The Dilution Paradox in Extracellular Vesicle Flow Cytometry.

Concentrations of extracellular vesicles (EVs) can be measured by flow cytometry, which detects fluorescence and light scattering signals from single particles. 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 published clinical EV studies was evaluated using an analytical model. Our results show that the minimum dilution factor to prevent swarm detection by the light scattering detector can exceed the maximum dilution factor that ensures labelled EVs outnumber fluorescent background events. This 'dilution paradox' precludes reliable detection of both fluorescence and light scattering signals of EVs for some samples. 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. The dilution paradox might be overcome by using fluorescence-triggering at a fixed sample dilution, removing non-EV particles from the sample, removing fluorescent particulates from staining reagents, and setting stricter inclusion criteria.