DNA content is a useful laboratory indicator of prognosis

Analysis of DNA content is a useful laboratory indicator of prognosis for many tumors including breast carcinoma. An abnormality in the DNA content of cells, called aneuploidy, is commonly associated with the chromosomal abnormalities found in malignant cells. When cells are labeled with fluorochromes that bind specifically and proportionately to the amount of cellular DNA, the signal obtained from each cell relates directly to the amount of DNA contained within its nucleus. Analysis of this information provides a means of detecting cell populations with abnormal DNA content (aneuploid populations) and also permits the measurement of the subpopulations of cells in the various phases of the cell cycle.
Specimens can also be reacted with fluorochrome-labeled lineage-specific antibodies (e.g., specific for cytokeratin) prior to analysis. In this way, epithelial cells only can be positively gated for analysis. This practice greatly increases the method's sensitivity in detecting minor populations of aneuploid tumor cells by effectively eliminating lymphocytes, stromal cells, and other contaminating material from the analysis.

Tumor cells are identified and gated based upon cytokeratin staining.
cytokeratin staining for tumor cells

To verify the morphology of the two populations, cells can be restained with Wright-Giemsa or H & E. Cells from each region can be relocated and visualized by the CompuSort™ process.
restained cell populations

Sample Preparation
Air-dried breast tumor smears or touch preparations were fixed in acetone for 1 minute at room temperature (RT) and air dried. Slides were reacted with 200 ul of a 1:20 dilution in PBS of FITC-conjugated anti-cytokeratin antibody (CAM5.2, Becton Dickinson, San Jose, CA) for 15 min. at RT in a humidified chamber. The smears were washed with PBS and reacted for 10 min. at RT with 2 ml of a 50 µg/ml solution of propidium iodide (PI, P-4170, Sigma, St. Louis, MO) in PBS containing 200 µg/ml RNAse (R-5250, Sigma). After decanting by inversion, the slides were coverslipped using 50 µl of a mixture of 25% PI (100 µg/ml in PBS) and 75% glycerol as the mounting medium. The slides were stored in the dark until analysis.
LSC Analysis
The prepared slides were placed onto the LSC stage, and the area of interest on the microscope slide were identified and selected for analysis using the 20X objective. Data collection utilized a red fluorescence threshold with a 100 pixel minimum cell size. Single cells were then displayed in a dot plot of PI integrated fluorescence vs. FITC integrated fluorescence. Gates were set on the cytokeratin-positive cells to display a histogram of tumor DNA. The cells were restained for visualization by removing the coverslip by immersing the slide in a PBS bath until the coverslip slid off. Histological staining procedures were performed and the slides re-coverslipped with 50 µl of a PBS/glycerol mixture.

(Procedure and data courtesy of Dr. Richard Clatch, Highland Park Hospital, Highland Park, IL. Ref: Arch Pathol Lab Med, vol. 121, June 1997.)