QUANTITATION OF MICROPARTICLES IN PLATELET SUSPENSIONS BY FLOW CYTOMETRY

Abstract

We have examined a platelet-poor, supernatant fraction from fresh and stored platelet suspensions with a FACS 440 (Becton-Dickinson) flow cytometer to study the distribution of small microparticles previously shown to be present in citrated plasma and serum (J. George et al., Blood 60: 834, 1982). Analysis by flow cytometry offers the advantage of discrimination of populations of particles by both light scattering and immunofluorescent properties. We found two distinctly different populations of particles: the predominant one had diameters in the range of 0.1 to 0.4um and was moderately autofluorescent (AF); the other was equally AF with particle diameters of 1.0 to 3.0um and probably included a few intact platelets. By adding a precise quantity of highly fluorescent beads of 0.9um diameter to each sample, relative concentrations of particles (small and/or large) could be quantified in platelet suspensions after various treatments using ratios of particle and bead counts. The lowest concentration of particles was found in samples from whole blood collected into CPDA-1 with PGE-1 and theophylline plus sodium azide (CPT-Az). Blood in CPDA-1 alone had twice the number of small and large particles; serum had a 20X higher particle concentration. A much larger number of particles was found in platelet concentrates (PC) stored for transfusion. Fresh PC had approx. 150X higher particle concentration than CPT-Az, rising to over 200X by the eighth day of storage at 22 C. Also, we noted a shift in distribution between particle populations in stored PC toward the larger size. The concentration of larger particles alone rose from 100X relative to CPT-Az to 350X after 8 days of storage. Similar changes in supernatant platelet factor 3 (PF3) activity were noted in stored PC in another study (A.P. Bode and D.T. Miller, Vox Sanguinis 51: 299, 1986), suggesting that supernatant PF3 activity may be related to one or the other population of particles seen by flow cytometry. This technique of examining and quantifying particles in platelet preparations by flow cytometry will facilitate and expand the characterization of platelet vesiculation and the released particles.