Evaluating Variability in Extracellular Vesicle Characterization Across Measurement Techniques

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.