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Nuclear grading of breast carcinoma by image analysis. Classification by multivariate and neural network analysis.

The use of nuclear grade as a prognostic indicator for breast carcinoma has been limited by interobserver variability. Advances in image analysis and automated cell classification offer one approach to this problem. The authors used the CAS-100 (Cell Analysis System. Elmhurst, IL) system to measure and analyze nuclear morphometric and texture features of cytologic preparations from 35 breast carcinomas (well, moderate, and poorly differentiated) as well as benign lesions. Morphometric and Markovian texture feature data from breast cancer nuclei of various grades comprised a training set, which was then used to establish classification criteria by multivariate (Bayesian) analysis and to train a neural network system. Both systems were tested for the ability to classify the nuclear grade of individual nuclei. There was good agreement between computer classification and the grade assigned by human observer to individual nuclei using either Bayesian or neural network analysis. Thirty-one unknown cases, which were assigned an overall grade by an observer, were then analyzed by computer, and an overall grade assigned based on the grade of nucleus most frequently present. Using this method, both classification systems were able to assign a "correct" grade to low-grade lesions (approximately 70% correct) more often than to high-grade tumors (approximately 20%). Difficulty in computer assignment of high-grade tumors was explained by nuclear heterogeneity in these tumors (i.e., although the percentage of high-grade nuclei was increased compared with that of low-grade tumors, high-grade nuclei frequently did not predominate). The authors present this study to demonstrate the feasibility of using image analysis as an objective means of nuclear grading. Further studies will be needed to establish criteria for assigning overall nuclear grade based on computer analysis of imaging data.

Artificial Intelligence↗

Application of a small microcomputer to cell image analysis.

Solutions to three problems in using small microcomputers for interactive cell image analysis are discussed. (1) To allow interactive processing of up to 62 X 88 pixels on inexpensive screens, data can be displayed in gray levels with an approximate logarithmic grading. Each pixel is composed of 32 screen coordinates, applying the dither matrix method to avoid artificial structures. (2) To mark special regions of interest in the image, a graphic cursor, handled from the keyboard, was implemented. (3) To evaluate parts of the image, as outlined by the cursor, the program must distinguish whether a particular pixel is outside, inside or on the border of the region. The developed algorithms permit practical interactive evaluation of cell images on a small microcomputer, with no image analysis implementation. However, it is necessary that the assembly language of the microprocessor be available for some sophisticated programming and that the operating system support graphic facilities with an appropriate resolution.

Animals↗

Image analysis for automatic segmentation of cytoplasms and classification of Rac1 activation.

BACKGROUND: Rac1 is a GTP-binding molecule involved in a wide range of cellular processes. Using digital image analysis, agonist-induced translocation of green fluorescent protein (GFP) Rac1 to the cellular membrane can be estimated quantitatively for individual cells. METHODS: A fully automatic image analysis method for cell segmentation, feature extraction, and classification of cells according to their activation, i.e., GFP-Rac1 translocation and ruffle formation at stimuli, is described. Based on training data produced by visual annotation of four image series, a statistical classifier was created. RESULTS: The results of the automatic classification were compared with results from visual inspection of the same time sequences. The automatic classification differed from the visual classification at about the same level as visual classifications performed by two different skilled professionals differed from each other. Classification of a second image set, consisting of seven image series with different concentrations of agonist, showed that the classifier could detect an increased proportion of activated cells at increased agonist concentration. CONCLUSIONS: Intracellular activities, such as ruffle formation, can be quantified by fully automatic image analysis, with an accuracy comparable to that achieved by visual inspection. This analysis can be done at a speed of hundreds of cells per second and without the subjectivity introduced by manual judgments.

Animals↗

Study of filamentous bacteria by image analysis and relation with settleability.

An automated procedure for the characterisation by image analysis of the morphology of activated sludge has been used to monitor the biomass in a pilot wastewater treatment plant during two runs inoculated with a different sludge and operated at two different temperatures. The bulking events were easily detected by image analysis. Correlations were found between settleability properties (Sludge Volume Index and settling velocity) and the morphological parameters (filament total length, filament number and floc size).

Bacteria↗

Neural net-based identification of cells expressing the p300 tumor-related antigen using fluorescence image analysis.

We report on preliminary investigations of the use of an image analysis system to perform preliminary algorithmic classification of images of fluorochrome-labeled cells followed by capture of gray-level images of potentially abnormal cells for analysis by a neural network. Cells were labeled with an antibody against a bladder cancer tumor-associated antigen, and the neural net was used to distinguish true-positive cells from negative cells, false-positive cells (autofluorescent or nonspecific labeling), and cell-sized artifacts. Gray-level cell images were digitized and processed for analysis by a feed-forward neural network using back-propagation. The network was trained and tested with two independent image sets. Various network configurations and activation functions were investigated, including a sinusoidal activation function. At high power, the network agreed completely with the human observer's classification. At low power, a strong clustering of cells classified by the network with expert classification was seen, while the neural network showed roughly 75% concordance with the human observer. In addition, a set of four features extracted from raw cell images were investigated. The features were: shape factor, texture, area, and average pixel intensity. A network trained with these features performed better than one operating with gray-level images. We conclude that using neural networks to recognize and classify images captured by an image analysis microscope is feasible.

Antigens, Neoplasm↗

A double fluorescence staining protocol to determine the cross-sectional area of myofibers using image analysis.

A double fluorescence staining protocol was developed to facilitate computer based image analysis. Myofibers from experimentally treated (irradiated) and control growing turkey skeletal muscle were labeled with the anti-myosin antibody MF-20 and detected using fluorescein-5-isothiocyanate (FITC). Extracellular material was stained with concanavalin A (ConA)-Texas red. The cross-sectional area of the myofibers was determined by calculating the number of pixels (0.83 mu m(2)) overlying each myofiber after subtracting the ConA-Texas red image from the MF-20-FITC image for each region of interest. As expected, myofibers in the irradiated muscle were smaller (P < 0.05) than those in the non-irradiated muscle. This double fluorescence staining protocol combined with image analysis is accurate and less labor-intensive than classical procedures for determining the cross-sectional area of myofibers.

Animals↗

Cytologic nuclear grade of malignant breast aspirates as a predictor of histologic grade. Light microscopy and image analysis characteristics.

OBJECTIVE: To determine if cytologic nuclear grade characteristics combined with image analysis assessment of morphometric nuclear parameters (1) correlate with the modified Scarff-Bloom-Richardson grading system and (2) discriminate between low and high nuclear grades of invasive ductal carcinoma. STUDY DESIGN: Fifty-four fine needle aspiration biopsies (FNABs) of breast carcinoma were evaluated for five morphologic nuclear grade characteristics. In addition, four morphometric, standardized object measurements were analyzed by an image analysis system. Corresponding biopsies of invasive ductal carcinoma (46 cases) were independently evaluated with the Scarff-Bloom-Richardson grading system, modified into low (scores 3-6) and high (scores 7-9) grades. RESULTS: An overall agreement of 82% was reached by three of four cytopathologists for each of five morphologic characteristics. There was a strong correlation (r = .8059, P < .0001) between cytologic nuclear grade and modified histologic grade. Only pleomorphism, nucleoli and sum optical density retained their statistical significance in distinguishing low from high grade ductal carcinomas. These three characteristics also had the strongest correlation with cytologic nuclear grade. CONCLUSION: Cytologic nuclear grade from aspirates of ductal carcinoma can be a predictor of the modified histologic grades of Scarff, Bloom and Richardson. Nuclear morphology reinforced by image morphometry may separate these tumors into low and high nuclear grade categories.

Biopsy, Needle↗

Biologic significance of quantitative estrogen receptor immunohistochemical assay by image analysis in breast cancer.

The authors assayed 209 stage I and II mammary carcinomas for the estrogen receptor (ER) with an immunocytochemical assay (ICA) and quantitated the nuclear stain with the SAMBA 4000 Cell Image Analysis System (Imaging Products International, Inc., Chantilly, VA). The cases had been followed for 54-214 months (average, 64 months). The results were correlated with the patients' overall and disease-free survival times. Three of the parameters obtained from the quantitative analysis were evaluated: labeling index, mean optical density, and quick score. Statistical analysis was performed with the Kaplan-Meier product limit estimator for quantitated values and Cox regression for risk of mortality and disease progression. Mean optical density of the nuclei at a cut-off value of 10 produced the strongest association between quantitative ERICA and overall and disease-free survival times in discriminating high- and low-risk groups (P = .016 and P = .018, respectively). When the mean optical density results were categorized into ranges of values of potential biologic significance according to overall survival times, patients with mean optical density greater than 15 were found to be at lowest risk; patients with values between 5 and 15 had a statistically significant intermediate risk; and the group with values less than 5 had the worst outcome (P = .018). The probability of disease-free survival also was statistically significant (P = .038) among the three groups with cut-off points of less than 10, 10-35, and greater than 35. These results showed that quantified optical density has better discriminating power for risk prediction than that obtained by the biochemical assay for ER at a cut-off value of 10 or 20 fmol/mg. Estrogen receptor ICA has been adequately shown to be as or more accurate than ligand-binding assays. Our results corroborate those studies and support the utility of ICA assays for ER by showing that quantitation performed by image analysis is an objective and reproducible method yielding clinical prognostic information of higher reliability than that given by the dextran-coated charcoal ER assay.

Adult↗

[PCNA expression and AgNORs image analysis as factors in judging the prognosis of lung cancer].

The expression of PCNA and AgNORs count or AgNORs' area in 86 cases of primary lung cancer and 10 cases of inflammatory pseudotumor were studied by S-P immunostaining of monoclonal antibody (PC-10) and image analysis system. The results showed that the proliferating index (PI) of PCNA positive cells was closely related to AgNORs count and AgNORs' area measured. Both were correlated with the histological type, and the TNM status, decreased with the increasing degree of histological differentiation. Survival analysis showed that the PI and the results of AgNORs image analysis in surgical patients who survived over 5 years were significantly lower than those who survived less than 3 years. The results suggest that immunostaining of PCNA and AgNORs image analysis are useful in judging the malignant degree and the prognosis of primary lung cancer.

Adenocarcinoma, Bronchiolo-Alveolar↗

Automatic enumeration of adherent streptococci or actinomyces on dental alloy by fluorescence image analysis.

The aim of the present study was to develop an automated image analysis method to quantify adherence of Streptococcus sanguinis or Actinomyces viscosus on surfaces of a currently used dental alloy. Counting such bacterial strains was difficult because of their arrangement, thus S. sanguinis being a coccus arranged in chains or pairs, and A. viscosus a long complexly arranged polymorph rod. Direct counting of fluorescently stained adherent bacteria was done visually and with image analysis methods. To differentiate these two morphotypes, two programs were developed: (i) for streptococci, thresholding and selection of the object maxima, and (ii) for actinomyces, two step thresholding and processing of the characteristic points of the object skeletons. The triplicate enumerations for each bacterial strain were not significantly different (p > 0.005) and correlations between visual counting and automated counting were significant (r = 0.91 for S. sanguinis and r = 0.99 for A. viscosus, p <00.0001). These rapid and reproducible methods, allowed us to count either cocci or rods, adherent on an inert substratum, in high density conditions.

Actinomyces viscosus↗

Transferrin secretion and hepatocyte ploidy: analysis at the single cell level using a semi-automatic image analysis method.

In a previous work it was shown that transferrin (Tf) secretion is directly related to the membrane surface area of hepatocytes (Péchinot D. et al. [31]). The aim of the present work was to search for a possible relationship between Tf secretion and hepatocytic ploidy using a semi-automatic image analysis method. A determination of Tf secretion by isolated normal adult hepatocytes was achieved at the single cell level, using a modified reverse hemolytic plaque test. A Feulgen reaction was also performed on these hepatocytes. It allowed the evaluation, for each secreting hepatocyte, of the quantity of Tf secreted and its nuclear characteristics. Discrimination between diploid (2c) and tetraploid (4c and 2c2c) hepatocytes was performed and the amount of Tf secreted by each subpopulation determined. It appeared that a 2-fold secretion ratio was not found between tetraploid and diploid hepatocytes. These results suggest, as Tf production is not directly proportional to the degree of ploidy of hepatocytes, that some not yet elucidated regulatory mechanisms may act on Tf gene expression.

Animals↗

Image analysis in the RGB and HS colour planes for a computer-assisted diagnosis of cutaneous pigmented lesions.

AIMS AND BACKGROUND: A study was carried out to evaluate the effectiveness of image analysis performed by the two color representation models when a computer-assisted diagnosis of melanoma is involved. METHODS: Color images of 40 skin pigmented lesions, which included 12 melanomas, were acquired by a standard color RGB video camera and stored in a PC for off-line processing. Image analysis was performed in the red green and blue color representation model and using hue and saturation color components. To describe shape and color characteristics of each lesion, including area, roundness and color variegation, 16 parameters were derived from red, green, blue, hue and saturation color planes and tested as possible variables useful to differentiate melanomas from benign nevi. RESULTS: The test gave a result of significance for six of the 16 derived image descriptors. The general trend of our data was in agreement with clinical observations according to which melanoma is usually darker, more variegated and less round than a benign nevus, whereas lesion dimension of melanomas and benign lesions was not significantly different. CONCLUSIONS: Our preliminary results suggested that image analysis performed on hue and saturation-derived and red green and blue-derived data could better discriminate melanoma from nevi than separately using the two color representation models.

Color↗

Improvements in slide preparation from archival material for automated DNA measurement by image analysis.

OBJECTIVE: To optimize slide preparation for DNA content measurement by automated image analysis and to obtain a rapid and easy method for routine use. STUDY DESIGN: Some improvements in previously described methods were achieved: automatic dewaxing, reduction of washing times, accurate adjustment of final concentration of nuclei, sedimentation and temperate drying of nuclei. RESULTS: The complete preparation of 12 slides required four intermittent working hours. Accurate adjustment of the final concentration and sedimentation of nuclei resulted in a constant and homogeneous distribution of nuclei. Cell loss was prevented and the most fragile structures preserved. CONCLUSION: High-quality preparations of nuclei lead to high-resolution histograms obtained from a large collection of events. This promotes automated image analysis method instead of flow cytometry when only archival material is available for DNA content measurement and proliferating fraction evaluation.

Cell Nucleus↗

Manual versus image analysis estimation of PCNA in breast carcinoma.

OBJECTIVE: To compare manual to image analysis estimation of proliferating cell nuclear antigen (PCNA) expression in paraffin sections of breast carcinomas. STUDY DESIGN: Paraffin sections of 51 breast carcinomas were stained with primary antibody to PCNA. Nuclear PCNA expression in 100 randomly selected tumor cells from marked areas was manually graded from 0 to 3. Antigen expression was also calculated by a cell analysis system (CAS-200, Becton Dickinson, Elmhurst, Illinois, U.S.A.) from marked and random microscopic fields. Obtained proliferative index (PI) from both methods was compared. RESULTS: Manually calculated PI correlated strongly with the CAS-200-calculated PI (P < .01). The highest correlation was seen between the CAS-200 PI value and manually calculated PI value using grade 2 and 3 nuclei. A particularly high correlation was noted between the number of positive nuclei and antigen staining area (P < .01) as estimated by the CAS-200. CONCLUSION: Nuclear expression of PCNA and other nuclear antigens can be accurately evaluated by an image analysis system. The speed and objectivity of such machines allow the evaluation of larger parts of tissues and provide more-representative antigen expression profiles.

Algorithms↗

Image analysis enhancement of the laser scanning cytometer.

The laser scanning cytometer offers a range of novel applications and the capacity for direct visual validation of experiments through sample analysis on a microscope slide. Linkage of the instrument to an image analysis system through standard connections and software enhances the capabilities of the instrument in image capture and manipulation. In this technical note, we describe a simple linkage between the LSC and the Kontron KS100 Image Analysis System, an example of a standard commercial image processing instrument.

Image Cytometry↗

A public domain image-analysis program for the single-cell gel-electrophoresis (comet) assay.

The single-cell gel electrophoresis (or comet) assay has gained widespread acceptance as a cheap and simple genotoxicity test, but it requires a computer-assisted image-analysis system. As commercial programs are expensive and inflexible, we decided to develop an image-analysis system based on public domain programs and make it publicly available for the scientific community. Our system is based on the scientific image-processing program NIH Image, and was written in its Pascal-like macro language. User interaction was kept as simple as possible, to enable the measurement of a large number of cells with a few keystrokes. Therefore, the time for image analysis is very low, even on slow computers. The comet macro can be obtained from http://mailbox.univie.ac.at/christoph.helma++ +/comet/, NIH Image is available at http://rsb.info.nih.gov/nih-image/. Both programs are free of charge.

Comet Assay↗

Determination of proliferation index in advanced ovarian cancer using quantitative image analysis.

It has been shown that the monoclonal antibody Ki-67 reacts with a nuclear antigen that is expressed only by proliferating cells. The feasibility of using image analysis to quantitate Ki-67 staining (proliferation index [PI]) of epithelial ovarian cancers was investigated. The PI was determined in 50 advanced-stage primary ovarian cancers. Frozen sections were immunostained with the Ki-67 monoclonal antibody, and the PI was calculated using static image analysis. Among 35 stage III ovarian carcinomas, the median PI was 8.9%, compared with 17.7% in 15 stage IV cancers (P = 0.06). There was no relationship between PI and histologic grade. The median survival time of 32 patients whose cancers had a high Ki-67 expression (> or = 7.5%) was 16.8 months, which differed significantly (P < 0.01) from the median survival of 31.5 months observed in patients whose tumors demonstrated low Ki-67 expression (< 7.5%). Quantitative image analysis of Ki-67-stained fresh-frozen ovarian cancers may provide useful prognostic information. Further studies are warranted to investigate the relationship between Ki-67 expression and other known clinicopathologic and genetic features of ovarian cancer.

Antibodies, Monoclonal↗

Comparison of planimetry and image analysis for the discrimination between normal and abnormal cells in cytological smears of suspicious lesions of the oral cavity.

Light microscope analysis of cytological smears of suspicious lesions of the oral cavity is used as a method for detecting early cancer in the oral cavity. The sensitivity of this approach can be improved by quantitative analysis of the cells in the cytological smears. We have compared the efficiency of planimetry and the Vids V system of image analysis, as quantitative methods for discriminating between normal and abnormal cells in cytological smears of suspicious lesions in the oral cavity. Both methods detected an increase in nuclear area and a decrease in cytoplasmic area in abnormal epithelial cells from dysplastic lesions of increasing severity. However, image analysis was better able to discriminate between benign and malignant cells on the basis of nuclear size. Thus the Vids V system of image analysis is more appropriate than planimetry for quantitative analysis of cytological smears from the oral cavity.

Cell Size↗