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A new microparticle size calibration standard for use in measuring smaller microparticles using a new flow cytometer

Summary.  Background: Microparticle size measurements are often calibrated on flow cytometers using polystyrene microspheres that forward scatter more light vs. particle diameter than cellular microparticles. Methods: We compared theoretical with measured forward angle light scattering on the LSRII, FC500 and Apogee A40 using polystyrene and silica microspheres vs. synthetic lipid vesicles and platelets, then compared plasma microparticle counts using different calibration strategies. Results: Polystyrene and silica microspheres with higher refractive indices forward scattered more light with a wavelength of 488 nm for a given size microparticle than did lipid vesicles or platelets. The LSRII and FC500 did not count many, and were unable to separate by size, polystyrene microspheres < 0.5 μm in diameter. On the Apogee A40, polystyrene microspheres could be separated by size down to 0.2 μm, and a polystyrene microsphere 0.4 μm in diameter produced the same forward scatter relative intensity as a 1‐μm lipid or cellular microparticle. Using the new calibrator, the Apogee A40 found 80 000–4 000 000 μL−1 total microparticles, 11 000–350 000 μL−1 annexin V positive microparticles and 6000–350 000 μL−1 platelet microparticles < 1 μm in plasma samples. Conclusions: The Apogee A40 was able to resolve size differences in polystyrene microspheres down to 0.2 μm and microparticles down to 0.4 μm. On the Apogee A40 we propose using a 0.4‐μm polystyrene microsphere as equivalent to a 1‐μm cellular microparticle for size calibration. Using this calibrator, the Apogee A40 detected higher numbers of total, platelet and annexin V positive microparticles than were found using a Megamix gate.