title: Enumeration of extracellular vesicles by a new improved flow cytometric method is comparable to fluorescence mode nanoparticle tracking analysis.
abstract: Extracellular vesicles (EVs) play a role in a variety of physiological and pathological processes. However, use of EVs as biomarkers has been hampered by limitations of current detection and enumeration methods. We compared fluorescence-threshold flow cytometry (FT-FC) to nanoparticle tracking analysis (NTA) for enumeration of cell culture-derived EVs. FT-FC and NTA utilising fluorescence mode (F-NTA) enumerated similar numbers of EVs stained with a membrane dye PKH67. Both methods were sufficiently sensitive to detect cell-derived EVs above the background of culture medium. Light scatter NTA (LS-NTA) detected 10-100× more particles than either fluorescence-based method but demonstrated poor specificity. F-NTA appeared to have better sensitivity for <100nm vesicles, however, the FT-FC method combined direct enumeration of EVs with high sensitivity and specificity in the >100nm range. Due to wider availability and higher degree of automation and standardisation, FT-FC is a reasonable surrogate to F-NTA for quantification of EVs.
  
  
  BACKGROUND
  Extracellular vesicles are small particles, which can act as tools for intercellular communication. One recent area of interest in EVs is their potentials as biomarkers. In this article, the authors investigated and compared fluorescence-threshold flow cytometry (FT-FC) to nanoparticle tracking analysis (NTA) for the detection of EVs and showed that FT- FC method could be more advantageous. This technique should provide a new alternative for the future.
authors.affiliation: Prince of Wales Clinical School, Adult Cancer Program, University of New South Wales, Sydney, Australia; Haematology, Institute of Clinical Pathology and Medical Research, Westmead, NSW, Australia.
authors.forename: Leonardo
authors.initials: L
authors.lastname: Pasalic
authors.affiliation: Children's Cancer Institute Australia for Medical Research, Sydney, Australia.
authors.forename: Rebekka
authors.initials: R
authors.lastname: Williams
authors.affiliation: Malvern Instruments Ltd, Amesbury, United Kingdom (Formerly NanoSight Ltd).
authors.forename: Agnieszka
authors.initials: A
authors.lastname: Siupa
authors.affiliation: Prince of Wales Clinical School, Adult Cancer Program, University of New South Wales, Sydney, Australia.
authors.forename: Heather
authors.initials: H
authors.lastname: Campbell
authors.affiliation: Children's Cancer Institute Australia for Medical Research, Sydney, Australia.
authors.forename: Michelle J
authors.initials: MJ
authors.lastname: Henderson
authors.affiliation: Prince of Wales Clinical School, Adult Cancer Program, University of New South Wales, Sydney, Australia; Haematology, Concord Repatriation and General Hospital, Sydney, Australia; ANZAC Research Institute, Concord, Sydney, Australia. Electronic address: vivien.chen@unsw.edu.au.
authors.forename: Vivien M Y
authors.initials: VMY
authors.lastname: Chen
mesh_terms: D015415:Biomarkers; D002450:Cell Communication; D045744:Cell Line, Tumor; D058948:Cell Tracking / Q000379:methods; D000067128:Extracellular Vesicles / Q000187:drug effects / Q000378:metabolism*; D005434:Flow Cytometry; D005456:Fluorescent Dyes / Q000008:administration & dosage / Q000737:chemistry; D006801:Humans; D053758:Nanoparticles / Q000008:administration & dosage* / Q000737:chemistry
chemical_list: D015415:Biomarkers; D005456:Fluorescent Dyes
keywords: Enumeration; Extracellular vesicles; Flow cytometry; Nanoparticle tracking analysis; PKH67