Hollow fiber flow field-flow fractionation enables high-quality Extracellular Vesicle isolation from minimal plasma samples in polycythemia vera liquid biopsy

Introduction: Extracellular vesicles (EVs) are crucial mediators of intercellular communication, transporting various macromolecules between cells. They are increasingly recognized for their roles in cancer progression, immune modulation, and therapeutic resistance. However, standard EV isolation methods often struggle to preserve EV heterogeneity and functional integrity. Methods: In this study, we used hollow-fiber flow field-flow fractionation (HF5) to isolate and characterize plasma-derived EVs from just 60 μL of plasma. HF5 is a cutting-edge, disposable microfluidic technique designed for advanced EV fractionation. EVs were from patients with polycythemia vera (PV), a rare hematological malignancy. We evaluated EVs isolated using HF5 against those obtained by size-exclusion chromatography (SEC), assessing their physico-chemical characteristics, surface marker expression and functionality in terms of up taking and inflammatory potential. Results: EVs isolated through HF5 closely resembled SEC-derived EVs in size, morphology, and classical EV markers, including platelet-specific proteins. HF5 consistently yielded purer EV preparations with reduced aggregation and greater reproducibility. Notably, HF5 achieved this using 8.3 times less plasma than SEC. HF5 EVs, along with inflammatory potential, showed superior cell up take to the SEC counterparts. Chemical analysis showed that HF5-EVs contained a higher protein concentration, while SEC-EVs had more aggregated material. Conclusion: HF5 integrates EV isolation and characterization, enhancing efficiency, and preserving sample integrity and functionality. Its minimal sample requirement and reproducibility make it particularly suitable for clinical and translational research. Our study demonstrated HF5 as a powerful better alternative to conventional EV isolation methods, with strong potential for standardized applications in biomarker discovery and cancer research.