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.

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Multidimensional flow cytometry identifies residual leukemia in more than half of the patients with AML who are in morphologic remission. The detection of leukemic blast cells in these patients by multidimensional flow cytometry is predictive of a more rapid relapse.

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Reduced levels of human myeloid nuclear differentiation antigen (MNDA) gene transcripts have been detected in both familial and sporadic cases of myelodysplastic syndromes (MDS). Numerous reports implicate elevated apoptosis/ programmed cell death and death ligands and their receptors in the pathogenesis of MDS. MNDA and related proteins contain the pyrin domain that functions in signaling associated with programmed cell death and inflammation. We tested the hypothesis that MNDA is involved in the regulation of programmed cell death in human myeloid hematopoietic cells. Clones of K562 cells (MNDA-null) that expressed ectopic MNDA protein were established using retroviral transduction. MNDA-expressing K562 clones were resistant to tumor necrosis factor-related apoptosis inducing ligand (TRAIL)-induced apoptosis, but were not protected from programmed cell death induced with genotoxic agents or H 2 O 2 . MNDA protein expression assessed in control and intermediate and high-grade MDS marrows showed several patterns of aberrant reduced MNDA. These variable patterns of dysregulated MNDA expression may relate to the variable pathophysiology of MDS. We propose that MNDA has a role regulating programmed cell death in myeloid progenitor cells, and that its down-regulation in MDS is related to granulocytemacrophage progenitor cell sensitivity to TRAIL-induced programmed cell death.

These results suggest that HIV-1-specific immune responses correlate with evidence of ongoing HIV-1 replication.

Many epitopes are phosphorylated during mitosis. These epitopes are useful biomarkers for mitotic cells. The most commonly used are MPM-2 and serine 10 of histone H3. Here we investigated the use of an antibody generated against a phospho peptide matching residues 774-788 of the human retinoblastoma protein 1 (Rb) to detect mitotic cells. Human cell lines were stained with DNA dyes and antibodies reactive with epitopes defined by antibody MPM-2, phospho-S10-histone-H3, and the phospho-serine peptide, TRPPTLSPIPHIPRC (phospho-S780-Rb). Immunoreactivity and DNA content were measured by flow and image cytometry. Correlation and pattern recognition analyses were performed on list mode data. Western blots and immunoprecipitation were used to investigate the number of peptides reactive with phospho-S780-Rb and the relationship between reactivity with this antibody and MPM-2. Costaining for bromodeoxyuridine (BrdU) was used to determine acid resistance of the phospho-S780-Rb epitope. Cell cycle related phospho-S780-Rb immunofluorescence correlated strongly with that of MPM-2. Laser scanning cytometry showed that phospho-S780-Rb immunofluorescence is expressed at high levels on all stages of mitotic cells. Western blotting and immunoprecipitation showed that the epitope is expressed on several peptides including Rb protein. Costaining of BrdU showed that the epitope is stable to acid. Kinetic experiments showed utility in complex cell cycle analysis aimed at measuring cell cycle transition state timing. The phospho-S780-Rb epitope is a robust marker of mitosis that allows cytometric detection of mitotic cells beginning with chromatin condensation and ending after cytokinesis. Costaining of cells with DNA dyes allows discrimination and counting of mitotic cells and post-cytokinetic ("newborn") cells. To facilitate use without confusion about specificity, we suggest the trivial name, pS780 for this mitotic epitope.