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

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 488nm 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 <0.5μm in diameter. On the Apogee A40, polystyrene microspheres could be separated by size down to 0.2μm, and a polystyrene microsphere 0.4μm 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 80000-4000000μL^-1 total microparticles, 11000-350000μL^-1 annexin V positive microparticles and 6000-350000μL^-1 platelet microparticles <1μm in plasma samples. Conclusions:The Apogee A40 was able to resolve size differences in polystyrene microspheres down to 0.2μm and microparticles down to 0.4μm. 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.