Technical Notes

DNA Ploidy and S-Phase Estimation

Clinical Significance:

The assessment of DNA content and the estimation of cell cycle characteristics provides vital information to the clinician in many human cancers such as breast ⁽¹⁾,
prostrate ⁽²⁾,
bladder ⁽³⁾,
ovarian ⁽⁴⁾,
HNSCC ⁽⁵⁾,
lung ⁽⁶⁾ and
non-Hodgkin's lymphomas ⁽⁷⁾.
Normal resting human cells have 46 chromosomes and are termed "diploid". During proliferation, the DNA content doubles; cells that are replicating DNA (in S-phase of the cell cycle) have an intermediate content of DNA. A characteristic of many malignancies is the presence of abnormal chromosome numbers or "aneuploidy". An analysis of the cell cycle parameters also provides vital information about the proliferative fraction of the cells; cells from a tissue with low proliferating fraction have a decreased percentage of cells in S-Phase and vice-versa.

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Methodology:

The relative DNA content of cells can be quantitatively and reproducibly analyzed by Flow cytometry using a fluorochrome such as Propidium Iodide (PI) that binds stoichiometrically to DNA at all stages of the cell cycle. Uniform suspension of single cells are prepared for DNA staining and flow cytometric analysis from solid tissue specimen, paraffin blocks or blood and bone marrow. The tissue is enzymatically treated to obtain a single cell suspension and the cells are permeabilized to allow PI to gain access to the nuclear DNA. The samples are loaded and run on a flow cytometer with electronic doublet discrimination capacity. The analysis of DNA histogram is performed according to the guidelines of the DNA Flow Cytometry task Force of the European Society for Analytical Cellular Pathology (8).

Each report is accompanied by the DNA histogram and interpretation of the ploidy status of each sub-population and the S-phase fraction.

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References:

1) Hedley D.W., Clark G.M., Cornelisse C.J. et al (1993) Cytometry 14, 482.
2) Shankey T.V., Kallioniemi O.P., Koslowski J.M. (1993) Cytometry 14, 497.
3) Wheeless L.L., Badalament R.A., deVere White R.W. et al. (1993) Cytometry 14, 478.
4) Friedlander M.L., Hedley D.W., Swanson C et al (1988) J. Clin. Oncol 6, 282.
5) Kokal W., Gardine R., Sheibani K. et al (1988) Am. J. Surg. 156, 276.
6) Yoneyama K., Erhardt K., Stenkvist B. et al (1987) Am. J. Clin. Oncol. 10, 330.
7) Ormerod M.G., Tribukait B., Giarretti W. et al. (1998) Anal. Cell Pathol. 17, 103.