Silica microspheres are superior to polystyrene for microvesicle analysis by flow cytometry

- One micron size MV gate using polystyrene standards includes platelets.
- One micron size MV gate using silica standards does not include platelets.
- Silica microspheres are better than polystyrene for sizing microvesicles.
- Standard curve obtained using microsphere standards is cytometer specific.

BackgroundCell-derived microvesicles (MVs) in biological fluids are studied for their potential role in pathological conditions. Flow cytometry is used to characterize MVs. Polystyrene microspheres are often used in flow cytometry to distinguish MV from cells by setting a 1-μm MV gate in a side-scatter (SSC) vs. forward-scatter (FSC) dot plot. Polystyrene microspheres, however, exhibit higher FSC and SSC than MVs of equal size. Consequently, some platelets are included within the MV gate, which incorrectly increases the reported percentage of platelet-derived MVs. Silica microspheres exhibit FSC that is closer to that of cellular vesicles and, therefore, should permit more accurate discrimination of MV from platelets.ObjectiveCompare silica with polystyrene microspheres to calibrate flow cytometers for definition of MV population and estimation of MV sizes.MethodsSilica and polystyrene microspheres of various sizes were used in flow cytometry assays to define MV populations and determine platelet and MV sizes in human plasma samples. Sizes determined by flow cytometry were compared to sizes determined by resistive pulse sensing (RPS) method.Results/ConclusionUse of 1.0-μm polystyrene microspheres to define the upper MV gate produced a median platelet contamination of 16.53% (8.24, 20.98) of the MV population; whereas, use of 1.0-μm silica microspheres excluded platelet events completely. Calibration with silica microspheres resulted in significantly better estimation of MV diameter than calibration with polystyrene microspheres. We conclude that silica microspheres are superior to polystyrene microspheres as standards to define MV populations without platelet contamination and to determine MV sizes by flow cytometry for a given cytometer.