The serum was removed and frozen at -80��C until measurement. Serum sCD40L levels were assayed by specific ELISA (Bender MedSystems, Vienna, Austria) according to the manufacturer’s instructions Sutent in the Atherosclerosis Research Laboratory of CIMA-University of Navarra (Pamplona, Spain).Plasma levels of TFVenous blood samples were collected in citrate collected plasma tubes and centrifuged within 30 minutes at 1,000*g for 15 minutes. The plasma was removed and frozen at -80��C until measurement. Assays for TF antigen were performed by specific ELISA (Imubind Tissue Factor ELISA?, American Diagnostica, Inc, Stanford, CT, USA) in the Laboratory Department of the Hospital Universitario de Canarias (La Laguna, Santa Cruz de Tenerife, Spain).

Serum levels of TNF-�� and IL-10Serum separator tubes (SST) were used to determine TNF-�� and IL-10 serum levels. Venous blood samples were taken and centrifuged within 30 minutes at 1,000 g for 15 minutes, and the serum was removed and frozen at -80��C until measurement. TNF-�� and IL-10 serum levels were measured by a solid-phase, chemiluminiscent immunometric assay kits (Immulite?, Siemens Healthcare Diagnostics Products, Llanberis, UK) in the Laboratory Deparment of the Hospital Universitario de Canarias (La Laguna, Santa Cruz de Tenerife, Spain).Statistical analysisIn a pilot study with 30 patients with severe sepsis, we found that surviving patients show lower circulating levels of sCD40L (3.83 �� 1.44 ng/mL) than non-survivors (4.37 �� 1.52 ng/mL).

We calculated to include 186 patients in a cohort study to demonstrate significant differences in the circulating levels of sCD40L between groups, for a power of 80% and a 5% type I error rate.Continuous variables are reported as medians and interquartile ranges. Categorical variables are reported as frequencies and percentages. Comparisons of continuous variables between groups were carried out using Wilcoxon-Mann-Whitney test. Comparisons between groups on categorical variables were carried out with chi-square Carfilzomib test. The association between continuous variables was carried out using Spearman’s rank correlation coefficient or Spearman’s rho coefficient. The cut-off 3.5 ng/mL was selected using a likelihood method as previously described [29]. Receiver operation characteristic (ROC) curves using lactate, APACHE score, sCD40L ��3.5 ng/mL as independent variables, and exitus at 30 days as a dependent variable were obtained. To calculate the standard error of the area under the curves we used the method of Delong et al. [30]. Survival curves at 30 days, using sCD40L levels �� or lower than 3.5 ng/mL, were represented using the Kaplan-Meier method and were compared by log-rank test.