PROTEIN C AND SOLUBLE THROMBOMODULIN IN RELATION TO TUMOR NECROSIS FACTOR-α IN CRITICALLY ILL SEPTIC PATIENTS AND THEIR CORRELATION WITH SOFA SCORE

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1 73 PROTEIN C AND SOLUBLE THROMBOMODULIN IN RELATION TO TUMOR NECROSIS FACTOR-α IN CRITICALLY ILL SEPTIC PATIENTS AND THEIR CORRELATION WITH SOFA SCORE Aida A Nazier*, Assem Abdel Razek**, Nadia E Zaki*, Wafaa A El-Neanaey and Ossama S Ibrahim* Departments of Internal Medicine (Hematology Unit)*, Anaesthesia and Intensive Care** and Clinical Pathology, Faculty of Medicine, University of Alexandria ABSTRACT The objective of this study was to investigate the influence of sepsis on protein C (PC) activity and soluble thrombomodulin (s-tm) in relation to tumor necrosis factor-α (TNF-α). Also, to correlate these parameters with the SOFA score and serum lactate concentration as predictors of morbidity and mortality in septic patients. Thirty two adult patients (17 with sepsis and 15 with severe sepsis) in the intensive care unit (ICU), as well as 10 healthy age- and sex- matched controls were accrued to the study. The results showed that the baseline values of PC activity were significantly lower in both groups of septic patients compared to the controls, whereas, the serum levels of s-tm, TNF-α and lactate were significantly higher in the former compared to the latter. Moreover, survivors in both patient groups had higher PC activity and lower serum levels of s-tm, TNF-α and lactate compared to non survivors, both on and day (3) of ICU admission with variable statistical significance reflecting the heterogeneity of sepsis and the well-known individual patient variation. In addition, day (3) samples showed significant increase in PC activity and reduction in serum levels of s-tm, TNF-α, lactate and SOFA scores in survivors and the reverse in non survivors compared to baseline levels. Also, baseline PC activity was correlated negatively with SOFA score but not with serum TNF-α in both groups of septic patients. It was also negatively correlated with serum TM and lactate in sepsis but not in severe sepsis. Conclusion: PC activity and serum levels of s-tm, TNF-α and lactate in addition to SOFA score are good predictors of clinical outcome in critically ill patients with sepsis. INTRODUCTION Sepsis is defined as systemic inflammatory response syndrome (SIRS) in the presence of documented or suspected infection. Severe sepsis is defined as sepsis associated with organ dysfunction. Septic shock is a subcategory of severe sepsis in which the cardiovascular system is dysfunctional and is defined as arterial hypotension despite adequate fluid resuscitation. (1) The pathogenesis of sepsis involves the activation of a number of host defense mechanisms including the cytokine network, leukocytes, complement and coagulation systems. (2) In addition to activation of coagulation in sepsis, there is an attenuation of natural anticoagulant responses and impaired fibrinolysis. The resulting shift toward a procoagulant state results in excessive thrombin generation, fibrin formation, and consumption of clotting factors and inhibitors such as antithrombin III and proteins C and S. (3) The protein C (PC) pathway provides a useful model for the impact of inflammation on coagulation. (4) The key features of this pathway is its ability to respond to the presence of thrombin. As the thrombin concentration rises, it binds to thrombomodulin (TM), an endothelial surface glycolprotein, leading to both rapid thrombin inactivation and change in the substrate preference of thrombin from procoagulants such as fibrinogen and factor V to anticoagulants such as PC especially in the capillaries. An endothelial protein C receptor (EPCR) augments PC activation by the thrombin-tm complex more than 10- fold in vivo. Conversely, once thrombin generation reverts to homostatic levels, the PC activation complex slows the rate of generation of activated protein C (APC) down to the physiologic levels. (4,5) APC was originally recognized as an anticoagulant that works up by proteolytic inactivation of factors Va and VIIIa. In addition, APC is profibrinolytic by inactivation of plasminogen activator inhibitor-1 (PAI-1). APC also possesses anti-inflammatory properties. In experimental animals challenged with endotoxin, APC inhibits the release of

2 74 proinflammatory cytokines such as tumor necrosis factor-α (TNF-α) and limits leukocyte extravasation to the tissues. However, this pathway is not static and could be impaired in a variety of clinically relevant situations including sepsis in which there is systemic endothelial dysfunction. (5,6) Recent trials have shown that mortality can be reduced among patients with severe sepsis through the use of a new therapy that inhibits both the procoagulant and the inflammatory cascades. Intravenous infusion of antithrombin, PC/APC, or tissue factor pathway inhibitor (TFPI) can reduce the frequency of lethal responses in non human primates and other animals. (7) Recombinant APC is the first biologic agent shown to reduce significantly the mortality in patients with severe sepsis. (8) The prognosis of septic patients is related to the severity of organ dysfunction. Scoring systems such as the Sequential Organ Failure Assessment (SOFA) score are useful tools for assessing and quantifying organ dysfunction and failure over time. (9) The SOFA is used to evaluate morbidity and is a good indicator of mortality in critically ill patients. (10) The aim of this work was to investigate the influence of sepsis on the PC activity and s-tm in relation to serum TNF-α. Also, to correlate these parameters with the SOFA score and serum lactate concentrations as predictors of morbidity and mortality in these patients. This could help to clarify the position of PC/APC and other novel therapeutic modalities in the intensive care armamentarium. PATIENTS AND METHODS This study was carried out on 42 individuals divided into 3 groups: Group I: 10 healthy age and sex-matched adult individuals as a control group, Group II: 17 adult patients (11 males and 6 females) with sepsis (1) (SIRS + infection): systemic reaction to infection defined by two or more of the following: (a) Temperature > 38 C or < 36 C, (b) Heart rate > 90 beats/min, (c) Respiration rate > 20 breaths/ min or PaCO 2 < 32mmHg, (d) Leukocytes > /L, < , or > 10 immature (band) forms. (1) Their ages ranged between 22 and 59 years with a mean of 46.88± years. Group III: 15 adult patients (10 males and 5 females) with severe sepsis: sepsis with new signs of organ dysfunction or decrease in organ perfusion (lactate acidosis> 3mmol/L, oliguria < 0.5 ml/kg body weight for at least 1 hour or < 30 ml/hour, hypotension < 90 mmhg or a decrease of > 40 mmhg), mental alteration). (1) Their ages ranged between 28 and 58 years with a mean of 49.33±11.94 years. The patients were selected from those admitted to the Intensive Care Unit, Main Alexandria University Hospital, during the period from April 2005 to September Exclusion criteria included age < 16 years or > 60 years, pregnancy, inherited or acquired bleeding or throbmotic disorders, hematological or non hematological malignancies, significant renal or hepatic dysfunction, medications altering bleeding or coagulation profiles, severe trauma and recent (within 24 hours) transfusion of fresh frozen plasma, blood coagulation products or platelets. All patients were subjected to: 1- Full history taking with stress on the onset of fever, bleeding or thrombotic manifestations, and symptoms of infection. 2- Thorough clinical examination with emphasis on detection of signs of inflammation and evidence of bleeding or thrombosis. 3- Routine laboratory investigations were done daily including renal, liver function tests, complete blood count, prothrombin time (PT) and Activated-partial thromboplastin time (A-PTT). Also, arterial blood gases were done every 12 hours. 4- Investigations of the septic patients on admission to the ICU including urine analysis, chest X-ray, cultures for bacteria and fungi, CT scans and/or MRI, when indicated. 5- Colorimetric assay for determination of serum lactate was done daily for the first 3 days after diagnosis using (Lactate PAP Fluid, Centronic GmbH. Germany). (11) 6- Upon diagnosis and after 48 hours the following tests were done:-

3 75 a- Measurement of protein C activity using (protein C reagent, Dade Behring/ Maburg. USA). (12) b- Estimation of serum thrombomodulin using (CD141 ELISA KIT, Diaclone. France). (13) c- Estimation of serum TNF-α using (TNF-α ELISA IM1121, IM1121, Beckman Coulter TM. France). (14) 7- Assessment of SOFA score was done daily for the first three days after diagnosis according to the criteria proposed by Ferreria et al. (9) Samples collection and storage Plasma samples were obtained by mixing one part of sodium citrate (3.8%) with 9 parts of venous blood and centrifuged immediately at approximately 3000 rpm for at least 10 minutes, the resulting plasma supernatent is used immediately for the estimation of PT and A- PTT while the remaining part stored in aliquots at -20 C until used for determineation of PC activity. Serum sample was obtained by centrifugation of clotted blood at approximately 3000 rpm for 15 minutes, then it was removed rapidly and stored in aliquots at -20 C until used for estimation of TNF-α, s-tm and lactate. STATISTICAL ANALYSIS Data were analyzed using SPSS V-10. RESULTS Table (1), illustrates the clinical data in the studied septic patients while Table (2), shows the statistical comparison between the studied parameters in controls and septic patients on. The baseline values of PC activity were significantly lower in both groups of septic patients compared to control group. Whereas the serum levels of s-tm, TNF-α and lactate were significantly higher in the former compared to the latter. Moreover, higher PC activity and lower serum levels of s-tm, TNF-α and lactate were observed in sepsis compared to severe sepsis but the difference was significant for all parameters except TNF-α. Table (3), demonstrates statistical comparison between survivors and non survivors in both groups of septic patients. On the mean values of PC activity were significantly higher in survivors than non survivors in sepsis (p = 0.013) but not in severe sepsis. While on day (3), the difference was significant in both groups of patients (p = and for sepsis and severe sepsis respectively). Also, the mean values of serum TM and TNF-α on were lower in survivors than non survivors but the difference was not statistically significant in both groups of patients. However, the significance was noted on day (3) (p =0.011 and in sepsis, p = and in severe sepsis for s-tm and TNF-α respectively). The mean values of serum lactate and SOFA score on were lower in survivors than non survivors but the difference was statistically significant only in sepsis (p = 0.002) for serum lactate, but not for SOFA. On comparing day (2) and day (3) values, the mean values of both parameters were significantly lower in survivors than non survivors in both groups of patients. A significant negative correlation was obtained between PC activity and SOFA score but not with serum TNF-α in both groups of patients. Also, PC activity was negatively correlated with serum TM and lactate in sepsis but not in severe sepsis (Table 4). DISCUSSION In the present study, the baseline values of PC activity were significantly lower in both groups of septic patients compared to the controls. This agrees with Dhainaut et al (15) who stated that a rapid and prolonged depletion of PC occurs in human sepsis, presumably due to increased consumption, degradation by serine protease inhibitors such as α 1 - antitrypsin, PC inhibitor and α 2 -macroglobulin, or decreased hepatic synthesis. This contributes to sepsis-induced coagulopathy and correlates with a poor prognosis. (16) In contrast, Mavrommatis et al (17) found that PC levels detected by ELISA technique were slightly and not significantly reduced in sepsis and severe sepsis, but the reduction was significant only in septic shock.

4 76 However, the definition of severe sepsis and septic shock was modified in their study since they enrolled patients who required the use of vasopressors to maintain arterial pressure in the normal range (i.e., patients with severe sepsis) within the septic shock group. In the present study, survivors, in both groups of septic patients, had significantly higher PC activity compared to non survivors, both on and day (3). The marginal significance (p = 0.056) observed on comparing the base line values of PC activity in survivors and non-survivors with severe sepsis could be explained by the fact that this situation is more hetrogenous and is associated with more exhaustion of coagulation inhibitors compared to sepsis. (17) Nevertheless, the difference became highly significant on day (3) Table (1): Clinical data of the studied septic patients Group II Group III Parameter (Patients with sepsis) (Patients with severe sepsis) (n=17) (n=15) No. % No. % Diagnosis AMI ± AF CHF ± acute pulmonary edema COPD + resp. failure CVS DKA Toxic coma Post-operative Trauma Infection Site: - RTI UTI Bed sores Peritonitis Culture: - Gram negative bacteria Gram positive bacteria Candida Mixed infections Bleeding tendency Thrombotic manifestations Outcome Survivors* Non survivors Abbreviations AMI ± AF : Acute myocardial infarction ± atrial fibrillation CHF : Congestive heart failure COPD : Chronic obstructive pulmonary disease. CVS : Cerebrovascular stroke DKA : Diabetic ketoacidosis RTI : Respiratory tract infection UTI : Urinary tract infection : 28-day survival.

5 77 Table (2): Comparison between studied parameters in septic patients and controls on. Variables PC activity(%) s-tm (ng/ml) TNF-α (pg/ml) Group I (Controls) (n=10) Group II (Sepsis) (n=17) Group III (Severe sepsis) (n=15) Mean± SD Mean± SD Mean± SD ± ± ± ± ± ± ± ± ± t-test P-value t 1 = p 1 = 0.000* t 2 = p 2 = 0.000* t 3 = p 3 = 0.000* t 1 = p 1 = 0.000* t 2 = p 2 = 0.023* t 3 = p 3 = 0.000* t 1 = p 1 = 0.000* t 2 = p 2 = 0.000* t 3 = p 3 = Lactate t 1 = p 1 = 0.000* (mmol/l) 1.46 ± ± ± t 2 = p 2 = 0.000* t 3 = p 3 = 0.000* SOFA score ± ± t 3 = 15.7 p 3< * Significance at P PC : Protein C. t 1 : Significance between group I and group II. s-tm : Soluble thrombomodulin. t 2 : Significance between group I and group III. TNF-α : Tumor necrosis factor-α. t 3 : Significance between group II and group III. (p = 0.002) reflecting the usefulness of follow-up measurements in predicting the clinical outcome in patients with severe sepsis. Similar results have been reported by Yan et al, (18) and Macias and Nelson. (19) Liaw et al, (20) studied 32 patients with severe sepsis and found no significant difference between survivors and non survivors with respect to baseline total PC antigen levels using ELISA technique. Although commercial kits are readily available for the measurement of PC, there were few available reports on endogenous APC levels in patients with sepsis, perhaps due to the lack of available assays that permit both rapid and accurate measure-ments of plasma APC levels. However, one method for quantifying plasma levels of APC termed enzyme capture assay was developed. Then it was modified by Liaw et al, (21) by generating a monoclonal antibody (HAPC- 1555) with high specificity to human APC and taking 3-19 hours to detect the physiologic level of APC. Liaw et al, (20) reported that endogenous PC activation is impaired in a subpopulation of adult septic patients and these might require APC therapy, whereas others with normal production of APC may benefit from the administration of inactive PC concent-rate. Moreover, they observed that the baseline APC levels in survivors were significantly higher than those in non survivors and they suggested that APC may have prognostic value in septic patients. Inflammatory cytokines including TNFα can down regulate expression of cell surface TM and EPCR by inhibing gene transcription and promoting shedding from the endothelium. In the latter mechanism, the released form is much less active than the endothelial form. (5,20) In the present study, the baseline values of s-tm were significantly higher in both groups of septic patients compared to the controls. On comparing survivors and non survivors, we observed no significant difference regarding s-tm levels on day (1), however, day (3) values were significantly higher in non survivors than survivors in both patients groups. This again reflects the importance of follow-up

6 Table (3): Comparison between survivors and non survivors in the studied septic patients. Variables studied Group II (patients with sepsis (no = 17) Group III (patients with severe sepsis (no = 15) Survivors (n=12) Non survivors(n=5) Z test P value Survivors(n=10) Non survivors(n=5) Z test P value PC activity (%) Day ± ± * 18.80± ± Day ± ± * 29.50± ± * z-test p-value 0.003* 0.043* 0.005* 0.042* s-tm (ng/ml) Day ± ± ± ± Day ± ± * 14.55± ± * z-test p-value * 0.005* 0.043* TNF-α (pg/ml) Day ± ± ± ± Day ± ± * 88.30± ± * z-test p-value 0.002* 0.043* 0.005* 0.043* Lactate (mmol/l) Day ± ± * 3.29± ± Day ± ± * 3.11± ± * Day ± ± * 2.509± ± * z-test Z Z Z P-value P * 0.042* 0.005* 0.043* P * 0.043* 0.005* 0.043* P * 0.043* 0.005* 0.043* SOFA score Day ± ± ± ± Day ± ± * 12.10± ± * Day ± ± * 9.50± ± * z-test Z Z Z P-value P * 0.034* 0.007* 0.024* P * * P * 0.039* 0.005* 0.001* Significance at P 0.05 Z 1 :Significance of serum lactate between day 1 and day 2. Z 4 : Significance of SOFA score between day 1 and day 2. Z 2 : Significance of serum lactate between day 2 and day 3 Z 5 :Significance of SOFA score between day 2 and day 3. Z 3 :Significance of serum lactate between day 1 and day 3. Z 6 : Significance of SOFA score between day 1 and day 3. 10

7 79 Table (4): Correlation between the studied parameters in both groups of septic patients on. Variables studied s-tm TNF-α Lactate SOFA score Protein C activity Group II (sepsis) r = P= 0.043* r = P = r = P = 0.003* r = P = 0.039* Group III (severe sepsis) r = P = r = P = r = P = r = P = 0.032* * Correlation is significant at P 0.05 levels assays in predicting the clinical outcome. Similar results were reported recently by Gando et al. (22) In contrast, Liaw et al, (20) observed no significant difference between septic patients who survived and those who died with respect to s-tm levels although individual patient variation was quite extreme. They suggested that a decrease in cell surface levels of TM and EPCR is a complex process and not only reflects shedding of the receptors but also decreases in gene transcription. Also, they proposed that severe sepsis is an extremely heterogenous condition and that factors involved in down regulation of the endothelium based PC activation complex in a subpopulation of patients remain to be identified. Concerning TNF-α, our results showed that baseline serum TNF-α levels were significantly higher in both groups of septic patients than the controls. However, on comparing survivors and non survivors within each group, significantly higher TNFα levels were observed in non survivors compared to survivors only on day (3) but not on samples. Interestingly, we also observed significant reduction in serum TNF-α on day (3) in patients who survived and significant elevation in those who died compared to baseline levels. This again emphasizes the validity of follow-up sampling in septic patients. These findings confirm the results of most previous reports. (23-25) On the other hand, Oberholzer et al, (26) found that the baseline values of TNF-α and the change over the initial 4 days were not useful in predicting the outcome in patients with severe sepsis. This could be related to enrollment of patients with septic shock in their series. Blood lactate levels tend to reflect the imbalance between oxygen demand and oxygen supply. Hyperlactemia in patients with sepsis is a marker of the severity of stress response. Increased lactate production during anaerobic and aerobic metabolism and decreased lactate clearance are likely contributors to hyperlactemia in septic patients. (27,28) In the current study, serum lactate concentrations were significantly higher in both groups of septic patients than controls and also were significantly higher in patients with severe sepsis than those with sepsis. The values were lower in survivors than non survivors in both groups, but the difference was statistically significant only in patients with sepsis. However, the difference was significant on comparing day (2) and (3) values between survivors and non survivors in both groups. Moreover, serum lactate levels significantly decreased in survivors and increased in non survivors during the follow-up samples on days (2) and (3) reflecting the prognostic significance of serial blood lactate concentrations in septic patients. (27,29) Our results are consistent with those obtained by Kobayashi et al, (27) and Nguyen et al. (29) They found no statistical difference in initial lactate levels between survivors and non survivors of septic patients. However, serum lactate progressively and significantly decreased in the survivors and remained high in the non survivors throughout the study period. Concerning the difference between sepsis and severe sepsis, no available literature could be obtained as most previous studies focused

8 80 on patients with severe sepsis and septic shock. In order to evaluate the usefulness of repeated measurements of the SOFA score for prediction of mortality in our septic patients in the ICU, we assessed the SOFA score for all patients on 3 consecutive days. We found that the mean values of SOFA on were significantly higher in patients with severe sepsis than those with sepsis and not statistically different between survivors and non survivors in both groups of septic patients, however, day (2) and (3) mean values were significantly lower in the former compared to the latter. Moreover, there was a significant reduction in survivors and increase in non survivors in the follow-up scores compared to SOFA scores reflecting improvement or deterioration in organ function, respectively. Our findings were in agreement with other investigators. (9,30-32) They reported that the mean SOFA scores, total maximum SOFA, delta SOFA (differences between subsequent scores) and admission total SOFA scores exhibited a good association with hospital mortality. Ferreira et al (9) demonstrated that, independent of the initial value, an increase in the SOFA score during the first 48 hours of ICU admission predicts a mortality rate of at least 50%. Furthermore, in a more recent study, Levy et al (32) suggested that outcomes for patients with severe sepsis are closely related to early (first 24 hours) improvement, or lack thereof, in organ function. Also, continued improvement before start of day (2) and start of day (3) was associated with further improvement in survival. In conclusion, PC activity and serum levels of TM, TNF-α and lactate are good predictors of clinical outcome in critically ill patients with sepsis. The baseline values of PC activity can aid in identifying septic patients with high risk of mortality but its predictive value for the use of recombinant APC infusion in such patients should be evaluated. However, serial measurements of all parameters in addition to the SOFA score are mandatory to predict the likelihood of survival among these patients. REFERENCES 1. Marshal JC, Parker MM, Ramsay G et al. Surviving sepsis campaign guidelines for management of severe sepsis and septic shock. Int Care Med 2004, 30: Hotchkiss RS, Karl IE. The pathophysiology and treatment of sepsis. N Engl J Med 2003; 348: Esmon CT. Crosstalk between inflammation and thrombosis. Maturitas 2004; 47: Esmon CT. The protein C pathway. Chest 2003; 124: Esmon CT. New mechanisms for vascular control of inflammation mediated by natural anticoagulant proteins. J Exp Med 2002; 196: White BM, Schmidt C, Murphy W, Livingstone D. activated protein C inhibits lipopolysaccharide- induced nuclear translocation of nuclear factor κb (NF-κB) and tumor necrosis factorα (TNF-α) production in the THP-1 monocytic cell line. Br J Haematol 2000; 110: 130:4. 7. Matthay MA. Severe sepsis, a new treatment with both anticoagulant and antiinflammatory properties. N Engl J Med 2001; 344: Pastores SM. Drotrecogin alfa (activated): A novel therapeutic strategy for severe sepsis. Postgraduate Med J 2003; 79: Ferreira FL, Bota DP, Bross A et al. Serial evaluation of the SOFA score to predict outcome in critically ill patients. JAMA 2001; 286: Okabayashi K, Wada H, Ohta S et al. Hemostatic markers and the sepsis related organ failure assessment score in patients with disseminated intravascular coagulation in an intensive care unit. Am J Haematol 2004; 76: Kaplan LA. Collection and storage of serum lactic acid samples at room temperature without deproteinization. Clin Chem 1980; 26: Girault C, Gufflet V, Robert A. The effect of lupus anticoagulant (LA) on clotting assay of protein C (PC). Thromb Haemost 1991; 66: 38.

9 Burrin DH. Immunochemical technique. In: Wilson K, Goulding KH. A biologist's Guide to Principles and Techniques of Practical Biochemistry. 3rd ed. Great Britain: Edward Arnold 1986; Waage A, Halstensen A, Expevix T. Association between tumour necrosis factor in serum and outcome in patients with meningococcal disease. Lancet 1987; 1: Dhainaut JF, Yan SD, Claessens YE. Protein C/activated protein C pathway: Overview of clinical trial results in severe sepsis. Crit Care Med 2004; 32: S194-S Liaw PCY. Endogenous protein C activation in patients with severe sepsis. Crit Care Med 2004; 32: S214-S Mavrommatis AC, Theodoridis T, Economou M et al. Activation of the fibrinolytic system and utilization of the coagulation inhibitors in sepsis: Comparison with severe sepsis and septic shock. Int Care Med 2001; 27: Yan SD, Helterbrand JD, Hartman DL et al. Low levels of protein C are associated with poor outcome in severe sepsis. Chest 2001; 120: Macias WL, Nelson DR. Severe protein C deficiency predicts early death in severe sepsis. Crit Care Med 2004; 32: S223-S Liaw PCY, Esmon CT, Kahnamoni K et al. Patients with severe sepsis vary markedly in their ability to generate activated protein C. Blood 2004; 104: Liaw PC, Ferrell G, Esmon CT. A monoclonal antibody against activated protein C allows rapid detection of activated protein C in plasma and reveals a calcium ion dependent epitope involved in factor Va inactivation. J Thromb Haemost 2003; 1: Gando S, Kameue T, Matsuda N et al. Serial changes in neutrophil-endothelial activation markers during the course of sepsis associated with disseminated intravascular coagulation. Thromb Res 2005; 116: Dougnac A, Riquelme A, Calvo M et al. Study of cytokines kinetics in severe sepsis and its relationship with mortality and score of organic dysfunction. Rev Med Chil 2001; 129: Lekkou A, Karakantza M, Mouzaki A et al. Cytokine production and monocyte HLA-DR expression as predictors of outcome for patients with communityacquired severe infections. Clin Diagn Lab Immunol 2004; 11: Gordon AC, Lagan AL, Aganna E et al. TNF and TNFR polymorphisms in severe sepsis and septic shock: a prospective multicentre study. Genes Immun 2004; 5: Oberholzer A, Souza SM, Tschoeke SK et al. Plasma cytokine measurements augment prognostic scores as indicators of outcome in patients with severe sepsis. Shock 2005; 23: Kobayashi S, Gando S, Morimoto Y et al. Serial measurement of arterial lactate concentrations as a prognostic indicator in relation to the incidence of disseminated intravascular coagulation in patients with systemic inflammatory response syndrome. Surg Today 2001; 31: Bokker J, de Lima AP. Increased blood lactate levels: an important morning signal in surgical practice. Crit Care 2004; 8: Nguyen HB, Rivers EP, Knoblich BP et al. Early lactate clearance is associated with improved outcome in severe sepsis and septic shock. Crit Care Med 2004; 32: Du B, Chen D, Liu D. Prediction of prognosis of patients with multiple organ dysfunction syndrome by sepsis-related organ failure assessment. Zhonghua Yi Xue Za Zhi 2001; 81: Vosylius S, Sipylaite J, Ivaskevicius J. Sequential organ failure assessment score as the determinant of outcome for patients with severe sepsis. Croat Med J 2004; 45: Levy MM, Macias WL, Vincent JL et al. Early changes in organ function predict eventual survival in severe sepsis. Crit Care Med 2005; 33: