Automated fluorescence gating and size determination reduce variation in measured concentration of extracellular vesicles by flow cytometry

Extracellular vesicles (EVs) are an upcoming biomarker for disease. However, the measured concentrations of EVs by flow cytometry are incomparable due to analytical variables. This study aimed to investigate how the choice of fluorophore, and thereby brightness, affects the measured concentration of EVs. Four commonly used fluorophores allophycocyanin, Brilliant Violet‐421, fluorescein isothiocyanate, and phycoerythrin, all conjugated to CD61 antibodies, were used to label platelet‐derived extracellular vesicles (PEVs) in human plasma. PEVs were measured by flow cytometry. The concentration of EVs was obtained by manually set fluorescence gates, automatically determined fluorescence gates, and automatically determined fluorescence gates combined with specific size gates. Manually set fluorescence gates by five independent experts resulted in a variation coefficient (CV) of 41% between the measured PEV concentrations labeled with the four different fluorophores. A new algorithm for automatic determination of fluorescence gates was applied to reduce inter‐operator variability. Applying this algorithm resulted in a CV of 58%. However, when the algorithm was combined with a size gate to correct for differences in brightness between fluorophores, the CV reduced to 25%. In this study, we showed that different fluorophores can detect similar concentrations of EVs by (1) determining fluorescence gates automatically, and (2) by adding a size gate to correct for differences in brightness between fluorophores. Therefore, our research contributes to further standardization of EV concentration measurements by flow cytometry.