DNA histograms

Fig. 13.8 G1 phase arrest of HOS cells exposed to either MTX or MTX-LDH for 20 h. The cell cycle was studied by FACS analysis of Pl-stained cells. DNA histograms are shown, with the x-axis representing the DNA content and the y-axis representing the cell number. Note that 320 pg/mI MTX are equivalentto 704pg/mlof MTX-LHD, based on MTX content in MTX-LDH.

The ability of some fluorescent dyes to bind DNA quantitatively is exploited in flow cytometry to determine the DNA content of a cell. Dyes such as propidium iodide that bind double-stranded DNA stoichiometrically can be used for the purpose. The intensity of red fluorescence is directly related to the amount of DNA bound by propidium iodide. By comparing the fluorescence intensity of the test specimen and, in turn, its DNA content to the fluorescence intensity of specimens containing normal diploid amounts of DNA, a DNA histogram can be generated. By computing a DNA index, which is the ratio of DNA content of a test specimen to the DNA content of a specimen containing a normal diploid population, information related to the presence of an aneuploid tumor population can be obtained. The DNA index of 1 would imply that the DNA in the test specimen is from a normal diploid population (2N DNA), whereas the DNA index of an aneuploid population will be greater or less than 1. Thus, the DNA index of a tetraploid (4N DNA) would be 2. 

In a DNA histogram, the fluorescence intensity, which is related to the quantity of DNA, is plotted on the x-axis, while the number of events or number of cells are plotted on the y-axis. 

In addition to providing information about the presence of an aneuploid cancer cell population, the DNA histograms can also be used to gain information on the relative number of cells in the various stages of the cell cycle. Cells in the... 

Fig. 6. Diagrammatic representation of a DNA histogram showing population of cells in cell...

Figure 6 is a schematic representation of a DNA histogram. The ability of the flow cytometer to rapidly count several thousand nuclei contributes to the sensitivity of this technique for DNA analysis. However, problems due to sample quality, staining, and instrumental artifacts should be recognized and minimized to insure accurate interpretation of data (B2). 

Monoparametric DNA Analysis Fixed cells were stained with PI 25 pg/niL, RNase 5 pg/mL, Nonidet P 40 0.125 pg/mL. Cells were kept at RT for 60 in the dark and were analyzed by BD FACSCalibur flow cytometer. Aggregates were gating out by DDM papameters and DNA content analysis was performed in >10,000-gated cells. DNA histograms were analyzed using ModFit LT . 

Joensuu, H. and Kallioniemi, O. P. (1989) Different opinions on classification of DNA histograms produced from paraffin-embedded tissue. Cytometry 10,711-717. van Dam, P. A., Watson, J. V., Lowe, D. G., and Shepherd, J. H. (1992) Flow cytometric measurement of cell components other than DNA virtues, limitations, and applications in gynecologic oncology. Obstet. Gynecol. 79,616-621. 

Peak reflect The peak reflect method is an algorithm for analyzing DNA histograms to determine the number of cells in the S phase of the cell cycle. In the peak reflect method, the shapes of the 2C and 4C peaks are assumed to be symmetrical, thus allowing subtraction of the contribution from these two peaks from the S-phase cells between them. 

S-FIT An S-FIT approximation is a mathematical algorithm for guessing at which cells in a DNA-content histogram are actually in the S phase of the cell cycle. The S-FIT algorithm bases this guess on the shape of the DNA histogram in the middle region between the G0/G1 and the G2/M peaks. 

Joensuu, H. and Kallioniemi, O. P. (1989) Different opinions on classification of DNA histograms produced from paraffin-embedded tissue. Cytometry 10,711-717. 

To get DNA histograms, after separating by tripsinization, cells were fixed with 20% ethanol. They were treated with 0.25% ribonuclease for 1 hr followed by the staining of intranuclear DNA with 0.005% propidium iodide. DNA histograms were drawn by flow cytometry (Cytofluorograf 50H, Ortho Co.) followed by cell cycle analysis. ... 

Cell growth was inhibited dose-dependently after exposure to the test solutions with concentrations from 10 ig/ml to 100 pg/ml (Fig. 1). Fig. 2 shows a normal cell surface as a control. Four min exposure to 50 Xg/ml of the drug caused a decrease in cell population of 20% and a partial loss of microvilli (Fig. 3). Flow cytometry did not reveal any abnormal DNA histograms (Fig. 4). 

Fig. 4. DNA histogram after exposure for 4 min to 100 pg/ml of amphotericin B. No abnormal pattern.

Four-min exposure with the highest concentration (1 mg/ml) did not inhibit cell growth (Fig. 8), damage the cell surface (Fig. 9) or cause any abnormal pattern in the DNA histogram (Fig. 10). 

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