An accessible method of flow cytometer scatter standardization for EV analysis

Background: Extracellular vesicle flow cytometry (EV-FC) results remain an area lacking standardization. Despite methods of scatter standardization being proposed, implementation is complex and requires proprietary or specialized information. Here we demonstrate an automated method of standardizing...

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Bibliographic Details
Published inJournal of extracellular vesicles Vol. 7; pp. 118 - 119
Main Authors Welsh, Joshua A, Horak, Peter, James, Wilkinson, Jones, Jennifer, d, Verity, Smith, David, Holloway, Judith, Englyst, Nicola
Format Journal Article
LanguageEnglish
Published Abingdon John Wiley & Sons, Inc 01.01.2018
Subjects
Biomaterials
Flow cytometry
Methods
Optics
Polystyrene
Silica
Standardization
United Kingdom > UK
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Summary:Background: Extracellular vesicle flow cytometry (EV-FC) results remain an area lacking standardization. Despite methods of scatter standardization being proposed, implementation is complex and requires proprietary or specialized information. Here we demonstrate an automated method of standardizing conventional flow cytometer scatter data by acquiring nanobeads of known diameter and refractive index, combined with freely available software we have developed. This method allows for accessible standardization of the scatter parameters for EV analysis and provides the ability to convert arbitrary axes to units of diameter or refractive index with the correct controls. Methods: Polystyrene & Silica NIST beads (ThermoFisher Scientific, Paisley, UK) ranging in diameter from 200 nm to 1600 nm were acquired using flow cytometry. Data was obtained on a Fortessa X-20, LSR Fortessa, Canto I, Attune NxT. SSC-H median of each bead population were compared to a precalculated database of predicted scatter for each of the beads from collection half-angles from 0.1 to 90 degrees in 0.1 degree increments. Analysed bead diameters were derived using the above modelling technique and were compared with manufacturer's bead specifications to determine the model accuracy to predict flow cytometer collection optics. This method was further applied to a jet-in-air sorter, the MoFlo Astrios-EQ, to evaluate its performance in systems with variable alignment and optical geometries. Results: Standardised flow cytometer scatter measurements predicting bead diameters and comparing them to bead specifications showed a median variation (25th percentile, 75th percentile) of 2.59% (0.55%, 5.28%). Summary/Conclusion: This work demonstrates that flow cytometer scatter measurements can be obtained using a user-friendly methodology without the requirement of specialised flow cytometer components. This method also further allows extrapolation to determine particle diameter or refractive index offering potentially new methods of EV and submicron biomaterial analysis.
ISSN:2001-3078