Venosafe plastic tubes

Blood samples for leptin, unacylated ghrelin, creatinine, glucose, urea, free fatty acids and electrolytes (Na²⁺, K⁺, Cl^–) were drawn from the antecubital vein after overnight fasting. Samples were collected into VenoSafe plastic tubes (VenoSafe^®, Terumo Europe, Leuven, Belgium) containing silica gel. Blood samples were centrifuged (3,500 rpm, 10 min) and serum was frozen at −20°C for later analysis. Leptin and ghrelin were determined with an ELISA-kit immunoassay system (Dynex DS 2, Dynex Technologies, Chantilly, VA, United States); creatinine, glucose and urea with a photometric enzymatic method (Konelab 20 Xti Clinical Chemistry Analyzer, Thermo Scientific, Vantaa, Finland); free fatty acids with an enzymatic colorimetric assay (Konelab 20 Xti Clinical Chemistry Analyzer, Thermo Scientific, Vantaa, Finland); and electrolytes (Na, K, Cl) with an ion selective electrode method (ISE; Konelab 20 Xti Clinical Chemistry Analyzer, Thermo Scientific, Vantaa, Finland).
The sensitivity and inter-assay coefficient of variation for these assays were: 0.2 ng/ml, 6.1% for leptin; 0.6 pg/ml, 17.3% for ghrelin; 2.32 μmol/l, 2.2% for creatinine; 0.1 mmol/l; 3.8% for glucose; 1.1 mmol/l, 5.8% for urea, 10 μmol/l, 5.8% for free fatty acids; 100 mmol/l, 0.7% for sodium; 2 mmol/l, 2.5% for potassium and 55 mmol/l, 3.3% for chloride.

Blood samples were processed, clinically annotated, anonymized, and aliquoted at the Biobank of the CHU UCL Namur. Whole-blood samples were collected by venipuncture using Venosafe Plastic Tubes (product n° VF-109SP, Terumo Europe). Clotting time was 30 min after which the samples were centrifuged at 3000g for 15 min at 4 °C and the serum fraction collected. Ten millimolar EDTA (pH 8) was added to stabilize the nucleosomes in the serum which were aliquoted, frozen, and stored at −80 °C.

PB was collected in heparin-coated tubes (Venosafe plastic tubes, Terumo Europe N.V., Leuven, Belgium) and PB mononuclear cells (PBMC) were isolated using high density centrifugation (Lympholyte; Cedarlane Laboratories, SanBio B.V., Uden, the Netherlands). PBMC (0.5×10⁶ cells) were stained using anti-human CD19 PerCP-Cy5.5 and CD4 APC to discriminate between B and T cells, respectively (BD Biosciences, Erembodegem, Belgium). B cell subpopulations and surface molecules were defined using following anti-human antibodies: IgD APC-Cy7, CD27 PE-Cy7, HLA-DR/DP/DQ (major histocompatibility complex (MHC)-II) FITC, CD80 PE and CD86 PE-CF594 (all from BD Biosciences, Erembodegem, Belgium). Following anti-human monoclonal antibodies were used for T cell analysis: CD45RA APC-H7, CD45RO PE-CF594, CXCR5 Alexa Fluor 488 and PD-1 PE-Cy7 (all from BD Biosciences, Erembodegem, Belgium), CD25 PerCP-Cy5.5 and CD127 PE (eBioscience, San Diego, USA). Following isotype controls were used: mouse IgG1 PErCP-Cy5.5, IgG1 PE, IgG1 PE-Cy7, IgG2aκ PE-CF594, IgG2bκ APC-H7, IgG1 APC, IgG2aκ FITC, IgG1κ PE-CF594, IgG1 PE-Cy7, IgG2aκ APC-H7 and rat IgG2b Alexa Fluor 488 (all from BD Biosciences, Erembodegem, Belgium). All flow cytometric analyses were performed on a FACSAriaII flow cytometer and analyzed with FACS Diva software (BD Biosciences).