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PubMed2005

Multi-resolution wavelet-transformed image analysis of histological sections of breast carcinomas.

Multi-resolution images of histological sections of breast cancer tissue were analyzed using texture features of Haar- and Daubechies transform wavelets. Tissue samples analyzed were from ductal regions of the breast and included benign ductal hyperplasia, ductal carcinoma in situ (DCIS), and invasive ductal carcinoma (CA). To assess the correlation between computerized image analysis and visual analysis by a pathologist, we created a two-step classification system based on feature extraction and classification. In the feature extraction step, we extracted texture features from wavelet-transformed images at 10x magnification. In the classification step, we applied two types of classifiers to the extracted features, namely a statistics-based multivariate (discriminant) analysis and a neural network. Using features from second-level Haar transform wavelet images in combination with discriminant analysis, we obtained classification accuracies of 96.67 and 87.78% for the training and testing set (90 images each), respectively. We conclude that the best classifier of carcinomas in histological sections of breast tissue are the texture features from the second-level Haar transform wavelet images used in a discriminant function.

Breast Neoplasms↗
PubMed1999

Nondestructive prediction method for yolk:albumen ratio in chicken eggs by computer image analysis.

The purpose of this study was to develop a nondestructive prediction method for the yolk: albumen ratio by computer image analysis for candling inspection. Twenty-two to 49 eggs per line were randomly sampled from four chicken lines. After weighing the eggs, the eggs were illuminated by an overhead projector beam through a small hole in dark room. Video images were taken of the eggs from four directions, the eggs rotated each time by 90 degrees. The eggs were broken for measuring egg traits, including the yolk:albumen ratio. The average value obtained from four directions was used for statistical analysis. The ratio of the number of pixels of light and dark parts (light: dark ratio), and the CV of red (R), green (G), and blue (B) components for the whole egg and for light and dark parts of the egg were calculated and defined as image analysis traits. Correlation coefficients between the yolk: albumen ratio and CV of R and G components of the whole egg were significant (0.42 to 0.79) in all the lines. The determination coefficient of multiple regression of the yolk:albumen ratio on the CV of R and G components of the whole egg and the light:dark ratio was 0.83. Observed and predicted yolk:albumen ratios were classified into five levels. The ratio of zero difference between observed and predicted values was 76.1%, and the percentage of 0 to +/- 1 difference between observed and predicted values was 100.0%. The image analysis method accurately predicted the yolk:albumen ratio without breaking the egg.

Albumins↗
PubMed1995

Uses of digital image analysis in electrophoresis.

The information in electrophoretic gels can be extracted visually, or by using specialised computer hardware and software which treat the gels as digital images. We discuss issues which arise in applying digital image analysis to electrophoresis, and comment on the range of computer packages available. We also illustrate how image analysis can make gel interpretation easier and more reliable. Methods covered include gel registration and warping, mathematical morphology and deconvolution to sharpen images.

Computers↗
PubMedDate not supplied

Quantitative image analysis of oesophageal squamous cell carcinoma from the high-incidence area of China, with special reference to tumour progression and papillomavirus (HPV) involvement.

BACKGROUND: Despite much research effort, the major prognostic factor of oesophageal squamous cell cancer (ESCC) remains the pathological stage of the disease as defined by the TNM classification, whereas tumour grading is of limited value in this respect, mainly due to its low reproducibility. A better means for disease prognostication based on improved understanding of the pathogenetic mechanisms is urgently required. MATERIALS AND METHODS: Among the cohort of 700 ESCC patients from the high-incidence area of China, previously subjected to extensive testing for Human papillomavirus (HPV) involvement by in situ hybridization (ISH) and PCR, a group of 273 patients was randomly selected for analysis of the primary tumour, adjacent mucosa and regional lymph nodes, by histopathology and quantitative image analysis. All these and the HPV data were subjected to extensive univariate and multivariate analysis to disclose independent predictors of progressive disease. RESULTS: For the analyses, the tumours were graded into two categories: well-moderately and poorly-differentiated. HPV DNA was detected in 116 (18.9%) of the carcinomas by ISH and in 15.2% by PCR. In univariate analysis, lymph node status (considered as the surrogate marker of progressive disease) was significantly (p < 0.01) predicted by the following nuclear parameters: nuclear area, G0/G1 ratio, HPV DNA status, integrated optical density (IOD), mean optical density (MOD) and S-Phase. In multivariate (stepwise backward LR) analysis, 6 variables remained as independent predictors of disease progression (at p < 0.05 level), the three most significant ones being nuclear perimeter, nuclear roundness and equivalent diameter (p < 0.01). CONCLUSION: A series of quantitatively measured nuclear parameters seem to bear a close correlation with ESCC differentiation and progression in univariate analysis and some of these variables proved to be significant independent predictors of disease progression in multivariate modelling as well. These data clearly advocate the use of quantitative image analysis in searching for additional prognostic factors of ESCC.

Carcinoma, Squamous Cell↗
PubMed2001

Computerized image analysis of Ki-67 in ductal breast carcinoma in situ.

OBJECTIVE: To develop and determine the staining protocols and computerized image analysis methods that are the most effective combination for performing quantitative analysis of Ki-67. STUDY DESIGN: We compared conventional bright-field light microscopy and refractive optical enhancement methods in combination with various immunodetection and filter enhancement methods, including immunogold in combination with epipolarization refractive optics and enzymatic conversion of chromogenic substrates in combination with optical filter enhancement. Initial Ki-67 tests were performed on lymph node tissues and cultured human breast cells and then applied to 200 ductal carcinoma in situ (DCIS) samples. DCIS acini were digitally acquired, and a region of interest was manually outlined in each one with a digital stylus to include only the cellular component; then the Ki-67 staining index was quantified by segmentation analysis. RESULTS: Although combining epipolarization analysis with immunohistogold staining was the most sensitive detection method, nonspecific binding was too high. The streptavidin-horseradish-peroxidase enzymatic conversion of 3,3'-diaminobenzidine (DAB) in combination with optical enhancement filters was the most effective method tested. Ki-67 stain was associated with dense fibrillar structures of the nucleoli in the less intensely staining nuclei and was most intense in paired nuclei. CONCLUSION: The method of measuring Ki-67 expression by DAB staining combined with optical enhancement filters and quantification via computer-assisted image analysis techniques produced objective and reproducible results. As such, this method can offer (1) less intraobserver and interobserver variability, (2) a digital archival record, and (3) a baseline for digital exchange of information between studies.

Antibodies, Monoclonal↗
PubMed1997

Reliability of an image analysis system for quantifying the radiographic trabecular pattern.

A reliability evaluation technique was used to examine the reliability of an image analysis system of the trabecular pattern and to determine the contribution of three possible sources of error variance. Two series of radiographs were taken of 14 lumbar vertebral slices (28 radiographs). Every radiograph was placed on a viewing box for digitization four times by a single operator (112 positions of radiographs) and from every position of a radiograph an area of 15 mm x 15 mm was digitized twice (224 samples for analysis). Ten geometrical characteristics of the trabecular pattern were studied and its orientation was analyzed in 12 directions. Reliability was determined by calculating Cronbach's alpha. This design enabled dividing the measurement error (1-alpha) into fractions associated with the X-ray procedure, the operator and the system. Using this reliability evaluation technique, it was found that the orientation variables are more reliable than the geometric variables. It was found that effort to increase the reliability should be directed toward improving the technical procedure of this image analysis system. Also, repeated measurements will increase the reliability. The number of repeated measurements based on a desired reliability can be calculated. This procedure of evaluation gives the opportunity to select a source of error variance which have to be reduced to increase reliability most effectively.

Adult↗
PubMed1995

Application of an image analysis system to the quantitation of tumor perfusion and vascularity in human glioma xenografts.

A semiautomatic method based on a computerized digital image analysis system was developed to quantitate the perfused fraction of blood vessels and the relative vascular area in cross-sections of human glioma xenografts, implanted subcutaneously in athymic mice or intracerebrally in nude rats. The fluorescent dye Hoechst 33342 was injected intravenously to detect perfused tumor vessels. An immunofluorescent staining of Collagen type IV visualized the vascular structures in the same tumor section. Whole tumor sections were automatically scanned twice on a computer-controlled motorized stage of a fluorescence microscope under two different settings of the image analysis system. At the beginning of a scanning session an interactive routine was used to determine the threshold value for segmentation of vascular structures from the darker background. After the first scan a composite image was created, from the individually processed microscopic images, containing the detected vascular structures. The second scan yielded another composite image with objects representing the perfused areas. When both composite images were combined the overlapping structures showed the perfused vessels. Differences in perfused fractions and relative vascular areas were found between different tumors. The reproducibility of this analysis system was tested and evaluated. The method developed here provides a fast and accurate technique for simultaneous quantitative analysis of tumor perfusion and vasculature.

Animals↗
PubMed1989

Evaluation of automated threshold selection methods for accurately sizing microscopic fluorescent cells by image analysis.

The accurate measurement of bacterial and protistan cell biomass is necessary for understanding their population and trophic dynamics in nature. Direct measurement of fluorescently stained cells is often the method of choice. The tedium of making such measurements visually on the large numbers of cells required has prompted the use of automatic image analysis for this purpose. Accurate measurements by image analysis require an accurate, reliable method of segmenting the image, that is, distinguishing the brightly fluorescing cells from a dark background. This is commonly done by visually choosing a threshold intensity value which most closely coincides with the outline of the cells as perceived by the operator. Ideally, an automated method based on the cell image characteristics should be used. Since the optical nature of edges in images of light-emitting, microscopic fluorescent objects is different from that of images generated by transmitted or reflected light, it seemed that automatic segmentation of such images may require special considerations. We tested nine automated threshold selection methods using standard fluorescent microspheres ranging in size and fluorescence intensity and fluorochrome-stained samples of cells from cultures of cyanobacteria, flagellates, and ciliates. The methods included several variations based on the maximum intensity gradient of the sphere profile (first derivative), the minimum in the second derivative of the sphere profile, the minimum of the image histogram, and the midpoint intensity. Our results indicated that thresholds determined visually and by first-derivative methods tended to overestimate the threshold, causing an underestimation of microsphere size. The method based on the minimum of the second derivative of the profile yielded the most accurate area estimates for spheres of different sizes and brightnesses and for four of the five cell types tested. A simple model of the optical properties of fluorescing objects and the video acquisition system is described which explains how the second derivative best approximates the position of the edge.

Cyanobacteria↗
PubMed2000

Image analysis and flow cytometric DNA studies of benign and malignant body cavity fluids: reappraisal of the role of current methods in the differential diagnosis of reactive versus malignant conditions.

Cytologic examination of body fluids is commonly performed in the clinical laboratory. Determination of the presence of malignancy may sometimes be difficult. In this study, we prospectively studied 60 body fluids with a panel of antibodies, including MOC-31, epithelial membrane antigen, carcinoembryonic antigen, B72.3, keratin, desmin, and CA-125. DNA and S-phase studies were performed both by flow cytometry and image analysis. Thirty-seven fluids were classified as benign and 23 were classified as malignant. The sensitivity of the antibodies for identification of carcinoma in descending order of percentage detection rate were MOC-31 (95%), epithelial membrane antigen (93%), B72.3 (84%), and carcinoembryonic antigen (80%). Desmin stained mesothelial cells in all cases. CA-125 gave similar results but was less specific. Flow cytometry detected 14 of 20 malignant fluids and image analysis 17 of 23 by identifying an aneuploid population. Benign reactive mesothelial cells were not aneuploid. Tetraploidy due to reactive mesothelial cells was found in 9 of 37 body fluids. Their S-phase fraction was low (average, 3.2%). Tetraploidy in malignant cells was distinguished from the reactive mesothelial cells by high S-phase (average, 25.95). S-phase had some use as a discriminating factor, because no benign reactive cases had more than 17%. However, 7 of 23 malignant cases had a value below 17%. DNA analysis by image was more sensitive and specific than flow. Either may be used when immunocytochemistry is nondiagnostic or cannot be performed.

Adult↗
PubMed1993

Use of cell image analysis in the detection of cancer from specimens obtained during endoscopic retrograde cholangiopancreatography.

The authors report a scoring system based on cell image analysis of specimens obtained during endoscopic retrograde cholangiopancreatography from either the biliary or pancreatic duct epithelium. Nine normal, 20 inflammatory, and 19 malignant epithelia were assessed using a cell-image processor that permits determination of morphometric (nuclear area), densitometric (nuclear DNA content), and textural (chromatin distribution) parameters on Feulgen-stained nuclei. The score is based on three densitometric parameters: the DNA histogram type, the nuclear DNA content, and the percent proliferating cell population. These parameters permit distinction of normal and inflammatory from malignant epithelium with 95% sensitivity and 100% specificity. Cell-image analysis of specimens obtained during endoscopic retrograde cholangiopancreatography is a useful clinical tool in the detection of malignant lesions of the pancreas and biliary epithelia.

Bile Duct Diseases↗
PubMed1995

Quantitative autoradiography of alpha 1 adrenoceptors with [3H]tamsulosin in human hypertrophied prostate using computerized image analysis.

Fourteen specimens of human hypertrophied prostate were evaluated for the distribution of alpha 1 adrenoceptors using autoradiography with a computerized image analysis system. The hypertrophied prostatic specimens, obtained at open prostatectomy, were dissected vertically to the urethra, and sectioned at 10 microns. They were immersed in 1 nM of specific alpha 1 ligand, [3H]tamsulosin chloride ([3H]tamsulosin) and exposed to autoradiographic film. The images were analysed by a computerized image analysis system. The total binding of [3H]tamsulosin in the whole section (n = 14) was 0.82 +/- 0.21 (mean +/- SE) nCi mg-1. The autographic data were correlated with data obtained in a membrane-binding assay. The prostatic tissue studied was divided into urethral, glandular and stromal zones, the latter two zones being further divided into the inner and outer areas. The total binding of [3H]tamsulosin in the urethral zone (n = 7) was 0.65 +/- 0.32 nCi mg-1. The glandular zone contained significantly more abundant alpha 1 adrenoceptors than the stromal zone and their densities (glandular vs stromal) were 1.15 +/- 0.19 nCi mg-1 (n = 14) vs 0.72 +/- 0.15 nCi mg-1 (n = 14), respectively (p < 0.05). The data from the whole section were not affected by prostatic weight. This method described enabled the distribution of the receptors in different sites to be evaluated both morphologically and quantitatively.

Adrenergic alpha-Antagonists↗
PubMed1994

Quantitative DNA measurement by flow cytometry and image analysis of human nonseminomatous germ cell testicular tumors.

Current clinical staging, which includes the use of serum tumor markers and imaging techniques, fails to identify the 30-40% of clinical stage I (CS I) nonseminomatous germ cell testicular tumor (NSGCT) patients who have occult metastatic disease. Therefore, there is a real clinical need to evaluate new biological parameters of the primary tumor that might be useful as predictors of occult metastatic disease. This study was undertaken to compare quantitative DNA measurements by flow cytometry and image analysis in CS I NSGCT, and to analyze the relevance of these parameters for predicting occult lymph node involvement. Different blocks of formalin-fixed, paraffin-embedded NSGCTs of 62 CS I patients who underwent retroperitoneal lymph node dissection between 1985 and 1989 were prepared according to the Hedley technique, and analyzed by quantitative cytometry. Thirty-six (58.1%) patients had histologically proven lymph node involvement (pathological stage II), whereas 26 (41.9%) patients (pathological stage I) had neither lymph node metastases according to retroperitoneal lymph node dissection (RPLND) specimens nor tumor recurrence during follow-up. Concordant results were found in 76.5% of the samples by both cytometric techniques. For flow cytometry, the percentages of aneuploid cells in the S- and the G2M + S-phase were the most robust predictive parameters for lymph node involvement, whereas for image analysis the 5c exceeding rate (5cER) had the most predictive significance. Based on the experience obtained in this study, both cytometric techniques provide additional information on tumor aggressiveness that might be useful in therapeutic selection of early stage NSGCT patients for either RPLND or surveillance only.

DNA, Neoplasm↗
PubMed1992

[Use of computer-assisted image analysis for the detection of immunohistochemical receptor status in breast cancer. Immunohistochemical densitometry receptor analysis--IRDA].

A method for the evaluation of the immunohistochemical staining score of steroid receptors in breast cancer using computer-based image analysis is presented. The optical density of tumour cell nuclei was measured using kryostat sections and, using a control section as reference, an objective threshold for specific staining was defined. In intensively stained sections, a good correlation with visual scoring was achieved, but in 9 out of 16 sections, which were regarded as receptor-negative by visual scoring, a specific receptor-positive staining was demonstrated by image analysis. Next to the percentage of positive cells (PP) and the mean staining intensity (SI), a histogram of the receptor concentration in positive nuclei was calculated. The method allows, besides an objective evaluation and documentation, an exact definition of a threshold for specific receptor measurement and furthermore shows the pattern of receptor concentration in the tumour cell nuclei. Hence, the method could contribute towards a more widespread use of immunohistochemical receptor analysis.

Breast↗
PubMed2004

Development of image analysis techniques as a tool to detect and quantify morphological changes in anaerobic sludge: I. Application to a granulation process.

Image analysis techniques were developed and applied to quantify the process of anaerobic granulation in an expanded granular sludge blanket reactor (EGSB) fed with a synthetic substrate based on glucose [60-30% COD (chemical oxygen demand)] and volatile fatty acids (40-70% COD) over 376 days. In a first operation period that lasted 177 days, the aggregation of dispersed sludge was quantitatively monitored through the recognition and quantification of aggregates and filaments. A parameter defined as the ratio between the filaments' length and the aggregates projected area (LfA) has proven to be sensitive to detect changes in the aggregation status of the anaerobic sludge. The aggregation time-defined as the moment when a balance between filaments' length and aggregates' size was established-was recognized through the LfA. The percentage of projected area of aggregates within three size ranges (0.01-0.1 mm, 0.1-1 mm, and >1 mm, equivalent diameter) reflected the granular size spectrum during the aggregation process. When sudden increases on the upflow velocity and on the organic loading rate were applied to the previously formed granules, the developed image analysis techniques revealed to be good indicators of granular sludge stability, since they were sensitive to detected filaments release, fragmentation, and erosion that usually leads to washout. The specific methanogenic activities in the presence of acetate, propionate, butyrate, and H(2)/CO(2) increased along the operation, particularly relevant was the sudden increase in the specific hydrogenophilic activity, immediately after the moment recognized as aggregation time.

Algorithms↗
PubMed2002

Automated image analysis and in situ hybridization as tools to study bacterial populations in food resources, gut and cast of Lumbricus terrestris L.

An image analysis procedure was developed for bacterial cells after staining with the DNA-intercalating dye 4'-6-diamidino-2-phenylindole (DAPI), and after in situ hybridization with Cy3-labeled, rRNA-targeted oligonucleotide probes. DAPI- and Cy3-images were captured separately from the same scenery with a cooled digital video camera with three CCD chips for the basic colors red (R), green (G) and blue (B). Using the appropriate filter sets, images of DAPI-stained cells were captured with the red channel shut down, while Cy3-stained cells were captured with the green and blue channels shut down. DAPI images and Cy3 images were subsequently merged to produce virtual color (RGB)-images. Processing of all color channels allowed to specifically enumerate DAPI-stained and hybridized bacteria, to measure their cell sizes, to subsequently calculate their biovolumes and to estimate their biomass. Using this procedure, significant differences were detected in bacterial populations in food resources, digestive tract and cast of the earthworm L. terrestris L. In leaves, bacteria were on average ten times more abundant and two times larger than in soil. In the digestive tract of L. terrestris, however, numbers and volumes of bacteria were comparable to those in soil indicating the disruption of cells originating from leaves before arriving in the foregut. Passage through the digestive tract of L. terrestris significantly reduced bacterial populations belonging to the alpha-, beta- and gamma-subdivisions of Proteobacteria. While these populations did not recover during incubation of cast, populations of the delta-subdivision of Proteobacteria and the Cytophaga-Flavobacterium cluster of the CFB phylum increased in cast. These results suggest a large impact of passage through the digestive tract of L. terrestris on bacterial community structure and demonstrate the usefulness of our image analysis procedure for the determination of cell sizes and biovolumes and thus biomass of specific bacterial populations in different terrestrial habitats.

Animals↗
PubMed1998

Measurement of the amounts of elastic fibers in the skin and temporal arteries of healthy aged individuals by automated image analysis.

BACKGROUND: A quantitative study of dermal and arterial elastic fibers as a function of age was carried out by computerized image analysis. OBJECTIVE: We investigated whether any parallelism can be established between the morphometric parameters of elastic fibers from the skin and the temporal artery in elderly subjects. METHODS: we quantitated the skin elastic fibers of the reticular dermis and the elastic fibers of the temporal artery using a specific staining procedure followed by automated image analysis in 16 subjects of age range 63-87 years. RESULTS: There was a good correlation between the area fraction occupied by the elastic fibers in the unexposed skin (inner part of the upper arm) and aging (r = 0.669, p < 0.01). The area fraction occupied by elastic fibers in unexposed skin was correlated with the area fraction occupied by elastic fibers in the deep part of the temporal artery (r = 0.498, p < 0.05). Actinic elastosis affected both tissues, but there was no correlation between the amount of elastotic material in the exposed skin and the area fraction of elastic fibers in the superficial part of the temporal artery. CONCLUSION: We provided evidence that in sun-protected tissues the area fraction occupied by elastic fibers in dermis and deep part of the temporal artery showed a significant correlation. We proposed that skin biopsies were a valuable diagnostic tool for predicting arterial wall abnormalities of elastic fibers.

Aged↗
PubMedDate not supplied

[Enumeration and size of planktonic bacteria determined by image analysis coupled with epifluorescence].

The interest of the image analysis procedure is the time-saving in automated planktonic bacterial counting and sizing, with the possibility of manual visual field control at all times. Bacterial biomass (in number and volume) and bacterial projected area histograms were determined with a microcomputer. Performance limits of image-analysed epifluorescence microscopy were: camera sensitivity, considering the very low fluorescence levels on stained bacteria; pixel-micron conversion factor, and the impossibility of the apparatus distinguishing between bacteria and fluorescent small particles. This method is not of interest for counting sediment bacteria.

Animals↗
PubMed1999

Artificial neural network-aided image analysis system for cell counting.

BACKGROUND: In histological preparations containing debris and synthetic materials, it is difficult to automate cell counting using standard image analysis tools, i.e., systems that rely on boundary contours, histogram thresholding, etc. In an attempt to mimic manual cell recognition, an automated cell counter was constructed using a combination of artificial intelligence and standard image analysis methods. METHODS: Artificial neural network (ANN) methods were applied on digitized microscopy fields without pre-ANN feature extraction. A three-layer feed-forward network with extensive weight sharing in the first hidden layer was employed and trained on 1,830 examples using the error back-propagation algorithm on a Power Macintosh 7300/180 desktop computer. The optimal number of hidden neurons was determined and the trained system was validated by comparison with blinded human counts. System performance at 50x and lO0x magnification was evaluated. RESULTS: The correlation index at 100x magnification neared person-to-person variability, while 50x magnification was not useful. The system was approximately six times faster than an experienced human. CONCLUSIONS: ANN-based automated cell counting in noisy histological preparations is feasible. Consistent histology and computer power are crucial for system performance. The system provides several benefits, such as speed of analysis and consistency, and frees up personnel for other tasks.

Cell Count↗