dSEC enables ultracentrifugation-free sample preparation for single-vesicle flow cytometry of amniotic fluid.

Single extracellular vesicle (EV) flow cytometry (FCM) of amniotic fluid-derived EVs (AF-EVs) holds great potential for prenatal diagnosis and mechanistic studies of fetal diseases and pregnancy complications. However, conventional sample preparation workflows rely heavily on ultracentrifugation (UC) for both EV isolation and unbound labels removal, limiting their clinical applicability. To address this limitation, this study aimed to develop a simple, high-purity, and UC-free workflow. First, dichotomic size-exclusion chromatography (dSEC), previously established by our group, was employed for AF-EV isolation. Multidimensional purity characterization was performed using nanoparticle tracking analysis, transmission electron microscopy, western blotting, and proteomic profiling confirming the suitability of dSEC for AF samples. A head-to-head comparison revealed that dSEC yielded significantly more particles than conventional UC, while maintaining a comparable level of purity. Following antibody incubation with AF-EVs, unbound fluorescent antibodies were efficiently removed via a second dSEC. Using this dSEC-based workflow, CD9+ EVs accounted for (38.00 ± 7.03)% of total AF-EVs. Moreover, after 7 days of storage at 4 °C, no significant changes were observed in the FCM-detected proportion of CD9+ EV, particle concentration, or relative fluorescence intensity distribution. In conclusion, this study demonstrates a dSEC-based sample preparation workflow for single-EV FCM analysis of AF, featuring UC independence, simple operation, high purity, and favorable stability. This workflow provides reliable technical support for the development, clinical translation, and application of single-EV FCM in AF.