IMMUNOPHENOTYPING OF ACUTE-LEUKEMIA BY FLOW CYTOMETRIC ANALYSIS - USEOF CD45 AND RIGHT-ANGLE LIGHT SCATTER TO GATE ON LEUKEMIC BLASTS IN 3-COLOR ANALYSIS

This article describes a procedure for performing routine three-color flow cytometric analysis for acute leukemia on lysed whole bone marrow preparations. This technique uses the combination of CD45 intensity a nd right-angle light scatter (RALS) to distinguish leukemic cells from normal lymphocytes, monocytes, neutrophils, eosinophils, and nucleate d red blood cells. On this display, leukemic cells occupy a unique bla st region characterized by intermediate CD45 density and low RALS, whi ch, in normal marrows, contains less than 5% of the total cells. This approach was applied to 39 cases of acute leukemia and 8 cases of myel odysplasia or myeloproliferative disorders. The estimate of blasts by flow cytometric analysis was correlated highly with morphologic leukem ic cell counts over a wide range. Moreover, the pattern seen on the CD 45-RALS display was different for different French-American-British su btypes of leukemia, suggesting that this pattern might be useful for c ategorization. When CD45-peridin chlorophyll alpha protein was combine d with other pairs of fluorescein isothiocyanate- and phycoerythrin-co njugated reagents, it was possible to set an analysis window on the le ukemic blasts and display dual-parameter tie, green vs. red fluorescen ce) data regarding expression of two additional markers on the leukemi c population. This gating strategy was superior to traditional forward -angle versus RALS displays in that it did a better job of isolating t he leukemic cells analytically.