Flow cytometric measurement of CD62P (P-selectin) expression on platelets: a multicenter optimization and standardization effort

BACKGROUND: One of the variables to determine the quality of platelets (PLTs) in vitro is measurement of CD62P expression. Different protocols are in use, however, making comparison of results virtually impossible. It was our aim to develop a uniform CD62P protocol that would yield comparable results in various laboratories. STUDY DESIGN AND METHODS: The effects of fixation, source and dilution of CD62P antibody, source of immunoglobulin G (IgG) isotypic antibody, and analysis of results were investigated. Once the optimal variables were defined, comparative studies were performed at five participating centers. In the final comparative study, eight split PLT concentrates were shipped to the centers, where samples were stained and fixed according to the uniform protocol. Analyses were performed using commercially available flow cytometers (BD Bio-sciences and Beckman Coulter). RESULTS: Uniformity between centers could be achieved by using a single clone for CD62P and IgG monoclonal antibody. A protocol was selected using fixation with 0.5 percent methanol-free formaldehyde. To increase conformity between flow cytometers, in the analysis of electronic data the thresholds of the isotypic control were set at 0.5 percent for the BID Biosciences and 2 percent for the Beckman Coulter flow cytometers. In the final comparative study, the 95 percent confidence intervals (CIs) for CD62P ranged between 8 and 21 percent in fresh and 20 to 40 percent in 8-day-old PLT concentrates. CONCLUSION: A uniform CD62P staining protocol and subsequent analysis can be used at multiple centers using different flow cytometers, yielding comparable results with acceptable 95 percent Cls.