Sanne de Bruin

191 Biotinylation of platelets for transfusion purposes: a novel method to label platelets in a closed system Biotinylation of platelets has previously been described under invalidated, experimental conditions. 1,13 Our protocol fulfils GPG requirements. Since US Food and Drug Adminis- tration (FDA) guidelines differ from European GPG requirements, the protocol needs to be validated according to the FDA standard before it can be implemented in the US. Our work can serve as a reference method. We adapted the previous protocol on various crucial points (Table 2). 13 After sterile fil- tration of the sulfo-NHS-biotin-solution, the procedure took place in a closed-system, minimizing the risk of bacterial contamination. We added an extra washing step, to minimize nonspecific biotinylation of plasma proteins in the PC. Our group showed that PAS-E can be used to optimize platelet storage. 19 We testedwhether PAS-E could be used to store the sulfo-NHS-biotin solution (for 42 days at (< -30°C). However, the quality of biotinylation decreased after storage of dissolved biotin in PAS-E. The biotin label re- mained stable when dissolved and stored at < -30ºC in PBS. Therefore, sulfo-NHS-biotin was dissolved and stored in PBS at a concentration of 50mg/L. Shortly before biotinyla- tion, the sulfo-NHS-biotin solution was diluted in PAS-E in a 1:9 ratio, to obtain a con- centration of 5 mg/L. Since the reactive group of dissolved sulfo-NHS-biotin is instable, the sulfo-NHS-biotin-solution should either be used within 30 minutes, or after frozen storage at < -30º C, to be used within 42 days of storage. After thawing, the solution should be used within 30 minutes, to avoid hydrolysis. Since >97% of all platelets were biotinylated using our protocol, we found 30 minutes of incubation to be sufficient to adequately biotinylate platelets, instead of the previously described 45 minutes. 1 TABLE 2. Adaptations on the previously described protocol Protocol described by van der Meer 13 Our protocol Advantage A platelet sample is centrifuged, the supernatant replaced by the biotin solution. Centrifuged and supernatant removed twice before incubation. Limits rest-biotinylation of proteins in PC. Incubation in saline in which biotin is added at a final concentration of 25 mg/L. The biotin solution was diluted 1:9 in PAS-E. Less activation of the platelets. Incubation for 45 minutes Incubation for 30 minutes. Time reducing. Resuspended in saline/ACD. Resuspended in PAS-E. Superior storage conditions, mimics PC more realistically. 250 ml 50 ml Minimal amount of traceable platelets. BioPLTs and sham samples showed equal decreases in platelet quality parameters as compared to control platelets. Hence, platelets were affected by the processing steps, 7