A Protocol for Improved Precision and Increased Confidence in Nanoparticle Tracking Analysis Concentration Measurements between 50 and 120 nm in Biological Fluids

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.

Keywords: extracellular vesicles; nanoparticle tracking analysis; particle enumeration; plasma; platelet releasate; platelets; serum.

Figure 1

The precision of nanoparticle tracking analysis (NTA) concentration measurements is enhanced by increasing the number of video replicates in all bin widths. For plasma, platelet releasate and serum the average relative standard error (RSE) per bin width from a total of 900 bootstrapped samples was reproducibly decreased by increasing n video replicates. As RSE is a measure of the statistical precision of a sample measurement, the reduction in RSE with increased video replicates indicated that more video replicates led to increased precision of NTA measurements.

Figure 2

Precision of nanoparticle tracking analysis concentration measurements from 32 healthy donors is increased by increasing the number of video replicates in all bin widths. For plasma and platelet releasate, the average relative standard error per bin width from 32 healthy donors was reproducibly reduced by increasing video replicates from n = 5 to n = 15.