Introduction
Organ failure contributes cumulatively to mortality in patients with sepsis [
1]. One of the mechanisms that is believed to contribute to the pathogenesis of organ failure in sepsis is microvascular thrombosis [
2‐
5]. Pathways involved in the prothrombotic state of critically ill patients include tissue factor-mediated thrombin generation and impaired anticoagulant and fibrinolytic mechanisms [
6]. Continuing consumption of platelets and coagulation factors may cause overt disseminated intravascular coagulation and carry risk for bleeding diathesis [
7]. In severe inflammation, coagulation-regulating systems appear to be defective, primarily as a result of endothelial dysfunction [
8,
9]. However, the effect of anticoagulant therapies on outcome is controversial [
10‐
13].
In clinical practice, routinely performed blood coagulation tests only incompletely mirror sepsis-induced coagulation abnormalities, and hypercoagulation in particular is not detected. More advanced laboratory analyses or experimental methods to monitor coagulation in critically ill patients, including tissue factor levels [
14], prothrombin fragments F
1 and F
2 [
15], thrombin-antithrombin (TAT) [
16,
17], and thrombomodulin expression [
18], have not yet been introduced into routine clinical management.
End-points of routine coagulation tests occur early in the hemostatic process, whereas thromboelastography measures the viscoelastic characteristics of blood clot formation in a whole blood assay, and it may therefore provide additional information on coagulation. Thromboelastography assesses the influence of plasmatic factors and platelets during all phases of the coagulation process. Thus, it permits one to evaluate the initiation of coagulation, the propagation of clot formation, and the final firmness of the blood clot.
Thromboelastography has gained importance in the management of bleeding disorders in trauma and surgical patients [
19‐
21]. It has also been used to evaluate alterations in hemostasis in
in vitro models of endotoxemia or at a single time point in patients with sepsis [
22‐
25]. However, the evolution of disorders of hemostasis measured by thromboelastography during severe sepsis has not been investigated.
The aim of this study was to evaluate the evolution of coagulation abnormalities using thromboelastometry (ROTEM® thromboelastometry; Pentapharm, Munich, Germany) in parallel with routine coagulation tests during the early phase of severe sepsis and septic shock, and to relate these abnormalities to organ dysfunction. We hypothesized that changes in ROTEM® variables may be related to the evolution of organ dysfunction. ROTEM® is a point-of-care device with limited susceptibility to shock and vibrations.
Materials and methods
The study protocol for the present prospective cohort study was approved by the regional governmental ethics committee (Ethik Kommission des Kantons Bern). Written informed consent was obtained from the patients or their relatives. Thirty patients admitted to a 30-bed multidisciplinary intensive care unit (ICU) with a diagnosis of sepsis (as defined by the recommendations of the Society of Critical Care Medicine/European Society of Intensive Care Medicine/American College of Chest Physicians/American Thoracic Society/Surgical Infection Society International Sepsis Definitions Conference [
26]) were enrolled in the study. Exclusion criteria were age under 18 years, pre-existent hematological disorders, current oral anticoagulants, or therapy to inhibit platelet aggregation.
General treatment
All patients with sepsis were monitored with a radial arterial line and a central venous line, and with a pulmonary artery catheter if they were unresponsive to initial volume loading. Treatment protocols were used for hemodynamic management, weaning from mechanical ventilation, analgesia and sedation, and insulin therapy. Thromboprophylaxis in all patients was achieved with graduated compression stockings or intermittent pneumatic compression devices in addition to low-dose unfractionated heparin 10,000 units/day, in accordance with the guidelines of the American College of Chest Physicians for critically ill patients [
27]. The heparin was administered as a continuous intravenous infusion.
Volume resuscitation with colloids in our patients was achieved with 6% hydroxyethyl starch 130/0.4 (Voluven®; Fresenius Kabi, Stans, Switzerland). Patients in the low Sequential Organ Failure Assessment (SOFA) group were administered a mean of 1,373 ± 1,929 ml over the whole study period; those in the high SOFA group received 2,523 ± 1,914 ml.
Data acquisition
Demographic data, source of infection, and length of stay in the ICU were recorded. The Simplified Acute Physiology Score II was determined on admission. The SOFA score was assessed daily for the first 3 days after inclusion in the study and on ICU discharge. The SOFA score was developed to quantify the severity of illness based on the degree of organ dysfunction [
28].
Blood sampling
Blood samples were taken from a radial arterial line after 10 ml blood had been discarded. Blood for thromboelastometry was drawn into a 5 ml syringe and immediately anticoagulated with 0.5 ml trisodium citrate 0.106 mol/l (Sarstedt, Nümbrecht, Germany).
Routine coagulation screening, including platelet count, International Normalized Ratio (INR), activated partial thromboplastin time (aPTT), levels of coagulation factors II, V, VII and X, and fibrinogen, were measured daily for the first 3 days and upon discharge. Routine coagulation laboratory measurements and analysis of coagulation factor activity were performed using the BCS® Analyzer and corresponding reagents (Siemens Health Care Diagnostics, Düdingen, Switzerland).
Thromboelastometry
Thromboelastometry was performed using a ROTEM
® analyzer (Pentapharm GmbH, Munich, Germany) every 12 hours for 48 hours and at ICU discharge. The method, technique and variables of thromboelastometry were described previously [
29]. Briefly, ROTEM
® measures viscoelastic properties of clot formation and fibrinolysis. Because of the use of a ball-bearing system for power transduction, it is less susceptible to movement and vibration.
Tests were performed using ROTEM cups and pins. The ROTEM® device was tested regularly for correct function using quality control serum (ROTROL®; Pentapharm GmbH).
We conducted intrinsically and extrinsically activated tests (INTEM and EXTEM), in accordance with the manufacturer's recommendations (INTEM test: 20 μl CaCL2 0.2 mol/l, 20 μl thromboplastin-phospholipid, 300 μl blood; EXTEM test: 20 μl CaCL2 0.2 mol/l, 20 μl tissue factor, 300 μl blood). In order to assess a possible effect of low-dose heparin administration to our patients, we also analyzed the effects of addition of heparinase to the blood samples (HEPTEM test) and compared the results with those of the INTEM test.
The influence of thrombocytes on clot firmness was estimated with a platelet-inactivating test (FIBTEM test: 20 μl CaCL2 0.2 mol/l, cytochalasin D, 20 μl tissue factor, 300 μl blood). Chemicals and reagents were purchased from Pentapharm GmbH.
The variables that were measured using ROTEM® thromboelastometry are the following. The clotting time (CT), the equivalent to the reaction time (r time) of conventional TEG® (Haemoscope, Skokie, IL, USA), represents the initiation of coagulation. The propagation of clot formation, reflecting thrombin generation and early fibrin polymerization, is characterized by clot formation time (CFT), comparable to the clotting time (k time) and alpha angle of conventional TEG®. CFT is thereby defined as the time necessary to attain a clot firmness of 20 mm. The maximal clot firmness (MCF), corresponding to the maximal amplitude of conventional TEG®, which describes the final strength of the clot, is influenced by the fibrinogen concentration and the platelet count.
Statistical analysis
SigmaStat version 3.5 (Systat Software, Inc., Chicago, IL, USA) was used for statistical analysis. After testing for normal distribution (Kolmogorov-Smirnov test), data were analyzed using analysis of variance for repeated measurements and the Student-Newman-Keuls test for post hoc comparisons or, where appropriate, Friedman analysis and the Dunn test. In order to compare coagulation profiles in groups of patients with different organ failure severity, patients were divided into two groups using median SOFA score. Differences between the two groups were analyzed using analysis of variance for repeated measurements with one grouping factor (high versus low SOFA score) and one within-subject factor (time). The correlation between routine coagulation and ROTEM® variables with the severity of organ dysfunction defined by the SOFA score was studied using the nonparametric Spearman correlation (r) with pooled data from the continuous time points. Comparison of the clotting times between the INTEM and HEPTEM assays was analyzed with a two-sided t-test. For all statistical tests, significance was assumed at P < 0.05. Values are expressed as mean ± standard deviation or median (interquartile range) where appropriate.
Discussion
The coagulation system is commonly activated during sepsis as a result of crossreactions with the inflammatory system [
5,
31]. In the present cohort study we assessed coagulation abnormalities in septic patients during the critical phase of the illness using ROTEM
® thromboelastometry. In order to evaluate further the relationship between perturbation of the coagulation system and organ dysfunction, we divided the cohort at the median of the initial SOFA score and compared the resulting two groups.
Improvements in SOFA scores of these patients were associated with shortened time to initiation of coagulation, accelerated clot formation, and improved firmness of the final clot at ICU discharge when compared with values on admission. Therefore, we hypothesize that the coagulation system has recovered toward the patients' individual baselines upon discharge from the ICU. Nevertheless, key thromboelastometric variables for the patients in our study remained within the range of normal reference values reported in a multicenter trial [
30]. In this context, no signs of overt disseminated intravascular coagulation with adverse bleeding events were observed. Of note, compared with the increase in clot firmness, the rise in the platelet count during the course of critical illness was disproportionately high, whereas fibrinogen levels remained constant. This underlines the observation that fibrinogen levels appear to have a much greater impact on MCF than do changes in platelet count [
32,
33].
The results of ROTEM
® thromboelastometry in the present study did not suggest hypercoagulability in terms of reduced CT, reduced CFT, or increased MCF compared with established normal ranges [
30,
34‐
37]. The mean of the MCF in the FIBTEM assay of the patients with a SOFA score less than 10 was slightly higher than the reported normal range. However, because the FIBTEM assay measures only an isolated component of the overall clot firmness, it may probably not be used as a reliable parameter for hypercoagulability. In this respect, our results differ from the thromboelastographic data reported in a study by Gonano and coworkers [
38], who described hypercoagulability based on their results of decreased
r and
k values using TEG
®. Two other studies employing sepsis models also came to the conclusion that lipopolysaccharide-induced hypercoagulability may be detected by thromboelastography [
22,
23]. Spiel and coworkers [
22] administered a bolus injection of endotoxin to healthy volunteers and demonstrated a transient decrease in the coagulation time, limited to the first 24 hours after injection, accompanied by increased markers of coagulation activation (increased levels of prothrombin fragments F
1+2) [
22]. Incubation of whole blood of healthy volunteers with endotoxin also produced a decrease in clotting time [
23]. Clearly, the two latter investigations measured the effect of lipopolyaccharide on hemostasis in highly standardized models reflecting the very early phase after induction of inflammation. However, the clinical scenario of critically ill patients admitted to an ICU differs from these situations.
In critically ill patients with severe sepsis and septic shock, the disease has reached a more advanced stage – with advanced inflammatory response and sustained exposure to the infectious pathogen – by the time they present at the ICU. In the clinical setting, assessment of activation of coagulation by thromboelastography may thus not present with shortening of CT and CFT but rather with a development in the opposite direction, in which coagulation factors are consumed. A concomitant decrease in the production of clotting factors due to hepatic dysfunction in sepsis may also play a role, however [
39].
Our data are supported by an analysis of the placebo group in the PROWESS (Recombinant Human Protein C Worldwide Evaluation in Severe sepsis) study, which demonstrated that increased severity of sepsis is accompanied by prolonged coagulation times, suggesting decreased activity of coagulation factors as a result of increased consumption [
40].
Activation of coagulation is a well-known pathophysiological process in sepsis [
5,
14‐
17,
31]. That accelerated clot formation and increased clot strength were not present in our study by no means excludes activation of coagulation in sepsis. The more likely explanation for our findings is that inappropriate activation of coagulation may be depicted by thromboelastography only at an early and possibly preclinical phase. However, once coagulation factors are depleted, direct thromboelastographic signs of hypercoagulation may be absent.
There is concern that the administration of colloids may have affected the coagulation system. Although the doses of fluid resuscitation with 6% hydroxethyl starch 130/0.4 differed between the two groups, the administered volumes have reportedly only minor effects on parameters of thromboelastography [
41,
42].
A low-dose regimen of unfractionated heparin was administered to the patients in the present study in order to prevent thromboembolic complications. This could have influenced thromboelastography analysis. However, in a heparinase assay (ROTEM
® HEPTEM) we analyzed clotting time as the variable of interest in this context and excluded such an effect [
43]. These results suggest that the low dose of heparin used for thromboprophylaxis may not be detected by thromboelastography.
A limitation of our study is that the reported changes of ROTEM® parameters had to be interpreted on the basis of external reference values. Future investigations in a controlled study may reveal clearer relationships between organ dysfunction and the coagulation system assessed by thromboelastography.
Conclusions
Key variables for ROTEM® remained within the reference ranges during the phase of critical illness in this cohort of patients with severe sepsis and septic shock without bleeding complications. Although average thromboelastometry variables did not provide additional information to standard coagulation tests, certain dynamics of ROTEM® variables were noted within the reference ranges. Improved organ dysfunction upon discharge from the ICU was associated with shortened coagulation time, accelerated clot formation, and increased firmness of the formed blood clot when compared with values on admission. With increased severity of illness, changes of ROTEM® variables were more pronounced.
Thromboelastography performed in patients with severe sepsis cannot reliably detect activation of coagulation in the sense of a hypercoagulable state. Further studies in patients with sepsis are warranted to investigate the role of thromboelastography in relation to bleeding and thromboembolic complications as end-points.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
FD made substantial contributions to the design of the study, analysis and interpretation of the data, and was involved in drafting the manuscript. UK, HF, and JSL made substantial contributions to the thromboelastometric measurements and to the acquisition of the data, and critically revised the manuscript for important intellectual content. JT critically revised the manuscript for important intellectual content. SMJ made substantial contributions to the concept and design of the study, and to the analysis and interpretation of the data, and was involved in drafting the manuscript. All authors read and approved the final manuscript.