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

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.