Simultaneous measurement of DNA content and cell‐surface immunofluorescence of human bone marrow cells using a single laser flow cytometer

Abstract

This paper describes the measurement of S phase DNA content in human bone marrow subpopulations using a single laser method for bivariate analysis of DNA content and cell‐surface immunofluorescence (s‐IF). Low density (<1.077 g/ml) bone marrow cells were labeled with a panel of unconjugated monoclonal antibodies (MoAb) for the lymphoid (CD2 + CD19), T‐lymphoid (CD2), B‐lymphoid (CD19), erythroid (anti‐glycophorin‐A), myelomonocytic (CD13, CD33; single and as cocktail) and monocytic (CD14) lineages. A fluorescein isothiocyanate (FITC)‐conjugated goat anti‐mouse label was used as second step. Unfixed, MoAb‐labeled cells were incubated for 24 h with a hypotonic propidium iodide solution for DNA staining. Cells were analysed on a single‐laser flow cytometer, operating at 488 nm. The effect of the combined staining protocol upon both s‐IF and DNA stainability was evaluated. Only a slight decrease (mean: 29.0%) in s‐IF intensity was observed after DNA staining. The percentages of immunofluorescent cells in the bone marrow samples of 10 normal individuals before and after DNA staining were essentially unchanged for all the MoAbs used. The DNA histograms of the immunophenotypically defined subpopulations were of excellent quality with a mean coefficient or variation of 1.8%. This procedure allows the assessment of very low levels of S‐phase DNA content, as measured in normal low density blood cells of 8 healthy volunteers (mean 0.07%). Considerable differences in percentages of cells with S‐phase content were seen between the four main bone marrow subpopulations (P<0.0001). The erythroid hematopoietic lineage showed the highest proportion of S‐phase DNA cells (26.4% ± 4.7%), the lymphoid lineage the lowest (2.5% ± 0.8%) while the monocytic lineage (6.1% ± 0.9%) and the myelomonocytic lineage (8.5% ± 1.4%) were in between. Within the lymphoid lineage the percentage of S‐phase cells was significantly lower in T‐cells (2.0% ± 0.7%) compared to B‐cells (5.1% ± 1.5%) (P<0.0001). We conclude that bivariate measurement of S‐phase DNA content and s‐IF is feasible with use of a single laser flow cytometer and that this approach provides important additional biological information. The value of this method to study hematologic malignancies is demonstrated in a bone marrow sample from a patient with erythroleukemia.

Sensitivity of combined DNA/immunophenotype flow cytometry for the detection of low levels of aneuploid lymphoblastic leukemia cells in bone marrow

“…Surprisingly, the S-phase of regenerating bone mar- a row cells is not significantly different from the S-phase of normal donors in our data. But in general, normal bone marrow lymphopoiesis has a lower proportion of cells in S-phase than myelo-or erythropoiesis (6,13). As shown with our data, this is also true after chemotherapy.…”

“…Because of the heterogeneity of bone marrow cells, the CV of G0/G1 peak is higher than that of isolated hematopoietic subpopulations (6). In addition, after chemotherapy the CV of G0/G1 peak is significantly greater as compared with normal donor bone marrow.…”

“…Bidirectional immunophenotype analysis (6,9,11,27) and in some cases tricolor immunophenotyping (22,31) was applied. In 85% of patients with acute myeloid leukemia, blast cells of different phenotypic subsets were found with multiparameter immunophenotyping (18).…”

hZAC encodes a zinc finger protein with antiproliferative properties and maps to a chromosomal region frequently lost in cancer

“…SaOs-2 cells were transiently transfected with different amounts of plasmid encoding mZac, hZAC, or p53, together with pRK5-CD20 encoding the CD20 antigen that was used as a marker for selection of the transfected cells. Twenty-four hours later, propidium-iodide staining was performed as previously described (17). Cell cycle distribution was determined with a FACScan f low cytometer (BecktonDickinson).…”

DRAQ5‐based DNA content analysis of hematolymphoid cell subpopulations discriminated by surface antigens and light scatter properties

“…This approach was previously reported with the use of UV-excitable Hoechst 33342 on viable cells (3), and PI with FITClabeled antibody detection of surface immunoglobulin in B-cell lymphomas (4,5). Simultaneous DNA and surface antigen labeling was also performed on unfixed bone marrow cells using lineage-specific primary monoclonal antibodies with a secondary FITC-conjugated antibody, followed by staining with hypotonic PI (6). PI-based procedures are relatively simple and can be performed using standard clinical instruments.…”