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To ensure single particle detection, plasma samples are diluted based on their particle concentration, which can differ 100\u2010fold 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\u2010in purified lipoproteins, (ii) at different dilutions, and (iii) using either scatter\u2010triggering or fluorescence\u2010triggering. 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 \u2018dilution paradox\u2019 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\u2010evaluated. The dilution paradox might be overcome by using fluorescence\u2010triggering at a fixed sample dilution, removing non\u2010EV particles from the sample, removing fluorescent particulates from staining reagents, and setting stricter inclusion criteria.<\/jats:p>","DOI":"10.1002\/jev2.70312","type":"journal-article","created":{"date-parts":[[2026,6,24]],"date-time":"2026-06-24T04:25:34Z","timestamp":1782275134000},"source":"Crossref","is-referenced-by-count":3,"title":["The Dilution Paradox in Extracellular Vesicle Flow Cytometry"],"prefix":"10.1002","volume":"15","author":[{"ORCID":"https:\/\/orcid.org\/0009-0008-4220-1800","authenticated-orcid":false,"given":"Joyce","family":"Rops","sequence":"first","affiliation":[{"name":"Department of Laboratory Medicine, Laboratory Specialized Diagnostics &amp; Research, Laboratory of Experimental Clinical Chemistry, Amsterdam UMC University of Amsterdam  Amsterdam the Netherlands"},{"name":"Biomedical Engineering &amp; Physics Amsterdam UMC, University of Amsterdam  Amsterdam the Netherlands"},{"name":"Atherosclerosis and Ischemic Syndromes Amsterdam Cardiovascular Sciences  Amsterdam the Netherlands"},{"name":"Department of Neurology Leiden University Medical Center  Leiden the Netherlands"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3201-7205","authenticated-orcid":false,"given":"Naomi","family":"Buntsma","sequence":"additional","affiliation":[{"name":"Department of Laboratory Medicine, Laboratory Specialized Diagnostics &amp; Research, Laboratory of Experimental Clinical Chemistry, Amsterdam UMC University of Amsterdam  Amsterdam the Netherlands"},{"name":"Biomedical Engineering &amp; Physics Amsterdam UMC, University of Amsterdam  Amsterdam the Netherlands"},{"name":"Department of Neurology Amsterdam University Medical Centers, University of Amsterdam  Amsterdam the Netherlands"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5083-7587","authenticated-orcid":false,"given":"Aleksandra","family":"G\u0105secka","sequence":"additional","affiliation":[{"name":"Department of Laboratory Medicine, Laboratory Specialized Diagnostics &amp; Research, Laboratory of Experimental Clinical Chemistry, Amsterdam UMC University of Amsterdam  Amsterdam the Netherlands"},{"name":"1st Chair and Department of Cardiology Medical University of Warsaw  Warsaw Poland"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3007-1849","authenticated-orcid":false,"given":"Leonie","family":"de Rond","sequence":"additional","affiliation":[{"name":"Department of Laboratory Medicine, Laboratory Specialized Diagnostics &amp; 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