Aim: Despite the growing number of disease-associated extracellular vesicle (EV) biomarkers being identified, the translation of EV diagnostics into routine clinical laboratory tests remains limited. The main challenge lies in the lack of simple, rapid, reproducible and high-throughput EV isolation and downstream analysis methods that can be easily integrated into clinical laboratory workflows. In this study, we evaluated a high-throughput bead-based immunoaffinity system (EXO-NET®) that captures a highly enriched subpopulation of EVs.
Methods: EVs were manually isolated from 500 µl of human pooled plasma using EXO-NET and compared with three other commercial EV isolation kits. EVs were characterized according to MISEV guidelines. High-throughput EV isolation was performed on two automated systems, Maxwell® and KingFisher®, with RNA extraction carried out using manual ReliaPrep™ RNA Miniprep Systems or Maxwell® RSC miRNA Plasma and Serum Kit for downstream analysis.
Results: RT-qPCR analysis established that expression levels of miR-21, miR-16, and let-7a were similar between the Maxwell system and the manual protocol using the ReliaPrep RNA isolation kit. The fully-automated Maxwell miRNA kit significantly improved the yield and recovery by 7-9 cycles, corresponding to an increase of ≥100 times compared to the manual protocol. Comparable expression of miR-21 and miR-16 was observed between the Maxwell and KingFisher systems.
Conclusions: The combination of high-throughput EV isolation using EXO-NET and the Maxwell high-throughput miRNA kit represents the first fully-automated high-throughput system for rapid, efficient and scalable enrichment of EVs. This approach holds great promise for facilitating the integration of EV diagnostics into routine clinical practice.