Isolation of extracellular vesicles from human plasma samples: The importance of controls.

Abstract

Extracellular vesicles (EV) are enriched with proteins and RNA cargo, promoting cell-to-cell communication. Biofluid derived EV cargo is used for discovering disease specific markers for diagnosis and disease monitoring. Blood is a complex fluid with an abundance of protiens and thus isolation of EVs is challenging. Therefore, methods for EV isolation, including commercial kits use thromboplastin D (TP-D) for pretreatment of plasma to increase EV purity and yield. This pretreatment can introduce contaminants. We performed a comparative study to evaluate the effect ofEV isolation methods focusing on (a) pretreatment of plasma with additives, which include: rabbit TP (rTP) versus human recombinant thromboplastin (huTP), to increase purity and yield (b) an additional centrifugation step prior to freezing plasma and (c) comparison of frozen versus fresh plasma EV isolations. Pretreatment with rTP generated a dynamic range of proteins, however, most of these proteins were contaminants, introduced from the rTP (99.1% purity). As an alternative, huTP was used, which did not introduce any significant contaminants, however, this did not increase yield or purity. Additionally, an extra 10,000g centrifugation did not improve either EV yield or purity. Finally, comparison of fresh or frozen plasma showed no significant difference, an important factor when sourcing plasma from biobanks. Appropriate controlsare required when adding any additives during EV isolationas even a small percentage of contaminants can have a major effect on results. Furthermore, biobanked plasma can be used with no major changes to processing.