The concentration of cells is a key component of modern blood tests. Given the biomarker potential of extracellular vesicles (EVs) in blood, we aimed to establish reference ranges for blood cell‐derived EVs using flow cytometry. To address the orders‐of‐magnitude variability in reported EV concentrations between different flow cytometers (FCMs), we first validated a calibration methodology to enable reproducible EV concentration measurements. The methodology was evaluated in an interlaboratory comparison study and shows that calibration reduces the median absolute deviation of EV concentrations measured on 25 different FCMs from 67 % to 25 %–31 %. The calibration methodology was then used to determine reference ranges of erythrocyte‐, leukocyte‐, and platelet‐derived EVs in human blood plasma in a cohort of healthy individuals ( n =  224). This study demonstrates that calibration enables comparable concentration measurements of blood cell‐derived EVs, thereby bringing EVs one step closer to clinical applications.

The National Institute of Standards and Technology (NIST) is developing analytical methods to characterize extracellular vesicles (EVs) to support the urgent need for standardized EV reference materials (RMs). 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 >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’s mission of developing reliable EV reference materials.

Extracellular vesicles (EVs) are membrane-surrounded vesicles that carry heterogeneous cellular components, including proteins, nucleic acids, lipids, and metabolites. EVs’ intravesicular and surface contents possess many biomarkers of physiological and pathological importance. Because of the heterogeneous cargo, EVs can mediate local and distal cell–cell communication. However, the way in which the genome signature regulates EV cargo has not been well studied. This study aimed to understand how genetics impact EV cargo loading. EVs were isolated from vector copy number cells with a fluorescent reporter (GFP) with varying inserted transgene copies and from NIST SRM 2373 cells (MDA-MB-231, MDA-MB-453, SK-BR-3, and BT-474), which contain varying copies of the HER2 gene. Spectradyne nCS1 was utilized to count EVs and measure size distribution. Imaging Flow Cytometry was used to analyze the surface protein content of single EVs and for total EV counts. The RNA content of the EVs was measured using ddPCR. Our results from stable reporter cell lines and breast cancer cell lines suggest that the gene copy number dictates the protein cargo of the EVs but not the RNA content. Increasing copies of a reporter gene (GFP) or a naturally occurring gene (HER2) from breast cancer cells correlated with increasing EV counts positive for the protein cargo compared to total EV counts until a copy threshold was reached. This study has broad implications for understanding EV biology in the context of cancer biology, diagnostics, EV biology/manufacturing, and therapeutic delivery.