Abstract
Nanoparticle tracking analysis (NTA) can be used to quantitate extracellular vesicles (EVs) in biological samples and is widely considered a useful diagnostic tool to detect disease. However, accurately profiling EVs can be challenging due to their small size and heterogeneity. Here, we aimed to provide a protocol to facilitate high-precision particle quantitation by NTA in plasma, the supernatant of activated purified platelets [the platelet releasate (PR)] and in serum, to increase confidence in NTA particle enumeration. The overall variance and the precision of NTA measurements were quantified by root mean square error and relative standard error. Using a bootstrapping approach, we found that increasing video replicates from 5 s × 60 s to 25 s × 60 s captures led to a reduction in overall variance and a reproducible increase in the precision of NTA particle-concentration quantitation for all three biofluids. We then validated our approach in an extended cohort of 32 healthy donors. Our results indicate that for vesicles sized between 50 and 120 nm, the precision of routine NTA measurements in serum, plasma, and PR can be significantly improved by increasing the number of video replicates captured. Our protocol provides a common platform to statistical compare particle size distribution profiles in the exosomal-vesicle size range across a variety of biofluids and in both healthy donor and patient groups.
Highlights
Extracellular vesicles (EVs) are released by most eukaryotic cells and are found in a diverse range of human biofluids
We sought to determine the effect of increasing video replicates on the precision of particle concentration quantitation using Nanoparticle tracking analysis (NTA) in the biofluids of plasma, serum, and the supernatant of activated purified platelets [the platelet releasate (PR)]
We examined the effect of increasing video replicates on the total variance of NTA measurement of particles in plasma, PR, and serum
Summary
Extracellular vesicles (EVs) are released by most eukaryotic cells and are found in a diverse range of human biofluids. They regulate a diverse range of biologic and inflammatory pathologic processes and have been implicated in myriad of diseases [1,2,3]. As such, they have emerged as a promising source of future biomarkers in biofluids with both diagnostic and prognostic value [4, 5]. Nanoparticle tracking analysis (NTA) is widely used to determine the particle size distribution of a sample [6,7,8,9]. The concentration of particles is determined by counting total particles and representing the concentration as a binned histogram [13]
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