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Application of automated image analysis to demonstrate the correlation between ras p21 expression and severity of gliomas.

We found a direct correlation between increasing ras p21 protein immunopositivity and severity of human glioma using computer-assisted, digital-image processing to quantify the amount of p21 immunoreactive to the monoclonal antibody RAP-5. We determined that there was a significant difference in reactivity between glioblastoma multiformes and more-differentiated astrocytomas (experiment-wise error less than 0.05). This result confirmed the conclusions made on the same tumors using standard light microscopy and visual examination. Immunohistochemistry quantized by automated image analysis may be a useful adjunct to current histopathological strategies since it decreases assay subjectivity and variation.

Antibodies, Monoclonal↗

[Grading of urinary bladder tumors by automated microscopic image analysis].

Studies were undertaken on biopsies from urinary papillomas and urothelial carcinomas of different grades (G1 to G3) along with normal urinary bladder mucosa from a total of 220 patients. They were performed by use of automated microscopic image analysis (system Robotron A 6471, software AMBA/R). As a result, the tumor grading was improved and lead to the separation of both papillomas and G1-carcinomas with high proliferative activity. In addition, it was possible to divide the G2-carcinomas into 2 groups which probably show a different biological behaviour.

Carcinoma, Transitional Cell↗

Large-cell acanthoma of the skin. A study by image analysis cytometry and immunohistochemistry.

Although large-cell acanthoma is a well-known clinicopathological entity, its biologic spectrum and nature are still subject to debate. We studied seven cases of large-cell acanthoma by image analysis cytometry for DNA content and by immunohistochemistry, using antibodies to proliferating cell nuclear antigen (PCNA)/cyclin. The data were compared with individual cases of seborrheic keratosis (SK), actinic keratosis (AK), and Bowen's disease (BD). The DNA distribution of large-cell acanthoma was variable. There were varying peaks at the DNA index values of 1 and 2 (diploid and tetraploid values), but all cases contained a significant aneuploid population between DNA index of 1 and 2. The mean DNA index was 1.44 (1.27-1.77); 1-20% of the cells exceeded 2, and 0-2% exceeded 3. The DNA index for lesions in the other differential diagnostic groups studied was as follows: SK, 1.0; AK, 1.4; BD, 1.8. The percentage of cells with positive nuclear staining for PCNA/cyclin was < 20% in all cases of large-cell acanthoma. The discrepancy between the high number of aneuploid and tetraploid cells observed on the DNA distribution curve and the lack of evidence for significant proliferation based on immunohistochemical stains suggest that these cells are resting cells with abnormal DNA clone. Although these results provide additional information about the biologic nature of large-cell acanthoma, they do not resolve the controversial nosologic status of lesions in this histologic group.

DNA↗

Automated image analysis of in vitro decondensation of human spermatozoa nuclei. III. Variable decondensation with and without incubation in the seminal fluid.

The effect of seminal fluid upon human spermatozoa was analyzed using in vitro chromatin decondensation and automated image analysis. A number of specimen portions processed after incubation in seminal fluid showed different total mean areas as compared to the corresponding portions processed immediately. Comparison of the results obtained with and without delay showed that the incubation in seminal fluid promoted decondensation in some cases, but retarded it in others. Thus, the seminal fluid stabilized the chromatin condensation in some spermatozoa, but not all. The stabilization may be due to the influence of prostatic zinc.

Cell Nucleus↗

Image analysis of murine chromatin fiber structure after in situ digestion with restriction endonucleases.

OBJECTIVE: To determine and quantify the differences produced in chromatin structure after selective in situ DNA digestion with restriction endonuclease (RE). STUDY DESIGN: Chromatin fiber structure from a murine cell line was analyzed under light and electron microscopy before and after in situ digestion with AluI, HinfI and HaeIII using digital image analysis (DIA). The proposed DIA-based method entails the generation of a binary image and a skeleton characteristic of the chromatin fiber. Morphologic features (surface, perimeter, shape, number of triple points and Euler number) of the chromatin structure can then be quantified from the digitized images. The results of these experiments were compared with those obtained from direct digestion of naked DNA using the same endonucleases in an attempt to correlate the distribution of restriction sites, according to the DNA fragment size obtained, with the extent of chromatin disorganization after RE in situ digestion. RESULTS: Homologous chromatin fiber regions are differentially affected by the action of each RE, although they are indistinguishable under light microscopy. CONCLUSION: The proposed analytical routine constitutes a valuable tool for uncovering subtle alterations in the structure of chromatin fiber that has been modified under different experimental conditions.

Animals↗

Tissue-specific variation of pea mitochondrial polypeptides detected by computerized image analysis of two-dimensional electrophoresis gels.

A tissue-specific variation of a total of 152 mitochondria-associated polypeptides is detailed for etiolated leaves, epicotyls and roots from the pea, Pisum satisum L., using computerized image analysis. These organ systems possess, respectively, 128, 100, and 96 mitochondria-associated polypeptides with 38, 8, and 10, respectively, being unique to each. Seventy-one polypeptides were observed to be present in all gels, but some of these also underwent a variety of quantifiable changes. Two polypeptides were identified that varied similarly to a polypeptide of interest (W), known to be abundant in pea epicotyls and potato tubers. It is postulated that these latter three polypeptides are therefore probably related functionally.

Electrophoresis, Gel, Two-Dimensional↗

Complementarity in the supramolecular design of arenaviruses and retroviruses revealed by electron cryomicroscopy and image analysis.

Arenaviruses are rodent-borne agents of diseases, including potentially lethal human hemorrhagic fevers. These enveloped viruses encapsidate a bisegmented ambisense single-stranded RNA genome that can be packaged in variable copy number. Electron cryomicroscopy and image analysis of New World Pichinde and Tacaribe arenaviruses and Old World lymphocytic choriomeningitis virus revealed pleomorphic enveloped particles ranging in diameter from approximately 400 to approximately 2,000 A. The surface spikes were spaced approximately 100 A apart and extended approximately 90 A from the maximum phospholipid headgroup density of the outer bilayer leaflet. Distinctive stalk and head regions extended radially approximately 30 and approximately 60 A from the outer bilayer leaflet, respectively. Two interior layers of density apposed to the inner leaflet of the viral lipid bilayer were assigned as protein Z and nucleoprotein (NP) molecules on the basis of their appearance, spacing, and projected volume. Analysis of en face views of virions lacking the GP-C spikes showed reflections consistent with paracrystalline packing of the NP molecules in a lattice with edges of approximately 57 and approximately 74 A. The structural proteins of retroviruses and arenaviruses assemble with similar radial density distributions, using common cellular components.

Animals↗

Image analysis and machine learning applied to breast cancer diagnosis and prognosis.

Fine needle aspiration (FNA) accuracy is limited by, among other factors, the subjective interpretation of the aspirate. We have increased breast FNA accuracy by coupling digital image analysis methods with machine learning techniques. Additionally, our mathematical approach captures nuclear features ("grade") that are prognostically more accurate than are estimates based on tumor size and lymph node status. An interactive computer system evaluates, diagnoses and determines prognosis based on nuclear features derived directly from a digital scan of FNA slides. A consecutive series of 569 patients provided the data for the diagnostic study. A 166-patient subset provided the data for the prognostic study. An additional 75 consecutive, new patients provided samples to test the diagnostic system. The projected prospective accuracy of the diagnostic system was estimated to be 97% by 10-fold cross-validation, and the actual accuracy on 75 new samples was 100%. The projected prospective accuracy of the prognostic system was estimated to be 86% by leave-one-out testing.

Biopsy, Needle↗

Image analysis, methanogenic activity measurements, and molecular biological techniques to monitor granular sludge from an EGSB reactor fed with oleic acid.

Morphological changes in anaerobic granular sludge fed with increasing loads of oleic acid were quantified by image analysis. The combination of this technique with data on the accumulation of adsorbed long chain fatty acid and with the molecular characterization of microbial community gave insight into the mechanisms of sludge disintegration, flotation and washout. It was found that the bacterial domain was more affected than the archaeal domain during this process. However, no acetoclastic activity and onlya residual hydrogenotrophic activity were detected in the sludge at the end of the operation.

Adsorption↗

[Study of the microcirculatory bed in inflammation by the method of automatic image analysis].

Peritonitis in rats was provoked by intraperitoneal injection of carrageenan (5 mg in 5 ml 0.9% NaCl). Twenty-four hours after injection microvessels of the intestinal mesentery were examined by intravital microscopy and automatic image analysis. Using the system of the texture analysis LEITZ-TAS the internal and external diameters of microvessels, the dilatation coefficient, the blood content index, intensity and extent of permeability disturbances of the vascular wall, and the number of mast cells per unit of the mesentery surface area were determined. During inflammation, the internal diameter of the capillaries increased from 6.6 + 2 to 7.4 + 0.2 microM and that of the venules from 15.2 + 0.9 to 18.8 + 1.1 microM. Dilatation of the arterioles was revealed by the dilatation coefficient which changed from 25 + 1.0 to 19 + 1.1%. At the same time the blood content index did not change significantly. During inflammation, the number of mast cells decreased 2.53 times as a result of degranulation. Permeability of the microvascular wall increased sharply (the intensity by 2.74 times, the extent by 15.3 times). The method applied enables one to obtain under intravital conditions a quantitative description of the microcirculatory status during inflammation.

Animals↗

Quantitative histological study of mucosal inflammatory cell densities in endoscopic duodenal biopsy specimens from dyspeptic patients using computer linked image analysis.

Inflammatory cell counting in endoscopic biopsy sections was carried out on duodenal mucosal samples from defined sites in patients with duodenal ulcer, duodenitis but no ulcer, non-ulcer dyspepsia, and asymptomatic controls using computer linked image analysis. The variables measured included polymorphonuclear and mononuclear cells per mm of superficial epithelium and per mm2 lamina propria. Duodenal ulcer crater margin and mucosal biopsy specimens from endoscopically inflamed mucosa in the group with duodenitis but no ulcer showed significantly higher inflammatory cell counts than endoscopically normal non-ulcer dyspepsia and control mucosa. Biopsy specimens from non-ulcer dyspepsia patients showed significantly higher lamina propria polymorphs than control group mucosa. Endoscopically normal duodenal ulcer and duodenitis but no ulcer mucosa also showed significantly higher acute and chronic inflammatory cell counts than controls. The prevalence of Helicobacter pylori in duodenal biopsy specimens was low (0-22%) and unrelated to local inflammatory response. Despite histological appearances, duodenal biopsy specimens from non-ulcer dyspepsia patients showed significantly higher inflammatory cell infiltration than control specimens, suggesting that at least some represent part of a spectrum of subclinical peptic disease.

Adult↗

Mean nuclear area and chromosomal DNA content of squamous cell carcinomas of the oral cavity using computerized image analysis.

Morphometric measurements of nuclei may be of prognostic value in some cancers. In this present study we have evaluated the mean nuclear area (MNA) of 50 squamous cell carcinomas of the oral cavity (SCCOC) using computerized image analysis. Since chromosomal DNA content is a reflection of the DNA content in the nucleus, we have evaluated the relationship between MNA and chromosomal DNA. Thirteen tumours had a MNA greater than 49.9 microns2 and 37 had a MNA less than this. Six tumours were classified as hypodiploid, 29 as diploid and 15 as aneuploid. There were 44 node-negative patients and six node-positive. When comparing MNA in these groups, 50 per cent of node-positive patients had a larger MNA whilst only 20 per cent of the node-negative group had a large MNA. The correlation coefficient between MNA and DNA indices was r = 0.75. The greater nuclear size is possibly a reflection of a more aggressive tumour biology in the node-positive patients. We conclude that a large MNA may be a marker of aggressive tumour biology in this group. In the future, we aim to evaluate the prognostic significance of MNA in patients with SCCOC.

Adult↗

Prosthetic hip failure: retrospective radiograph image analysis of the acetabular cup.

A method has been developed for quantifying movement and wear of the acetabular component (cup) of total hip replacements (THR) from routine postoperative and review radiographs. The method uses both interactive and automatic computer image analysis techniques. Dimensions of the prosthesis are used to scale the measurements and so overcome variation in radiographic alignment. The application of the method is illustrated by retrospective investigations of cup migration and wear using review radiographs taken over a follow-up of at least 12 years.

Acetabulum↗

A study of the solar effect on actinic keratoses by quantification of elastic fibres using an image analysis system.

It is widely accepted that elastotic changes of the skin are primarily an indicator of cumulative sun exposure of the dermis and are a characteristic finding of actinic keratoses. To date, there have been few reports that measure the amount of elastic tissue objectively and quantitatively, especially in actinic keratoses. The computerized image analysis method has proved useful recently in determining the area of elastic fibres. Using this method, we objectively quantified the elastotic tissue in actinic keratoses and evaluated the relationship between the degree of dermal elastosis, epidermal atypia and histological types of actinic keratoses. Of the 28 actinic keratoses studied, the average percentage area of the elastic fibre was 40.48 +/- 14.48 (mean +/- SD) percentile. There was a 3.65-fold increase in the amount of elastic fibre in actinic keratoses compared with that of seborrhoeic keratoses occurring on the face (p < 0.00001). In addition, the more severe the atypia, the greater the area of elastic fibres in a representative section of the dermis. In conclusion, we observed that on quantitative assessment of elastic tissue in actinic keratoses, the percentage area of the elastic fibres in a representative section of the dermis ranges from 34.86 to 46.11%. This result may provide information for use in histological diagnosis of actinic keratoses and evidence for the possible role of sunlight in the pathogenesis of actinic keratosis.

Adult↗

Variable pitch in frozen-hydrated sickle hemoglobin fibers: an image analysis model study.

The intracellular polymerization of deoxyhemoglobin S (HbS) into helical fibers is the primary pathological event which gives rise to sickle cell disease. The structure of these fibers has previously been studied by electron microscopy of negatively stained specimens. We are extending these studies with unstained frozen-hydrated HbS fibers (cryo-EM), which afford better visualization of the internal details of the fiber structure than can be achieved by negative staining, but have lower signal-to-noise ratio images. The pitch of the HbS fiber structure varies locally along any given particle. Because rotation about the particle axis thus is partially decoupled from translation along the axis, the pitch and angular rotation of a fiber unit cell cannot be inferred by symmetry (as is the case with constant pitch helices). Image analysis procedures are presented which are capable of explicitly identifying the pitch and angular rotation of individual HbS fiber unit cells having low signal-to-noise ratios. Fiber images are divided into segments one unit cell long (63 A) which are analyzed in two steps. First each unit cell is aligned with constant pitch electron density reference models by cross-correlation. Correlation coefficients are then used to determine angular rotation and pitch. This procedure was tested, and found to be robust, using model images corrupted to simulate experimental problems normally encountered in the analysis of cryo-electron micrographs. The effects of limited resolution, low signal-to-noise ratio, scaling errors, and rotational and axial misalignment are described.

Computer Simulation↗

Knowledge-based multi-modality three-dimensional image analysis of the brain.

With the recent advances in medical imaging, three-dimensional anatomical and metabolic images of the brain are now available through MR/CT and PET/SPECT imaging modalities. Computerized multi-modality three-dimensional brain image registration and analysis can provide important correlated information for improving diagnosis and studying the pathology of disease. Such analysis may also provide help in planning brain surgery. Further, an anatomical model based quantification and analysis of internal structure can be used to develop a computerized anatomical atlas. Conventional anatomical atlases provide rigid spatial distribution of internal structures extracted from a single subject. The proposed computerized anatomical atlas provides probabilistic spatial distributions which can be easily updated to incorporate the variability of brain structures of subjects selected from pre-defined groups. This paper first presents a review of the current trends in knowledge-based segmentation, labeling, and analysis of MR brain images and then describes the Principal Axes Transformation based registration of three-dimensional MR brain images to develop composite models of selected internal brain structures. The composite models can be used as a computerized anatomical atlas in model-based segmentation and labeling of MR brain images. Three-dimensional labeled MR images of the brain can also be registered and correlated with PET images for analyzing the metabolic activity in the anatomically selected volume of interest. On the other hand, a volume of interest can be selected using the metabolic information and then analyzed for correlated anatomical information using the registered MR-PET images.

Algorithms↗

A 'macro' image analysis of fura-2 fluorescence to visualize the distribution of functional glutamate receptor subtypes in hippocampal slices.

The distribution of functional glutamate receptor subtypes was visualized as regional changes in intracellular Ca2+ concentration induced by administration of a specific agonist for each subtype. An image of the fura-2 fluorescence from a whole hippocampal slice was directly excited at 340 or 380 nm through an optical fibre and observed via a low-magnification objective lens (x4). A heterogeneous distribution of N-methyl-D-aspartate (NMDA) and non-NMDA subtypes was demonstrated by the ratio of fluorescence intensities before and after administration of each agonist. This 'macro' image analysis can reveal distributions of multiple functional receptors in a living brain slice.

Animals↗