The absolute counting of red cell-derived microparticles with red cell bead by flow rate based assay

Abstract

Background: Activation of red blood cell is associated with the formation of red cell-derived microparticles (RMPs). Analysis of circulating RMPs is becoming more refined and clinically useful. A quantitative Trucount™ tube method is the conventional method uses for quantitating RMPs. In this study, we validated a quantitative method called "flow rate based assay using red cell bead (FCB)" to measure circulating RMPs in the peripheral blood of healthy subjects. Methods: Citrated blood samples collected from 30 cases of healthy subjects were determined the RMPs count by using double labeling of annexin V-FITC and anti-glycophorin A-PE. The absolute RMPs numbers were measured by FCB, and the results were compared with the Trucount™ or with flow rate based calibration (FR). Statistical correlation and agreement were analyzed using linear regression and Bland-Altman analysis. Results: There was no significant difference in the absolute number of RMPs quantitated by FCB when compared with those two reference methods including the Trucount™ tube and FR method. The absolute RMPs count obtained from FCB method was highly correlated with those obtained from Trucount™ tube (r2 - 0.98, mean bias 4 cell/μl, limit of agreement [LOA] - 20.3 to 28.3 cell/μl), and FR method (r2 = 1, mean bias 10.3 cell/μl, and LOA - 5.5 to 26.2 cell/μl). Conclusion: This study demonstrates that FCB is suitable and more affordable for RMPs quantitation in the clinical samples. This method is a low cost and interchangeable to latex bead-based method for generating the absolute counts in the resource-limited areas. © 2008 Clinical Cytometry Society.