Sanne de Bruin

182 Chapter 7 Standardized platelet biotinylation procedure The biotinylation procedure is depicted in Figure 1B. Biotinylation of platelets was per- formed in a closed system. The full protocol for biotinylation of platelets is provided in Appendix 1. A 50 ml portion of PC was transferred to a small transfer bag. Transfusion of a biotinylated PC aliquot of 50 ml would theoretically achieve a bioPLT enrichment of 2.5-7% in healthy subjects. Plasma proteins that could interfere with biotinylation were reduced by washing the platelet concentrate twice for PCs stored in plasma or once for PCs stored in 65% PAS-E. Prior to centrifugation, samples were acidified by ACD-A (Anticoagulant citrate dextrose Solution A, Terumo BCT) to prevent irreversible clumping during centrifuging (1700 x g for 10 minutes). For the first washing step, 100 ml of 8.5:1.5 PAS-E / ACD-A solution was added to the 50 mL of platelet concentrate. 150 ml of a 9:1 PAS-E/ACD-A solution was added or the second washing step. The washed platelets were incubated with the 100 mL biotin solution, for 30 minutes, under gentile agitation, at 22º Celsius. Twelve ml of ACD-A was added prior to centrifugation (1700 x g for 10 minutes), after which the biotinylated platelets were resuspended in PAS-E, to their original volume of 50ml. To confirmbiotinylation, samples of both biotinylated and unbiotinylated platelets were counterstained with Streptavidin 488 (1:200), Alexa Fluor 488 conjugate (Thermo Fisher Scientific, catalogue number: S32354), incubated for 30 minutes at room temperature, washed (1700 x g for 10 minutes) and measured by flow cytometry on a LSRII + HTS (BD Biosciences). Data were analyzed with FlowJo v(CFC). Validation experiment For the validation procedure, aliquots of six in plasma stored PCs were biotinylated at day 1 and day 7 of storage. At these time points a ‘sham’-sample was also obtained from the same unit, in which all processing steps were identical to the biotinylated samples, except the incubation with sulfo-NHS-biotin, which was replaced by incubation with a 1:9 PBS-PAS-E solution. Platelets that were biotinylated after one day of storage were subsequently stored for two more days and tested for biotinylation and quality param- eters (hence, at day three of storage after donation). To show applicability to various types of PCs, also three platelet concentrates that were obtained via apheresis and three PCs stored in 65% PAS-E were biotinylated at day 1 of storage. Storage of bioPLT Two methods of storage of bioPLT were tested: first 50 mL bioPLT and sham platelets were stored for three days after processing and then tested for quality parameters (Supplement Figure 1B). Since this led to inacceptable high platelet activation a second method was tested. We hypothesized that storage of a small sample in this storage bag caused the high platelet activation. Therefore, 50 ml of bioPLTwas returned to the