PERFORMANCE OF CALIBRATION STANDARDS FOR ANTIGEN QUANTITATION WITH FLOW-CYTOMETRY

In the frame of the activities initiated by the Task Force for Antigen Quantitation of the European Working Group on Clinical Cell Analysis (EWGCCA), an experiment was conducted to evaluate microbead standards used for quantitative flow cytometry (QFCM). An unified window of anal ysis UWA) was established on three different instruments (EPICS XL [Co ulter Corporation, Miami, FL], FACScan and FAGS Calibur [Becton Dickin son, San Jose, CA] with QC3 microbeads (FCSC, PR). By using this defin ed fluorescence intensity scale, the performance of several monoclonal antibodies directed to CD3, CD4, and CD8 (conjugated and unconjugated ), from three manufacturers (BDIS, Coulter [Immunotech], and DAKO) was tested. In addition, the QIFI system (DAKO) and QuantiBRITE (BDIS), a nd a method of relative fluorescence intensity (RFI, method of Giorgi) , were compared. mAbs reacting with three more antigens, CD16, CD19, a nd CD38 were tested on the FACScan instrument. Quantitation was carrie d out using a single batch of cryopreserved peripheral blood leukocyte s, and all tests mere performed as single color analyses. Significant correlations were observed between the antibody-binding capacity (ABC) values of the same CD antigen measured with various calibrators and w ith antibodies differing in respect to vendor, labeling and possible e pitope recognition. Despite the significant correlations, the ABC valu es of most monoclonal antibodies differed by 20-40% when determined by the different fluorochrome conjugates and different calibrators. The results of this study indicate that, at the present stage of QFCM cons istent ABC values may be attained between laboratories provided that a specific calibration system is used including specific calibrators, r eagents, and protocols. (C) 1998 Wiley-Liss,Inc.