From platelets to particles: Optimizing extracellular vesicle isolation in a National Blood Centre

BACKGROUND AND OBJECTIVES: Platelet-derived extracellular vesicles (PEVs) are submicron, membrane-bound particles released upon platelet activation, with a recognized role in haemostasis, inflammation and immunoregulation. PEVs remain insufficiently characterized in blood products. This study compared four isolation methods to evaluate their impact on PEV yield, purity and characteristics, aiming to identify a practical approach for transfusion service workflows. MATERIALS AND METHODS: PEVs were isolated from expired single-donor aphaeresis platelet concentrates (n = 12) using methods based on different isolation principles: ultracentrifugation (UC), size exclusion chromatography (SEC), mixed size/charge separation (hybrid) and an affinity-based spin column method (affinity). Size, number and biochemical marker expression of all extracellular vesicle (EV) isolates were assessed. RESULTS: PEVs were successfully isolated by all methods, although at varying yields. The overall size distribution of all methods was similar, although SEC and affinity methods isolated PEVs with the largest diameters. PEV isolated by the affinity method had the lowest lipid:protein ratio, consistent with high purity. No differences in expression of EV marker CD9 or platelet activation marker CD42b were found. CONCLUSION: Comparison of the physical and biochemical characteristics of the PEVs isolated by each method reveals that the affinity method was superior to other methods. In addition, its simplicity, cost effectiveness and accessibility make it a practical option for blood transfusion services to further explore the role of PEVs.