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Preanalytical, analytical, and biological variation of blood plasma submicron particle levels measured with nanoparticle tracking analysis and tunable resistive pulse sensing

Morten Mørkab, Shona Pedersenabc, Jaco Bothaa, Sigrid Marie Lunda & Søren Risom Kristensenabc
Background: Nanoparticle tracking analysis (NTA) and tunable resistive pulse sensing (TRPS) enable measurement of extracellular vesicles (EVs) in blood plasma but also measure other particles present in plasma. Complete isolation of EVs from similarly sized particles with full EV recovery is currently not possible due to limitations in existing isolation techniques.
Aim: This study aimed to evaluate preanalytical, analytical, and biological variation of particle measurements with NTA and TRPS on blood plasma.
Methods: Blood from 20 healthy subjects was sampled in the fasting and postprandial state. Platelet free plasma (PFP) was analyzed immediately and after a freeze-thaw cycle. Additionally, the effect of prandial state and a freeze-thaw cycle on EV-enriched particle fractions obtained via size-exclusion chromatography (SEC) was examined.
Results: We observed analytical linearity in the range of 1.0–10.0 × 108 particles/mL for NTA and 1.0 × 108–1.8 × 109 particles/mL for TRPS. The analytical variation was generally below 10%. A considerable intra- and inter-individual variation was demonstrated with estimated reference intervals of 1.4 × 1011–1.2 × 1012 particles/mL for NTA and 1.8 × 108–1.6 × 109 particles/mL for TRPS. Food intake and to a lesser extent a freeze-thaw cycle affected particle populations in PFP and, similarly, in EV-enriched fractions.
Conclusion: In this study NTA and TRPS enabled acceptably precise concentration and size measurement of submicron particles in PFP. An appreciable intra- and inter-individual biological variation was observed. In studies on particle populations in PFP or EV-enriched fractions, we recommend analysis of fresh, fasting samples.
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