Sanne de Bruin

195 Biotinylation of platelets for transfusion purposes: a novel method to label platelets in a closed system Appendices Appendix 1. Protocol to biotinylate platelets 1.0 Purpose The aim of this protocol is to describe a technique for biotin-labeling of fresh or stored platelets with Sulfo-NHS-LC-Biotin, in a closed system. This protocol describes how to biotinylate 50 ml of platelet concentrate. 2.0 Equipment, supplies, and reagents 2.1. Sterile docking station, sealing device. 2.2. Calibrated, variable speed, swinging bucket centrifuge. 2.3. Device to remove supernatant of centrifuged platelets 2.4. Calibrated analytical electronic balance, accurate to ±0.00002 g. 2.5. Calibrated generic electronic scale, accurate to 0.5 g. 2.6. Kochers, clamps. 2.7. 0.22 µ sterilizing filter unit. 2.8. PBS: Phosphate buffered saline. 2.9. ACD-A: anticoagulant citrate dextrose solution, Formula A. 2.10. PAS-E : Platelet Additive Solution. 2.11. Product bags for platelets, 150 ml. 3.0 Preparation of biotin solution Note: Dissolved biotin needs to be used directly, or immediately after thawing of frozen product, as dissolved biotin is vulnerable to hydrolysis. Bound biotin is stable. 3.1. Biotin is dissolved in PBS at a concentration of 50 mg/L, and transferred in a 150 ml bag. 3.2. Filtrate biotin-solution by passing a 0.22 µ sterilizing filter. 3.3. Store within 15 minutes at -30º, or use within 30 minutes. 4.1 Preparation of required solutions 4.5. Prepare 150 ml of 15% ACD-A in 85% PAS-E in a sterile, labeled bag. 7