Single laser flow cytometric detection of lymphocytes binding three antibodies labelled with fluorescein, phycoerythrin and a novel tandem fluorochrome conjugate

Abstract

In order to implement three-color surface immunofluorescence on a single laser flow cytometer, we combined a new fluorescent tandem conjugate (TC, R-phycoerythrin plus Texas red, available as DuoCHROME) with fluorescein (FITC)- and phycoerythrin (PE)-labelled monoclonal antibodies for the simultaneous detection of three antigens on individual lymphoid cells. Considerable amounts of fluorescence leaked from the PE component of the TC complex into the PE detector, which necessitated compensation of the PE signal. Single and double stainings of peripheral lymphocytes with FITC-, PE- and/or TC-labelled antibodies revealed that the individual labels all identified populations of similar size, and that subsets of T cells could be properly and accurately detected with TC as one label. Triple staining was used to study phenotypically activated HLA-DR+ cells within functionally important subsets of CD4+ T cells, i.e., CD45R+ CD4+ (naive, 'suppressor inducer' T) and UCHL1+ CD4+ (memory, 'helper' T) cells. In the blood of healthy subjects as well as patients with primary biliary cirrhosis, CD4+ UCHL1+ (i.e., memory) T cells were significantly more activated than CD4+ UCHL1- cells. The reciprocal CD45R+ CD4+ (naive) T subset expressed HLA-DR to a much lesser extent. In patient samples, this difference in CD4+ T subset activation was more pronounced than among normal lymphocytes. The described triple immunofluorescence staining technique facilitates true multicolor analysis of individual cells on a single laser flow cytometer. Such multiparameter investigations may prove to be important for the further understanding of the phenotypic and functional diversity of immunocompetent cells.