title: Preanalytical, analytical, and biological variation of blood plasma submicron particle levels measured with nanoparticle tracking analysis and tunable resistive pulse sensing.
abstract: 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.
  
  
  OBJECTIVE
  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 × 10(8) particles/mL for NTA and 1.0 × 10(8)-1.8 × 10(9) 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 × 10(11)-1.2 × 10(12) particles/mL for NTA and 1.8 × 10(8)-1.6 × 10(9) 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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: a Department of Clinical Biochemistry , Aalborg University Hospital , Denmark ;|b AF Study Group , Aalborg University Hospital , Denmark ;
authors.forename: Morten
authors.initials: M
authors.lastname: Mørk
authors.affiliation: a Department of Clinical Biochemistry , Aalborg University Hospital , Denmark ;|b AF Study Group , Aalborg University Hospital , Denmark ;|c Department of Clinical Medicine , Aalborg University , Aalborg , Denmark.
authors.forename: Shona
authors.initials: S
authors.lastname: Pedersen
authors.affiliation: a Department of Clinical Biochemistry , Aalborg University Hospital , Denmark ;
authors.forename: Jaco
authors.initials: J
authors.lastname: Botha
authors.affiliation: a Department of Clinical Biochemistry , Aalborg University Hospital , Denmark ;
authors.forename: Sigrid Marie
authors.initials: SM
authors.lastname: Lund
authors.affiliation: a Department of Clinical Biochemistry , Aalborg University Hospital , Denmark ;|b AF Study Group , Aalborg University Hospital , Denmark ;|c Department of Clinical Medicine , Aalborg University , Aalborg , Denmark.
authors.forename: Søren Risom
authors.initials: SR
authors.lastname: Kristensen
mesh_terms: D002623:Chemistry Techniques, Analytical / Q000379:methods; D002850:Chromatography, Gel; D000067128:Extracellular Vesicles; D005260:Female; D006801:Humans; D008297:Male; D053758:Nanoparticles; D010316:Particle Size; D010949:Plasma
keywords: Plasma; cell-derived microparticles; extracellular vesicles; lipoproteins; reference values