A systematic evaluation of isolation techniques and freeze-thaw effects on plasma extracellular vesicle heterogeneity and subpopulation profiling

A Proximity-dependent Barcoding Assay (PBA) was applied to evaluate three distinct EV isolation methods—asymmetrical flow field-flow fractionation (AF4), automated centrifugal microfluidic disc system combined with functionalized membranes (Exo‑CMDS), and size-exclusion chromatography (SEC)—comparing their efficiency in isolating EVs from both freshly frozen and freeze-thawed plasma samples by detecting 262 EV membrane proteins and analyzing them at both bulk and single-EV levels. In this study, plasma was isolated from peripheral blood of a healthy subject through sequential centrifugation and storage at –80 °C, with a subset undergoing freeze-thaw cycles every 3 to 7 days. Subsequently, extracellular vesicles (EVs) were extracted using three distinct isolation methods, and 262 EV membrane proteins were profiled using Proximity Barcoding Assay (PBA) technology at both bulk and single-EV levels.