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Background  Tetraspanin expression of extracellular vesicles (EVs) is often used as a surrogate for their detection and classification, a practice that typically assumes their consistent expression across EV sources.    Results  Here we demonstrate that there are distinct patterns in colocalization of tetraspanin expression of EVs enriched from a variety of in vitro and in vivo sources. We report an optimized method for the use of single particle antibody-capture and fluorescence detection to identify subpopulations according to tetraspanin expression and compare our findings with nanoscale flow cytometry. We found that tetraspanin profile is consistent from a given EV source regardless of isolation method, but that tetraspanin profiles are distinct across various sources. Tetraspanin profiles measured by flow cytometry do not totally agree, suggesting that limitations in subpopulation detection significantly impact apparent protein expression. We further analyzed tetraspanin expression of single EVs captured non-specifically, revealing that tetraspanin capture can bias the apparent multiplexed tetraspanin profile. Finally, we demonstrate that this bias can have significant impact on diagnostic sensitivity for tumor-associated EV surface markers.    Conclusion  Our findings may reveal key insights into protein expression heterogeneity of EVs that better inform EV capture and detection platforms for diagnostic or other downstream use.    Graphical abstractSupplementary Information  The online version contains supplementary material available at 10.1186/s12951-021-00987-1.

Tetraspanins are unevenly distributed across single extracellular vesicles and bias sensitivity to multiplexed cancer biomarkers