Multiparameter flow cytometry.

Immunophenotyping is an important method for identifying cells, and there is no single field for which it is applied more often than in hematology. Although immunohistochemistry or fluorochrome-labeled antibodies are often used for microscopic identification of cells, flow cytometry has the advantage of identifying cell subsets more rapidly by using multiple antibodies simultaneously, and these subsets can be sorted for further characterization. Microscopic analysis is the method of choice if morphological information is desired, but flow cytometry is the choice for identifying and quantifying cell subsets and evaluating their frequency in a heterogeneous population. Even though polyclonal antibodies are still used occasionally, monoclonal antibodies (MAbs) to epitopes on membrane or internal antigens are the reagents used for identification. We now know that any given protein is often found on several different kinds of cells, and one antibody cannot be used to identify any particular cell lineage. Instead, each cell subpopulation has a very unique repertoire of proteins, and a mixture of antibodies to them can be used for explicit identification. When the function of the protein is known, additional information about the cell is obtained. In this chapter, I will describe the methods for labeling cells with up to four antibodies simultaneously. This number is chosen because it is possible to measure all of them with a single laser. With human lymphocytes as the example, we now recognize over 80 subpopulations and, although these subpopulations are not all mutually exclusive, they do represent specific functional subsets that interact together to produce the hematopoietic system and exemplify the power of flow cytometry for resolving them.