Karyometric features in nuclei near colonic adenocarcinoma. Statistical analysis.

The normal mucosa adjacent to colonic adenocarcinoma (marginal or transitional mucosa) has been shown to have subtle alterations of architecture, surface glycoproteins and proliferative activity. To evaluate possible changes in nuclear configurations in this marginal mucosa, a large set of cytometric features was evaluated using a computer-assisted video analysis system. Preliminary statistical analysis of the measurements identified six nuclear features useful for discriminating marginal mucosa nuclei from normal (control) mucosa nuclei: total optical density (OD), nuclear area, chromatin texture (from gray value cooccurrence matrix), chromatin coarseness, average OD of nuclear staining and peripheral tendency of the chromatin in the nucleus. An analysis of variance revealed that both patient-to-patient and gland-to-gland variation would limit the usefulness of any one feature as a screening tool. As a group, however, these six features should be investigated further as markers of preneoplastic changes in histologically normal-appearing mucosa.     

胰腺内镜超声图像纹理特征提取与分类研究

提出了胰腺内镜超声图像的纹理特征提取与分类方法,可应用于胰腺癌内镜超声图像的计算机辅助诊断。对胰腺内镜超声图像采用数字图像处理算法提取9大类共69个纹理特征。使用类间距作为可分性判据,实现特征的初步筛选,之后使用顺序前进搜索算法进一步筛选特征,并由支撑向量机实现分类。对216例病例随机选取训练集和测试集,通过多次随机实验表明。本文提出的算法实现了较高的分类准确率,为胰腺癌的临床诊断提供有价值的参考意见。     

The study of chromatin and chromosome structure on preparations of interphase nucleus derivatives resulting from nuclear wall removal.III Structural heterogeneity of chromatin and argyrophilic zone of the nucleolus in stretched membrane-free nuclei and chromatin bodies from human peripheral lymphocytes

Viewed by light microscopy, the majority of lymphocytes in smears of human peripheral blood display a deep staining (with any chromatin- or DNA-specific dye) of the nucleus consisting of densely aggregated chromatin in addition to one or several small nucleoli with a dot- or spot-like argyrophilic zone. Amembraneous nuclei and "free chromatin" structures were isolated from intact lymphocytes gently treated with Triton X-100. Surface stretching of both these nuclei and structures, shortly fixed in methanol--glacial acetic acid (3:1), resulted in spatial separation of thin and thick chromatin or argyrophilic fibres, nucleoli, intranuclear bodies, polymorphous aggregations of chromatin or argyrophilic fibres and incidentally observed splitted or beaded thick chromatin fibres and the chromocenter. The light microscopic pattern of chromatin fibres of stretched amembraneous nuclei, isolated from peripheral lymphocytes, well compares with that of deconvolved images of intact lymphocyte nucleus obtained with optical tomography.     

Development of the sperm head in the caddis fly Potamophylax rotundipennis (Insecta,Trichoptera)

The restructuring of the sperm head has been examined in a caddis fly, Potamophylax rotundipennis (Limnephilidae), using light and electron microscopy. The roughly spherical nuclei of young spermatids are transformed into needle-shaped elements in advanced spermatids. During this process, the nuclei transiently become sickle-shaped. Prominent structural changes occur within the nucleus during spermiogenesis. The chromatin of spherical and slightly elongated nuclei has an amorphous appearance, then coarse granules become apparent, chromatin threads are visible in fully elongated nuclei and finally lamellar elements appear. During the changes in chromatin texture, a dense layer, the chromatin rim, develops transiently. This feature of the chromatin surface is interpreted as the structural expression of exchanges between nucleus and cytoplasm. A microtubular manchette is formed at the cytoplasmic face of the nuclear envelope. Whereas the manchette covers the full perimeter of the nucleus in early stages of elongation, gaps in the palisade of microtubules appear before the nuclear diameter decreases and needle-shaped nuclei develop. It is possible that the intermittent deployment of manchette microtubules is involved in reducing the nuclear diameter towards the end of nuclear elongation. The delayed detachment of the chromatin from the posterior pole of the nucleus, observed at the onset of nuclear clongation, points to local modifications of the nuclear envelope responsible for the connection of the centriole adjunct and the flagellum with the posterior pole of the nucleus.     

Chromatin texture from hematoxylin stained thyroid lesions.

Quantitative aspects of cytology and histology should be considered in diagnostic standardisation processes. The present paper summarises the cytological differences detected in 75 thyroid lesions using a computerized textural analysis. Cells stained with progressive hematoxylin and taken from paraffin blocks were overlaid with the extracted texture. This technique was based on the lineal detection of a grey level gradient of the common logarithm of the integrated optical density (IOD) of each nucleus. Diffuse and nodular goiters (36 cases) were demonstrated to be composed of small cells containing high density texture that, on microscopical visual inspection, gave a "salt and pepper" appearance. The adenomatous goiters (2 cases) and adenomas (26 cases) were composed of low texture cells with a visual "blurry or smudgy" chromatin, while the atypical adenomas with capsular invasion (4 cases) were characterised by a "woodworm" nuclear appearance that produced the highest texture of the series. Finally, encapsulated folliculo-papillary carcinomas (3 cases) were composed of large clear nuclei with high IOD, low texture, and scattered lines that resulted in an "empty grape skin" aspect. Our findings seam to confirm the suitability of computerized textural techniques that aid in recognizing cell microscopic features objectively. The one used in the present work, based on a mathematical function of the DNA content of each individual nucleus (IOD), fulfills all microscopy detection criteria.     

Markers for malignancy in the nuclear texture of histologically normal tissue from patients with thyroid tumors

The chromatin of nuclei from histologically normal-appearing glands in tissue sections from patients with follicular adenoma, papillary carcinoma and follicular carcinoma of the thyroid exhibited changes that were statistically significant when compared to the nuclear chromatin in tissue sections of thyroid from normal individuals. Certain chromatin texture features assumed values approximating those found in tumor cell nuclei. Staining was affected only slightly, and morphometric features, such as nuclear area, roundness and ellipticity, were statistically not different from those in normal controls. In patients with Hürthle-cell tumors, such marker features could not be found. Results from measurements on 30 patients (approximately 1,000 nuclei) are reported.     

The use of fractal features from the periphery of cell nuclei as a classification tool.

A polygonization-based method is used to estimate the fractal dimension and several new scalar lacunarity features from digitized transmission electron micrographs (TEM) of mouse liver cell nuclei. The fractal features have been estimated in different segments of 1D curves obtained by scanning the 2D cell nuclei in a spiral-like fashion called "peel-off scanning". This is a venue to separate estimates of fractal features in the center and periphery of a cell nucleus. Our aim was to see if a small set of fractal features could discriminate between samples from normal liver, hyperplastic nodules and hepatocellular carcinomas. The Bhattacharyya distance was used to evaluate the features. Bayesian classification with pooled co-variance matrix and equal prior probabilities was used as the rule for classification. Several single fractal features estimated from the periphery of the cell nuclei discriminated samples from the hyperplastic nodules and hepatocellular carcinomas from normal ones. The outer 25-30% of the cell nuclei contained important texture information about the differences between the classes. The polygonization-based method was also used as an analysis tool to relate the differences between the classes to differences in the chromatin structure.     

Surface Roughness Detection of Arteries via Texture Analysis of Ultrasound Images for Early Diagnosis of Atherosclerosis

There is a strong research interest in identifying the surface roughness of the carotid arterial inner wall via texture analysis for early diagnosis of atherosclerosis. The purpose of this study is to assess the efficacy of texture analysis methods for identifying arterial roughness in the early stage of atherosclerosis. Ultrasound images of common carotid arteries of 15 normal mice fed a normal diet and 28 apoE^−/− mice fed a high-fat diet were recorded by a high-frequency ultrasound system (Vevo 2100, frequency: 40 MHz). Six different texture feature sets were extracted based on the following methods: first-order statistics, fractal dimension texture analysis, spatial gray level dependence matrix, gray level difference statistics, the neighborhood gray tone difference matrix, and the statistical feature matrix. Statistical analysis indicates that 11 of 19 texture features can be used to distinguish between normal and abnormal groups (p<0.05). When the 11 optimal features were used as inputs to a support vector machine classifier, we achieved over 89% accuracy, 87% sensitivity and 93% specificity. The accuracy, sensitivity and specificity for the k-nearest neighbor classifier were 73%, 75% and 70%, respectively. The results show that it is feasible to identify arterial surface roughness based on texture features extracted from ultrasound images of the carotid arterial wall. This method is shown to be useful for early detection and diagnosis of atherosclerosis.     

Distinguishing cortical adrenal gland adenomas from carcinomas by their quantitative nuclear features

OBJECTIVE: To explore data from a set of cases of adrenal cortical adenomas with different endocrine syndromes and carcinomas to determine whether quantitative image analysis of nuclear features might be used to separate the groups. STUDY DESIGN: Fifteen adrenal cortical tumors in which clinical information and optimally preserved, paraffin-embedded tissue were available were used. There were 10 adenomas and 5 carcinomas. Among the adenomas, five were associated with primary hyperaldosteronism (Conn's syndrome) and five with Cushing's syndrome. Five-micrometer-thick sections were stained with hematoxylin and eosin. In each case 50 nuclei were measured, and a number of morphometric and densitometric features were extracted. The data were subjected to multivariate analysis. RESULTS: Quantitative analysis showed that nuclei from adrenal carcinomas are larger than those from adenomas. Total optical density (OD) had a near-diploid distribution in the adenomas, while it was clearly aneuploid in the carcinomas. The pixel OD histograms were almost identical for all groups. Differences in nuclear chromatin texture were found between adenomas and carcinomas and also between the two adenoma categories. Multivariate analysis showed good discrimination between carcinomas and adenomas (Wilks lambda = .628, P < .0001) and between adenomas. However, based on Bayesian decision boundaries, 20-25% of carcinoma nuclei could be expected to be in the range of adenoma, and about 12% of Cushing's adenoma nuclei and 15% of Conn's adenoma nuclei would be classified as carcinoma. CONCLUSION: Computer-assisted analysis of nuclear characteristics proved useful in identifying and describing differences between groups of tumors arising in the adrenal cortex.     

Ultrastructural and cytochemical changes of the head components of human spermatids and spermatozoa

The ultrastructural study of chromatin condensation simultaneously with the evolution of the perinuclear organelles was conducted in the spermatids and epididymal and ejaculated spermatozoa of man with the aid of the “en bloc” alcoholic PTA staining and the EDTA regressive method. The round nuclei of young spermatids (steps 1, 2) were characterized by the persistence of nucleoli that were PTA positive, and the presence of a subacrosomal layer of well-stained peripheral chromatin. In the beginning of the phase of nuclear elongation (step 3), the central chromatin also became dense, like the peripheral chromatin, while the nuclear ring and the associated manchette and the two anlages of the postacrosomal dense lamina and the posterior ring appeared. During steps 4 and 5, the sliding of the nuclear ring and the manchette, the growth of the postacrosomal dense lamina, and the progression of the posterior ring towards the base of the nucleus were seen along with structural and cytochemical modifications of the chromatin. In the flattened nuclei of step 4 spermatids, coinciding with the loss of the nucleolar components, the chromatin achieved maximum compactness in the entire nucleus and was PTA positive. In the spermatids of step 5, the disappearance of peripheral dense chromatin and the specific staining of the chromatin granules marked the beginning of the second stage of transformation of the basic nucleo-proteins. The condensed nuclei of the mature spermatids were partially stained by PTA in step 6 and totally unstained in step 7. The PTA staining revealed the persistence of PTA-positive chromatin areas in the nuclei of certain spermatids otherwise mature. The morphological aspect of the chromatin then remained the same in the nuclei of epididymal and ejaculated spermatozoa. These observations suggest that in man, as in other mammals studied, new proteins accumulate in the elongating nuclei of spermatids and are replaced at the phase of maturation by sperm-specific nucleoproteins. The defects in condensation of the chromatin that occur during spermiogenesis could be related to the modalities of accumulation of intermediate nucleoproteins.     

Morphological changes of sperm nuclei during spermatogenesis in the brown alga Cystoseira hakodatensis (Fucales, Phaeophyceae)

Morphological changes and chromatin condensation of sperm nuclei were observed during spermatogenesis in the fucalean brown alga Cystoseira hakodatensis (Yendo) Fensholt. Ultrastructural studies have shown that the mature spermatozoid has an elongated and concave nucleus with condensed chromatin. The morphological changes and the chromatin condensation process during spermatogenesis was observed. Nuclear size decreased in two stages during spermatogenesis. During the first stage, spherical nuclei decreased in size as they were undergoing meiotic divisions and the subsequent mitoses within the antheridium. During the second stage, the morphological transformation from a spherical into an elongated nucleus occurred. Afterwards, chromatin condensed at the periphery in each nucleus, and chromatin‐free regions were observed in the center of the nucleus. These chromatin‐free regions in the center of nucleus were compressed by the peripheral chromatin‐condensed region. As the result, the elongated and concave nucleus of the mature sperm consisted of uniformly well‐condensed chromatin.     

Image analysis of mesothelioma. II. Discrimination of mesothelioma from metastatic serous ovarian adenocarcinoma

OBJECTIVE: To examine the utility of karyometric measurements in the differentiation of mesothelioma from metastatic serous ovarian adenocarcinoma. STUDY DESIGN: High-resolution images of 1,631 nuclei from 32 cases of mesothelioma and 742 nuclei from 15 cases of ovarian adenocarcinoma were recorded. A stepwise discriminant analysis and nonparametric classifier were applied. RESULTS: Nuclei from these two diagnostic categories appear very similar and occupy feature space with significant overlap. A nonparametric classification procedure provided acceptable correct classification. CONCLUSION: For certain regions in feature space, cases could be unequivocally classified.     

Hydrolysis profiles of formalin fixed paraffin-embedded tumors based on IOD (integrated optical density) and nuclear texture feature measurements.

The aim of the study was to determine optimal hydrolysis time for the Feulgen DNA staining of archival formalin fixed paraffin-embedded surgical samples, prepared as single cell suspensions for image cytometric measurements. The nuclear texture features along with the IOD (integrated optical density) of the tumor nuclei were analysed by an automated high resolution image cytometer as a function of duration of hydrolysis treatment (in 5 N HCl at room temperature). Tissue blocks of breast carcinoma, ovarian serous carcinoma, ovarian serous tumor of borderline malignancy and leiomyosarcoma were included in the study. IOD hydrolysis profiles showed plateau between 30 and 60 min in the breast carcinoma and leiomyosarcoma, and between 40 and 60 min in the ovarian serous carcinoma and ovarian serous tumor of borderline malignancy. Most of the nuclear texture features remained stable after 20 min of hydrolysis treatment. Our results indicate that the optimal hydrolysis time for IOD and for nuclear texture feature measurements, was between 40 and 60 min in the cell preparations from tissue blocks of three epithelial and one soft tissue tumor.     

Image preprocessing improves Fourier-based texture analysis of nuclear chromatin

OBJECTIVE: To investigate whether preprocessing of digitized images can improve the image analysis of chromatin of cytologic preparations using Fast Fourier Transformation (FFT). STUDY DESIGN: In a preprocessing step the nuclear borders of the segmented nuclei were smoothed, thus avoiding the Airy ring artifact. We tested this method, comparing the inertia values of digitalized cardiomyocyte nuclei of rats of different ages. Furthermore, we created in silicio nuclear images with chromatin alterations at or nearby the nuclear edge in order to investigate the robustness of our method. RESULTS: After preprocessing, the FFT-derived variable inertia discriminated significantly better the chromatin structure of the nuclei at different ages in every frequency range. The investigation on simulated nuclei revealed that within the frequency ranges from 1.8 microm to 0.72 microm smoothing of the borders does not interfere with the detection of chromatin changes at the nuclear border. CONCLUSION: Smoothing of borders in segmented images can improve the analysis of Fourier-derived variables of the chromatin texture.     

Distribution of DNase I-sensitive sites in simian virus 40 nucleoprotein complexes from disrupted virus particles.

Nucleoprotein complexes (core particles) released from simian virus 40 (SV40) virions were compared with similar complexes (SV40 chromatin) extracted from nuclei of infected cells. Core particles were sensitive to cleavage by DNase I at about the same enzyme concentration required to cleave SV40 chromatin. DNase I preferentially cleaved SV40 chromatin adjacent to the viral origin of replication; however, cleavage of core particles occurred with much less selectivity. The difference between these nucleoproteins was not due to a structural alteration induced by the virion disruption procedure, since SV40 chromatin retained its pattern of DNase I-sensitive sites when subjected top this treatment. On the other hand, core particles did not acquire the nuclease-sensitive feature typical of SV40 chromatin when they were exposed to infected cell nuclei and the Triton X-100-EDTA extraction procedure. Hence, the nuclease-sensitive feature was lost or altered during the normal process of virion assembly and maturation.     

Novel, contrast gradient-oriented, automated chromatin texture analysis. I. Feasibility study on nuclei from benign and malignant breast epithelial cell lines in fine needle aspirates.

Coarse granularity of nuclear chromatin texture is a prominent feature of most malignant cell lines. We have chosen the abrupt transition from eu- to heterochromatic foci (high contrast gradient [CG]) as a novel parameter for coarseness. This feature was quantified using automated image analysis of single nuclei in smears stained by the May-Grünwald-Giemsa technique. The principle of this approach consists of eliminating, with the help of subtraction between two image lowpass filters, the small grey level differences among pixels, so that only high CG values are retained on the digitized image. The sum of these distinctive microareas is then taken as a fraction of the area of the peripherally eroded nucleus, and this ratio is designated as contrast gradient index (CGI) per nucleus. This method was tested on fine needle aspirates from 11 patients with benign breast disease (BBD) and 14 with mammary carcinoma (CA). For each specimen, 60 nuclei were analyzed, with a measuring time per nucleus of about 1 min. A high significant distinction between epithelial cell populations in BBD and CA, respectively, was obtained by variance analysis of all CGIs per nucleus (p = 2 x 10(-18). The median and the mean values of CGI per specimen were the next best discriminators, followed by the modes and the standard deviation of CGI per specimen. The percentage of nuclei per specimen with CGI values of greater than 12 was also significantly greater in CA than in BBD.     

An ectopic copy of the Drosophila ftz associated SAR neither reorganizes local chromatin structure nor hinders elution of a chromatin fragment from isolated nuclei.

In vitro assays using detergent extracted nuclei allow the operational definition of SARs--specific sequences in the chromosome which are thought to interact with a structural matrix or scaffold. This interaction results in the formation of large stable protein-DNA complexes. We have used P-element transformation to introduce a characterized SAR into the Drosophila genome. The standard assay, which uses detergent extracted nuclei, shows that the ectopic SAR is indeed bound to the scaffold. However, in unextracted nuclei, a chromatin fragment containing the SAR sequence is eluted from the nucleus as readily as a fragment which lacks an SAR. Furthermore, an analysis of the accessibility of the neighbouring chromosomal restriction sites in unextracted nuclei indicates that the introduction of this ectopic SAR does not influence the local structure of chromatin. We conclude that the ectopic SAR site is not attached to a nuclear scaffold or matrix in vivo.