LSC Application: DNA Content of Bladder Carcinoma

Urothelial carcinoma of the bladder has variable biologic behavior and malignant potential. To improve diagnostic reproducibility and accuracy, laser-scanning cytometry (LSC) may be used as an objective and measurable adjunct to classical morphology. LSC combines rapid DNA content analysis of large numbers of cells with morphologic verification. Therefore, it offers features of both image and flow cytometry.
.....Previous studies have shown that DNA content analysis can aid in predicting tumor behavior. Bladder tumors that are DNA aneuploid have statistically significant increased rates of recurrence and poorer five-year survival as compared to DNA diploid tumors. In addition, the 5C exceeding rate (percentage of cells with DNA content greater than 5C) has been shown to provide important prognostic information in urothelial carcinoma (Figure 1).
.....The ability to relocate cells for morphologic examination is an important feature of DNA content analysis by LSC. In bladder carcinoma, this feature is particularly important to rule out: (1) false-aneuploidy produced by umbrella cells and (2) the presence of DNA-tetraploidy associated with viral infection (polyoma virus).

DNA content analysis Figure 1. DNA content analysis of a bladder washing from a patient with transitional cell carcinoma. A DNA aneuploid stemline is observed at DI = 1.3. Cells with a DI > 5C were relocated for morphologic examination, as shown below.

relocated aneupoloid cells

Sample Preparation
Standard cytologic preparations (Cytospin, Thinprep, etc.) can be used for DNA content analysis by LSC. Preparations are reacted for 10 minutes at room temperature with approximately 2 ml of a 50 µg/ml solution of propidium iodide (PI) in PBS containing 200 µg/ml RNAse. After decanting by inversion, the slides are coverslipped and analyzed immediately. For long term storage of slides, a 50 µL mixture of 25% PI and 75% glycerol can be used as a mounting medium.
LSC Analysis
After establishing a scan area, slides are analyzed using a 40X objective and 5 mW of Argon laser power. A minimum of 15,000 cells (or the entire cell preparation) is examined. A cell gallery is created by relocation of cells from each of the major peaks in the histogram of integrated red fluorescence. The morphologic composition of relocated cells is examined for purposes of quality assurance. The percentage of cells exceeding 5C is determined by setting an appropriate histogram gate. By relocating 5C exceeding cells, morphologic composition (singlets versus clumps) can be confirmed.
Procedure and data from Dr. James B. Hendricks, Children’s Hospital Research Institute, New Orleans, LA.