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To elucidate the reasons for the malignant histologic appearance of melanocytic nuclei within benign Spitz nevi (SN), we evaluated nuclear DNA distribution and nuclear size using a computerized image analysis system. In each case of 28 SN and 34 malignant melanomas (MM), about 100 randomly sampled nuclei were analyzed, prepared as monolayers from paraffin-embedded tissues. Large nuclei in MM (nuclear area > mean nuclear area of normal melanocytes + 4 delta) were significantly more likely to be aneuploid (DNA content > or = 5c) than large nuclei in SN chi2 test, p < 0.0001). Only two of 990 large SN nuclei exhibited DNA values higher than 5c, whereas 236 of 2,024 large MM nuclei were aneuploid or polyploid. Accordingly, in multivariate analysis, five features of DNA distribution proved to be most important for objective discrimination between MM and SN: 2c deviation index, 5c exceeding rate, standard deviation of the nuclear DNA content, and both the 85th and the 95th percentiles of DNA distributions. On the basis of these features, we could define a linear discriminant function that allowed a correct diagnosis in 94% of the cases. Our data demonstrate that diagnostically misleading large nuclei in SN are euploid, in contrast to MM. It is thus possible to discriminate SN and MM with high accuracy using DNA cytometry. Because paraffin-embedded tissue can be used, this technique could be a valuable complement to routine histology in equivocal cases.

Human erythropoiesis is an appropriate model for analyzing correlations between functional activity of cells during maturation and nuclear morphology as determined by high-resolution image analysis. In a previous study a relationship had been found between nuclear condensation and the proliferative activity of the cells. This present study analyzed the correlation between nuclear morphology and the protein synthesis rate. The latter was assayed by measuring the 14C-1-leucine incorporation rate utilizing the technique of quantitative 14C-autoradiography. The labeled cells were first classified according to conventional cytological criteria into four groups of increasing maturity. Following grain counting the nuclei were Feulgen-stained, and after removal of the silver grains, the local nuclear optical densities were evaluated by scanning. There was a nonlinear relationship between the protein synthesis rate and features representing the degree of chromatin condensation. This nonlinearity was explained by the mediator function of RNA, predominantly mRNA. The amount of protein produced at a given time depends on the transcriptional activity of the chromatin, the frequency of mitotic divisions partitioning the mRNA, and the half-life of the mRNA. It was concluded that the chromatin texture of erythroblasts reflects three different metabolic activities: the rate of DNA strand duplication, the transcriptional activity for structural proteins enabling a cell to grow and cycle, and for functional proteins, particularly hemoglobin. Since these three activities appear to be synchronized, it is understandable that functional as well as textural features can be used to perform a supervised classification by means of multivariate analysis. Application of the five most significant features allowed a consistent classification of the erythroblasts into the various cytological compartments in 76.7% of all cases, while the classification error of the observer amounted to 13.7%.

Chinese hamster fibroblasts were investigated for the existence of correlations between proliferative activity and nuclear morphology. As a proliferative parameter, the rate of DNA synthesis of individual cells was determined by quantitative 14C-autoradiography. In a second step the images of the Feulgen-stained nuclei were digitized for extraction of features of morphology and texture. These features were correlated with the corresponding DNA synthesis rate values. The following relationships were found: Round nuclei have higher rates of DNA synthesis than flat ones. The more chromatin is packed at the nuclear rim, possibly representing heterochromatin, the lower the rate of DNA synthesis. The DNA synthesis rate also correlates with the graininess of chromatin. Larger areas of condensed chromatin are associated with lower rate values. A fine and irregular network of chromatin, as is typical of immature cell types, is associated with a high rate of DNA synthesis. Although these results are presently confined to the cell line investigated, parallels seem to exist to other cell types, such as erythropoietic cells, which await further investigation.

The nuclear morphology of preneoplastic and unaltered hepatocytes in diethylnitrosamine-treated rats was investigated. Two-micrometer-thick sections of methacrylate-embedded liver were scanned with a TV camera and evaluated in a computer using multivariate analysis methods. The preneoplastic cell populations (islands) were distinguished from unaltered hepatocytes by histochemical demonstration of glycogen storage in specimens from starved animals. After the hemalaun-stained liver sections were scanned randomly, the sections were stained for glycogen, and the previously registered cells were identified visually using a scanning stage for relocation. This objective identification of unaltered and preneoplastic hepatocytes formed the basis for the selection of a training set for feature evaluation and supervised classification. Image analysis for quantitative nuclear morphology was applied to the hemalaun-stained cells. The results showed that condensed chromatin was reduced and nuclear area was increased in the nuclei of glycogen-storing cells. Differences in the nuclear structure were also found. A multivariate analysis including seven features gave a correct classification result of about 82%.

Image analysis was performed using Feulgen-stained smears of L cells that were either confluent or had received a nutritional stimulus to proliferate. The cells were grown directly on slides in order to minimize damage to them during manipulation and to avoid trypsin treatment. A series of experiments was performed by varying the dose and the time of the stimulus. The nuclei were interactively sampled and automatically segmented. Most of the information about the fine chromatin structure was gained after subtracting the original image from a median-filtered image. The results indicate that there were changes in the Feulgen-stained nuclear morphology and densitometry shortly after transition of quiescent G0 cells to the proliferating G1 compartment. Multidimensional cluster analysis provided model-independent discrimination between the resting and the stimulated cells; the latter presented increased nuclear dispersion, indicating increased template activity of the cells. The results obtained are applicable both to obtaining a better understanding of the mechanisms of regulation during the G0-G1 transition and to deriving morphologic criteria applicable in diagnostic routines.