Sanne de Bruin

180 Chapter 7 contamination compared to other blood products, a method for biotinylating platelets in a closed system was developed. In this manuscript, we describe the results of a method to produce a biotinylated plate- let product in a closed system, in accordance to GPG, with minimal platelet activation, which can be used in clinical research in humans. Material and Methods Platelet concentrates Platelet concentrates (PCs) were manufactured and stored by Sanquin Blood Bank, ac- cording to the Dutch Blood Bank standards. Whole blood (WB) collections (500ml) were obtained from volunteer, non-remunerated donors. WB was centrifuged and separated after overnight hold into red cell concentrates, plasma and buffy coats. To obtain a PC, pooled buffy coats from five donors were re-suspended in 100% plasma or 65% PAS-E (Terumo BCT, Inc Lakewood CO USA) and 35% plasma and leukoreduced by filtration. Single donor apheresis PCs were obtained according to the manufacturer’s instructions (Trima, Terumo BCT). PCs were stored under gentle agitation, at 20-24°C. Informed consent to use their blood for research purposes was obtained from all donors. The validation protocol was approved by the Sanquin department of Quality Assurance. Preparation of the biotin solution Sulfo-NHS-biotin was dissolved in phosphate-buffered saline (PBS,140.3 mmol/L NaCl, 10.9 mmol/L Na 2 HPO 4 .2H 2 O, 1.8 mmol/L NaH 2 PO 4 .2H 2 O, pH 7.4, Fresenius Kabi) to a concentration of 50 mg/L (EZ-link Sulfo-NHS--Biotin, 100mg; Thermo scientific). The sulfo-NHS-biotin-solution was sterilized by passing it through a 0.22 µMfilter (Fresenius HemoCare, Fresenius Kabi) using a sterile connection device (Sterile TurbingWelder-Ter- umo BCT, TSCD II) and a 600-mL container (Compoflex, Fresenius Kabi). After filtration of the sulfo-NHS- biotin-solution, the biotinylation took place in a closed system, to prevent microbiological contamination. To obtain the final concentration of 5 mg/L, the sulfo-NHS-biotin solution was diluted 1:9 in Platelet Additive Solution (PAS-E: 0.030% MgCl.12H 2 O, 0.037% KCl, 0.105% NaH 2 PO 4 .2H 2 O, 0.318% C 6 H 5 Na 3 O 7 .2H 2 O, 0.405% NaCl, 0.442 %, C 2 H 3 NaO 2 .3H 2 O, 0.769%Na 2 HPO 4 .12H 2 O, pH: 7.1-7.5) (Terumo BCT), and divided in portions of 100 mL, and used within 30 minutes after diluting