In order to better characterize the new rapid staining method for flow cytofluorometry proposed by Krishan, we have tested its stability and several other properties, and have carried out a quantitative comparison of the fluorescence histograms obtained using propidium iodide or the acriflavine-Feulgen staining procedure.Using a human hematopoietic cell line in the logarithmic phase of growth, and analyzing the data by means of a mathematical method we have devised, we found that the fluorescence intensity of cells stained with propidium iodide remains stable for at least 48 h; it is insensitive to dye concentration between 0.025 and 0.10 mg/ml (37-150 tzM); it is not affected by incubation with ribonuclease before staining; propidium iodide in 0.1% sodium citrate remains stable for at least 20 days; and quantitative estimates of the fractions of cells in the different phases of the cell cycle are in good agreement with those obtained from acriflavine-Feulgen staining and from autoradiography after pulse labeling with tritiated thymidine. We conclude that this method is useful for the measurement of relative DNA content by flow cytofluorometry, although modifications in the technique are necessary for some cell types which grow in monolayers.Because it can be used to measure selected properties of large numbers of individual cells in a population rapidly and conveniently, flow cytofluorometry has become increasingly popular as a research tool, and its potential for clinical application is being intensively explored. In principle, the intercellular distribution of any cellular component can be measured provided an appropriate fluorescent dye can be used which binds stoichiometrically to the component in sufficient quantity to be detectable by the available instrumentation. Most applications of the method have until now been concerned with the determination of the relative distribution of DNA content among the cells of a population. A method that permits one to carry out rapid determinations of relative DNA content has important implications not only as a more convenient and potentially more accurate means of cell cycle analysis of populations in culture, but also for monitoring the status and kinetic response to therapy of patients with leukemia or other neoplasms (8,9,12,13).Until very recently, the fluorescent staining methods used by most investigators were the acri-

Summary. Two adults with rapidly progressive acute myeloblastic and myelomonoblastic leukemia were given single injections of tritiated thymidine, and measurements were made of the growth rates of their leukemic and normal hematopoietic cells by radioautographic methods. Although almost all leukemic blasts in both marrow and blood were metabolically active as shown by their ability to incorporate tritiated uridine and leucine in Vitro, only 5.6% and 6.1% ol the blasts in the marrow and even fewer in the blood incorporated tritiated thymidine. The mitotic indexes of the marrow blasts were 0.66% and 0.52%; no circulating blasts were dividing. The mean generation times of the actively proliferating blasts were estimated to be 49 and 83 hours. This cannot be equated with the doubling time of the total leukemic population as there is evidence that many blasts fail to continue dividing and die. The mean durations of the phases of the blasts' mitotic cycles were as follows: DNA synthesis (S) = 22 and 19 hours, premitosis (G2) = 3 hours, mitosis (M) = 0.47 and 0.62 hour (minimal estimates), and postmitosis (G1) = 24 and 61 hours. In both patients the maximal mean transit time of the blasts in the blood was 36 hours, and the minimal numbers of actively dividing blasts present were 1.6 and 2.6 X 109 per kg of body weight.Estimates were also made of the rates of proliferation and maturation of the residual normal erythrocytic and granulocytic cells in these two patients. Although total production was markedly diminished because of reduction in the number of normal elements, the relatively few remaining normal cells appeared to be dividing and maturing at rates that are about the same or only slightly slower than those found in normal subjects.We conclude that the main reason leukemic blasts displace normal hematopoietic precursors in acute leukemia is that the blasts largely fail to differentiate. Many die but many others persist in the marrow and elsewhere as primitive cells and continue to proliferate. As the blasts accumulate, they

The murine erythroleukemia cell (MELC) line in suspension culture can be induced to differentiate to erythroid cells by various compounds, including dimethyl sulfoxide (Me2SO). Analysis of the cell cycle, during differentiation induced by Me2SO, using thymidine incorporation, thymidine labeling index, and relative DNA content per cell as measured by flow microfluorometry, demonstrates a transient inhibition of entry of (2), accumulation of globin mRNAs (3-6), a and f globin synthesis (7), increase in heme synthesis (8), synthesis of erythrocyte-specific proteins (9), loss in capacity for cell division (2, 10), and the appearance of erythrocytespecific membrane proteins (11).The rate of DNA synthesis, proportion of cells in S-phase, and the pattern of DNA accumulation have been measured and compared in MELC grown in the presence and absence of Me2SO and other inducing agents, e.g., butyric acid (12) and dimethylacetamide (13). DNA synthesis has been measured using incorporation of isotopically labeled thymidine in pulse-labeling experiments; cells in S-phase were assayed by autoradiography of thymidine pulse-labeled cells; DNA accumulation per cell was determined using propidium iodide staining with flow microfluorometric analysis. By MATERIALS AND METHODS Cells. Strain 745-A, which is infected with Friend virus complex, was kindly provided by Charlotte Friend and has been maintained in culture in our laboratory as described elsewhere (14). Isolation of the Me2SO-sensitive cell line (DS19) and resistant cell line (DR10) was described by Ohta et al. (15). Cell concentration and proportion of hemoglobin-containing (benzidine-reactive) cells were determined as previously described (14).Pulse Labeling with Thymidine. DNA synthesis was determined as incorporation of [3Hjthymidine (20 Ci/mmol, New England NuclearCorp.) into cells (2 X 105 cells/per ml) incubated for 20 min at 370 at a concentration of 20 ,Ci/ml of culture medium. Thymidine pulse labeling in this manner was performed on aliquots removed from culture at the times indicated for each experiment. Following incubation with radioisotope, cells were washed with phosphate-buffered saline, pH 7.4, containing 2 mM thymidine, and precipitated with 10% trichloroacetic acid. Precipitates were collected on Millipore filters and their radioactivity was measured in Aquasol. A separate aliquot of cell suspension was removed from the culture and, washed with the same phosphate-buffered saline, and smears were prepared employing the cytocentrifuge. These slides were processed for radioautography (16

Twelve methods for analysing FCM-histograms were compared using the same set of data. Some of the histograms that were analysed were simulated by computer and some were taken from experiments. Simulated data were generated assuming asynchronously growing cell populations and (i) measurement coefficients of variation (CV) from 2 to 16%; (ii) constant measurement CV or CV's increasing from G, to G , phase, and (ii) varying fractions of cells in each phase. Simulated data were also generated assuming synchronous cell populations i n which a block in early S phase was applied and released. DNA histograms were measured for L-929 cells at various times after mitotic selection. Labelling indices were also measured for these cells at the same time.The fractions of cells in the G,, S, and (G2 + M) phases were calculated by each analytical method and compared with the actual fractions used for simulation, or in case of exp&rimental data., with autoradiographic results. Generally, all methods yielded reasonably accurate fractions of cells in each phase with relative errors in the range of 1&20%. However, most methods tended to overestimate G, fractions and underestimate S fractions. In addition, variations in the shape of the S phase distribution often caused considerable errors. Phase fractions were also calculated for histograms of kinetically perturbed populations, simulated as well as experimental The errors were only slightly larger than for histograms from asynchronously growing cell populations.Several methods for estimating cell-cycle-traverse parameters of DNA histograms generated from flow cytometry (FCM) have been published in the last few years (Dean

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