Quantitative light scattering of extracellular vesicles for flow cytometry standardization
||E. van der Pol, R. Nieuwland, A. Sturk, T.G. van Leeuwen, and F.A.W. Coumans|
||International Society for Extracellular Vesicles 2015, Washington D.C., United States|
||April 23, 2015|
Introduction: Although flow cytometry is the most widely used method to study single vesicles, comparison of flow cytometry results between laboratories remains challenging. At present, most laboratories select vesicles by setting an inclusion gate based on the scatter signal from two polystyrene bead sizes, resulting in a coefficient of variation (CV) of the vesicle concentration of 91%. This moderate reproducibility is attributed to the variety of optical configurations in flow cytometers and the refractive index difference between vesicles and polystyrene. Due to this refractive index difference, polystyrene beads scatter light >10-fold more efficiently than vesicles.
Methods: A well-defined polystyrene beads mixture (metves.eu) and two vesicle standards are measured on 45 flow cytometers in 31 laboratories worldwide. The relation between particle size, refractive index, and scatter is obtained for the specific optical configuration of each instrument by describing the data from beads with Mie theory (exometry.com). This relation is used to set three vesicle size gates, taking into account the latest insights on the refractive index of vesicles.
Results: Preliminary data show that our calibration procedure reduces the CV of the vesicle concentration with 21% compared to the current standard. Circa 30% of all instruments is unable to detect 400 nm FITC-labeled polystyrene beads. For comparison, a 400 nm polystyrene bead scatters light more efficiently than a 1 μm urinary vesicle. In addition, the size of the smallest detectable vesicle differs 2-fold between instruments of the same type. Final data and results are expected in February and June 2015, respectively.
Conclusion: Well-defined beads and Mie theory are used to standardize the size range detected by flow cytometry, thereby improving the CV of the vesicle concentration with 21% compared to the current standard. This unique approach provides a profound understanding of vesicle detection by flow cytometry, which is essential to inter-laboratory data comparison.