Quantitative morphologic assessment of nuclei extracted from paraffin for DNA flow cytometry

We present a method for quality control of flow cytometric DNA content studies using nuclei extracted from paraffin-embedded tissue. This method is based on a quantitative morphologic assessment of extracted nuclei. Cell nuclei prepared from 22 paraffin-embedded tumors known to contain discrete diploid and aneuploid stemlines were deposited onto poly-L-lysine-coated glass slides. Nuclei were stained with Diff-quic and examined by light microscopy. Two hundred nuclei were counted and classified based on morphologic appearance into tumor and nontumor groups. Classification criteria included differences in nuclear size, nuclear chromatin structure, the degree of nuclear chromatin condensation, and the presence of nucleoli. Excellent agreement was found between two independent observers (R = 0.989) on the classification of nuclei. The relative number of tumor nuclei on the morphologic preparation was compared with the relative number of aneuploid cells in the DNA histogram. Good agreement was observed (R = 0.975) in all but three cases in which the relative number of tumor nuclei was underrepresented by the percentage of aneuploid nuclei in the DNA histogram. In each case, further analysis by image cytometry demonstrated a diploid and aneuploid component of the tumor cell population. This quantitative method of morphologic examination of the preparation ultimately analyzed by flow cytometry offers several distinct advantages, including: (a) identification of peaks in the DNA histogram, (b) assessment for selective loss of cell nuclei in the extraction process, and (c) identification of biologic heterogeneity in tumor populations.