Ethylene diamine tetraacetic acid (edta)

Venous blood samples were obtained after fasting overnight peripheral venipuncture into two 4.5mL sodium citrate tubes (Becton Dickinson, Plymouth, UK) to measure circulating EMPs, into two 4mL tubes with EDTA (Becton Dickinson, Plymouth, UK) to measure circulating PCs and into two 4mL tubes with EDTA for other biochemical determinations. The latter were centrifuged immediately 10min (800g, 4°C) and, after centrifugation, plasma was aliquoted and stored at -80°C until analysis. Plasma levels of vascular endothelial growth factor (VEGF), interleukin-6 (IL-6), ultra-sensible C-reactive protein (hsCRP), transforming growth factor beta (TGF-β), brain natriuretic peptide (BNP), fibrinogen, hepatocyte growth factor (HGF), letpine, adiponection, cyclic guanosine monophosphate (cGMP), soluble tumor necrosis factor-a receptor type I (sTNFa-RI), soluble intercellular adhesion molecule-1 (sICAM-1) and soluble tyrosine kinase receptor Axl (sAXL) were determined by ELISA using commercially available kits (DuoSet, R&D Systems, Abingdon, UK).

Tubes for sample collection and storage were distributed by Integrated BioBank of Luxembourg (IBBL). Blood samples were collected in the following polypropylene tubes: 10 mL EDTA [Becton, Dickinson and Company (BD), ref. 367525] for plasma, 4 mL EDTA (BD, ref. 368861) for whole blood, and 10 mL clot activator tubes (CAT) (BD, ref. 367896) for serum. CSF was collected in 10 mL polypropylene tubes (Sarstedt, ref. 62.610.018). Blood samples for DNA were not centrifuged and stored at maximal −20°C. All other samples were centrifuged at room temperature at 2,000 × g (min 1,800 × g, max 2,200 × g) and stored at −80°C. A maximum of 2 h was allowed between collection and freezing. A more detailed description of the SOP used for the collection of samples for the central biobank can be found elsewhere (13 (link)). For every subject 2 mL CSF, 2 mL serum, and 2 mL plasma were stored in 0.5 aliquots (in 0.5 mL Matrix 2D Thermo tubes) and 4 mL blood was stored for DNA isolation. Primary specimens and samples derivatives were coded with a three-letter center code and a subject number. Samples were at first stored locally, and then shipped on dry ice to IBBL for long-term storage. DNA extraction was performed at the IBBL. Samples and associated data were processed and stored at IBBL in compliance with ISO 9001:2008, NF S96-900: 2011, and ISO 17025:2005 standards and the ISBER Best Practices.