Flow cytometric analysis of extracellular vesicle subsets in plasma: impact of swarm by particles of non-interest

BACKGROUND: Extracellular vesicles (EVs) in plasma are increasingly recognized as potential biomarkers. EV analysis for diagnostic purposes should be robust and should allow analysis of EV subsets in a wide range of abundance and in a large number of patient samples. Flow cytometry offers possibilities to meet these criteria as it allows multi-parameter analysis of individual EVs. However, analysis of plasma EVs is challenging due to their size and heterogeneity and the presence of other submicron-sized particles in plasma that could interfere in EV-analysis. OBJECTIVES: Explore whether fluorescence-based flow cytometric analysis of EV subsets is suitable when the EVs of interest are present in low abundance in a background of non-labelled or differently labelled EVs and particles. METHODS: Fluorescently labeled EVs of interest were spiked at different ratios in full plasma, purified plasma components, or (non-)fluorescent polystyrene beads, and subsequently analyzed by flow cytometry using fluorescence threshold triggering. RESULTS: We found that light scatter detection of low-abundant or rare EV subsets during fluorescence threshold triggering were severely affected by particles of non-interest due to coincidence and swarm. Importantly, we show that interfering particles labelled with different fluorophores induced false-positive fluorescent signals on the particles of interest. These unwanted effects could only be discerned and controlled by performing serial dilutions and analyzing light scatter and fluorescence parameters. CONCLUSIONS: We demonstrate how particles of non-interest in plasma can impact the light-scatter and fluorescence detection of low-abundant EVs of interest during fluorescence-based flow cytometric analysis, and provide means to prevent erroneous data interpretation. This article is protected by copyright. All rights reserved