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DNA ploidy studies of benign and malignant tumours: comparison of flow cytometry and image analysis techniques using two types of cytological specimen.

DNA ploidy studies were carried out on Feulgen stained smears and cytocentrifuge preparations from 35 malignant tumours and four benign neoplasms using the CAS image analyser. The smears were prepared from scrapings from fresh tumour tissue whereas the cytocentrifuge preparations were prepared from single nuclear suspensions from paraffin-embedded cell blocks from the same tumour. Histograms obtained by image analysis of the tumour scrapes were compared with those obtained on the cytocentrifuge preparations. Concordant results were obtained in four benign tumours (100%) and 32 malignant tumours (91%). The results obtained by image analysis were also compared with results obtained by flow cytometry of the tumour tissue. Discordant results were obtained for three malignant tumours. Possible reasons for the discrepancy include sampling error, tumour heterogeneity and selective loss of cell populations during processing.

Base Sequence↗

Assessment of leukocyte alkaline phosphatase by image analysis.

We have shown that it is possible to automate the assessment of leukocyte alkaline phosphatase by using an azo dye cytochemical staining procedure and a commercial, highly sophisticated image analysis instrument originally designed specifically as a differential white cell counter. The data to date indicate that values obtained by this approach are at least as precise and accurate as current manual techniques. Instrumental analysis avoids the subjectivity associated with manual interpretation of staining intensity and should permit meaningful interlaboratory comparisons. The stability of the stained smears upon exposure to immersion oil or Polymount mounting medium proved to be an unexpected bonus. In addition to such functional data on leukocytes as illustrated by this report, these instruments, with appropriate staining methods and software, can also provide clinically useful quantitative data on red cells, as have been described for reticulocytes. We hope to see more clinical applications in the future for these expensive and target-oriented image analysis instruments. They are capable of automatically providing objective quantitative information on a cell by cell basis--providing feature data that cannot be obtained by other means.

Alkaline Phosphatase↗

Quantitative determination of murine dermal elastic fibers by color image analysis: comparison of three staining methods.

We compared three different staining methods to determine if the dermal elastic fiber content of the HRS/Skh-1 hairless mouse could be accurately measured by color image analysis. Comparisons were made among Kligman's modification of Luna's mast cell stain for elastin, Unna's orcein stain with or without potassium permanganate preoxidation, and Gomori's aldehyde fuchsin stain with potassium permanganate preoxidation. The color image analysis system could be used to identify and quantify murine dermal elastin fibers in sections stained by all three methods. Gomori's aldehyde fuchsin stain with preoxidation demonstrated twice the content of dermal elastic fibers demonstrated by either Kligman's modification of Luna's mast cell stain or Unna's orcein stain with or without preoxidation. Gomori's aldehyde fuchsin method with preoxidation should be considered the stain of choice for evaluating murine dermal elastic fiber content.

Animals↗

Proficiency of the Tradescantia-micronucleus image analysis system for scoring micronucleus frequencies and data analysis.

The Tradescantia-micronucleus (Trad-MCN) bioassay is an efficient short-term test for genotoxicity of pollutants. In order to increase the efficiency and to standardize the micronucleus (MCN) scoring process, an automated scoring system was developed using the principle of image analysis in computer science. This assemblage is called the Tradescantia-micronucleus image analysis (Trad-MCNIA) system. The MCN frequencies scored by this system were compared with those scored by human observation for its proficiency. A set of low MCN frequency (around 5 MCN/100 tetrads) slides prepared from a control group, a set of medium MCN frequency (around 20 MCN/100 tetrads) slides prepared from sodium azide treated plant cuttings and a set of high MCN frequency (around 50 MCN/100 tetrads) slides prepared from X-ray treated materials were used for this study. In the low MCN frequency slides, the Trad-MCNIA system scored about the same value as human observation. In the medium and high frequency slides, MCN frequencies scored by the system were lower than those scored by human observers. This discrepancy was corrected by increasing the power of the objective of the microscope in the system. The MCN frequencies scored by the system attained 90% congruity with those scored by human observers after the correction. The scoring speed of the system was about 3.5 times as fast as that by human observers, and the data could be statistically analyzed immediately after the data scores were recorded. Further improvements can be made by upgrading the video camera and the computer speed.

Azides↗

Use of image analysis for sludge characterisation: studying the relation between floc shape and sludge settleability.

This paper starts by presenting a fully automatic image analysis procedure for characterisation of flocs and filaments in activated sludge images. Thereafter the attention is directed towards the results of four lab-scale experiments, in which image information is related to sludge settleability in terms of sludge volume index. This relation is statistically confirmed by applying a principal component analysis to the data. In addition, the redundancy in the data sets is studied with regard to floc shape descriptors and the monitoring potential of image analysis is demonstrated by means of a multiple linear regression exercise.

Image Processing, Computer-Assisted↗

[Puncture cytology of the breast: correlation of visual and image analysis studies with hormone receptor status].

It is possible to predict to some extent the hormone receptor status of carcinomas of the breast via cytomorphology of fine needle biopsy specimens. In most cases the cytologically highly differentiated carcinomas are receptor-positive and the poorly differentiated ones receptor-negative if the cases are arranged in the sequence of increasing conspicuousness of cellular criteria of malignancy. Since visual microscopic examination is subjective by nature, quantification was carried out by means of high-resolution image analysis using Pappenheim stained slides. The quantitative image analysis grading system basing on this method proved to be even more efficient than visual diagnosis.

Biopsy, Needle↗

Influence of staining on fast automated cell segmentation, feature extraction and cell image analysis.

FAZYTAN, a system for fast automated cell segmentation, cell image analysis and extraction of nuclear features, was used to analyze cervical cell images variously stained by the conventional Papanicolaou stain, the new Papanicolaou stain and hematoxylin and thionin only; the last two dyes are used as the nuclear stains in the two versions of the Papanicolaou stain. Other dyes were also tried in cell classification experiments. All cell images in the variously stained samples could be described by the same nuclear features as had been adapted for the discrimination of conventional-Papanicolaou-stained cells. Variances were lower for thionin-stained cells as compared with hematoxylin-stained cells. By application of spectrophotometry, it was confirmed that the spectra of the cytoplasmic counterstains are superimposed on those of the nuclear stains. It appears that a variety of dyes are suitable as cytologic stains for cell classification by the FAZYTAN system, provided that they achieve sufficiently strong nuclear-cytoplasmic contrast by precisely delineating the chromatin texture.

Cell Nucleus↗

Relationship of DNA ploidy to histology and prognosis in rhabdomyosarcoma. Comparison of flow cytometry and image analysis.

BACKGROUND: Although DNA ploidy correlates with prognosis in certain childhood cancers, e.g., neuroblastoma, its significance in rhabdomyosarcoma (RMS) is unclear and controversial. METHODS: Ploidy by flow cytometry (FCM) and image analysis (IA) in 26 of 27 children with RMS (17 embryonal, 3 mixed embryonal/alveolar, 5 alveolar, 1 anaplastic, 1 ectomesenchymoma) and 4 adults with pleomorphic RMS were evaluated. Statistical comparisons were analyzed between DNA content and gender, age, localization, Intergroup Rhabdomyosarcoma Study (IRS) group, and histopathologic subtype. Survival analyses were performed by the Kaplan-Meier test using the approximate chi-square statistic for the log rank test. RESULTS: The concordance rate between FCM and IA was 26 of 30 (87%); FCM was not performed in one tumor. Image analysis was more sensitive than FCM in detecting aneuploidy. Furthermore, DNA content was associated significantly with histologic subtype (P = 0.031); embryonal histology commonly was hyperdiploid (mean, 1.44; median, 1.27), whereas alveolar histology usually was near-tetraploid (mean, 1.83; median, 1.95). All four adult patients with pleomorphic RMS were aneuploid, with one showing multiple DNA peaks. No correlation between DNA content and survival was observed in the children with RMS. However, IRS group (P = 0.011) and patient age (P = 0.036) were independent prognostic indicators significantly related to survival. All adult patients died of their disease. CONCLUSIONS: Although ploidy correlates with histologic subtype, DNA content is not significantly predictive of prognosis in patients with RMS. Age at diagnosis and IRS group are independent predictors of clinical outcome in children with RMS.

Adolescent↗

Quantitative enzymatic, immunologic and histochemical studies of clinically relevant human kidney alterations using image analysis.

Tissue sections of kidneys from 172 patients with various pathologic conditions, such as hydronephrosis, interstitial nephropathies, ischemia, chronic graft rejection and renal cancer, were evaluated by an image analysis technique. Structurally defined kidney alterations were monitored for enzymatic, immunologic and other histochemical changes. Indicator enzymes of the proximal tubule, alanine-aminopeptidase (AAP), alkaline phosphatase (AP), beta-glucoronidase (beta-Gl) and gamma-glutamyltranspeptidase (GGTP), were used as parameters for screening. Enzyme concentrations were found to be significantly decreased in kidney sections of patients with various renal diseases (AP less than 15%, AAP less than 55% and beta-Gl less than 60%) as compared to normal kidney tissues (100%). AAP concentration was measured quantitatively by specific immunofluorescence using an antienzyme antibody. Immunofluorescence of AAP was comparable to that of AAP calculated by the colorimetric technique (substrate: DL-alanine-beta-naphthylamide-HCl) and decreased to less than 50% in altered kidney tissues. Furthermore, kidney cancer (less than 20%) and kidney tissue adjacent to tumours (less than 65%) displayed significantly decreased levels of kidney marker enzyme activity. This study suggests that (1) the diseased kidney is characterized by a defined change in key enzymes of the cell surface and (2) renal cancer exhibits partial depletion of these constituents. Image analysis of the pattern of enzyme activity appears to be a useful tool in the analysis of renal pathology.

Alkaline Phosphatase↗

Application of image analysis and neural networks to the pathology diagnosis of intraductal proliferative lesions of the breast.

We studied whether a computer-assisted system using a combination of data collection by image analysis and analysis by neural networks can differentiate benign and malignant breast lesions. Forty-six intraductal lesions of the breast were studied by pathologists and by the computer-assisted system. Histological evaluation was performed independently by three pathologists, and the lesions were classified into pathologically malignant (n = 12), undetermined (n = 13), and benign (n = 21). Computerized nuclear image analysis was performed using the CAS200 (Cell Analysis Systems, Elmhurst, IL) system to obtain data on nuclear morphometric and textural features. A neural network was constructed using the morphometric and texture data obtained from teaching cases of malignant and benign lesions. Then data for unknown cases were classified by the constructed neural network into neural network-malignant (n = 11), -undetermined (n = 5), and -benign (n = 30). The agreement rate between the diagnosis by pathologists and judgment by the computer-assisted system was 75%, excluding pathologically undetermined lesions. There were four false-negative but no false-positive results. False-negative cases had nuclei that were quite different from those of the teaching cases. The agreement rate obtained using either morphometric data or texture data only was lower than that using a combination of both. Selection of appropriate teaching data and incorporation of both morphometric and textural parameters seemed important for obtaining more accurate results. The present data suggest that development of a computer-assisted histopathological diagnosis system for practical use may be possible.

Breast↗

Automated image analysis for counting unstained cultured neurones.

A fully automated image analysis technique was developed for counting the number of live or fixed, unstained neurones present in a representative region of a cell culture dish. A dish containing cultured mouse hippocampal neurones was placed on the motorized stage of an inverted microscope, and the neurones were visualized using Hoffman modulation contrast optics. The resulting image was digitized, and processed by subtracting the background illumination, low pass filtering, thresholding, then deleting objects whose areas fell outside a specified range. Two threshold levels were used, each with its own area range, and the two resulting binary images were combined. The number of objects in the combined image was counted. The number of cells in each field was also counted manually, and the processing was repeated on a series of 100 fields covering a representative region of the dish. The automated counts were highly correlated with the manual counts for each of the 12 culture dishes examined in this study. The correlation coefficient was calculated for the manual and automated counts from each dish, and the values ranged from 0.91 to 0.97. Six of the dishes were treated with the envelope protein of the human immunodeficiency virus (gp120), which reduces survival of neurons in this system. The six treated dishes were found to have significantly fewer neurones than the six control dishes, using either manual or automated counting techniques.

Animals↗

Dynamic digital image analysis: emerging technology for particle characterization.

The feasibility of applying dynamic imaging analysis technology to particle characterization has been evaluated for application in the water sector. A system has been developed which captures in-situ images of suspended particles in a flowing sample stream and analyzes these images in real time to determine particle size and concentration. The technology can measure samples having a wide range of particle sizes (approximately 1.5 to 1,000 microm equivalent circular diameter) and concentrations (<1 to >1 million/ml). The system also provides magnified images of particles for visual analysis of properties such as size, shape and grayscale level. There are no sample preparation requirements and statistically accurate results are produced in less than three minutes per sample. The overall system architecture is described. The major design challenges in developing a practical system include obtaining adequate contrast for the range of particle materials found in typical water samples and achieving this under operating conditions permitting an adequate sample processing rate for real time feedback of results. Performance of the instrument is reported in reference to industry accepted particle standards and applications as an analytical tool for the water industries are considered.

Automation↗

Immunohistochemical estrogen receptor assay: quantitation by image analysis.

The immunohistochemical estrogen receptor (ER) assay quantitated by image analysis was compared with the biochemical dextran-coated charcoal (DCC) assay in 97 primary breast carcinomas. Frozen sections immunostained for ER with a monoclonal antibody (ER-ICA; Abbott Laboratories, Chicago, IL) and the peroxidase-antiperoxidase technique were scored semiquantitatively by microscopy (HSCORE) and quantitatively [percentage of nuclear area immunopositivity (PNA)] using the CAS 200 image analyzer (Elmhurst, IL). There was a significant and identical correlation between DCC and both HSCORE and PNA. According to sensitivity/specificity calculations, the cut-off points of 10 for HSCORE and 1% for PNA were chosen with positive and negative predictive values of 97.7% and 74.5%, respectively. Low DCC-positive cases between 10 and 50 fmol of ER/mg of protein were immunohistochemically ER positive only in 61.5%. Immunohistochemical results of greater than 150 HSCORE and greater than 15% PNA corresponded to high DCC results of greater than 150 fmol/mg of protein. Immunohistochemical assay quantitated by image analyzer (PNA) is a comparable alternative to biochemical ER, especially when insufficient fresh tissue is available.

Breast Neoplasms↗

Image analysis for quantitation of solid tumor drug sensitivity.

BACKGROUND: A method of assessing chemosensitivity of tissue has been described by Rotman et al. The aim of this study was to use image analysis to provide a more rapid and quantitative means of assessing drug effect on tissue proliferative capacity. METHOD: Fluoropyrimidine sensitive Ward rat colon adenocarcinoma tumor was implanted onto collagen impregnated cellulose fibers suspended on metal grids at an air-fluid interface and kept in a 95% air/5% CO2 incubator at 37 degrees C. The fluorescent microscopic image captured by a silicon intensified target (low light detecting) camera and linked to an image processing unit was measured for fluorescent brightness and tumor image area. Blinded 5-Fluorouracil (5-FU) drug treatment was begun 8 days after tumor explantation on the collagen-cellulose matrix. Tumor image area and fluorescent brightness were measured at 24 h pretreatment, 48 h posttreatment, and at 48 h post drug removal. RESULTS: Nontreated tumor cultures demonstrated an increase in area and fluorescent brightness with time following tumor implantation on the collagen gel. Dose responsiveness was seen with increasing concentrations of 5-FU. At the highest clinically achievable concentration of 5-FU (500 microM), there was a 39% decrease in area compared with the nontreated group, 113%. Linear dose responsiveness was not demonstrated between 50 and 150microM 5-FU. CONCLUSIONS: Fluoropyrimidine activity was demonstrated with the implemented image analysis system. The in vitro tumor sensitivity to FU using collagen gel was consistent with responsiveness of tumors in vivo borne by rats.

Adenocarcinoma↗

The effect of a knowledge-based, image analysis and clinical decision support system on observer performance in the diagnosis of approximal caries from radiographic images.

The aim of this study was to investigate the effect of a knowledge-based image analysis and clinical decision support system (CariesFinder, CF) on diagnostic performance and therapeutic decisions. The study material consisted of radiographic images of 102 approximal surfaces, 35 sound, 67 caries (25 caries and cavitated and 42 caries). Sixteen general practitioners were presented with (1) radiographic film images and (2) digital filmless images with the results of CF. The viewers were asked to respond whether approximal caries was present and whether a restoration was indicated. Responses were analyzed for accuracy, sensitivity, specificity and agreement. Further, the practitioners were ranked according to the accuracy of their restorative decisions and assigned to ten overlapping groups of 7 practitioners. For each group the diagnostic and therapeutic decisions were then examined for unanimity. The parameters of accuracy, sensitivity and specificity were then established for each group based on only unanimous, correct decisions. The diagnostic and therapeutic accuracy of CF alone was equal or superior to the decisions of the practitioners viewing film images alone. For unanimous decisions, CF alone was more accurate than the most accurate group of practitioners and made fewer incorrect decisions to restore non-cavitated surfaces than the practitioners. In general, dental practitioners viewing the results of CF significantly increased their ability to diagnose caries correctly, their overall diagnostic accuracy, and their ability to recommend restorations for cavitated surfaces. There was a decrease in the accuracy of their restorative decisions overall and in the specificity in particular.

Analysis of Variance↗

Spatially selective laser irradiation method controlled by real-time image analysis: optical aspects.

We present a spatially selective irradiation method for laser treatment of biological surfaces. The purpose is to irradiate only the pathological targets and to preserve the healthy surrounding parts. We are interested here in the optical arrangement of the device and in the evaluation of its physical limitations before it is used for medical purposes. The method uses real-time image analysis applied to the video imagery of the surface to be treated. The result of the image analysis generates the control signal to a spatial light modulator, allowing us to project an adequate repartition of laser irradiance on the surface to be treated. Experimental results demonstrate a spatial selectivity of approximately 10 microm for a 6.7 mm x 5 mm field. The optical system has no moving parts (unlike galvanometric scanners) and is able to irradiate multiple targets simultaneously with different doses.

Equipment Design↗

Electron microscopy and image analysis of two-dimensional crystals and single molecules of alcohol oxidase from Hansenula polymorpha.

The octameric protein alcohol oxidase from the yeast Hansenula polymorpha was studied by electron microscopy and image analysis. Two-dimensional crystals were formed by applying the protein, in a phosphate buffer containing poly(ethylene glycol) and EDTA, to a carbon-coated formvar film which had been glow-discharged in pentylamine at least several hours earlier. The crystals show p4 symmetry and have a unit cell of 12.5 X 12.5 nm2, containing one molecule. Image analysis of the crystals and of single molecules yielded two different views. From these it can be deduced that the subunits have an elongated shape and form two layers of four, stacked face to face. A tentative model of the structure is presented.

Alcohol Oxidoreductases↗

Comparative study between biochemical and histological methods and image analysis in liver iron overload.

Iron overload has been measured in 100 hepatic biopsies by three different methods: (i) biochemical assay of the liver iron concentration (LIC), (ii) histological grading (HISTO) and (iii) automated image analysis with a Leitz Texture Analysis System by estimating two parameters, (a) the total iron area (TIA) and (b) the first grey level step (FGLS) at which the iron is detected. Image analysis appears as a specific, sensitive, quick, reproducible and valid method.

Autoanalysis↗