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.
