Chromatin Texture Analysis Research Articles

Can the analysis of chromatin texture and nuclear fractal dimensions serve as effective means to distinguish non-invasive follicular thyroid neoplasm with papillary-like nuclear features from other malignancies with follicular pattern in the thyroid?: a study

ABSTRACT Objective Thyroid carcinoma ranks as the 9th most prevalent global cancer, accounting for 586,202 cases and 43,636 deaths in 2020. Computerized image analysis, utilizing artificial intelligence algorithms, emerges as a potential tool for tumor evaluation. Aim This study aims to assess and compare chromatin textural characteristics and nuclear dimensions in follicular neoplasms through gray-level co-occurrence matrix (GLCM), fractal, and morphometric analysis. Method A retrospective cross-sectional study involving 115 thyroid malignancies, specifically 49 papillary thyroid carcinomas with follicular morphology, was conducted from July 2021 to July 2023. Ethical approval was obtained, and histopathological examination, along with image analysis, was performed using ImageJ software. Results A statistically significant difference was observed in contrast (2.426 (1.774–3.412) vs 2.664 (1.963–3.610), p = .002), correlation (1.202 (1.071–1.298) vs 0.892 (0.833–0.946), p = .01), and ASM (0.071 (0.090–0.131) vs 0.044 (0.019–0.102), p = .036) between NIFTP and IFVPTC. However, morphometric parameters did not yield statistically significant differences among histological variants Conclusion Computerized image analysis, though promising in subtype discrimination, requires further refinement and integration with traditional diagnostic parameters. The study suggests potential applications in scenarios where conventional histopathological assessment faces limitations due to limited tissue availability. Despite limitations such as a small sample size and a retrospective design, the findings contribute to understanding thyroid carcinoma characteristics and underscore the need for comprehensive evaluations integrating various diagnostic modalities.

Nuclear morphometry and chromatin texture changes in hepatocellular carcinoma samples may predict outcomes of\xa0liver transplanted patients

BackgroundNuclear changes are typical in the carcinogenesis of hepatocellular carcinoma (HCC). Morphometry and chromatin texture analysis are quantitative methods for their quantification. In this study, we analyzed nuclear morphometry and chromatin texture parameters in samples of hepatocellular carcinoma from liver transplant patients and their associations with clinicopathologic variables.MethodsSamples of HCC and adjacent tissue from 34 individuals were collected in tissue microarray blocks. Stained slides were microphotographed using an optical microscope and nuclear parameters analyzed in ImageJ (FracLac plug-in). ROC curve analysis was used to find accurate cut-offs for differentiation of neoplastic and non-neoplastic cells. The inter-rater agreement was also evaluated.ResultsNuclear morphometric and textural differences were observed between the samples of HCC and adjacent tissue of liver transplant patients. Lower mean gray value (p = 0.034) and Feret diameter (p = 0.024) were associated with higher Model for End-Stage Liver Disease (MELD) scores. Nuclei with larger area (p = 0.014) and larger Feret diameter (p = 0.035) were associated with lower survival. Lower aspect ratio was associated with HCC recurrence after the transplant (p = 0.048). The cut-off of 1.13 μm (p = < 0.001) for aspect ratio and cut-off of 21.15 μm (p = 0.038) for perimeter were established for the differentiation of neoplastic and non-neoplastic cells. The morphometric analysis was reproducible to area, circularity, Feret diameter, mean gray value and aspect ratio between observers (p = < 0.001).ConclusionsNuclear morphometric differences between the HCC and the adjacent tissue samples were associated with prognostic variables (MELD scores, recurrence and survival) and may predict liver transplant patients’ outcomes.

DNA Methylation Changes in Valproic Acid-Treated HeLa Cells as Assessed by Image Analysis, Immunofluorescence and Vibrational Microspectroscopy

Valproic acid (VPA), a well-known histone deacetylase inhibitor, has been reported to affect the DNA methylation status in addition to inducing histone hyperacetylation in several cell types. In HeLa cells, VPA promotes histone acetylation and chromatin remodeling. However, DNA demethylation was not checked in this cell model for standing effects longer than those provided by histone acetylation, which is a rapid and transient phenomenon. Demonstration of VPA-induced DNA demethylation in HeLa cells would contribute to understanding the effect of VPA on an aggressive tumor cell line. In the present work, DNA demethylation in VPA-treated HeLa cells was assessed by image analysis of chromatin texture, the abundance of 5-methylcytosine (5mC) immunofluorescence signals and Fourier transform-infrared (FT-IR) microspectroscopy centered on spectral regions related to the vibration of–CH3 groups. Image analysis indicated that increased chromatin unpacking promoted by a 4-h-treatment with 1.0 mM VPA persisted for 24 h in the absence of the drug, suggesting the occurrence of DNA demethylation that was confirmed by decreased 5mC immunofluorescence signals. FT-IR spectra of DNA samples from 1 mM or 20 mM VPA-treated cells subjected to a peak fitting analysis of the spectral window for–CH3 stretching vibrations showed decreased vibrations and energy of these groups as a function of the decreased abundance of 5mC induced by increased VPA concentrations. Only the 20 mM-VPA treatment caused an increase in the ratio of -CH3 bending vibrations evaluated at 1375 cm-1 in relation to in-plane vibrations of overall cytosines evaluated at 1492 cm-1. CH3 stretching vibrations showed to be more sensitive than–CH3 bending vibrations, as detected with FT-IR microspectroscopy, for studies aiming to associate vibrational spectroscopy and changes in DNA 5mC abundance.

Single Molecule Localization Microscopy of Mammalian Cell Nuclei on the Nanoscale.

Nuclear texture analysis is a well-established method of cellular pathology. It is hampered, however, by the limits of conventional light microscopy (ca. 200 nm). These limits have been overcome by a variety of super-resolution approaches. An especially promising approach to chromatin texture analysis is single molecule localization microscopy (SMLM) as it provides the highest resolution using fluorescent based methods. At the present state of the art, using fixed whole cell samples and standard DNA dyes, a structural resolution of chromatin in the 50–100 nm range is obtained using SMLM. We highlight how the combination of localization microscopy with standard fluorophores opens the avenue to a plethora of studies including the spatial distribution of DNA and associated proteins in eukaryotic cell nuclei with the potential to elucidate the functional organization of chromatin. These views are based on our experience as well as on recently published research in this field.

Macrophage-tumor cell fusions from peripheral blood of melanoma patients.

BackgroundWhile the morbidity and mortality from cancer are largely attributable to its metastatic dissemination, the integral features of the cascade are not well understood. The widely accepted hypothesis is that the primary tumor microenvironment induces the epithelial-to-mesenchymal transition in cancer cells, facilitating their escape into the bloodstream, possibly accompanied by cancer stem cells. An alternative theory for metastasis involves fusion of macrophages with tumor cells (MTFs). Here we culture and characterize apparent MTFs from blood of melanoma patients.MethodsWe isolated enriched CTC populations from peripheral blood samples from melanoma patients, and cultured them. We interrogated these cultured cells for characteristic BRAF mutations, and used confocal microscopy for immunophenotyping, motility, DNA content and chromatin texture analyses, and then conducted xenograft studies using nude mice.FindingsMorphologically, the cultured MTFs were generally large with many pseudopod extensions and lamellipodia. Ultrastructurally, the cultured MTFs appeared to be macrophages. They were rich in mitochondria and lysosomes, as well as apparent melanosomes. The cultured MTF populations were all heterogeneous with regard to DNA content, containing aneuploid and/or high-ploidy cells, and they typically showed large sheets (and/or clumps) of cytoplasmic chromatin. This cytoplasmic DNA was found within heterogeneously-sized autophagic vacuoles, which prominently contained chromatin and micronuclei. Cultured MTFs uniformly expressed pan-macrophage markers (CD14, CD68) and macrophage markers indicative of M2 polarization (CD163, CD204, CD206). They also expressed melanocyte-specific markers (ALCAM, MLANA), epithelial biomarkers (KRT, EpCAM), as well as the pro-carcinogenic cytokine MIF along with functionally related stem cell markers (CXCR4, CD44). MTF cultures from individual patients (5 of 8) contained melanoma-specific BRAF activating mutations. Chromatin texture analysis of deconvoluted images showed condensed DNA (DAPI-intense) regions similar to focal regions described in stem cell fusions. MTFs were readily apparent in vivo in all human melanomas examined, often exhibiting even higher DNA content than the cultured MTFs. When cultured MTFs were transplanted subcutaneously in nude mice, they disseminated and produced metastatic lesions at distant sites.Conclusions and HypothesisApparent MTFs are present in peripheral blood of patients with cutaneous melanomas, and they possess the ability to form metastatic lesions when transplanted into mice. We hypothesize that these MTFs arise at the periphery of primary tumors in vivo, that they readily enter the bloodstream and invade distant tissues, secreting cytokines (such as MIF) to prepare “niches” for colonization by metastasis initiating cells.

Evaluation of Quantitative Digital Pathology in the Assessment of Barrett Esophagus-Associated Dysplasia.

Barrett esophagus (BE) is a precursor lesion that confers an increased risk of esophageal adenocarcinoma. Two issues confront the diagnosis of patients with BE: (1) sampling error at the time of endoscopy and (2) variability among pathologists in grading dysplasia. The purpose of our study was to evaluate quantitative digital pathology (QDP) as a marker of dysplasia and stratification from low-grade to high-grade dysplasia to intramucosal adenocarcinoma in BE. Sixty-one esophageal biopsy specimens with BE were selected and divided into six groups according to the dysplasia grade. QDP image analysis was carried out by an in-house automated quantitative system on sections. The values of 110 nuclear features that analyze the morphology and chromatin texture were generated for each nucleus. A progressive correlation was found between nuclear morphometric features and chromatin features with BE dysplasia. The chromatin texture was the best discriminator of the class diagnosis. There was a significant difference between the chromatin features of isolated low-grade dysplasia vs low-grade dysplasia that was associated with higher grade lesions in other biopsy tissue fragments. QDP is a promising tool in the new era of digital pathology. Pending clinical validation studies, analysis of chromatin texture could contribute to the differential diagnosis of BE class and the detection of concomitant high-grade lesions if not sampled.

Image analysis of hyperchromatic crowded cell groups in SurePath cervical cytology

The discrimination of hyperchromatic crowded cell groups (HCCGs) in cervical cytology is a difficult and error-prone interpretive task. While the classic features of dyskaryosis are of undoubted value, the contribution of size, shape and colour intensity of HCCGs is less certain. This study employed morphometric analysis to determine whether HCCG area, shape and colour intensity are useful in categorising them. Seventy-five digital images from each of six categories of HCCG were subjected to image analysis. Ten variables relating to HCCG size, shape and colour intensity were assessed by discriminant function analysis. A further 28 cases were employed as a test set to determine the classification accuracy of the discriminant model. All samples were SurePath liquid-based cytology preparations. Nine of the 10 variables contributed significantly to the model (P<0.001) but no single variable had sufficient discriminative ability. Classification accuracy was highest for abnormal endocervical HCCGs and lowest for squamous metaplastic cells (64.0 vs. 17.3% correct classification rate). The accuracy of the model for distinguishing normal and abnormal HCCGs was 70.0%, which was significantly higher than chance (P<0.0001), but this reduced to 64.3% for the test cases, which was no better than chance (P>0.05). The area, shape and colour intensity of HCCGs, either alone or in combination, have little discriminative value. Practitioners and trainers should focus on the well-established features of dyskaryosis, such as chromatin pattern, nuclear membrane irregularities and group architecture. In terms of morphometric analysis, DNA ploidy and chromatin texture analysis may be more fruitful avenues of investigation.

Chromatin changes in papillary thyroid carcinomas may predict patient outcome

New insights in prognostic predictions are urgently needed for papillary thyroid carcinoma (PTC). The present study aimed to investigate whether computerized analysis of chromatin texture allows the identification of PTC patients with a poor prognosis. We randomnly selected paraffin-embedded blocks from surgical specimens of 103 classic cases of PTC. During follow-up, 68 of the patients were classified as free of disease, whereas 35 presented with recurrences. Characteristics of chromatin were obtained from digitized images of at least 100 randomly selected tumor nuclei per patient. An independent series of 30 goiters was used to validate our observations. Stage, age and distant metastases were found to serve as independent prognostic factors for survival. In addition, multivariate Cox regression confirmed variable cluster prominence as an independent prognostic factor. By comparing malignant and benign nodules, we found that the PTC lesions presented with higher nuclear perimeters, nuclear areas, Minkowski fractal dimensions, optical densities and nuclear longest chords. From our results we conclude that, in conjunction with clinical and histopathological data, morphometric data may provide relevant prognostic information in PTC patients.

자궁경부암 진단을 위한 3차원 세포핵 질감 특성값 유의성 평가에 관한 연구

본 연구의 목적은 세포핵의 3차원 염색질 질감 특성값이 암의 진행정도를 인식하는데 있어 유용한 특성값인지 평가하는데 있다. 특히, 제안한 방법이 악성이라고 진단된 세포진 도말 표본에서 정상으로 보이는 세포의 염색질 패턴에서의 미세한 차이를 인식할 수 있는지 살펴보고자 한다. 분류등급 정상(Normal), 저등급 편평 상피내 병변(LSIL, Low grade Squamous Intraepithelial Lesion), 고등급 편평 상피내 병변(HSIL, High grade Squamous Intraepithelial Lesion)에서 각각 100개씩의 세포 볼륨데이터로부터 3차원 GLCM(Gray Level Co occurrence Matrix)에 기반한 질감 특성값과 3차원 Wavelet 변환에 기반한 질감 특성값을 추출하고 분류기를 생성한 후 각 분류기에 대한 분류정확도를 비교하였으며, 2차원 세포진 영상에서의 세포핵 질감 특성값과 비교하기 위해 동일한 실험 볼륨데이터의 투영된 2차원 영상을 이용하여 같은 방법으로 2차원 세포핵 질감 특성값을 추출하고 분류기를 생성한 후 분류정확도를 비교하였다. 2차원 세포핵 질감 특성값과의 비교연구에서 3차원 세포핵 질감 특성값이 등급별 분류에 있어 보다 효율적인 것을 확인 할 수 있었으며 이는 3차원 염색질 질감 특성값이 자궁경부 세포의 정량화에 대한 정확성과 재현성을 개선할 수 있음을 의미한다. The aim of this study is to evaluate whether 3D nuclear chromatin texture features are significant in recognizing the progression of cervical cancer. In particular, we assessed that our method could detect subtle differences in the chromatin pattern of seemingly normal cells on specimens with malignancy. We extracted nuclear texture features based on 3D GLCM(Gray Level Co occurrence Matrix) and 3D Wavelet transform from 100 cell volume data for each group (Normal, LSIL and HSIL). To evaluate the feasibility of 3D chromatin texture analysis, we compared the correct classification rate for each of the classifiers using them. In addition to this, we compared the correct classification rates for the classifiers using the proposed 3D nuclear texture features and the 2D nuclear texture features which were extracted in the same way. The results showed that the classifier using the 3D nuclear texture features provided better results. This means our method could improve the accuracy and reproducibility of quantification of cervical cell.

Digital stain separation for histological images

It is often desirable to perform digital image analyses on sections prepared for human interpretation, e.g. nuclear chromatin texture analysis or three-dimensional reconstructions using sections requiring human delineation of structures of interest. Unfortunately such analyses are often more effective using stains with less complex contrast. Here an automated selective 'de-staining' method for digital images is presented. The method separates an image into its red, green and blue and hue, saturation and intensity components. A mask of stained tissue is prepared by automatic percentile thresholding. A single weighted inverted colour channel is then added to each of the three primary colour channels separately by an iterative algorithm that adjusts the weights to give minimum variance within the mask. The modified red, green and blue channels are then recombined. This method is automatic requiring no pre-definition of stain colours or special hardware. The method is demonstrated to 'de-stain' nuclei in haematoxylin and eosin (H&E) sections (and a separate haematoxylin image can be derived from this). An image of isolated brown reaction product is produced with immunoperoxidase preparations counterstained with haematoxylin. Furthermore trichrome (haematoxylin van Gieson, picrosirius red) and other common stains may be separated into their components with modifications of the same algorithm. Although other methods for colour separation do exist (e.g. spectral pathology and colour deconvolution) these require special apparatus or precise calibration and foreknowledge of pure dye colour spectra. The present method of digital stain separation is fully automatic with no such prerequisites.

Discrimination between benign and malignant prostate tissue using chromatin texture analysis in 3‐D by confocal laser scanning microscopy

Analysis of chromatin texture may improve both the diagnosis and the assessment of the prognosis of prostate cancer. Confocal laser scanning microscopy (CLSM) allows performing measurements in nuclei reconstructed in 3-D. The aim of this study was to evaluate the clinical usefulness of 3-D texture analysis of prostate tissue. Image stacks of eight prostate cancer sections were obtained by CLSM of both benign and malignant areas. Texture feature values were computed for individual nuclei. The discriminative power of the texture features was established by receiver operating characteristic (ROC) analysis and linear discriminant analysis (LDA). Texture features were identified that could discriminate between benign and malignant nuclei. LDA correctly classified 89% of the nuclei of the pooled set of benign and malignant nuclei. 3-D nuclear texture features allow discrimination of most benign and malignant prostate nuclei. We estimate that the classification rates can be increased by improving the image quality.

Prediction of radiosensitivity in human bladder cell lines using nuclear chromatin phenotype

Nuclear texture analysis measures phenotypic changes in chromatin distribution within a cell nucleus, while the alkaline Comet assay is a sensitive method for measuring the extent of DNA breakage in individual cells. The authors aim to use both methods to provide information about the sensitivity of cells to ionizing radiation. The alkaline Comet assay was performed on six human bladder carcinoma cell lines and one human urothelial cell line exposed to gamma-radiation doses from 0 to 10 Gy. Nuclear chromatin texture analysis of 40 features was then performed in the same cell lines exposed to 0, 2, and 6 Gy to explore if nuclear phenotype was related to radiation sensitivity. Comet assay results demonstrated that the cell lines exhibited different levels of radiosensitivity and could be divided into a radiosensitive and a radioresistant group at >6 Gy. Using stepwise discriminant analysis, a subset of important nuclear texture features that best discriminated between sensitive and resistant cell lines were identified A classification function, defined using these features, correctly classified 81.75% of all cells into their radiosensitive or radioresistant groups based on their pretreatment chromatin phenotype. Posttreatment chromatin changes also varied between cell lines, with sensitive cell lines showing a relaxed chromatin conformation following radiation, whereas resistant cell lines exhibited chromatin condensation. The authors conclude that the alkaline Comet assay and nuclear texture methodologies may prove to be valuable aids in predicting the response of tumor cells to radiotherapy.

Nuclear chromatin texture and sensitivity to DNase I in human leukaemic CEM cells incubated with nanomolar okadaic acid.

It is now known that the analysis of chromatin texture can be used in oncology as a sensitive detection method, either to define diagnostic classifications or to locate a lesion along a defined trend curve. However, the functional significance of these variations in textural features remains sometimes unclear. Several drugs have been shown to be able to modulate chromatin structure. Among them, the phosphatase inhibitor okadaic acid at low concentration can increase accessibility to DNA in chromatin of carcinoma cells. This paper demonstrates that short exposures (0-3 h) to a 10-nM dose of okadaic acid induced an increased sensitivity to DNase I digestion in human CEM leukaemic cell nuclei and that this sensitization was associated to variations of nuclear texture characteristics, as evaluated by image cytometry. CEM cells treated with okadaic acid for 0-3h displayed changes in chromatin supraorganization with a more homogeneous and fine chromatin texture, as compared to control cells. This suggests that the appearance of an open configuration of chromatin structure as evaluated by biochemical methods corresponds to a more decondensed texture of nuclei measured by image cytometry. Longer exposures (6-24h) of CEM cells to 10 nM okadaic acid lead to apoptosis. As reported previously for camptothecin-treated HL60 cells, okadaic acid-treated CEM cells display biphasic nuclear chromatin texture changes, i.e. a decondensation phase followed by the appearance of typical apoptotic cells with a smaller nuclear area and a highly condensed chromatin. Finally, using the multidrug-resistant CEM-VLB cell line, it was confirmed that these multidrug-resistant cells also display cross-resistance to okadaic acid, as this compound was unable to induce either increased DNase I sensitivity, apoptosis, or altered nuclear texture in this particular cell line.

Value of morphometry, texture analysis, densitometry, and histometry in the differential diagnosis and prognosis of malignant mesothelioma.

Malignant mesothelioma is a tumour with increasing incidence due to widespread use of its causative agent, asbestos, in the past decades. The poor survival necessitates a correct differentiation from other lesions at the same site, such as hyperplastic mesothelium and carcinomas metastatic to pleura or peritoneum. Since genetic and immunohistochemical markers are not absolutely differentiating, the diagnosis is based on the histology complemented with (immuno)histochemistry. However, as the tumour presents itself in numerous heterogeneous histological forms, visual evaluation is extremely difficult. In order to evaluate the prognostic and diagnostic performance of syntactic structure analysis (SSA), chromatin texture analysis, densitometry, and morphometry, an automated KNN-classification system has been used to compare Feulgen-stained tissue sections of hyperplastic mesothelium, malignant mesothelioma, and pulmonary adenocarcinoma. In addition, we also studied most discriminative aspects in the differentiation, typing, and prediction of survival. The results indicate that for the diagnosis of malignant mesothelioma, chromatin texture parameters outperform SSA, densitometry, and morphometry (recognition score=96.8 per cent). Most discriminative parameters highlight spatial patterns of the chromatin distribution that are hard to appraise visually and directly show the benefits of a quantitative approach. Typing of the tumour is best described by SSA parameters, relating to the spatial arrangement of the cells in the tissue (recognition score=94.9 per cent). In survival time classifications, chromatin texture yields the highest recognition score (82.9 per cent), although accurate estimations are unreliable due to a large degree of misclassification.

Nuclear chromatin texture analysis of nonmalignant tissue can detect adjacent prostatic adenocarcinoma

BACKGROUND The aim of this study was to investigate the possibility of identifying prostatic adenocarcinoma by nuclear chromatin texture feature analysis of adjacent histologically benign-looking tissue. METHODS Two hundred and forty prostatectomy specimens were selected from the archives of the Department of Pathology, University of Innsbruck. These consisted of 67 cases of benign prostatic hyperplasia (BPH) and 173 cases of prostatic adenocarcinoma (PAC). The specimen collection was divided randomly into a training set and test set. Cytospin preparations of disaggregated cells prepared from paraffin-embedded material were stained specifically for DNA by the Feulgen method. For the cancer cases, only tissue that histologically appeared nonmalignant, from the vicinity of the lesion, was used in the sample preparation. Only normal-appearing diploid cell nuclei were analyzed from both the BPH and PAC groups. A discriminator comprised of three nuclear texture features to separate BPH from PAC cases was derived from the training set of cases, and then applied to the independent test set cases. RESULTS PAC cases were separated from BPH cases with a sensitivity of 90% and a specificity of 97% on the independent test set of cases. CONCLUSIONS This retrospective investigation demonstrates that by high-resolution image cytometry it is possible to detect the presence of prostatic adenocarcinoma with very high reliability when examining prostate samples that only contain histologically normal-looking cells. This method could become clinically relevant in identification of cancers missed by histologically negative core needle biopsies. Prostate 41:12–19, 1999. © 1999 Wiley-Liss, Inc.

Nuclear chromatin texture analysis of nonmalignant tissue can detect adjacent prostatic adenocarcinoma

The aim of this study was to investigate the possibility of identifying prostatic adenocarcinoma by nuclear chromatin texture feature analysis of adjacent histologically benign-looking tissue.Two hundred and forty prostatectomy specimens were selected from the archives of the Department of Pathology, University of Innsbruck. These consisted of 67 cases of benign prostatic hyperplasia (BPH) and 173 cases of prostatic adenocarcinoma (PAC). The specimen collection was divided randomly into a training set and test set. Cytospin preparations of disaggregated cells prepared from paraffin-embedded material were stained specifically for DNA by the Feulgen method. For the cancer cases, only tissue that histologically appeared nonmalignant, from the vicinity of the lesion, was used in the sample preparation. Only normal-appearing diploid cell nuclei were analyzed from both the BPH and PAC groups. A discriminator comprised of three nuclear texture features to separate BPH from PAC cases was derived from the training set of cases, and then applied to the independent test set cases.PAC cases were separated from BPH cases with a sensitivity of 90% and a specificity of 97% on the independent test set of cases.This retrospective investigation demonstrates that by high-resolution image cytometry it is possible to detect the presence of prostatic adenocarcinoma with very high reliability when examining prostate samples that only contain histologically normal-looking cells. This method could become clinically relevant in identification of cancers missed by histologically negative core needle biopsies.

Nuclear chromatin texture analysis of nonmalignant tissue can detect adjacent prostatic adenocarcinoma.

The aim of this study was to investigate the possibility of identifying prostatic adenocarcinoma by nuclear chromatin texture feature analysis of adjacent histologically benign-looking tissue. Two hundred and forty prostatectomy specimens were selected from the archives of the Department of Pathology, University of Innsbruck. These consisted of 67 cases of benign prostatic hyperplasia (BPH) and 173 cases of prostatic adenocarcinoma (PAC). The specimen collection was divided randomly into a training set and test set. Cytospin preparations of disaggregated cells prepared from paraffin-embedded material were stained specifically for DNA by the Feulgen method. For the cancer cases, only tissue that histologically appeared nonmalignant, from the vicinity of the lesion, was used in the sample preparation. Only normal-appearing diploid cell nuclei were analyzed from both the BPH and PAC groups. A discriminator comprised of three nuclear texture features to separate BPH from PAC cases was derived from the training set of cases, and then applied to the independent test set cases. PAC cases were separated from BPH cases with a sensitivity of 90% and a specificity of 97% on the independent test set of cases. This retrospective investigation demonstrates that by high-resolution image cytometry it is possible to detect the presence of prostatic adenocarcinoma with very high reliability when examining prostate samples that only contain histologically normal-looking cells. This method could become clinically relevant in identification of cancers missed by histologically negative core needle biopsies.

Chromatin texture analysis in living cells.

In recent years, there has been an increasing interest in applications of fluorescence measurements to studies on many physiological mechanisms in living cells. However, few studies have taken advantage of DNA quantification by fluorometry for dynamic assessment of chromatin organization. This type of approach involves both optimal conditions for DNA staining and the use of image cytometry. In this context, this report describes the application of an internal grey-level segmentation method for the assessment of real time modifications of chromatin organization in living cells. These developments are based on a specific, stoichiometric method for nuclear DNA content measurement. Preliminary data obtained from Hela cells suggests the possibility of following variations of nuclear texture (heterogeneity, granularity, condensation, radial distribution) related to the cell cycle progression of cells that are maintained alive.

Application of confocal scanning laser microscopy for an automated nuclear grading of prostate lesions in three dimensions.

Confocal scanning laser microscopy provides the opportunity to obtain three-dimensional (3-D) images by piling up consecutive confocal planes. This technique was applied to capture 3-D images from 100-microns-thick tissue blocks from prostate lesions (hyperplasia, dysplasia, adenocarcinomas). Automated methods were implemented to perform a nuclear grading of 3-D cell nuclei from these specimens. Special attention was focused on the development of a new approach to 3-D chromatin texture analysis. This method uses mathematical morphology operations to tessellate the chromatin into homogeneous domains. The nuclear features (volume, shape, texture) were subjected to a discriminant analysis. Using a set of five features, the classification of cell nuclei yielded an accuracy of 96.3%. The results indicate the potential of 3-D imaging and analysis techniques for an automated nuclear grading of prostate lesions.

Nuclear pattern recognition by two-parameter texture analysis

The present paper describes a simple procedure for the analysis of chromatin texture. High-resolution digitized images of nuclei are first standardized to render gray values invariant to staining and illumination conditions. Subsequently, the nucleus is subdivided by a square grid into 0.4 × 0.4 μm 2 quadrats and standard deviations of gray values within each quadrat are estimated. Finally, the overal mean and standard deviation of quadrat standard deviations are calculated. These values may be considered as pure descriptors of the nuclear texture, as they represent the distribution of chromatin changes, disregarding any absolute densitometric and morphometric feature. Using the above descriptors it is possible to recognize at least seven chromatin patterns in a mixed population of developing and degenerating neurons. Results are visually verified by mapping the original pictures at the corresponding bivariate plot points. Comparison with the Markovian texture analysis is discussed.