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This study used orthogonal techniques, cryogenic electron microscopy (Cryo-EM), particle tracking analysis (PTA), asymmetrical flow field-flow fractionation (AF4), and microfluidic resistive pulse sensing (MRPS), to evaluate particle size distributions (PSDs) and particle number concentrations (PNCs) of human mesenchymal stem cells (MSCs) and LNCaP prostate cancer cell EVs. Proteomic profiles were assessed by mass spectrometry (MS), and microRNA (miRNA) content of LNCaP EVs was evaluated by small RNA-seq at two independent laboratories. A commercial green fluorescent protein exosome served as a control, except in Cryo-EM, proteomic, and miRNA analyses. Cryo-EM, regarded as the gold standard for morphological resolution, served as PSD reference. PSDs from all methods skewed larger than Cryo-EM, with MRPS closest, AF4 most divergent, and PTA intermediate with broader distributions. All techniques reported broad PSDs (30 nm to &gt;350 nm) with PNCs decreasing with increasing particle size, except for AF4. Quantitative discrepancies in PNCs reached up to two orders of magnitude across methods and cell sources. MS identified global and EV-specific proteins, including syntenin-1 and tetraspanins CD9, CD63, and CD81. RNA-seq revealed notable inter-laboratory variation. These findings highlight the variability across measurement platforms and emphasize the need for reproducible methods to support NIST\u2019s mission of developing reliable EV reference materials.<\/jats:p>","DOI":"10.3390\/biom16010066","type":"journal-article","created":{"date-parts":[[2025,12,31]],"date-time":"2025-12-31T16:34:23Z","timestamp":1767198863000},"page":"66","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":9,"title":["Intermethod Characterization of Commercially Available Extracellular Vesicles as Reference Materials"],"prefix":"10.3390","volume":"16","author":[{"given":"Sumeet","family":"Poudel","sequence":"first","affiliation":[{"name":"National Institute of Standards and Technology, Gaithersburg, MD 20899, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Diane L.","family":"Nelson","sequence":"additional","affiliation":[{"name":"National Institute of Standards and Technology, Gaithersburg, MD 20899, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5242-7629","authenticated-orcid":false,"given":"James H.","family":"Yen","sequence":"additional","affiliation":[{"name":"National Institute of Standards and Technology, Gaithersburg, MD 20899, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5731-6143","authenticated-orcid":false,"given":"Yuefan","family":"Wang","sequence":"additional","affiliation":[{"name":"Department of Pathology, School of Medicine, Johns Hopkins University, Baltimore, MD 21231, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-8726-7098","authenticated-orcid":false,"given":"Hui","family":"Zhang","sequence":"additional","affiliation":[{"name":"Department of Pathology, School of Medicine, Johns Hopkins University, Baltimore, MD 21231, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0002-4650-2826","authenticated-orcid":false,"given":"Zhiyong","family":"He","sequence":"additional","affiliation":[{"name":"National Institute of Standards and Technology, Gaithersburg, MD 20899, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2065-4218","authenticated-orcid":false,"given":"Ashley Beasley","family":"Green","sequence":"additional","affiliation":[{"name":"National Institute of Standards and Technology, Gaithersburg, MD 20899, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0524-8403","authenticated-orcid":false,"given":"Wyatt N.","family":"Veerland","sequence":"additional","affiliation":[{"name":"National Institute of Standards and Technology, Gaithersburg, MD 20899, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1992-8450","authenticated-orcid":false,"suffix":"IV","given":"Thomas E.","family":"Cleveland IV","sequence":"additional","affiliation":[{"name":"National Institute of Standards and Technology, Gaithersburg, MD 20899, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sean E.","family":"Lehman","sequence":"additional","affiliation":[{"name":"National Institute of Standards and Technology, Gaithersburg, MD 20899, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3474-779X","authenticated-orcid":false,"given":"Kurt D.","family":"Benkstein","sequence":"additional","affiliation":[{"name":"National Institute of Standards and Technology, Gaithersburg, MD 20899, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-8166-4121","authenticated-orcid":false,"given":"Bryant C.","family":"Nelson","sequence":"additional","affiliation":[{"name":"National Institute of Standards and Technology, Gaithersburg, MD 20899, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2456-898X","authenticated-orcid":false,"given":"Lili","family":"Wang","sequence":"additional","affiliation":[{"name":"National Institute of Standards and Technology, Gaithersburg, MD 20899, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2025,12,31]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"301","DOI":"10.1007\/s10571-016-0366-z","article-title":"Introduction to Extracellular Vesicles: Biogenesis, RNA Cargo Selection, Content, Release, and Uptake","volume":"36","author":"Abels","year":"2016","journal-title":"Cell. 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