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DNA content, apoptosis and mitosis in transplanted human hearts.

Aim of the study: To analyze the changes in DNA content, the percentage of apoptosis and the nuclear mitotic frequency of myocytes in transplanted human hearts. Methods: Twenty-three transplanted hearts were obtained from 22 patients. The mean interval between transplantation and death was 649 days (ranging from 13 to 2558 days). Ten control hearts were selected from individuals whose death was not due to primary heart disease. Tissue samples were obtained from the mid section of the lateral wall of left and right ventricles. DNA content was evaluated on isolated myocardial cells using image cytometry. In situ detection of apoptosis was performed by the terminal deoxynucleotidyl transferase-mediated digoxigenin-dUTP nick-end labeling (TUNEL) technique. Mitotic figures were examined by staining the nuclear DNA with YOYO-1 iodide. Myocytes were distinguished from stromal cells by using antibodies reacting with a-sarcomeric actin. Results: Comparing with control hearts, the myocytic changes after cardiac transplantation are characterized by: 1) a decrease in mononucleated myocytes and an increase in binucleated and multinucleated myocytes; 2) a decrease in diploid myocytic nuclei and a distinct augmentation of intermediate ploidies; 3) an increase in myocytic nuclei in DNA ploidies higher than 4c; 4) a marked augmentation of percentage of apoptotic myocytes and 5) an increased frequency of nuclear mitosis of myocytes; this fact appears as a declining phenomenon after six months of cardiac transplantation. Conclusion: After cardiac transplantation the DNA content of myocytes shows two completely different aspects: 1) a distinct increase in subdiploidy and intermediate ploidies related to myocyte injury induced by apoptosis and necrosis; 2) an increase in multinucleation, polyploidization and mitotic proliferation. Both myocyte growth and myocyte injury alter the function of the allograft and contribute to adaptation or failure of the graft. Furthermore, a relevant difference of age between the recipient and the donor may lead to a more marked myocyte damage and a lower myocyte growth. This tendency provides an evidence that age matching could be an important aspect in selecting the donor for the recipient.

Journal Article↗

Detection of compact low-chromatin areas in cell nuclei images.

When analysing some DNA stained human cell nuclei using a light microscope or an quantitative image cytometer, compact low-chromatin areas (CLCA) can be observed. We are still not certain about the meaning and source of this phenomena. To enable the detection of CLCA by an image cytometer, a special image processing algorithm has to be developed and new nuclear cell features have to be designed. The presented image processing algorithm automatically detects CLCA in nuclei cell images taken from different tissues. The algorithm is composed of many basic image processing operations. The sequence of operations is determined by a priory knowledge about the properties of CLCA as a set of heuristic roles. The calculated CLCA features are CLCA area, perimeter, average intensity, compactnes and edge sharpness. The matching of the automatic CLCA detection and manual detection (performed by a biologist) was tested using 1400 cell nuclei. The results show a 83.4% match for the nuclei without CLCA and a 93.8% match for the cells with CLCA.

Algorithms↗

[In Process Citation]

OBJECTIVE: The objective of the study was to analyse the prognostic impact of several criteria for median survival and recurrence free interval in patients with uterine sarcomas. Factors included to our analysis were the staging according to FIGO, DNA content (ploidy), mitotic index, histology, the kind of primary therapy, grading and menopausal status. MATERIAL AND METHODS: Retrospectively, clinical data of 78 patients (41 leimyosarcomas, 23 carcinosarcomas = homologous malignant mixed Mullerian tumors, 14 endometrial stromal sarcomas--10 low grade, 4 high grade) were analysed. Additionally, in 36 of the cases mitoses were counted. Furthermore, DNA ploidy was determined using image cytometry on paraffine sections stained according to the Feulgen method. Receptor status was determined using immunohistochemical staining. Two and five year survival rates were 22% and 15%. There were 21 cases with local relapse and 27 cases with metastasis. RESULTS: The staging according to FIGO was the main prognostic factor, significantly influencing median survival time (p = 0.001) as well as the recurrence free interval (p = 0.03). There was a significant difference between median survival time compared to mitotic index (p = 0.014) and DNA ploidy (p = 0.02). A mitotic index < 10/10 HPF and diploidy were related with a better prognosis. Receptor status did not have an impact on median survival time. CONCLUSIONS: As our results suggest, DNA ploidy and mitotic index are likely to provide additional information for prognosis in patients with uterine sarcomas and could be used as criteria for selecting patients for adjuvant therapy.

Journal Article↗

Effects of cyclosporin A on contractile activity and cytoskeleton in chick embryo cardiomyocytes.

The effects of cyclosporin A (CsA), a clinically used immunosupressive drug, on contractile activity of chick cardiomyocytes grown as small aggregates or explants suspended on a network of elastic glass fibres or cultured in a monolayer were analysed in vitro with computer-aided image cytometry methods. At therapeutic concentrations (200-1500 ng/mL), CsA induced changes in the frequency and amplitude of the beating activity of cardiomyocytes 15 min after application. Longer treatment of cardiomyocytes, for 20-24 h, additionally induced changes in their shape and cytoskeleton organization (F-actin and alpha-actinin distribution). These results indicate that CsA is able to affect directly the contractile activity, morphology, and cytoskeleton architecture of heart cells.

Animals↗

Improved correction of quantitative nuclear DNA (ploidy) measurements in tissue sections.

OBJECTIVE: To evaluate, using a computer model, the advantages for ploidy measurements of selecting only center-containing sections of nuclei in ultrathin, very thick or relatively thick tissue sections. STUDY DESIGN: The computed corpuscle sectioning program was run on a personal computer. Its synthetic data were corrected by a variety of correction algorithms. RESULTS: When only center-containing sections of nuclei were selected in ultrathin sections, spherical nuclei could be corrected perfectly, and mildly prolate ellipsoidal nuclei with a selection bias in favor of elliptical nuclear section profiles could be corrected with high fidelity. Ultrathin sections most faithfully represented the true height of the peak of highest ploidy and showed better peak discrimination than other choices of section thickness, but small sample size, wavy sections, markedly inhomogeneous intranuclear DNA distribution and oblate ellipsoidal nuclei represented significant limitations of this approach. As nuclear prolation increased, peak definition worsened, and the peak of highest ploidy was falsely shortened. Results were unaffected by errors in the estimation of section thickness when an internal diploid standard was used. The effect of variable internuclear DNA concentration in mildly or moderately prolate ellipsoidal nuclei was nil. The choice of correction algorithm was unimportant, except that the reference curve method was better able to analyze oblate ellipsoidal nuclei, wavy sections and nuclei with inhomogeneous intranuclear DNA, and provided superior insight into nuclear and section parameters. Thick and very thick sections did not require correction and, unlike ultrathin sections, were immune to markedly inhomogeneous intranuclear DNA distribution, to nonspherical nuclear shape and to focal variation in section thickness (waviness), but (in relatively thick more than in very thick sections) the height of the peak of highest ploidy was falsely shortened, often markedly, and peak definition was worse. CONCLUSION: Choice of section thickness and selection of only center-containing nuclear sections for analysis with a bias in favor of elliptical nuclear section profiles in ultrathin sections are very important for optimal results; the choice of correction algorithm is less important.

Algorithms↗

Chromatin texture in high grade prostatic intraepithelial neoplasia and early invasive carcinoma.

OBJECTIVE: To evaluate individual nuclei from high grade prostatic intraepithelial neoplasia (PIN) lesions with early invasive carcinoma foci in the area of microinvasion and in the gland in which the microinvasion originated. STUDY DESIGN: High-resolution, digitized images of nuclei from defined locations were recorded and segmented, and karyometric variables were computed. These included a set of 93 features, which form a nuclear signature characterizing the spatial and statistical distribution of the nuclear chromatin. Nuclei in the glandular epithelium were recorded sequentially, along the basal cell layer, at increasing distances from the point of microinvasion and by random selection in the region of microinvasion. RESULTS: At a distance > 60 nuclear locations from the point of microinvasion, the nuclear signatures corresponded to those seen in high grade PIN. Between 40 and 20 nuclear locations removed from the microinvasion focus the signatures began to change gradually until at a distance of 15-5 locations they strongly resembled the signatures seen in adenocarcinoma. The total optical density decreased to values seen in adenocarcinoma, and the nuclear chromatin had finer granularity. While nuclei in high grade PIN followed a widely dispersed total optical density distribution suggestive of wide-ranging aneuploidy, the nuclei in the region of microinvasion exhibited a less dispersed and bimodal total optical density distribution. CONCLUSION: The chromatin texture signatures showed a clear trend: there was an obvious attenuation as the measured nuclei approached the microinvasion area. The decrease in total optical density at the microinvasion might suggest the emergence of one or two clones that can be responsible for the invasive phenotype.

Adenocarcinoma↗

Quantitative oral exfoliative cytology. Effect of alcohol on normal buccal mucosa.

OBJECTIVE: To assess the effect of chronic alcohol intake on the DNA distribution and cell area of normal oral mucosal cells. STUDY DESIGN: Smears were taken from clinically normal buccal mucosa of 50 patients attending an alcohol-problem service (i.e., chronic alcohol use) and average alcohol units per week recorded. DNA distribution histograms and total cell area values were then compared to those obtained from smears taken from a control group (which included social drinkers) of patients attending for routine dental treatment. Nuclear DNA content was assessed on 100 randomly selected, Feulgen-stained nuclei using a Seescan TV image analysis system, and total cell area was assessed on 50 Papanicolaou-stained cells using the Vids V image analysis system. RESULTS: The DNA distribution histograms were essentially diploid in appearance for the alcohol group, although there was an increase in nuclear DNA content in the occasional nucleus. A highly significant reduction in total cell area was found for the alcohol group when compared to the controls. CONCLUSION: The chronic ingestion of alcohol is associated with a reduction in total cell area but appears to have little effect on nuclear DNA content. Our previous research using the same technique showed that oral cancers are frequently nondiploid. Thus, a nondiploid DNA distribution histogram for smears taken from a clinically suspicious lesion in someone who consumes excessive amounts of alcohol is unlikely to be due to alcohol use alone and should indicate biopsy.

Adult↗

Use of three-dimensional microscopy with multiple oblique illumination in evaluating cytopathology preparations.

OBJECTIVE: To analyze whether the use of multiple oblique illumination (MOI), in contrast to axial illumination (AI), improves imagery in bright field microscopy. Specimens containing thick cell clusters, such as cervical Pap smears, are often misread because of out-of-focus cell clusters. We hypothesized that visualization of these specimens with MOI would enhance this information as compared with AI. STUDY DESIGN: Micrometer images and Pap smears were analyzed in focus, and 8 and 40 microns out of focus by MOI and AI. All fields were captured to a remote computer, histograms were constructed, and mean light intensity was calculated. Mathematical formulation was used to define the histogram distribution of the micrometer images. Statistical analysis was performed using one-way ANOVA and Newman-Keuls test. RESULTS: K(focus) was improved (P < .01) and at 20 and 40 microns out of focus, mean light intensities were greater (P < .003, P < .05, respectively) with MOI as compared to AI. CONCLUSION: MOI improves out-of-focus information by increasing light penetration through the specimen and enhancing contrast and resolution.

Female↗

Quantitative immunoprofiles of breast cancer performed by image analysis.

OBJECTIVE: To determine the biopathologic profiles of breast cancer for greater knowledge of tumor natural history and clinical outcome. STUDY DESIGN: In 99 in situ (ISC) and 2718 infiltrating breast carcinomas (IC), biologic markers (estrogen receptor [ER], progesterone receptor [PR] proliferation index, cerbB-2/NEU, p53, bcl-2 and DNA ploidy) were evaluated with an image analysis system (CAS 200/486). In 105 mixed invasive cancers with size < or = 1 cm, a separate analysis of in situ (ISCm) and invasive component (ICm) was obtained. A clinical study of 836 invasive breast cancers was performed. RESULTS: Different biophenotypes were obtained: among ISCs, cribriform type exhibited biologic behavior similar to that of normal breast tissue (ER+, PR+, proliferation index [PI] low, NEU-, p53-, bcl-2+) the opposite profile was displayed by comedo type, and intermediate phenotypes were observed in noncomedo and lobular types. Comparing ISC and ISCm, PI and p53 expression had the highest levels in ISCm with respect to other groups. NEU overexpression exhibited a decreasing value from ICm to IC. Younger women (< or = 40 years) with IC demonstrated a worse biologic profile (high PI, p53+, ER- and size > 2 cm). In multivariate analysis, PI and NEU in node-negative patients, and NEU, PR and size in node-positive ones emerged as prognostic parameters. CONCLUSION: The results underline the importance of the quantitative biologic profile for defining tumor behavior and patient management.

Adult↗

Image analysis of p53 and cyclin D1 expression in premalignant lesions of the oral mucosa.

OBJECTIVE: The expression of p53 and cyclin D1 proteins was analyzed by image analysis in oral premalignant lesions and normal oral mucosa. STUDY DESIGN: Punch biopsies from the normal oral mucosa were obtained from 20 normal donors and 41 patients with oral dysplastic leukoplakias. After controlled formaldehyde fixation and paraffin embedding, immunohistochemistry was used to detect cyclin D1 and p53. Image analysis was performed using stain intensity levels established by determining color thresholds (nuclear score) in the basal and parabasal layers. RESULTS: Analysis of sections showed a similar pattern: only two normal donors had a few intensely positive p53 cells in the basal layer of the floor of the mouth and the tongue epithelia. Similarly, only three donors had intensely positive cyclin D1 cells in the normal epithelia of the same sites. Most cells fell in the range of negative or marginal stain (lower quartiles or terciles of nuclear score). These data on normal mucosa were compared with low grade oral leukoplakias (LGD) with mild to moderate dysplasia and with high grade leukoplakias (HGD) with severe dysplasia. Both markers were differentially expressed in precursor lesions versus normal epithelia. Statistical analysis of our data shows that the intensity of the immunohistochemical stain, as reflected in the nuclear scores of p53, is a reliable parameter that can differentiate between LGD and HGD of the oral mucosa. This was especially true when higher nuclear scores were compared. In contrast, low nuclear scores are more effective in differentiating normal epithelia from dysplastic epithelia. Although cyclin D1 immunohistochemistry does not stain as intensely as p53 stain, similar conclusions can be derived from those data. CONCLUSION: Image analysis of these two markers proved useful in distinguishing normal oral epithelia from low grade and high grade leukoplakias. With further developments in this field it is hoped that image analysis procedures could be used in different types of studies in which variations of protein expression in tissue sections could have prognostic implications or could be useful to determine subtle effects of curative or preventive treatment.

Adolescent↗

Three-dimensional image analysis of hepatic restructuring in liver cirrhosis.

OBJECTIVE: To investigate the three-dimensional structure, including the angioarchitecture, of the cirrhotic liver and clarify morphogenesis of the cirrhotic nodule. STUDY DESIGN: The three-dimensional liver structure of nontumor areas in two partially hepatectomized cases of hepatitis C virus-positive liver cirrhosis with hepatocellular carcinoma was examined by computerized reconstruction from serial tissue sections. RESULTS: Our image analysis revealed the following: (1) The parenchyma consisted of two kinds of cirrhotic nodules. The first was the nodule centrifugally formed around the portal veins, and their flows drained into the hepatic veins inside and around the nodule. The second was the nodule derived from the first. The latter was divided into the former by bridging fibrosis-induced intranodular septation. (2) The stroma consisted of the newly formed fibrovascular tissue--i.e., the septum and intranodular inflow and outflow vascular systems and the preexisting one. CONCLUSION: Our computerized reconstruction suggested, from an angioarchitectural point of view, that the first and second kind of cirrhotic nodule might be named the stable and the unstable nodule, respectively, and that the first kind of cirrhotic nodule could be derived from the regenerative nodule appearing in the course of chronic hepatitis.

Carcinoma, Hepatocellular↗

Mean weighted nuclear volumes in colonic adenocarcinomas. Evaluation of prognostic significance.

OBJECTIVE: To investigate the correlation of stereologically estimated mean weighted nuclear volumes of tumor cell nuclei (MWNV) with other prognostic factors and survival in colonic adenocarcinomas. STUDY DESIGN: The study group consisted of 42 patients with colonic adenocarcinomas who were treated by a standardized protocol and had a mean follow-up of 89 months. The point sample intercepts method was used to estimate MWNV. In addition, immunohistochemical expression of p53 and Ki-67 was evaluated semiquantitatively. RESULTS: The calculated nuclear volumes correlated significantly with histologic grading and with the extent of tumor progression (organ confined/nonorgan confined). There was no correlation with p53 expression or proliferative activity determined by MIB-1 positivity. No correlation could be demonstrated with sex, age or clinical outcome of the patients. CONCLUSION: Assessment of MWNV in colonic adenocarcinomas does not provide additional information concerning the clinical course.

Adenocarcinoma↗

Learning vector quantizer in the investigation of thyroid lesions.

OBJECTIVE: To investigate the capability of the learning vector quantizer (LVQ) in the discrimination of benign from malignant thyroid lesions. STUDY DESIGN: The study was performed on May-Grünwald-Giemsa-stained smears taken by fine needle aspiration (FNA). Using a custom image analysis system, 25 features that describe the size, shape and texture of approximately 100 nuclei were measured from each case. Statistical features were extracted from each case, and a linear regression analysis was performed to detect the statistically significant features. The cases were distributed by category, as follows: 100 cases of goiter and follicular adenomas, 11 cases of follicular carcinoma, 35 cases of papillary carcinoma, 24 cases of oncocytic adenoma, 8 cases of oncocytic carcinoma and 20 cases of Hashimoto thyroiditis. About 30% of the cases from each class were used for training two LVQ classifiers. The remaining 139 cases, out of a total of 198, were used as the test set. A classifier was used to discriminate into four classes and a second into two classes. RESULTS: The application of LVQ neural networks allows good discrimination between benign and malignant lesions (O.A. = 97.8). However, reliable discrimination of the cytologic types of the lesions was not obtained. CONCLUSION: These results indicate that the use of neural networks combined with image morphometry may offer useful information on the potential for malignancy of thyroid lesions and may improve the diagnostic accuracy of FNA of the thyroid gland, especially in cases of follicular neoplasms classified as suspicious for malignancy and in cases of oncocytic tumors.

Adenoma↗

Three-dimensional image analysis of the hepatic restructuring process in chronic active hepatitis.

OBJECTIVE: To elucidate the process of hepatic restructuring in the course of chronic hepatitis from a morphologic viewpoint. STUDY DESIGN: The three-dimensional (3-D) liver structure was investigated by computer-aided reconstruction in five cases (one autopsy and four surgical cases) of chronic active hepatitis (type C), including early to late stages of restructuring. RESULTS: Our 3-D reconstruction revealed the following. At an early stage, portal and periportal inflammation and fibrosis widened the portal tracts, giving rise to the formation of portal-to-portal and portal-to-hepatic venous connections, although most central veins were still located at an almost normal site in the hepatic lobules. In a middle stage, bridging fibrosis developed to create a network of interstitium where the central veins were rather decreased in number, with regenerative nodules multiplying in the parenchyma. At the late stage, the lobular structure was destroyed, and the parenchyma consisted uniformly of regenerative nodules, with remaining but rearranged lobules among them. CONCLUSION: The above changes of liver structure suggest that in cirrhogenesis from chronic hepatitis, a combination of nodular regeneration and formation of an interstitial network come to replace the normal lobular structure, hastening the development of liver cirrhosis.

Adult↗

Lack of correlation between morphometric analysis and presence of microsatellite alterations in proliferative ductal lesions of the breast.

OBJECTIVE: To apply morphometric studies using an image analyzer to a previously reported group of proliferative duct lesions and to compare results to see if there was a correlation between nuclear size range and monoclonality. STUDY DESIGN: Fifteen proliferative lesions of the breast from 12 subjects who had no history of breast malignancy were retrieved from archival pathology specimens. Evidence of monoclonality was studied using a panel of polymerase chain reaction primers to examine microsatellite alterations on microdissected paraffin-embedded specimens. Variation in nuclear size was studied using an image analyzer. RESULTS: Of six proliferative breast lesions with demonstrated genetic instability, three showed cytologic evidence of uniformity in nuclear size, a cytologic feature of malignancy. One of the six, which showed microsatellite alterations at three loci, demonstrated a wide range of nuclear size variation. Of the eight proliferative lesions that showed no genetic instability, three showed very uniform nuclear size, and five showed significant variations in nuclear size. One lesion, which fell into the "uncertain but probably genetic instable" category, showed diverse nuclear size ranges. CONCLUSION: This study demonstrated that there is no correlation between monoclonality and monomorphic cell cytology of histologic proliferative breast lesions.

Breast↗

Image morphometric nuclear grading of intraepithelial neoplastic lesions with applications to cancer chemoprevention trials.

A new image morphometric method of nuclear grading is described and assessed in the context of the evaluation of histological samples from ductal carcinoma in situ of the breast and cervical intraepithelial neoplasia. The method results in a continuous scaled variable, or nuclear grading scale, expressed in SD units from measured normal nuclei from breast or cervix. For a given histological preinvasive neoplastic lesion, the mean nuclear grade of measured nuclei was shown to be analogous to the histopathological nuclear grade of the same lesion assigned subjectively by the pathologist. In a chemoprevention trial of the effect of difluoromethylornithine given for 1 month to subjects with cervical intraepithelial neoplasia grade 3, pathologists could see no difference in 14 histological sections taken before and after difluoromethylornithine treatment. However, the image morphometric method detected a systematic effect of lowered mean nuclear grade and a decrease in the variability of nuclear grade expression. Twelve of 14 samples showed a lower posttreatment mean nuclear grade (P<0.05), and 13 of the 14 samples showed a decrease in the SD of their nuclear grade distributions (P<0.01). This study demonstrates the use of image morphometric nuclear grading in a chemoprevention setting. It may be very useful in supplementing the pathologist's histopathological grading by providing objective, quantitative assessments.

Antineoplastic Agents↗

Conceptual comparison of two computer models of corpuscle sectioning and of two algorithms for correction of ploidy measurements in tissue sections.

OBJECTIVE: To compare two computer models of corpuscle sectioning and two algorithms for correction of ploidy measurements in tissue sections. STUDY DESIGN: Two models of corpuscle sectioning (the computed corpuscle sectioning program [CCSP] [Analyt Quant Cytol Histol 1997;19:376-386] and the ellipsoid sectioning program [ESP]) were run on a personal computer to generate synthetic corpuscle section data that model the sectioned nuclei in a tissue section. These synthetic data were analyzed by two algorithms for correction of ploidy measurements in tissue sections: the reference curve method (RCM) (Analyt Quant Cytol Histol 1997;19:376-386) and the method of McCready and Papadimitriou (MMP) (Analyt Quant Cytol 1983;5:117-123) for a variety of choices of section thickness and of nuclear section profile selection criteria. RESULTS: Previous recommendations (Analyt Quant Cytol Histol 1999;21:103-112) for optimization of ploidy analysis in tissue sections (selection of only center-containing sections of nuclei in ultrathin sections with a selection bias in favor of elliptical nuclear section profiles) are valid regardless of which corpuscle sectioning model and correction algorithm are employed. Perimeter correction may be desirable or necessary in some cases. The RCM has very significant advantages over the MMP, and the CCSP is more applicable to actual ploidy analysis than is the ESP. CONCLUSION: The RCM always should be used to correct ploidy measurements in tissue sections. The MMP should not be used as the sole method but, when used, should be used with and interpreted in the context of the RCM.

Algorithms↗

Value of morphometric nuclear image analysis using the Feulgen reaction in renal cell carcinoma.

OBJECTIVE: To evaluate retrospectively the ability of morphometric nuclear image analysis to predict survival in patients with renal cell carcinoma. STUDY DESIGN: The subjects were 40 patients with previously untreated renal cell carcinoma. Pathologic stage was determined using Robson's stage system. Nuclear grade was assigned according to the criteria of Fuhrman et al. We used the Feulgen staining technique, which has been widely used for the histochemical assessment of nuclear DNA content. A minimum of 300 nuclei were analyzed from each subject. Five variables in morphometric nuclear image analysis were measured: nuclear area, nuclear perimeter, nuclear ellipticity, nuclear regularity and DNA content. Cox's proportional hazard model was applied to identify prognostic usefulness with respect to survival time. RESULTS: All nuclear morphometric variables but nuclear regularity correlated with tumor grade. According to univariate survival analyses, Robson stage and nuclear ellipticity revealed a prognosis on survival with statistical significance. After adjustments for age and sex, nuclear ellipticity remained the only significant prognostic factor related to survival (P < .01). The survival rates were relatively high for patients with nuclear ellipticity > 773 as compared to those with nuclear ellipticity < 773 (P < .05). CONCLUSION: These findings indicate that morphometric nuclear image analysis using the Feulgen reaction is a reliable and efficient technique and that nuclear ellipticity is the most discriminating morphometric variable for predicting the prognosis of renal cell carcinoma patients.

Adult↗