Quantitative Flow Cytometry Measurements in Antibody Bound per Cell Based on CD4 Reference
Lili Wang, Heba Degheidy, Fatima Abbasi, Howard Mostowski, Gerald Marti, Steven R. Bauer, Robert Hoffman, Adolfas K. Gaigalas
Multicolor flow cytometer assays are with fluorescently labeled antibodies routinely used in clinical laboratories to measure the cell number of specific immunophenotypes and to estimate expression levels of specific receptors/antigens either on the cell surface or intracellularly. The cell number and specific receptors/antigens serve as biomarkers for pathological conditions at various stages of a disease. Existing methods and cell reference material for quantitative expression measurements have not yet produced results which are of neither clinical interest nor are the results instrument independent across all fluorescence channels of cytometers. This chapter details a procedure for quantifying surface and intracellular biomarkers by calibrating the output of a multicolor flow cytometer in unit of antibody bound per cell (ABC). The procedure includes the following critical steps (a) quality control (QC) of the flow cytometer, (b) fluorescence intensity calibration using hard dyed microspheres assigned with fluorescence intensity values, (c) compensation for fluorescence spillover between adjacent fluorescence channels, and (d) application of a biological reference calibrator to establish an ABC scale. The chapter also points out current efforts for implementing quantification of biomarkers in a manner which is independent of instrument platforms and reagent differences.