For the Online Version of the Manuscript: Crivellari Et Al

For the online version of the manuscript: Crivellari et al. Human protein C zymogen concentrate in patients with severe sepsis and multiple organ failure after adult cardiac surgery.

--Statistics and laboratory methods used to measure protein C and other parameters.

--Table 1: Microbiological findings.

--Table 2: laboratory variables

--Figure 3: fluid balance

Statistical Analysis

For repeated measurements Friedman’s two-way analysis of variance was used. Post hoc testing of values versus baseline was performed by Wilcoxon-testing for paired samples; p < 0.05 (two-sided) was considered statistically significant. Data for continuous variables are given as median and interquartile range. Statistical analyses were performed using Systat 7.0 for Windows (SPSS Inc., Chicago, IL).

Laboratory methods

Serial venous samples (4.5 ml) were collected in siliconized Vacutainer tubes (Becton-Dickinson, Plymouth, UK) containing (0.5 ml) tri-sodium citrate (0.129 M) and in tubes containing 0.5 ml of a mixture of tri-sodium citrate and benzamidine-HCl (200mM) at the following times: before the bolus dose, 6 hours after bolus and every 12 hours thereafter up to 78 hours. Within 1 hour from collection, platelet poor plasma was obtained by centrifugation for 10 minutes at 2,000 x g at room temperature. Prothrombin time (PT, Hemoliance Recombiplastin, Instrumentation Laboratory, Lexington, MA), activated partial thromboplastin time (aPTT, STA aPTT Kaolin, Diagnostica Stago, Asnier sur Seine, France ), fibrinogen (FG, clotting assay, STA Fibrinogen, Stago), and D-dimer (STA Liatest D-D, Stago) determinations were performed on fresh citrated plasma samples with an automated coagulometer (STA, Stago). Plasma aliquots were snap-frozen with methanol and dry ice and stored at – 70°C for additional measurements in citrated plasma of protein C anticoagulant activity (STA Protein C, Stago), and antithrombin (amidolytic activity, STA Antithrombin, Stago). Blood samples collected in tri-sodium citrate and benzamidine-HCl were also centrifuged as described above with plasma aliquots snap-frozen and stored at -70°C. Within one month, prothrombin fragment 1+2 (F1+2, Enzygnost F1+2, Dade-Behring, Marburg), thrombin-antithrombin III complex (TAT Enzygnost TAT micro, Dade-Behring, Marburg), and interleukins (IL) 6, 8 and 10 (R&D systems, Minneapolis, MN, USA) were measured with commercially available ELISA kits. Quantification of activated protein C (APC) levels was by an home-made ELISA as described by Liaw et al [18]. Briefly, 96-well vinyl microtiters plates (Costar) were coated with the monoclonal antibody HAPC 1555 (5 µg/ml in coating buffer: 20mM HEPES, pH7.5, 150mM NaCl, 5mM CaCl2). Benzamidine-HCl plasma samples were anticoagulated and recalcified by addition of heparin 2 IU/ml, HEPES 20 mM, pH 7.5 and CaCl2, 10 mM (final concentrations) and incubated (100 µl) at room temperature for 30 min with the coated antibody. Wells were washed thrice and the chromogenic activity of bound APC was measured by addition of 100 µl S2366 (0.5 mM) (Chromogenix, Mölndal, Sweden) in coating buffer. Substrate hydrolysis was monitored at 405 nm. A standard curve was generated with increasing amounts of APC (Xigris, Eli Lilly, Indianapolis, IN), from 0 to 50 ng/ml, spiked into coating buffer.

Table 1 online: Microbiological findings for the online version of the manuscript.

Table 2 online. Medians and interquartile range of laboratory variables for the online version of the manuscript. P values are according to Friedman’s two-way analysis of variance. The asterisks (*) indicate the first time point showing a statistically significant difference over baseline levels (p ≤ 0.03)

Abbreviations:

PT : prothrombin time;

aPTT: acitvated partial thromboplastin time;

AT: antithrombin;

PC:C: protein C anticoagulant activity;

APC: circulating activated protein C;

F1.2: prothrombin fragment 1.2;

IL: interleukin.

Fig 3 (for the online version of the manuscript)