QUANTITATIVE MORPHOLOGIC ASSESSMENT OF NUCLEI EXTRACTED FROM PARAFFINFOR DNA FLOW-CYTOMETRY

We present a method for quality control of flow cytometric DNA content studies using nuclei extracted from paraffin-embedded tissue. This me thod is based on a quantitative morphologic assessment of extracted nu clei. Cell nuclei prepared from 22 paraffin-embedded tumors known to c ontain discrete diploid and aneuploid stemlines were deposited onto po ly-L-lysine-coated glass slides. Nuclei were stained with Diff-quic an d examined by light microscopy. Two hundred nuclei were counted and cl assified based on morphologic appearance into tumor and nontumor group s. Classification criteria included differences in nuclear size, nucle ar chromatin structure, the degree of nuclear chromatin condensation, and the presence of nucleoli. Excellent agreement was found between tw o independent observers (R = 0.989) on the classification of nuclei. T he relative number of tumor nuclei on the morphologic preparation was compared with the relative number of aneuploid cells in the DNA histog ram. 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, fu rther analysis by image cytometry demonstrated a diploid and aneuploid component of the tumor cell population. This quantitative method of m orphologic examination of the preparation ultimately analyzed by flow cytometry offers several distinct advantages, including: (a) identific ation of peaks in the DNA histogram, (b) assessment for selective loss of cell nuclei in the extraction process, and (c) identification of b iologic heterogeneity in tumor populations.