). Glycaemic variability, infections and mortality in a medical-surgical intensive care unit. Crit Care Resusc. Mar;16(1):13-23.

59. Donati A, Damiani E, Luchetti M, Domizi R, Scorcella C, Carsetti A, Gabbanelli V, Carletti P, Bencivenga R, Vink H, Adrario E, Piagnerelli M, Gabrielli A, Pelaia P, Ince C. (2014). Microcirculatory effects of the transfusion of leukodepleted or non-leukodepleted red blood cells in patients with sepsis: a pilot study. Crit Care. Feb 17;18(1):R33. doi: 10.1186/cc13730.

60. Vellinga NA, Boerma EC, Koopmans M, Donati A, Dubin A, Shapiro NI, Pearse RM, Machado FR, Fries M, Akarsu-Ayazoglu


281

T, Pranskunas A, Hollenberg S, Balestra G, van Iterson M, van der Voort PH, Sadaka F, Minto G, Aypar U, Hurtado FJ, Martinelli G, Payen D, van Haren F, Holley A, Pattnaik R, Gomez H, Mehta RL, Rodriguez AH, Ruiz C, Canales HS, Duranteau J, Spronk PE, Jhanji S, Hubble S, Chierego M, Jung C, Martin D, Sorbara C, Tijssen JG, Bakker J, Ince C; microSOAP Study Group. (2015). International study on microcirculatory shock occurrence in acutely ill patients. Crit Care Med. Jan;43(1):48-56. doi: 10.1097/CCM.0000000000000553.

61. Boncagni F, Francolini R, Nataloni S, Skrami E, Gesuita R, Donati A, Pelaia P. Epidemiology and clinical outcome of Healthcare-Associated Infections: a 4-year experience of an Italian ICU. (2015) Minerva Anestesiol. 81(7),765-775

62. Damiani E, Adrario E, Luchetti MM, Scorcella C, Carsetti A, Mininno N, Pierantozzi S, Principi T, Strovegli D, Bencivenga R, Gabrielli A, Romano R, Pelaia P, Ince C, Donati A (2015). Plasma free hemoglobin and microcirculatory response to fresh or old blood transfusions in sepsis. PLoS One. 2015 May 1;10(5):e0122655. doi: 10.1371/journal.pone.0122655. eCollection 2015.

63. Damiani E, Pierpaoli E, Orlando F, Donati A, Provinciali M. (2015) Sidestream dark field videomicroscopy for in vivo evaluation of vascularization and perfusion of mammary tumours in HER2/neu transgenic mice. Clin Exp Pharm Physiol. 42 (2), 225-229.

64. Damiani E , Donati A, Serafini G, Rinaldi L, Adrario E, Pelaia P, Busani S, Girardis M. Effect of performance improvement programs on compliance with sepsis bundles and mortality: A systematic review and meta-analysis of observational studies (2015). PLoS ONE. 6 May 2015; 10(5): e0125827.

65. Donati A, Carsetti A, Damiani E, Adrario E, Romano R, Pelaia P (2015). Fluid responsiveness in critically ill patients. Indian J Crit Care Med 2015;19:375-6.

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