FLOW CYTOMETRIC QUANTITATION OF IMMUNOFLUORESCENCE INTENSITY - PROBLEMS AND PERSPECTIVES

Quantification of immunofluorescence intensity serves to estimate the number of defined molecules expressed on or in cells. Clinical applica tions of this diagnostic tool are increasing, e.g,, aberrant expressio n of various antigens (Ag) by leukemic blasts or lymphoma cells, inten sity of CD38 expression by CD8(+) T-lymphocytes to monitor activation status, and intensity of CD62P to detect platelet activation. In this report we discuss the quality-control measures required for quantitati on of fluorescence intensity, and we review seven concepts that have b een developed to quantify fluorescence intensity during the past 15 ye ars. Initial work addressed the conversion of logarithmic channel numb ers into units of relative fluorescence. The design and use of calibra tion beads labeled with predefined amounts of dye allowed instrument-i ndependent expression of fluorescence intensity in units of molecules of equivalent soluble fluorochrome (MESF). This method was refined by the combined use of such standards with monoclonal antibodies (mAb) co njugated 1:1 with phycoerythrin (PE), allowing translation of fluoresc ence intensity into numbers of antibodies bound per cell. Alternativel y, the use of 1:1 PE-conjugated mAb under the assumption that CD4(+) l ymphocytes reproducibly bind 50,000 CD4 mAb molecules was proposed to convert units of relative fluorescence intensity into units of antibod ies bound per cell. The use of antibody-binding capacity as a surrogat e marker for quantification of Ag expression was addressed more direct ly by the development of antibody-binding standards. The quantitative indirect immunofluorescence assay is based on beads labeled with vario us amounts of CD5 mAb that calibrate the binding of the secondary anti body in units of antibody-binding capacity. Alternatively, goat anti-m ouse-labeled calibration beads have been developed. Published results obtained with the latter calibrators showed an unexpected inaccuracy. The different ways in which calibrators and cells under study bind mAb (i,e,, Fab mediated versus Pc mediated) may have contributed to this variation. Recently, the use of stabilized cell populations expressing Ag in a specified range of concentrations has been proposed as an Ag- specific calibration system of mAb binding. We identify several issues on the level of instrumentation, reagents, and cells under study that should be solved to allow standardization of quantitative assessments of immunofluorescence intensity, (C) 1998 Wiley-Liss, Inc.