Extracellular vesicles (EV) are secreted from both healthy and diseased tissues and are detectable in most body uids, where their measurement can be of prognostic/diagnostic value. We aimed to evaluate pre-analytical and analytical variables in the measurement of EV in human plasma using nanotracking analysis (NTA) and tunable resistive pulse sensing (TRPS).
Commercial beads (200 nm and 400 nm in diameter) and human plasma from volunteer donors were used in this study. A total of 36 bead and 175 plasma measurements were undertaken by NTA and TRPS. The pre- and analytical conditions tested on plasma samples were: fresh, stored for 1 day or 1 week at 4°C or -80°C. The other variables included testing of neat or pelleted EVs and choice of diluent.
The 200 nm and 400 nm beads, when tested alone or combined showed coef cient of variations (CV) of <10% at all dilutions. The CVs for triplicate results of plasma samples varied between 3-43%, with samples frozen for 1 week showing the least CV spread (5-15%). The EV counts from pellets or supernatants obtained after centrifugation of plasma at 21000g for 1 h, were different to the neat samples. Although both the supernatant and pellet fractions contained EVs of approximately 100 nm in size, only the pellet contained larger vesicles of 300 nm, and only the supernatant contained vesicles <100 nm.. Different EV counts were obtained for the same plasma aliquot using NTA and TRPS.
Measurements by NTA can be performed on fresh and/or frozen samples provided similar storage and centrifugation conditions are used. The CVs for plasma samples are high indicating the need for standardized conditions. TRPS requires at least two different nanopore lters for measurements and plasma as diluent in lower chamber for optimal results. NTA and TRPS measure EVs by different approaches and the knowledge of the size range for measurement by each method is critical.