DNA stainability in aneuploid breast tumors: comparison of four DNA fluorochromes differing in binding properties

The aim of this study was to evaluate whether or not the differences in chromatin structure between diploid stromal cells or lymphocytes, which are often used as DNA ploidy standard, and aneuploid breast tumor cells can significantly affect the estimates of the DNA index of these tumors. To this end, the DNA content estimates of 34 aneuploid breast tumors, differing in size, degree of differentiation, and presence or absence of estrogen and progesterone receptors and metastases, were compared using four common DNA fluorochromes: DAPI, Hoechst 33342, propidium iodide, and acridine orange. These dyes differ in their mode of interaction with DNA (binding to minor groove or intercalation) and for each of them binding to DNA is restricted to a different degree by nuclear proteins. It was expected, therefore, that if differences in chromatin structure play a role in DNA content estimates, the DNA index of the measured tumors may vary depending on the dye. The cell nuclei were isolated from the tumors using a detergent-based procedure and stained with each of the dyes and the DNA index was estimated using peripheral blood lymphocytes as a DNA content standard. For each of the tumors, the DNA index estimates with all four dyes correlated very well. When the results obtained with individual dyes were compared in pairs, the correlation coefficients (r) of DNA indices were all above 0.96 (correlation at p less than 0.001). The best concordance was seen between specimens stained with Hoechst 33342 and DAPI (r = 0.99), and the least between those stained with Hoechst 33342 and propidium iodide (r = 0.96). The data indicate that DNA content analysis of unfixed nuclei, utilizing the above fluorochromes, is not significantly biased by differences in chromatin structure of the measured cells.