MULTICOLOR FLOW CYTOMETRIC ANALYSIS OF THE CD45 ANTIGEN PROVIDES IMPROVED LYMPHOID-CELL DISCRIMINATION IN BONE-MARROW AND TISSUE BIOPSIES

Samples from bone marrow or non-hematopoietic tissue such as solid org an biopsies often contain an excess of non-leukocytes exhibiting lymph ocyte-like light scatter characteristics, making it sometimes difficul t to define satisfactory light scatter lymphocyte gates. To circumvent this, we describe here a multiparametric method of identifying lympho id cells by expression of the CD45 antigen, in conjunction with light scatter parameters. A 'third color'-conjugated anti-CD45 antibody was included with every FITC/PE double staining, thereby permitting live o r list mode analysis gating on CD45 positive cells. The triple-stainin g technique was applied to (a) human bone marrow, showing that special attention has to be given to the enumeration of B cells, and (b) to l iver biopsies, where gating on CD45 fluorescence and orthogonal light scatter was shown to clearly resolve all lymphocyte subsets from debri s. All cell types examined in tissue biopsies as well as T and NK cell s in bone marrow were best distinguished by gating on bright CD45 expr ession in conjunction with low orthogonal light scatter, while accurat e identification of marrow B cells relied upon including all levels of CD45 intensity. The multicolor gating procedure, aimed mainly at immu ne-monitoring of non-malignant tissues, is applicable to most kinds of single cell samples, and may prove to be an aid for lymphocyte gating in cases where leukocyte populations are not clearly resolved on a li ght scatter basis alone.