Flow cytometric DNA analysis of colon adenocarcinomas: a comparative study of preparatory techniques

DNA quantitation of solid tumors requires either nuclear extraction or disaggregation into single cells for flow cytometric (FCM) analysis. We investigated methods for disaggregating colon carcinomas comparing mechanical and enzymatic techniques in fresh tumors and compared these to extraction of nuclei from paraffin blocks. Mechanical dissociation resulted in the highest yield of tumor cells, especially tumor cells with abnormal DNA content. In addition, mechanically disaggregated intact cells, particularly when dual labeled with cytokeratin, produced optimum DNA histograms. Cytokeratin staining allowed gating on epithelial cell populations and excluded contaminating diploid inflammatory and stromal cells, improving our ability to calculate synthesis phase fractions (SpF). Human colon tumor nuclei extracted from paraffin-embedded tissue resulted in greater variation of DNA histograms and less sensitivity in identifying aneuploid cells compared to mechanically dissociated intact cells from fresh tumors. The quality of the histograms and the ability to identify aneuploid cell populations were improved by modifying the standard "Hedley" technique by additional enzyme incubation. Mechanical dissociation of fresh tumors was the preferred technique for FCM DNA analysis. Nuclear extraction from paraffin blocks resulted in some loss of sensitivity in identifying aneuploid cell lines and decreased ability to measure SpF.