Size and concentration analyses of extracellular vesicles by nanoparticle tracking analysis: a variation study

Area of Science:

• Biotechnology
• Analytical Chemistry
• Nanotechnology

Background:

• Current extracellular vesicle (EV) characterization methods lack standardization, hindering data comparability.
• Nanoparticle tracking analysis (NTA) is widely used for determining EV size and concentration.
• Analytical variation in NTA can arise from EV heterogeneity and instrument settings.

Purpose of the Study:

• To assess analytical variation in NTA for EV characterization.
• To evaluate the impact of instrument settings, operators, and software versions on NTA measurements.
• To highlight the need for standardized NTA protocols in EV research.

Main Methods:

• Determined intra-assay and inter-assay variations (% CV) for EVs and artificial samples using NanoSight NS500 instruments.
• Compared results using identical software settings versus instrument-optimized settings.
• Investigated the effects of different operators and software versions on NTA measurements.

Main Results:

• Intra-assay CVs ranged from 1-12% for both EVs and artificial samples.
• Day-to-day variations were 2-25%, with significant differences between instruments using identical settings.
• Instrument-optimized settings reduced inter-instrument discrepancies; operator and software variations were less significant than day-to-day variations.

Conclusions:

• Performance variations between NTA instruments can lead to significant divergences in EV size and concentration measurements.
• Standardized NTA execution and monitoring of variation parameters are essential for reliable EV characterization.
• Optimized instrument settings can mitigate some analytical variability in NTA.