Comparison of fixation procedures for fluorescent quantitation of DNA content using image cytometry

DNA quantitation using fluorescent dyes is of interest in image cytometry in that it is nondestructive in its mode of staining and is compatible with techniques such as FISH and immunofluorescence, allowing multicolor analysis of a wide range of cellular markers of interest. Optimal preparation techniques were sought using human peripheral blood lymphocytes and fine needle samples of breast carcinomas. Unfixed (air-dried only), ethanol, ethanol/acetic acid, and paraformaldehyde/ethanol fixations were tested. Unfixed or fixed cells were placed on slides as a drop or by cytocentrifugation, stained with 4',6-diamidino-2-phenylindole dihydrochloride and DNA content was measured using image analysis. Histogram quality was evaluated using G0G1 peak coefficient of variation, and compared to those generated by flow cytometry. Drop preparations of ethanol/acetic acid fixed and cytospin preparations of paraformaldehyde/ethanol fixed cells appeared to give the best histograms for image analysis, which were inferior in quality to those generated by flow cytometry. Comparison of breast carcinoma histograms generated by flow and image showed the same DNA aneuploid populations but with slightly higher DNA indices measured by image analysis.