Comparison of Methods for Assessing the Concentration of Extracellular Vesicles Isolated From Different Biological Fluids

Accurate quantification of extracellular vesicles (EVs) remains a significant challenge in biomedical research. Although various analytical methods have been developed, their reliability is often limited by the presence of non-vesicular nanoparticles and biological contaminants, particularly in biological fluids. Moreover, for some sources of EVs, such as uterine aspirates and gastric juice, quantitative evaluation has been scarcely addressed. The aim of the study is to perform a comparative analysis of three EV quantification methods: total protein content measurement, nanoparticle tracking analysis (NTA), and esterase activity assessment using the commercial FluoroCet exosome quantitation kit in EVs isolated from various biological fluids: blood plasma, ascitic fluid, uterine aspirates, gastric juice, and conditioned medium of ovarian and non-small cell lung cancer cell lines. For EV samples derived from conditioned medium, all three methods demonstrated strong correlation, supporting their validity for EV quantification in highly purified samples. In contrast, plasma, ascitic fluid, and uterine aspirates exhibited discrepancies between methods, likely attributable to the presence of non-vesicular nanoparticles. Notably, EVs from gastric juice demonstrated a strong correlation between protein content and esterase activity, indicating a prevalence of vesicle-associated proteins and a potentially unique EV composition in this fluid. The findings underscore the necessity for a multifactorial approach to EV quantification, taking into account factors such as sample origin and limitations inherent to the specific method employed. These results may serve as a basis for the development of standardized protocols for EV quantification, which is particularly relevant for clinical sample analysis.