Sanne de Bruin

183 Biotinylation of platelets for transfusion purposes: a novel method to label platelets in a closed system retained original PC, resulting in the original volume of approximately 330ml consisting of labeled and unlabeled platelets (Supplement Figure 1C). Therefore triple staining was necessary to distinguish platelet activation of the unlabeled and labeled platelets, at day of biotinylation and day 4 to day 7 of storage. Additional conditions To evaluate the effect of storage of the sulfo-NHS-biotin solution on biotinylation, a part of the sulfo-NHS-biotin-PBS- solution was stored within 30 minutes after dilution at -30 (± 5) °C. After42 days the frozen sulfo-NHS- biotin-PBS- solution was thawed at 37°C in 10 minutes to approximately 20°C . After thawing, the sulfo-NHS-biotin-PBS- solution was diluted 10 times with PAS-E (at 20°C) to a final concentration of 5 mg/L, aliquoted to portions of 100 ml, and used within 30 minutes. To analyze the effect of irradiation on the biotin label, biotin-labeled platelets were irradiated after labeling with a dose of 25 Gray (according to standard blood bank reg- ulations). Samples were obtained prior and after irradiation to assess the effect of this treatment on the biotin label. Platelet quality parameters Ranges for quality parameters were pre-defined according to local blood bank guide- lines and are expressed in Table 1. Blood gas analysis was performed to determine the pH of the platelet concentrates. (Rapidlab 1265, Siemens Medical Solution Diagnostics). Platelet counts were measured on an Advia 2120 (Siemens Medical Solutions Diagnos- tics). The morphology of the platelets was assessed both non-invasive (swirl) and invasive (microscopically). To perform the platelet swirling test, the motion of the platelets was assessed visually by gently moving the bag in front of a light source. The swirl was re- corded as positive (3), moderate (2), impaired (1) or absent (0). The test was performed by an independent, experienced, laboratory staff member, who was blinded for the intervention. Platelet morphology was also assessed by light microscopy, for which 50 μl of platelet concentrate wasmixedwith 250 μl 0.5%glutaraldehyde (Merck, Darmstadt Germany) in PBS. The fixed platelets were visualized with a 1000 times magnification (Axio, Zeis, Breda, the Netherlands). Baseline platelet activation was assessed by CD62P expression. 14 The samples were incubated for 20 minutes at RT with PE-mouse anti-human CD62P (BD Pharmingen Biosciences) and FITC mouse antihuman CD61 (BD Pharmingen Biosciences. For the 7