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

Abstract

Background Detection of extracellular vesicles (EVs) by flow cytometry has poor interlaboratory comparability, owing to differences in flow cytometer (FCM) sensitivity. Previous workshops distributed polystyrene beads to set a scatter-based diameter gate in order to improve the comparability of EV concentration measurements. However, polystyrene beads provide limited insights 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 in order to measure the concentration of CD61-phycoerythrin-positive platelet EVs. Results Of the 46 evaluated FCMs, 21 FCMs detected the 600-1200-nm EV diameter gate. The 1200-3000-nm EV diameter gate was detected by 31 FCMs, with a measured EV concentration interlaboratory variability of 81% as compared with 139% with 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 min differed six-fold in measured flow rates between instruments. Conclusions EV diameter gates improve the interlaboratory variability as compared with 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.

Extracellular vesicles from human saliva promote hemostasis by delivering coagulant tissue factor to activated platelets

“…The diameter of EVs stained for TF and CD24 are mostly 200 nm and larger, as shown by TEM. In suspension, the diameter of EVs will be slightly larger because EVs shrink to dehydration and fixation by about 14% when being prepared for TEM . Therefore, a major fraction of the single EVs staining for TF and/or CD24 is indeed above the detection limit and thus can be measured by our flow cytometer.…”

Hollow organosilica beads as reference particles for optical detection of extracellular vesicles

“…An alternative to setting EV size gates with HOBs is to relate the scattering intensity of solid polystyrene and silica reference beads to that of EVs by the use of Mie theory . Mie theory accounts for RI differences, but requires complex software and knowledge of the optical configuration of the flow cytometer.…”

“…Samples were measured for 1 min at a flow rate of 3.01 μL min −1 and with a sheath pressure of 150 mbar. Rosetta Calibration (Exometry, Amsterdam, the Netherlands) was used to relate SSC to the size and RI of nanoparticles according to Mie theory . To validate this relationship, the SSC of silica beads (Kisker Biotech) was measured at a concentration of 10 7 mL −1 .…”

Centrifugation affects the purity of liquid biopsy‐based tumor biomarkers

“…The concentration of beads before and after centrifugation was measured on side scatter of a flow cytometer (FACSCalibur; BD, Franklin Lakes, NJ, 15 mW 488 nm laser, flow rate~60 μl/min calibrated by weight (22), SSC 400 V, gain 0, threshold 0), and data were analyzed using FlowJo (v10; FlowJo LLC, Ashland, OR). The concentration of beads was measured by flow cytometry before and after centrifugation at 300g for 20 min or 2,700g for 22 min using a Rotina 380R centrifuge (Hettich, Tuttlingen, Germany) or 15,800g for 60 min using a SW 41 Ti rotor and Optima L-80 XP ultracentrifuge (Beckman Coulter, Fullerton, CA), all at 20 C and with deceleration set to the minimum possible value.…”