Evaluating Variability in Extracellular Vesicle Characterization Across Measurement Techniques.
J Extracell Biol 2026 Jun; 5(6):e70154.

Abstract

Reliable, reproducible, and standardized characterization of extracellular vesicles (EVs) remains a central challenge due to methodological variability across analytical platforms and intrinsic EV heterogeneity within and across biofluids. Here, we evaluated EVs isolated using ultracentrifugation from dedifferentiated liposarcoma-conditioned media and commercially available healthy donor urine. Physical characterization and surface tetraspanin profiling of isolated EVs were performed using three widely used instruments: NanoFCM, CytoFLEX Nano, and ZetaView Evolution. All platforms consistently detected small EVs; however, absolute size distributions, particle concentrations, and tetraspanin expression levels varied, reflecting differences in optical configuration, detection principles, and fluorescence background sensitivity. Additionally, EV-associated DNA was quantified using two different methods, revealing further variability in both the DNA yield and quantity. Together, the observations highlight the method-dependent nature of EV analyses and underscore the importance of carefully considering instrument-specific metrics for interpreting EV data across biofluids.

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Authors+Show Affiliations

Singh PKDepartment of Mechanical and Aerospace Engineering The Ohio State University Columbus Ohio USA.
Usmani AFDepartment of Mechanical and Aerospace Engineering The Ohio State University Columbus Ohio USA.
Halder DDepartment of Mechanical and Aerospace Engineering The Ohio State University Columbus Ohio USA.
Miller JDepartment of Mechanical and Aerospace Engineering The Ohio State University Columbus Ohio USA.
Kanagasabai TDepartment of Mechanical and Aerospace Engineering The Ohio State University Columbus Ohio USA.
Sarchet PComprehensive Cancer Center The Ohio State University Columbus Ohio USA.
Weller KComprehensive Cancer Center The Ohio State University Columbus Ohio USA.
Klarmann GJ4DBio3 Center for Biotechnology Department of Radiology and Bioengineering Uniformed Services University of the Health Sciences Bethesda Maryland USA. The Geneva Foundation Tacoma Washington USA.
Pollock REComprehensive Cancer Center The Ohio State University Columbus Ohio USA.
Calore FComprehensive Cancer Center The Ohio State University Columbus Ohio USA. Department of Cancer Biology and Genetics The James Comprehensive Cancer Center The Ohio State University Columbus Ohio USA.
Prakash S0000-0002-5263-0843Department of Mechanical and Aerospace Engineering The Ohio State University Columbus Ohio USA. Comprehensive Cancer Center The Ohio State University Columbus Ohio USA.

Pub Type(s)

Journal Article

Language

eng

PubMed ID

42256441