Evaluating Variability in Extracellular Vesicle Characterization Across Measurement Techniques

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.