title: Immunophenotyping of acute leukemia by flow cytometric analysis. Use of CD45 and right-angle light scatter to gate on leukemic blasts in three-color analysis.
abstract: 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 and right-angle light scatter (RALS) to distinguish leukemic cells from normal lymphocytes, monocytes, neutrophils, eosinophils, and nucleated red blood cells. On this display, leukemic cells occupy a unique blast region characterized by intermediate CD45 density and low RALS, which, 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 myelodysplasia or myeloproliferative disorders. The estimate of blasts by flow cytometric analysis was correlated highly with morphologic leukemic cell counts over a wide range. Moreover, the pattern seen on the CD45-RALS display was different for different French-American-British subtypes of leukemia, suggesting that this pattern might be useful for categorization. When CD45-peridin chlorophyll alpha protein was combined with other pairs of fluorescein isothiocyanate- and phycoerythrin-conjugated reagents, it was possible to set an analysis window on the leukemic blasts and display dual-parameter (ie, green vs. red fluorescence) data regarding expression of two additional markers on the leukemic population. This gating strategy was superior to traditional forward-angle versus RALS displays in that it did a better job of isolating the leukemic cells analytically.
authors.affiliation: Department of Pathology, Duke University Medical Center, Durham, North Carolina.
authors.forename: M J
authors.initials: MJ
authors.lastname: Borowitz
authors.forename: K L
authors.initials: KL
authors.lastname: Guenther
authors.forename: K E
authors.initials: KE
authors.lastname: Shults
authors.forename: G T
authors.initials: GT
authors.lastname: Stelzer
mesh_terms: D001853:Bone Marrow / Q000473:pathology*; D005434:Flow Cytometry / Q000379:methods*; D006801:Humans; D016130:Immunophenotyping / Q000379:methods*; D015470:Leukemia, Myeloid, Acute / Q000473:pathology*; D017493:Leukocyte Common Antigens / Q000032:analysis; D054198:Precursor Cell Lymphoblastic Leukemia-Lymphoma / Q000473:pathology*
chemical_list: D017493:Leukocyte Common Antigens