Sanne de Bruin

184 Chapter 7 triple staining platelets were also incubated with Streptavidin 647 (1:200), Alexa Fluor 647 conjugate (Thermo Fisher Scientific, catalogue number: S32357). Immunoglobulin G1 Mouse PE conjugated (Immunotech SAS, Beckman Coulter, Marseille France) was used as isotype control for the CD62P activation. To assess platelets’ ability to become activated, platelets were incubated simultaneously with CD61 and CD62P. Agonists used were 625 nM of thrombin receptor-activating peptide, (TRAP-6-amide/trifluoracetate salt, Bachem AG, Switzerland) or 125 µl/ L of adenosine di-phosphate (ADP, Chronolog, Havertown, USA). The reaction was stopped after 20 minutes by adding formaldehyde 1% (Merck) in PBS. 15 Annexin V binding was assessed as amarker for phosphatidylserine (PS), as described. 16 Flow cytometrywas performed using a LSRII + HTS (BD Biosciences). Data were analyzed with FlowJo v(CFC). The nucleotide content of the platelets was assessed in neutralized perchloric acid extracts, which were stored at -80°C, until batch analysis with high-performance liquid chromatography using a cation exchange, column as described previously. 4,17 Platelet concentrates were cultured by BacT/ALERT(Bio Merieux), both before and after the biotinylation procedure, to rule out bacterial contamination. Statistical analysis Statistical analyses were performed in R, version 3.5.0. Variables were assessed for nor- mality and corresponding statistical tests were performed (paired t-test for parametric data, Mann-Whitney U test for non-parametric data). Differences were considered to be statistically significant if the p-value was < 0.05. Results Biotinylation of platelet concentrates Six pooled buffy-coat derived PCs in plasma, three pooled buffy-coat derived PCs in 65% PAS-E/35%plasma and three apheresis PCs in plasma were biotinylated as described in the protocol (Appendix 1). After the biotinylation-procedure, 98.4% ± 0.9% and 99.0% %± 0.9%of platelets were labeled with biotin at day 1 and day 7 of storage respectively (Figure 2). There was no difference in biotinylation of PCs obtained from pooled buffy coats and stored in plasma as compared to apheresis PCs and PCs stored in PAS-E. The unbiotinylated fraction and the biotinylated fraction of the PC could be visualized as two distinctive populations on flow cytometry. We confirmed that biotin labeling of platelets is still successful after 42 days of storage of the dissolved biotin solution at