Immunofluorescent analysis of blood cells by flow cytometry

Historically, immunofluorescence was one of the first applications of flow cytometry. In the conventional method, forward light scatter and fluorescence are measured for each cell in the flowing sample stream. The size-related forward scatter measurement permits the fluorescence measurement to be made on cells within a particular size range. Fluorescence intensity above a fixed threshold is interpreted to mean a cell is stained or positive. Provided purified cells are used, and that the stained cells are brightly fluorescent, this conventional method provides useful results that are easy to interpret. In this paper we have reported our recent investigations of restrictions, fundamental limitations and basic extensions evidenced in our application of a new method of immunofluorescent analysis of whole blood preparations. These include: limitations due to autofluorescence and nonspecific staining, techniques for optimal staining, and the appropriate evaluation of fluorescent histogram data. Our data indicate that the method reviewed here offers a rapid technique for evaluating T cells and their subclasses with the potential, due to its ease of performance, for application to repeated use in longitudinal studies.