Flow cytometry CD45 gating for immunophenotyping of acute myeloid leukemia

A flow cytometry method has been introduced into the routine age values within the malignant blast cell populations or investigation of whole bone marrow samples following red within the total nucleated cells present in each sample. In blood cell lysis on the basis of a primary CD45/side scatter order to give numbers of malignant cells and proportions of (SSC) gating procedure. Blast cells were first identified by cells within this cohort, a reliable practical discrimination CD45/SSC gating in 74 cases of acute myeloid leukemia (AML) between malignant blasts and normal cell types would be and the results were compared to a conventional FSC/SSC gating procedure and to MGG-staining smears. The percentages required. In this paper, as previously recommended by of blast cells in these samples as defined by the morphological Borowitz et al, 6 we suggest that such a discrimination can be analysis of MGG smears correlated better with the values readily facilitated by introducing primary gating for CD45 determined by CD45/SSC gating (r 5 0.94) than with the blast antigen expression and side scatter (CD45/SSC). We also sugcell counts recorded with FSC/SSC gating (r 5 0.76). These gest that this step should replace the first gating step for forfindings were not surprising because while CD45 expression ward scatter and side scatter (FSC/SSC), as this latter procedure was regularly lower on leukemic blasts than on normal lymphoid and monocytic cells, the FCS/SSC characteristics of these does not discriminate well between leukemic blasts, lymphopopulations were overlapping. In 53 samples, the blast cell cytes and monocytes. populations were also analyzed with a panel of FITC-conju- In this paper we present the application of double and triple gated monoclonal antibodies that were utilized in double labe- immunofluorescence (IF) analysis of bone marrow samples ling with CD45-PE. We show that the CD45/SSC gating pro- taken from patients during the presentation of AML. With the cedure improved phenotypic determination of the blast cells in systematic use of leukocyte common antigen (CD45) marker three ways: (1) by discriminating between leukemic blast cells and residual normal cells; (2) by excluding normal cells from in combination with lineage-specific markers, a good disthe phenotypic analysis of leukemic blast cells; and (3) by crimination can be achieved between the blast cell popuidentifying blast cell heterogeneity in many cases of leukemia lations and the normal cells. This discrimination is based on on the basis of different CD45 display. Moreover, this immuno- the fact that the precursor cells in the bone marrow, as well phenotyping procedure on whole bone marrow samples also as the leukemias which derive from these cell types, express allowed an efficient discrimination between the various cell lin- low and intermediate values of CD45, while lymphocytes and eages and facilitated the analysis of leukemic blasts present in low proportions. monocytes express high levels of CD45. 7