Red cell microparticle enumeration: Validation of a flow cytometric approach

Background and Objectives There is growing interest in the clinical application of red blood cell (RBC) microparticle (MP) enumeration as they have been postulated to be effectors of coagulation and inflammation following transfusion and in sickle cell disease. No uniform approach in MP enumeration exists and a key limitation is the lack of an internal validation process. We present and validate a flow cytometric approach where an internal standard is utilized. Materials and Methods Glycophorin A⁺ Annexin V⁺ events were enumerated using MPs isolated from RBC units or plasma samples obtained from volunteers. A mixture of absolute counting (7·6μm) and calibration beads (0·5, 0·9 and 3μm) at a fixed ratio was added to each sample. Results RBC MPs were initially selected based upon a fluorescence threshold, and the 0·5- and 0·9-μm beads defined the upper and lower light scatter distribution of MPs. The ratio of 7·6:3-μm bead events was used as an internal standard to validate the precision of MP enumeration across samples (coefficient of variation=2·5-7·2%) and remained constant in both platelet-rich plasma (PRP) and platelet-free plasma (PFP). RBC MP counts increased in both PRP and PFP obtained from whole blood stimulated with ionophore and increasing calcium concentrations, with PRP showing higher MP counts than PFP at every concentration studied. Conclusion This method is a useful strategy to detect RBC MP counts across bio-samples provided that the flow cytometer can reliably discriminate the size of the calibration beads.