Flow cytometric chemosensitivity analysis of blasts from patients with acute myeloblastic leukemia and myelodysplastic syndromes: the use of 7AAD with antibodies to CD45 or CD34.
BACKGROUND
Flow cytometry is potentially suited to the chemosensitivity analysis of peripheral blood or bone marrow subpopulations in patients with leukaemia and myelodysplastic syndromes.


METHODS
The use of the fluorescent dye 7-amino-actinomycin (7AAD) on unfixed cells to measure loss of viability at a range of cytosine arabinoside (ara-C) doses was evaluated. A six-tube flow cytometric assay for measuring the sensitivity to ara-C of CD45/side-scatter-gated or of CD34-positive leukemic blasts with 7AAD was established, using fixed stained normal mononuclear cells as an internal standard for quantitation of viable cells following culture.


RESULTS
7AAD dose response curves for 10 patients with acute myeloblastic leukemia (AML) showed a wide range of sensitivities at 2.5-5 microM araC (3.7-97%, mean 54% of control cell viability at 2.5 microM and 4.1-94.6 %, mean 27% at 5 microM). Parallel assays for ATP bioluminescence agreed reasonably well with the 7AAD method, r(s) = 0.78. The chemosensitivity of CD45/SSC-gated blast cells at 2.5 microM araC showed no consistent relationship with the ungated cell populations, such that CD45/SSC-gated blast sensitivity of seven samples ranged from 86% more to 38% less than that of the total population. Similarly, the chemosensitivities of the CD34-gated subpopulations ranged from 51% more to 78% less than those of the total populations.


CONCLUSIONS
These results emphasize the necessity of measuring the chemosensitivity of the population of interest rather than of the sample as a whole in heterogeneous clinical material.
Pallis
M
M
Department of Haematology, Nottingham City Hospital and University of Nottingham, Nottingham, United Kingdom.
Syan
J
J
Russell
N H
NH
D000906:Antibodies; D018952:Antigens, CD34 / Q000032:analysis / Q000276:immunology*; D002452:Cell Count / Q000379:methods; D003561:Cytarabine / Q000494:pharmacology; D003609:Dactinomycin / Q000031:analogs & derivatives*; D005434:Flow Cytometry / Q000379:methods*; D005456:Fluorescent Dyes; D006801:Humans; D015470:Leukemia, Myeloid, Acute / Q000276:immunology / Q000473:pathology*; D017493:Leukocyte Common Antigens / Q000032:analysis / Q000276:immunology*; D008027:Light; D009190:Myelodysplastic Syndromes / Q000276:immunology / Q000473:pathology*; D012542:Scattering, Radiation; D012680:Sensitivity and Specificity; D013234:Stem Cells / Q000737:chemistry / Q000187:drug effects / Q000473:pathology; D014407:Tumor Cells, Cultured / Q000737:chemistry / Q000187:drug effects / Q000473:pathology
D000906:Antibodies; D018952:Antigens, CD34; D005456:Fluorescent Dyes; D003561:Cytarabine; D003609:Dactinomycin; C025942:7-aminoactinomycin D; D017493:Leukocyte Common Antigens