Additionally outcome parameters such as survival until hospital discharge, consecutive organ failure (definitions: acute kidney failure = increase in creatinine www.selleckchem.com/products/Bortezomib.html ��0.3 mg/dL, or ��1.5-fold climb in baseline creatinine within 48 hours; acute liver failure = increase in International Normalized Ratio ��0.5 within 72 hours; acute heart failure = clinical signs of cardiac decompensation or cardiogenic shock), severity of disease (with the help of Sequential Organ Failure Assessment (SOFA)) and neurological outcome (on the basis of Glasgow Outcome Scale (GOS)) of survivors were recorded in both groups (Table (Table11).Table 1Baseline characteristics of resuscitated and control patientsLaboratory methodsBlood sampling and handlingBlood samples in resuscitated patients were collected immediately after admission to the ICU and 24 hours after ROSC, using an arterial line.

In controls and healthy subjects, 20 mL blood was drawn from the arterial line in case of coronary catherization or by venipuncture on the second day and in healthy controls, respectively. Samples were drawn slowly, handled carefully and analysed immediately after sampling. For venipuncture, we used a 21-gauge butterfly needle and discarded the first 7.5 mL.AntibodiesThe following monoclonal antibodies (mAb) were purchased from Beckman Coulter (Marseille, France): anti-human CD61-FITC, anti-human CD14-FITC. Anti-human CD11b-FITC was purchased from Becton Dickinson (San Jose, CA, USA), annexin V and the mouse IgG1-PE and IgG1-FITC were from BD Biosciences Pharmingen (San Diego, CA, USA) and anti-CD62E-PE was obtained from Southern Biotech (Birmingham, AL, USA).

Flow cytometry analysisFlow cytometry was performed using a three-colour FACSCalibur? cytometer (BD Biosciences; San Jose, CA, USA) running Cell-Quest? version 3.3 (BD Biosciences, San Jose, CA, USA), acquisition and analysis software. Instrument setup with individual settings for each antibody and calibration were performed with CaliBRITE? beads (BD Biosciences, San Jose, CA, USA), according to the manufacturer’s recommendations.Sample preparation for measurement of MMPs, procoagulant PMPs, and EMP-platelet conjugatesBlood samples were collected in citrated tubes. According to the protocol of Hughes et al., samples were centrifuged for 10 minutes at 240 g at room temperature to obtain platelet-rich plasma and MPs were distinguished from platelets on the basis of their characteristic flow cytometric profile [40].

Supernatant was diluted 1:50 with Tyrode buffer and was incubated for 30 minutes at room temperature in the dark with fluorochrome-labelled antibodies. After the fixation of the tubes with 300 ��L of CellFix? (BD Biosciences, Erembodegem-Aalst, Belgium), samples were ready to be analysed AV-951 by flow cytometry. MPs were counted on the basis of the events, positive for the cell-specific mAbs (see below) and values are given in counts/��L.