Standardization of extracellular vesicle measurements by flow cytometry through vesicle diameter approximation

Abstract

BACKGROUND Detection of extracellular vesicles (EVs) by flow cytometry (FCM) has poor inter-laboratory comparability due to differences in FCM sensitivity. Previous workshops distributed polystyrene beads to set a scatter-based diameter gate to improve the comparability of EV concentration measurements. However, polystyrene beads provide limited insight into the diameter of detected EVs. OBJECTIVES To evaluate gates based on the estimated diameter of EVs instead of beads. METHODS A calibration bead mixture and platelet EV samples were distributed to 33 participants. Beads and a light scattering model were used to set EV diameter gates to measure the concentration of CD61-phycoerythrin positive platelet EVs. RESULTS Of the 46 evaluated FCMs, 21 FCMs detected the 600-1,200 nm EV diameter gate. The 1,200-3,000 nm EV diameter gate was detected by 31 FCMs, with a measured EV concentration inter-laboratory variability of 81% compared to 139% by the bead diameter gate. Part of the variation in both approaches is caused by precipitation in some of the provided platelet EV samples. Flow rate calibration proved essential because systems configured to 60 μL/minute differed 6-fold in measured flow rates between instruments. CONCLUSIONS EV diameter gates improve the inter-laboratory variability compared to previous approaches. Of the evaluated FCMs, 24% could not detect 400 nm polystyrene beads, and such instruments have limited utility for EV research. Finally, considerable differences were observed in sensitivity between optically similar instruments, indicating that maintenance and training affect the sensitivity. This article is protected by copyright. All rights reserved.