From 55 patients and 20 healthy volunteers another 8 ml was collected into heparin-coated tubes (Vacutainer) and used for the isolation of peripheral blood mononuclear cells (PBMCs). For 83 patients, blood sampling was repeated on the day of worsening of sepsis stage. For 30 patients who progressed into septic shock, blood was sampled daily for seven days starting sellekchem immediately after the start of vasopressors. Tubes were transported by a courier service within the same day to the Laboratory of Immunology of Infectious Diseases of the 4th Department of Internal Medicine at ATTIKON University Hospital of Athens. Tubes were centrifuged and serum was kept frozen at -70��C until assayed. IgM was estimated in duplicate by an enzyme-linked immunosorbent assay (e-Bioscience Inc.

, San Diego, CA, USA) following the manufacturer��s instructions; the lower detection limit was 20 ng/ml. All estimations were performed and reported by two technicians who were blinded to clinical information.The central laboratory of the study participates in the UK NEQAS quality control system for leukocyte immunophenotyping (registration number 40926). In this laboratory, the absolute count of B lymphocytes was measured as described elsewhere [12]. Briefly, red blood cells were lysed with ammonium chloride 1.0 mM. White blood cells were washed three times with phosphate-buffered saline (PBS) (pH 7.2) (Merck, Darmstadt, Germany) and subsequently incubated for 15 minutes in the dark with the monoclonal antibody anti-CD19 at the flurochrome fluorescein isothiocyanate (FITC, emission 525 nm, Immunotech, Marseille, France) using fluorospheres (Immunotech) for the determination of absolute counts.

One IgG isotypic negative control at the fluorocolor FITC was analyzed for every patient. Cells were analyzed after running through the EPICS XL/MSL flow cytometer (Beckman Coulter, Inc., Miami, FL, USA) with gating for mononuclear cells based on their characteristic forward scatter/side scatter (FS/SS) scattering.The isolation of PBMCs was limited to 55 patients because these samples should come from patients hospitalized at study sites close to the central laboratory. This allowed the time from blood collection until processing to be less than 30 minutes. As such, PBMCs were studied from patients hospitalized at the ATTIKON University Hospital that is close to the central laboratory of the study.

Production of IgM was studied according to a procedure described elsewhere Drug_discovery [13]. Heparinized venous blood was layered over Ficoll Hypaque (Biochrom, Berlin, Germany) and centrifuged for 20 minutes at 1400 g. Separated PBMCs were washed three times with ice-cold PBS (pH: 7.2) (Biochrom) and counted in a Neubauer chamber. Their viability was more than 99% as assessed by trypan blue exclusion of dead cells.