Multicolor flow cytometric analysis of the CD45 antigen provides improved lymphoid cell discrimination in bone marrow...

Samples from bone marrow or non-hematopoietic tissue such as solid organ biopsies often contain an excess of non-leukocytes exhibiting lymphocyte-like light scatter characteristics, making it sometimes difficult to define satisfactory light scatter lymphocyte gates. To circumvent this, we describe here a multiparametric method of identifying lymphoid 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 or list mode analysis gating on CD45 positive cells. The triple-staining 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 liver biopsies, where gating on CD45 fluorescence and orthogonal light scatter was shown to clearly resolve all lymphocyte subsets from debris. All cell types examined in tissue biopsies as well as T and NK cells in bone marrow were best distinguished by gating on bright CD45 expression in conjunction with low orthogonal light scatter, while accurate identification of marrow B cells relied upon including all levels of CD45 intensity. The multicolor gating procedure, aimed mainly at immune-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 light scatter basis alone.