septic shock: Prognostic value and association with a distinct serum cytokine profile

    Thrombocytopenia in critically ill patients with severe sepsis/septic shock: Prognostic value and association with a distinct serum cytokine profile Panagiotis Tsirigotis, Spiros Chondropoulos, Frantzeska Frantzeskaki, Maria Stamouli, Konstantinos Gkirkas, Anastasia Bartzeliotou, Nikolaos Papanikolaou, Maria Atta, Ioannis Papassotiriou, George Dimitriadis, Ioanna Dimopoulou PII: DOI: Reference:

S0883-9441(15)00561-4 doi: 10.1016/j.jcrc.2015.11.010 YJCRC 52003

To appear in:

Journal of Critical Care

Please cite this article as: Tsirigotis Panagiotis, Chondropoulos Spiros, Frantzeskaki Frantzeska, Stamouli Maria, Gkirkas Konstantinos, Bartzeliotou Anastasia, Papanikolaou Nikolaos, Atta Maria, Papassotiriou Ioannis, Dimitriadis George, Dimopoulou Ioanna, Thrombocytopenia in critically ill patients with severe sepsis/septic shock: Prognostic value and association with a distinct serum cytokine profile, Journal of Critical Care (2015), doi: 10.1016/j.jcrc.2015.11.010

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Thrombocytopenia in critically ill patients with severe sepsis/septic shock:

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Prognostic value and association with a distinct serum cytokine profile

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Panagiotis Tsirigotisa, Spiros Chondropoulosa, Frantzeska Frantzeskakib, Maria Stamoulia, Konstantinos Gkirkasa, Anastasia Bartzeliotouc, Nikolaos Papanikolaoua,

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Department of Internal Medicine, „‟ATTIKO‟‟ General University Hospital,

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a nd

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Maria Attaa, Ioannis Papassotiriouc, George Dimitriadisa, Ioanna Dimopouloub

National and Kapodistrian University of Athens, Athens, Greece b nd

2 Department of Critical Care, „‟ATTIKO‟‟ General University Hospital, National

and Kapodistrian University of Athens, Athens, Greece Department of Clinical Biochemistry, “Aghia Sophia” Children‟s Hospital, Athens,

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c

Correspondence

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Greece

Panagiotis Tsirigotis, MD,

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Associate Professor of Hematology 2nd Department of Internal Medicine

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„‟ATTIKO‟‟ General University Hospital Rimini-1, Haidari

PO: 12462, Athens, Greece Tel: +30-210-5832317 Fax: +30-210-5832728 E-mail: [email protected]

Conflict of interest statement: No relevant conflicts of interest Running title: Thrombocytopenia in severe sepsis/septic shock

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Abstract Purpose: to evaluate the incidence, association with serum cytokine profile, and

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prognostic value of thrombocytopenia, in critically ill patients with severe

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sepsis/septic shock.

Methods: A cohort of 105 consecutive patients admitted in intensive care unit (ICU)

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was included in our analysis. Serum levels of ICAM, VCAM, IFN-γ, IL-8, and

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suPAR were measured.

Results: Thrombocytopenia was observed in 53% of patients at the time of admission. Platelet counts (PLT) showed a statistically significant negative correlation with serum levels of ICAM, suPAR, and IL-8 (p<0.0001). In multivariate analysis,

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high APACHE II score, high serum suPAR, and low PLT counts were associated with increased mortality, and ROC analysis was used to determine the best cut-off value

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for mortality prediction. Each variable with a value above or below the pre-defined

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cut-off levels were given 1 point. Patients were categorized in risk groups based on total point score. High risk (2-3), intermediate risk (1), and low risk group (0 points)

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consisted of 43%, 22%, and 35% and 28-day mortality was observed in 69%, 26%, and 3% of the patients in each group respectively. Conclusion: Thrombocytopenia is associated with poor prognosis and a distinct serum cytokine profile.

Key words: thrombocytopenia, suPAR, sepsis, endothelium, ICAM, IL-8

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1. Introduction

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Sepsis is the clinical syndrome resulting from the inflammatory response of the host

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against various invading pathogens such as bacterial infections. Sepsis can be

associated with hematological abnormalities.

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complicated by hemodynamic instability, multi-organ dysfunction and is commonly Patients with severe sepsis usually

coagulation

(DIC),

drug

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develop thrombocytopenia due to various reasons such as, disseminated intravascular induced

myelosuppression,

heparin

induced

thrombocytopenia (HIT), drug induced immune destruction, hemodilution, massive transfusion, hemophagocytosis syndrome, etc [1]. However, in many instances, severe

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thrombocytopenia accompanies sepsis without an identifiable cause. In these cases, activation and consumption of platelets due to contact with a damaged endothelium

process,

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studies

have

shown

a

negative

impact

of

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pathogenetic

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has been postulated as the pathogenetic mechanism [2]. Irrespective of the underlying

thrombocytopenia in sepsis outcome [3, 4]. Serum cytokine profiling of patients with

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severe sepsis/septic shock is a valuable tool for better understanding of the underlying pathogenetic mechanism [5]. Patients with significant mortality risk as well as patients with high probability for evolution to organ dysfunction can be identified by the different cytokine profile [6]. Urokinase-type plasminogen activator (uPA) is a protein involved in the conversion of plasminogen to the active fibrinolytic enzyme plasmin. uPA is activated upon binding to its cognate receptor (uPAR) which is expressed on the surface of various cell types including neutrophils, lymphocytes, monocytes, macrophages, and endothelial cells. After shedding from cell surface, uPAR can be found in a soluble form in blood (suPAR) and other tissue fluids. suPAR has a pleotropic function:

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Except from regulating fibrinolysis, it is involved in inflammatory response and in various cell functions, including adhesion, migration, degradation of extracellular

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matrix, and angiogenesis [7]. Previous data suggest that serum suPAR is a predictor

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of outcome in critically ill patients and a marker of the degree of inflammation [8].

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Serum levels of intercellular adhesion molecule 1 (ICAM-1), vascular cell adhesion molecule (VCAM-1), and interleukin-8 (IL-8) have been used as markers of

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endothelial dysfunction which is a key factor in pathogenesis of sepsis. ICAM-1, VCAM-1, and IL-8 are expressed at the surface of the endothelial cell in response to injury or activation by pro-inflammatory cytokines and result in leukocyte adhesion, increased transendothelial migration, and increased endothelial permeability [9, 10].

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The aim of this prospective, observational study was to describe the incidence, prevalence and prognostic value of thrombocytopenia, in critically ill patients with

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severe sepsis and/or septic shock. We also studied the relationship between

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thrombocytopenia and patients‟ inflammatory response as expressed by the cytokines,

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suPAR, ICAM and VCAM, IL-8, and IFN-γ.

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2. Materials and Methods

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2.1. Patients

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Our study included 105 consecutive patients with severe sepsis and/or septic shock

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treated in the ICU of our institute from October/2009 to September/2012. Exclusion criteria were HIV infection, mechanical ventilation for more than 72 hours prior ICU

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admission, brain death, and no need for mechanical ventilation during ICU stay. This study was designed with the aim to examine the correlation between thrombocytopenia and patient‟s inflammatory response, as well as the association with sepsis outcome. Therefore patients with thrombocytopenia due to a known cause

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not related to sepsis such as: 1) due to suspected drug etiology, 2) hematologic malignancy, 3) HIT syndrome, 4) DIC directly related to a malignant disorder, and/or

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5) pancytopenia due to previous administration of chemotherapy, were excluded from

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the study. Systemic inflammation response syndrome (SIRS), sepsis, severe sepsis, and septic shock were defined according to standard criteria [11]. Data collection was

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performed prospectively and outcome was defined as ICU mortality. Briefly, 23 out of 105 patients had undergone a previous operation the days before admission to ICU. Fifteen patients had an abdominal infection including peritonitis, diverticulitis, cholangitis, 6 patients had bacteremia without an identifiable primary site of infection, 2 patients had bacterial endocarditis, 4 patients had CNS infection including meningitis and encephalitis, 58 patients had lower respiratory tract infection, 5 patients had soft-tissue infection, and 15 patients had urinary tract infection. Baseline clinical characteristics and laboratory results collected during the first 24 hours of admission were used for the estimation of the Acute Physiological and Chronic Health Evaluation (APACHE II) score [12]. The sequential organ failure

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assessment score (SOFA) score recorded on the day of admission was used as a variable in statistical analysis [13].

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Patients were categorized according to platelet count (PLT) estimated at the time of

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admission. PLT count at admission was considered as the lowest PLT count observed during the 3 first days after admission in ICU. Thrombocytopenia was defined as a


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PLT below 150X103/μl. Thrombocytopenia was considered as mild (100X103/μl (50X103/μl
or

severe

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moderate

(PLT<50X103/μl) depending on PLT counts. Written informed consent was given by all patients or by relatives of patients not able to give consent. Patient‟s characteristics

2.2. Serum cytokines

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are shown in more detail in Table 1.

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Luminex xMAP technology (Luminex Corporation, Austin, TX, USA) was used to

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measure serum concentrations of IFNγ, IL-8, ICAM and VCAM, (ProcartaPlexTM immunoassays, eBioscience, Inc. San Diego, CA, USA). The inter- and intra-assay

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CVs in all assays performed were 3.5–8.5% and 4.6–12%, respectively. Serum suPAR levels were determined using a commercial enzyme-linked immunosorbent assay (ELISA) (suPARnostic Standard kit; ViroGates A/S, Birkerød, Denmark). This assay utilizes a double monoclonal antibody that measures circulating suPAR, including full-length and cleaved forms of the receptor. According to the standards provided by the manufacturer, a curve was constructed and the results were expressed as ng/mL, in a range between 0.6 and 20.0ng/mL. The intra- and inter-assay CVs ranged from 1.3% to 4.7% and 1.7% to 5.1%, respectively, whereas the sensitivity limit was 0.1ng/mL. The cytokines mentioned above were selected based on the results of a previous retrospective study, performed by our group, including 42 patients with

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severe sepsis. Serum levels of IFNγ, IL-8, ICAM, VCAM, and suPAR among a large panel of cytokines showed a negative correlation with PLT counts (data not

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published), and therefore these cytokines were selected for further testing in our

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prospective trial.

2.3. Statistics

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Comparison between groups was performed by using Fisher‟s exact test for dichotomous or categorical variables and by using Mann–Whitney test for continuous variables. Spearman correlation was used to estimate the degree of association between two variables. Comparison of cytokines levels among groups of patients with

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different numbers of platelets and absolute neutrophil count (ANC) at admission was performed with one way analysis of variance (ANOVA). Time to death was estimated

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as the time from admission to ICU to death from any cause. For estimating mortality

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at 28-day, time to death was defined as the time from ICU admission to death, or discharge before day +28. Progress was recorded until day +100 after admission or

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until discharge from ICU, whichever occurred first. The following variables were entered in a multiple logistic regression model: 1) age and sex, 2) cancer as an underlying condition, vs. not, 3) septic shock vs. severe sepsis, 4) APACHE II score, 5) SOFA score on admission (day +1), 6) bacteremia vs. not, 7) infection due to gram-, vs not, 8) pre-existing comorbidities, 9) serum IFNγ, ICAM, VCAM, IL-8, and suPAR levels (estimated on day of admission), and 10) PLT count on admission. Receiver operating curve (ROC) method was used to determine the best cut-off value of various parameters for the prediction of ICU mortality by estimating the areaunder-the-curve (AUC) values and selecting the maximal sum of sensitivity and

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specificity. Kaplan-Meier and log rank test were used for estimating the probability of

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ICU mortality. Statistical analyses were performed using Medcalc and R software.

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3. Results

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3.1. Incidence of thrombocytopenia

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Fifty-six out of 105 (53%) patients were thrombocytopenic at the time of admission in

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ICU. In more detail, mild, moderate and severe thrombocytopenia was observed in 15 (14%), 7 (7%), and in 34 (32%) patients respectively. Patients with severe

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thrombocytopenia had higher APACHE II score, higher SOFA score on admission, higher serum ICAM, IL-8 and suPAR levels, higher incidence of bacteremia and they had higher probability to present with septic shock as compared with patients who admitted to ICU with normal platelet counts. Finally patients with severe

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thrombocytopenia had statistically significantly higher hospital mortality in

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comparison with patients without thrombocytopenia (Table 1).

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3.2. Serum cytokine levels in patients with pre-existing comorbidities and a history of a malignant disorder

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There was no difference in serum cytokine levels between groups of patients with diabetes, chronic renal failure (CRF), chronic obstructive pulmonary disease (COPD), chronic heart failure (CHF), cerebrovascular disease (CAD), and in patients without any pre-existing comorbidity. Similarly, there was no difference in serum cytokine levels between patients with and without a history of a malignant disorder (data not shown).

3.3. Parameters correlated with platelet count at admission Spearman correlation was performed in order to estimate the degree of association between PLT number at admission in the ICU and other variables (mentioned in the

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section statistical analysis). Parameters with significant correlation with platelet counts were: 1) APACHE II score, [rho=-0.538, (95% CI, -0.662 to -0.387),

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p<0.0001] 2) SOFA score at day +1 of admission, [rho=-0.557, (95% CI, -0.677 to -

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0.410), p<0.0001] 3) serum ICAM levels, [rho=-0.506, (95% CI, -0.636 to -0.348),

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p<0.0001] 4) serum suPAR levels, [rho=-0.613, (95% CI, -0.720 to -0.477), p<0.0001], and 5) serum IL-8 levels, [rho=-0.424, (95% CI, -0.569 to -0.252),

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p<0.0001].

Comparison of serum cytokine levels across groups of patients with different degrees of thrombocytopenia was performed by using one way analysis of variance. Patients with severe thrombocytopenia had significant higher serum ICAM (p<0.001), IL-8

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(p=0.013), and suPAR (p<0.001) levels respectively as compared with patients with normal PLT and mild to moderate thrombocytopenia. Spearman's coefficient of rank

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correlation and results of one-way analysis of variance for each cytokine are shown in

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Figure 1.

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3.4. Parameters correlated with neutrophil count at admission Spearman correlation was performed in order to estimate the degree of association between ANC at admission in the ICU and other variables. No correlation between ANC at admission and serum cytokine levels was observed in our study. Similarly, no correlation between ANC and PLT count at admission was observed (data not shown).

3.5. ICU mortality at 28-days. Prognostic stratification Thirty-eight out of 105 patients died during the first 28 days in ICU for an overall 28day mortality of 36%. The median time to 28-day death after ICU admission was day

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+14.5, (range, 5 - 28). In multivariate analysis higher APACHE II score, thrombocytopenia, and higher serum suPAR levels were statistically significantly

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associated with a higher risk of 28-day mortality. Results of multivariate analysis are

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shown in Table 2.

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ROC analysis was performed for the estimation of optimal cut off levels of APACHE II score, PLT count, and serum suPAR levels for prediction of 28-day mortality. The

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area under the curve (AUC) for APACHE II, PLT and suPAR was 0.719, 0.840, and 0.787 respectively. Results are shown graphically in Figure 2. The sensitivity and specificity of APACHE score >18 in predicting hospital mortality was 63% and 73% respectively. The sensitivity and specificity of PLT count < 120.000 and serum

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suPAR levels > 7.6 in predicting 28-day mortality was 82%, 75% and 82%, 73% respectively. Comparison of ROC curves shows that the predicting ability of PLT was

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higher as compared with APACHE II score (PLT v.s APACHE, p=0.029), while there

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was no difference in the predicting ability between PLT and suPAR, as well as between APACHE II and suPAR, (data not shown).

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Taking into consideration the above cut off levels, patients were categorized in different groups based on APACHE II score (APACHE II>18), PLT counts (PLT<120.000) and serum suPAR levels (suPAR>7.6). Patients with none, 1, 2, or 3 of the variables above or below the predefined cut-off levels were given 0, 1, 2, or 3 points, respectively. In multivariate analysis, the new scoring system remained the only and most significant factor associated with statistically significantly increased 28-day mortality [OR=7.1, (95% CI, 3.1 to 16.1), p<0.0001]. High risk group consisted of 45 (43%) patients with score=2 or 3, low risk group consisted of 37 (35%) patients with score=0, while intermediate risk group consisted of 23 (22%) patients with score=1. Intermediate risk group was associated with increased 28-day

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mortality as compared with low risk group [Hazard Ratio (HR)=10.3 (95% CI, 1.2 to 85.7), p=0.03]. High risk group was associated with increased 28-day mortality as

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compared with intermediate [HR= 4.0 (95% CI, 1.6 to 9.8), p=0.001] and low risk

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group [HR=40.9 (95% CI, 5.5 to 301.1), p=0.0002] respectively. The cumulative

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incidence of 28-day mortality was 3%, 26% and 69% in low, intermediate, and high

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risk group respectively (Figure 3).

3.6. Overall ICU mortality. Causes of death

Fifty-seven out of 105 patients died during their ICU stay for an overall ICU mortality of 54%. The median time to death after ICU admission was day +19, (range, 5-89).

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The cause of death was sepsis related in all 57 patients. Twenty-one out of these 57 patients survived the initial septic event but subsequently developed secondary

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infection such as ventilator associated pneumonia, catheter related sepsis, etc and

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finally died before discharge from ICU. Repeated multivariate analysis with primary end point the overall ICU mortality showed again that higher APACHE II score,

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thrombocytopenia, and higher serum suPAR levels were statistically significantly associated with a higher risk of mortality (data not shown). The new scoring system retained its prognostic significance in predicting overall ICU mortality (data not shown).

3.7. Platelet kinetics during the course of sepsis Eighteen out of 49 patients with normal PLT at admission developed thrombocytopenia during the course of sepsis, while PLT count remained normal in 31 out of 49 patients. Reversal of low PLT counts was observed in 13 out of 56 thrombocytopenic patients, while 43 patients never achieved normal PLT count.

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Development of thrombocytopenia and/or not reversal to normal PLT counts during the course of sepsis (53 patients) is associated with statistically significantly increased

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overall ICU mortality (48 died out of 53 patients), as compared with patients who

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never developed thrombocytopenia or with patients who had reversal of

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thrombocytopenia (9 died out of 52 patients), (p<0.0001).

In multivariate analysis, development of low PLT counts, or not reversal of

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thrombocytopenia during the course of sepsis [OR=14.2, (95% CI, 4.8 – 41.8), p<0.0001] and serum suPAR levels at admission [OR=1.1, (95% CI, 1.0 – 1.2),

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p=0.029] were the most significant predictors for overall ICU mortality.

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4. Discussion

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Thrombocytopenia is very common among patients with severe sepsis. The incidence

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of thrombocytopenia on ICU admission has been estimated around 20% to 30% of

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patients, while a similar percentage is developing thrombocytopenia during ICU hospitalization. However, the incidence of severe thrombocytopenia shows a greater

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variability between 5% and 20% of patients with sepsis [14]. In our study we observed a higher incidence of thrombocytopenia. PLT count below the normal limit was observed in 53% of the patients at the time of admission to ICU, while the incidence of severe thrombocytopenia was 32%. However, during hospitalization, 18

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out of 49 patients with normal PLT at admission developed thrombocytopenia increasing the total incidence of thrombocytopenia during ICU stay to 75%.

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Thrombocytopenia in critically ill patients is a marker of disease severity. Various

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studies have shown that patients with low platelet counts have higher APACHE II scores and increased hospital mortality as compared with patients with normal PLT

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counts [15-17]. A large prospective multicenter observational study including 1238 patients with severe sepsis was performed in Canada between March 2003 and November 2004 with primary objective to determine mortality rates. Hospital mortality was 38.1% and in multivariate analysis thrombocytopenia was identified as an independent variable significantly associated with increased mortality [18]. In consistence with these results, in our study we confirmed a significant correlation between thrombocytopenia and mortality. In a prospective trial studying the epidemiology, etiology and outcome of thrombocytopenia in critically ill patients hospitalized in ICU, sepsis was the most common cause of thrombocytopenia observed in 75%, followed by DIC observed in

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41% of patients. Thrombocytopenia due to hemodilution after massive transfusion and drug-induced thrombocytopenia were observed in 13% and 11% of the patients

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respectively. Thrombotic microangiopathy, hemophagocytic syndrome, HIT were

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observed in small percentages of thrombocytopenic patients [19]. The etiology of

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thrombocytopenia during sepsis is more complex than was previously considered. Activation of coagulation occurs during sepsis and in many cases thrombocytopenia is

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observed in the context of DIC. However many patients with severe thrombocytopenia and sepsis do not fulfill the criteria for overt DIC raising concerns about the pathogenetic mechanisms [20].

Microvasculature endothelial cell dysfunction is considered as a major feature of

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sepsis pathophysiology. Components of the bacterial cell collectively named as pathogen associated membrane patterns (PAMPs) bind to specific receptors on the

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surface of immune and endothelial cells and trigger the inflammatory cascade.

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Activated endothelial cells acquire structural and functional changes, such as the expression of adhesion molecules, including ICAM and VCAM [21]. The latter

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procedure is resulting in platelet activation, initiation of coagulation cascade and leucocyte migration to extravascular space. Important consequences of endothelial dysfunction are endothelial cell apoptosis and detachment, increased vascular permeability, resulting in capillary leaking, redistribution of fluid, hypotension, hypoxia, and finally multi-organ failure. Moreover, disruption of the endothelial integrity is promoting platelet adhesion and subsequent consumption due to extensive and inappropriate activation in the microvasculature [22]. Therefore, endothelial cell/platelet interaction, as expressed by inflammatory mediators, might be a possible pathogenetic mechanism of sepsis-related thrombocytopenia.

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Indeed in our study using Spearman and ANOVA analysis we observed a significant negative correlation between PLT counts and serum levels of biomarkers associated

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with immune activation and endothelial dysfunction such as suPAR, ICAM and IL-8.

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The results of our study support the concept that the severity of thrombocytopenia is

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directly related to the degree of inflammation. On the contrary, in our study we did not observe any significant correlation between ANC and serum cytokine levels

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meaning that ANC is not a marker of the severity of the inflammatory process. Previous studies examined peripheral blood cytokine levels in patients with sepsis, in an effort to identify biomarkers helping in prognostic stratification and in clinical management of critically ill patients [23–26]. Recent studies focused on the

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significance of serum suPAR levels in predicting hospital mortality in patients with sepsis [27–30]. Analysis of data showed that high serum suPAR levels were

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associated with significantly increased sepsis-related mortality. Moreover the

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predictive ability of suPAR was independent of other parameters associated with mortality such as APACHE II score.

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The immune response of the host in sepsis is divided in two sequential phases. The onset of sepsis is characterized by a dominant hyper-inflammatory response resulting in a pro-inflammatory cytokine storm syndrome. This phase is followed by an antiinflammatory response leading to immune cell apoptosis with the aim to restore immune homeostasis and therefore to avoid uncontrolled inflammation. However, the sequence of events is not always finely balanced and a protracted anti-inflammatory response might occur, leading to a state of immune deficiency. At this phase, patients fail to mount normal immune responses, and suffer from viral–reactivation, opportunistic infections, or ventilator pneumonia, finally leading to recurrent episodes of sepsis and death [31]. In many previous trials, the impact of various parameters on

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sepsis outcome was examined by considering early mortality (7-day, 14-day, or 28day mortality) as a reasonable end point. Although early mortality is directly related

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to the pathogenicity of the initial triggering event, overall ICU mortality is more

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representative of the severity of the septic process as a whole. Taking these data into

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account, we decided to examine the impact of various parameters on early (28-day), but also on overall ICU mortality. In accordance with previous data, in our study, 38

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out of 105 (36%) patients died during the first 28-days, while 19 more patients died after day +28 increasing the cumulative incidence of overall ICU mortality to 54%. A significant proportion of patients who survived the initial infectious event, finally died from secondary septic episodes as a further proof of the concept that delayed sepsis

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mortality is attributed to the late immune-suppression state. In our study we observed that thrombocytopenia, high serum suPAR levels and high

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APACHE II score were the only variables significantly associated with increased

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hospital mortality (28-day and overall ICU mortality). ROC analysis was used to determine the best cut-off value of APACHE II score, PLT and suPAR for the

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prediction of hospital mortality. Using the above cut-off levels, a new prognostic system was proposed. Patients were stratified in different risk groups and overall ICU mortality was observed in 15%, 48%, and 93% of patients, while mortality at 28-days was observed in 3%, 26%, and 69% of patients in low, intermediate and high risk group respectively. The predictive ability of the new prognostic system was tested in multivariate analysis, and the total score remained as the only and most significant factor associated with statistically significantly increased hospital mortality. The importance of a single measurement of suPAR at admission as a strong predictor of mortality is further supported by the fact that serum suPAR levels remain constant during the first days of the septic process [27].

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In conclusion, in our study we showed that thrombocytopenia is a common finding in patients with severe sepsis/septic shock. Although DIC is usually present, the

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pathogenesis of thrombocytopenia in patients with sepsis remains poorly understood.

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Severity of thrombocytopenia parallels the severity of inflammation and subsequent

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mortality, as manifested by the significant negative correlation observed between platelet counts and serum levels of ICAM, suPAR, and IL-8. In multivariate analysis,

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high APACHE II score, high serum suPAR and low PLT counts were statistically associated with significantly increased mortality. Patient stratification based on PLT, suPAR, and APACHE II score helps in discrimination of groups with statistically different mortality risk. New prognostic systems with high predicting ability need to

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be developed. The new systems should incorporate simple measurements of highly reproducible variables that tend to remain rather constant instead of displaying

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extensive fluctuations over short periods of time. The proposed new prognostic

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system, need to be further validated in larger, prospective multicenter trials, including centers from different countries and patients with diverse ethnicity before this

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approach can be recommended.

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therapy

the

answer?

Virulence

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LEGENDS FOR FIGURES

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Figure 1: a, b, c) Spearman analysis showed statistically significant negative

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correlation between PLT counts and serum ICAM, IL-8, and suPAR levels,

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respectively. d, e, f) Result of one-way analysis of variance is shown for serum ICAM, IL-8, and suPAR levels in patients with normal, mild to moderate and severe

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thrombocytopenia, respectively: Serum ICAM, IL-8, and suPAR levels were significantly higher in the group of patients with severe thrombocytopenia compared to the levels of the group with normal and mild to moderate thrombocytopenia,

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respectively.

Figure 2: Receiver operating curve analysis testing the ability of APACHE II score,

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serum suPAR levels, and PLT counts for prediction of 28-day mortality.

Figure 3: Cumulative incidence of 28-day mortality in different prognostic groups.

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High risk group (2-3 points), Intermediate risk group (1 point), and low risk group (0 points).

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Table 1: Patients characteristics Thrombocytopenia at admission Mild

Moderate

No of patients

49/105 (47%)

15/105 (14%)

7/105 (7%)

Age, median, range

64, (21-85)

79, (45-89)

61, (53-82)

Sex, male/female

32/17

7/8

Surgery

11/49

4/15

Malignancy

5/49

1/15

Comorbidities&

33/49

Diabetes

Statistics Severe

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Normal PLT at admission (PLT>150.000)

Clinical features

--------

66, (28-90)

n.s

6/1

17/17

n.s

1/7

7/34

n.s

2/7

9/34

n.s

12/15

3/7

19/34

n.s

12/49

3/15

1/7

8/34

n.s

COPD

10/49

2/15

0/7

5/34

n.s

CHF/CAD

9/49

3/15

2/7

6/34

n.s

CRF

7/49

2/15

1/7

3/34

n.s

APACHE score, median, range

16, (5-24)

18, (8-31)

19, (15-22)

21, (9-30)

p<0.0001

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34/105 (32%)

6, (1-12)

8, (6-14)

7, (5-11)

9, (5-16)

p< 0.001

Severe sepsis/ septic shock

36/13

3/12

4/3

8/26

p<0.0001

Bacteremia*

6/49

1/15

2/7

13/34

p=0.007

Gram-negative sepsis**

34/49

2/15

3/7

17/34

n.s

IFNa, median, range (pg/ml)

1.0 (1.0-71.0)

3.3 (1.0-47.3)

1.0 (1.0-23.7)

3.2 (1.0-126.4)

n.s

ICAM, median, range (ng/ml)

80.1 (18.3-747.9)

123.3 (36.4-414.6)

175.7 (95.9-515.7)

226.1 (49.5-835.2)

p<0.0001

IL8, median, range (pg/ml)

7.2 (0.2-174.2)

14.9 (5.3-62.7)

23.1 (3.9-72.1)

38.1 (4.3-1140.4)

p<0.0001

VCAM, median, range (ng/ml)

47.8 (8.0-428.5)

47.3 (25.9-648.7)

37.2 (11.3-339.8)

47.8 (5.6-1061.3)

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5.1 (1.4-10.5)

10.7 (3.5-113.9)

8.4 (6.6-33.7)

13.5 (3.3-57.6)

p<0.0001

ICU mortality, (%)

11/49 (22%)

10/15 (67%)

5/7 (71%)

31/34 (91%)

p<0.0001

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SUPAR, median, range (ng/ml)

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&At least one of the following: Diabetes, chronic obstructive pulmonary disease (COPD), chronic heart failure/cerebrovascular disease (CHF/CAD), chronic renal failure (CRF), *At the time of admission, **Infection due to Gram(-) bacteria detected in blood, urine, BAL, or any other infected tissue at the time of admission.

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Table 2: Early mortality at 28 days. Multiple logistic regression analysis Parameters Odds ratio lower 95% CI upper 95% CI APACHE score 1.17 1.07 1.28 Thrombocytopenia 4.68 1.43 15.25 suPAR levels 1.13 1.01 1.29

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p-value 0.03 0.01 0.04