Extracellular vesicle characteristics in stored red blood cell concentrates are influenced by the method of detection

Extracellular vesicles (EVs), including microvesicles and exosomes, are small phospholipid vesicles (≤1 μm in diameter) that are present in blood products, accumulate during storage, and have a potential transfusion-related immunomodulatory role. Knowledge of EVs in stored blood products is limited due to the challenges and difficulties in detecting these heterogeneous submicron-sized vesicles. The aim of this study was to assess the impact of different approaches to characterize EVs in stored RBC products. Quantification and size-profiling of EVs in leukoreduced red cell concentrates (RCCs) were examined on day 3, 7, 21, and 42 of storage using tunable resistive plus sensing (TRPS), flow cytometer (FC), and dynamic light scatting (DLS) methods. Using the TRPS method, the concentration of EVs < 200 nm significantly increased throughout storage (p < 0.05). This change in exosome concentration was not detectable with FC or DLS due to limitations in their ability to resolve particles <200 nm and/or accurately determine EV concentration. Both the TRPS and FC demonstrate that the concentration of EVs ≥ 200 nm significantly increases in RCCs by day 42/43 compared to EVs present on day 3 (p < 0.001). As the DLS measures the average size of particles in suspension, only an increase in the zeta-average size was observed during storage. EV size and concentration in RBC products is significantly influenced by the length of storage. Overall, this study shows that combining technologies may be important to improve the characterization and study of EVs in stored RCCs.