Detection of microparticles in human plasma using flow cytometry: limitations and optimization techniques. (P3265)

Abstract

Abstract Microparticles (MPs) range in size from 40 nm to 1 um. Flow cytometry (FCM) is the most commonly used method for analyzing MPs; however, accurate characterization of MPs remains challenging due to their small size and lack of discrete positive populations. Here we report the use of optimization techniques for overcoming these limitations. To reduce background, we decreased antibody concentration, increased sample dilution, and reduced sample flow rate. Antibodies (Abs) to CD14, CD16, CD19, and CD235a showed high levels of aggregation resulting in false positive events in the MP FSC/SSC gate, while Abs to CD28, CD62L, and CD41a did not have significant aggregates. Switching Ab manufacturers was effective only at resolving background for one Ab, CD235. Utilizing a two pronged approach that included 1) pre-filtration of antibodies to remove aggregates, followed by 2) detergent lysis of a replicate sample to account for remaining false positive events, we were able to effectively limit false positive non-MP events. We show that our antibody filtration method removes aggregates without compromising the effectiveness of the dye to identify/label antigen-expressing cells. Furthermore, we found our pre-filtration method to be slightly more effective at removing aggregates than a centrifugation method described in the literature. In conclusion, use of these optimized techniques enhances the accuracy and reliability of MP detection using FCM.