Approaches to Quantitation in Flow Cytometry

Abstract

Flow cytometry is used to measure the fluorescence intensity (FI) from labels bound to antigens present on the surface of T and B cells. The T and B cells are associated with the human adaptive immune system and the amounts of surface antigens, such as CD4, CD8, and CD20, are used for diagnostic purposes. To estimate the number of a specific antigen expressed on the surface, the cells are incubated in a solution containing antibodies specific to that antigen and the antibodies are conjugated to fluorophores that provide the fluorescence signals used to detect the presence of the antibodies on the cell surface. The fluorophore on the antibody, microsphere, or cell is called a label. The antibody gives biological specificity and the fluorophore provides a mechanism for readout. Quantitation of cytometer measurements is accomplished by the comparison of the fluorescence signal of labeled cells with the fluorescence signal of labeled reference microspheres. The fluorescence signals from labeled cells and labeled microspheres are converted to a fluorescence yield (FY) where FY is defined as the product of the number of fluorophores and the fluorophore quantum yield. The comparison of fluorescence provides the basis for an estimate of the number of molecules of equivalent soluble fluorophores (MESF) associated with the labels bound on the cell surface. A procedure is outlined for assigning MESF values to microspheres followed by a demonstration of the assignment of MESF values to microspheres with immobilized R-phycoerythrin. The MESF values are used to obtain an estimate of the antibodies bound to the cell (ABCe). The final step in the quantitation process is the conversion of the ABCe values to an estimate of the number of specific antigens on a surface of a cell. With proper care, the ABCe value may be a good indicator of the total number of specific antigens present on the cell surface. Examples are given of the determination of the number of CD4 and CD20 antigens on lymphocytes. The MESF quantitation strategy has been applied to the calibration of multicolor flow cytometers.