title: Particle size distribution of exosomes and microvesicles determined by transmission electron microscopy, flow cytometry, nanoparticle tracking analysis, and resistive pulse sensing.
abstract: BACKGROUND
  Enumeration of extracellular vesicles has clinical potential as a biomarker for disease. In biological samples, the smallest and largest vesicles typically differ 25-fold in size, 300,000-fold in concentration, 20,000-fold in volume, and 10,000,000-fold in scattered light. Because of this heterogeneity, the currently employed techniques detect concentrations ranging from 10(4) to 10(12) vesicles mL(-1) .
  
  
  OBJECTIVE
  To investigate whether the large variation in the detected concentration of vesicles is caused by the minimum detectable vesicle size of five widely used techniques.
  
  
  METHODS
  The size and concentration of vesicles and reference beads were measured with transmission electron microscopy (TEM), a conventional flow cytometer, a flow cytometer dedicated to detecting submicrometer particles, nanoparticle tracking analysis (NTA), and resistive pulse sensing (RPS).
  
  
  RESULTS
  Each technique gave a different size distribution and a different concentration for the same vesicle sample.
  
  
  CONCLUSIONS
  Differences between the detected vesicle concentrations are primarily caused by differences between the minimum detectable vesicle sizes. The minimum detectable vesicle sizes were 70-90 nm for NTA, 70-100 nm for RPS, 150-190 nm for dedicated flow cytometry, and 270-600 nm for conventional flow cytometry. TEM could detect the smallest vesicles present, albeit after adhesion on a surface. Dedicated flow cytometry was most accurate in determining the size of reference beads, but is expected to be less accurate on vesicles, owing to heterogeneity of the refractive index of vesicles. Nevertheless, dedicated flow cytometry is relatively fast and allows multiplex fluorescence detection, making it most applicable to clinical research.
authors.affiliation: Laboratory of Experimental Clinical Chemistry, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands; Biomedical Engineering and Physics, Academic Medical Center, University of Amsterdam, Amsterdam, the Netherlands.
authors.forename: E
authors.initials: E
authors.lastname: van der Pol
authors.forename: F A W
authors.initials: FA
authors.lastname: Coumans
authors.forename: A E
authors.initials: AE
authors.lastname: Grootemaat
authors.forename: C
authors.initials: C
authors.lastname: Gardiner
authors.forename: I L
authors.initials: IL
authors.lastname: Sargent
authors.forename: P
authors.initials: P
authors.lastname: Harrison
authors.forename: A
authors.initials: A
authors.lastname: Sturk
authors.forename: T G
authors.initials: TG
authors.lastname: van Leeuwen
authors.forename: R
authors.initials: R
authors.lastname: Nieuwland
mesh_terms: D015415:Biomarkers / Q000378:metabolism; D055252:Cell-Derived Microparticles / Q000378:metabolism; D055354:Exosomes / Q000378:metabolism*; D005434:Flow Cytometry; D006801:Humans; D046529:Microscopy, Electron, Transmission; D053758:Nanoparticles / Q000737:chemistry; D055095:Optics and Photonics; D010316:Particle Size; D012031:Refractometry; D015203:Reproducibility of Results
chemical_list: D015415:Biomarkers
keywords: cell-derived microparticles; exosomes; optical devices; reference standards; secretory vesicles