Comparison of eight modifications of Hedley's method for flow cytometric DNA ploidy analysis of paraffin-embedded tissue

Flow cytometric DNA analysis of nuclear suspensions from formalin-fixed, paraffin-embedded tissue often fails to detect aneuploid cell populations present in corresponding fresh tissue. Nuclear suspensions were prepared by 8 different modifications and standard Hedley's method using 50-microns sections of tissue blocks from 8 breast and 8 colonic carcinomas, all previously known to be DNA aneuploid by analysis of fresh tumor. Pepsin solutions of three different enzymatic activities were used to release nuclei using three different tissue digest formats. DNA aneuploidy was demonstrated overall in 7 of 72 different colon tumor experiments and 25 of 72 breast cancer experiments. Modifications yielded aneuploid populations not detected by the standard Hedley method; DNA aneuploidy of 4 breast and 2 colon cancers was detected by modifications compared to 2 breast and 1 colon cancer demonstrated by the standard. No single method consistently demonstrated DNA aneuploidy. High histogram baselines, presumably from debris, contributed to the marked loss of sensitivity in detecting most DNA aneuploid populations. Detection of DNA aneuploidy was most closely associated with specific cases, regardless of the method of nuclear suspension preparation. Recovery of DNA aneuploid nuclei seems to depend primarily on tissue processing or innate characteristics of the tumor cells, not on the method used to prepare the nuclear suspension.