Calibration of Flow Cytometers Enables Reproducible Measurements of Extracellular Vesicle Concentrations and Reference Range Establishment

The concentration of cells is a key component of modern blood tests. Given the biomarker potential of extracellular vesicles (EVs) in blood, we aimed to establish reference ranges for blood cell-derived EVs using flow cytometry. To address the orders-of-magnitude variability in reported EV concentrations between different flow cytometers (FCMs), we first validated a calibration methodology to enable reproducible EV concentration measurements. The methodology was evaluated in an interlaboratory comparison study and shows that calibration reduces the median absolute deviation of EV concentrations measured on 25 different FCMs from 67 % to 25 %-31 %. The calibration methodology was then used to determine reference ranges of erythrocyte-, leukocyte-, and platelet-derived EVs in human blood plasma in a cohort of healthy individuals (n = 224). This study demonstrates that calibration enables comparable concentration measurements of blood cell-derived EVs, thereby bringing EVs one step closer to clinical applications.