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An algorithm for automatic segmentation of PAP-stained cell images and its digital implementation is described. First the image is filtered in order to eliminate the granularity and small objects in the image which may upset the segmentation procedure. In a second step, information on gradient and compactness is extracted from the filtered image and stored in three histograms as functions of the extinction. From these histograms, two extinction thresholds are computed. These thresholds are suitable to separate the nucleus from the cytoplasm, and the cytoplasm from the background in the filtered image. Masks are determined in this way, and finally used to analyse the nucleus and the cytoplasm in the original image.

Human lymphocytes, transferred in culture into medium conditioned with phytohemagglutinin, were studied with the combined use of quantitative 14C-uridine autoradiography and high-resolution image analysis. During the first 24 h before the onset of DNA synthesis it was possible to study pure GO and GI populations. The return of the proliferating cells into the Go state was clearly monitored in an RNA synthesis ratelDNA content plot during 1-week growth. On days 3-6, the proliferation decreased progressively, and more cells accumulated in the quiescent Go state. Geometric, densitometric, and texMuch experimental evidence that leads to the common belief that Go and GI cells are different has been reported, (4,11,15,16,20). In particular, it has been shown that the transition from quiescence to proliferation (Go + G1 transition) is associated with the switching on of genes and that the ability of chromatin isolated from resting differentiated cells to synthesize new RNA (the so-called template activity) is lower than and different from that observed with chromatin from stimulated cells. The return from proliferation to quiescence is not a welldocumented phenomenon, and the mechanisms and time courses of this transition are not well known.In previous studies (13,141 in which confluent mouse fibroblasts in culture were stimulated to proliferate and used to mimic the Go-Gl transition, the combination of quantitative 14C-uridine autoradiography and high-resolution image analysis proved to be a n interesting methodology, since it allowed quantitative correlation of the autoradiographic outcome of a cellular function, the RNA synthesis rate, with the structural appearance of the chromatin at the light-microscope level.Among the chromatin structural parameters, the skewness of the distribution of the nuclear optical density values (13) proved to be of particular interest. The same methodology was used in the present study to investigate phytohemagglutinin-activation (Go -+ GI transition) and the return to quiescence (GI -+ Go trantural parameters evaluated from the Feulgenstained nuclei of the same cells indicated that the decondensationlcondensation processes of the nuclear chromatin during the Go 8 G1 transitions paralleled the RNA synthesis activity. The results help to associate descriptors of nuclear morphology, as derived from image analysis, with functional parameters during activation and return to quiescence of human lymphocytes.

The histologic and immunohistologic differential diagnosis between pseudolymphomas (PL) and malignant lymphomas (ML) of the skin can be difficult. Since DNA cytometry has been found to be of both diagnostic and prognostic value in various neoplasms, its ability to discriminate between ML and PL in Feulgen-stained imprints of 17 PL and 49 ML skin biopsies was examined by high-resolution image analysis. The reliability of the following algorithms of DNA distribution was evaluated: 1) 2cDI (2c-deviation index), which reflects the variation of the nuclear DNA values around the diploid DNA peak; 2) percentage of cells having a DNA value greater than or equal to 5c (5cER; 5c-exceeding rate); 3) percentage of cells presenting with a DNA value greater than or equal to 4c (4cER). A 2cDI of 0.1 was found to be the most reliable marker for the differentiation between PL and ML. On the basis of this feature, 16 of 17 cases of PL and 46 of 49 cases of ML were correctly classified. The sensitivity, specificity, and efficiency of this feature were 94%. A 5cER greater than or equal to 1% had a specificity of 100%, but the sensitivity was only 43%. For the 4cER, a sensitivity of 61% and a specificity of 94% were found. In conclusion, the calculation of the 2cDI and the 5cER based on high-resolution image analysis provided additional helpful diagnostic features, and therefore should be included as a diagnostic tool. If the 5cER is at least 1%, the diagnosis of a ML can be confirmed with a specificity of 100%.

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 l4C-Ileucine 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 Feulgenstained, and after remove1 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%.Key terms: High-resolution image analysis, quantitative autoradiography, erythropoiesis, protein synthesis, chromatin structure.In previous studies we have investigated to what extent the functional status of a cell is associated with distinct features of the nuclear image. It became obvious that G1, S, and G2 cells are distinguishable by their chromatin pattern (3) and that the rates of DNA and RNA synthesis are also correlated with the nuclear texture as determined by high-resolution image analysis (9,17). In a recent analysis of human erythropoiesis (10) we demonstrated a relationship between the degree of chromatin condensation and the rate of DNA synthesis of individual erythroblasts. With increasing nuclear condensation the rate of DNA synthesis and the proliferative activity decreased. However, it appeared likely that the proliferative activity in this cell system is not the only variable associated with features of nuclear texture. We assumed that further functional properties were related to the nuclear morphology, particularly the template activity of...

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