Performance of calibration standards for antigen quantitation with flow cytometry
Article first published online: 6 JAN 1999
Copyright © 1998 Wiley-Liss, Inc.
Special Issue: Quantitative Fluorescence Cytometry: An Emerging Consensus
Volume 33, Issue 2, pages 188–196, 1 October 1998
How to Cite
Lenkei, R., Gratama, J. W., Rothe, G., Schmitz, G., D'hautcourt, J. L., Årekrans, A., Mandy, F. and Marti, G. (1998), Performance of calibration standards for antigen quantitation with flow cytometry. Cytometry, 33: 188–196. doi: 10.1002/(SICI)1097-0320(19981001)33:2<188::AID-CYTO13>3.0.CO;2-Q
- Issue published online: 6 JAN 1999
- Article first published online: 6 JAN 1999
- Manuscript Accepted: 12 JUN 1998
- Manuscript Received: 5 JUN 1998
- DG XII, European Commission, Brussels, Belgium. Grant Number: Concerted Action Contract BMH4-CD97–2611 in the framework of the Biomed-2 Programme
- 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 analysis (UWA) was established on three different instruments (EPICS XL [Coulter Corporation, Miami, FL], FACScan and FACS Calibur [Becton Dickinson, San Jose, CA]) with QC3 microbeads (FCSC, PR). By using this defined 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), and a method of relative fluorescence intensity (RFI, method of Giorgi), were compared. mAbs reacting with three more antigens, CD16, CD19, and CD38 were tested on the FACScan instrument. Quantitation was carried out using a single batch of cryopreserved peripheral blood leukocytes, and all tests were 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 with antibodies differing in respect to vendor, labeling and possible epitope recognition. Despite the significant correlations, the ABC values 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 consistent ABC values may be attained between laboratories provided that a specific calibration system is used including specific calibrators, reagents, and protocols. Cytometry 33:188–196, 1998. © 1998 Wiley-Liss, Inc.