The dilution paradox in extracellular vesicle flow cytometry

The concentration of extracellular vesicles (EVs) can be measured by flow cytometry, which detects fluorescence and light scattering signals from single particles in a sample. To ensure single particle detection, plasma samples are diluted based on their particle concentration, which can differ 100-fold between samples. Here we studied how sample dilution affects EV detection by flow cytometry. EVs were measured in human plasma (i) with different concentrations of spiked-in purified lipoproteins, (ii) at different dilutions, and (iii) using either scatter-triggering or fluorescence-triggering. In addition, the reliability of two previously published clinical EV studies was evaluated using an analytical model. To prevent swarm detection, reliable scatter-based EV detection requires a minimum dilution factor, which can exceed the maximum dilution factor ensuring that labelled EVs exceed the fluorescent background events in number. This “dilution paradox” precludes reliable EV detection with scatter-triggering for samples requiring different dilutions. In the two clinical studies, 30% and 65% of the measurements were unreliable, depending on the label used. Therefore, results from previous studies should be re-evaluated. In the future, the dilution paradox might be overcome by using fluorescence-triggering at a fixed sample dilution, removing non-EV particles from the sample, and including fasting patients when measuring plasma.