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Clinical usefulness of computerized colposcopy: image analysis and conservative management of mild dysplasia.

OBJECTIVES: The purposes of this study were to evaluate the clinical usefulness of computerized colposcopy and image analysis, to investigate the correlation between lesion size and grade of dysplasia, and to examine and record the colposcopic changes associated with progression or regression of cervical dysplasia. METHODS: Sixty-eight patients with a fully visualized squamocolumnar junction and a histopathologic diagnosis of mild dysplasia were serially monitored for a period of 1 year using computerized colposcopy and image analysis. All patients had baseline computer-assisted measurements of their cervical lesions and repeat measurements at 3-month intervals. RESULTS: The mean age of the patients was 31 years, and the mean size of the colposcopically visualized lesions was 58 mm2. During the 12-month observation, 5.9% of the lesions increased in size, 32.4% decreased in size, 13.2% remained unchanged, 20.6% disappeared, and 27.9% completely changed location. In patients with an increase in lesion size (N = 4), a repeat biopsy was performed, revealing a progression to moderate dysplasia. Treatment was withheld from patients whose lesions disappeared (N = 14), decreased in size (N = 22), or remained unchanged (N = 9). Active therapy was unnecessary in 66% of cases and repeat biopsy was avoided in 94.1%. CONCLUSIONS: Computerized colposcopy provides objective information and may be an adjunct to cytology and histopathology in some cases. Computerized colposcopy replaces subjective colposcopic evaluation with objective computer assessment and may hold promise for conservative management of cervical dysplasia.

Adult↗

Quantitative study of germinal center area in normal and diseased tonsils using image analysis.

The aim of this study was to measure the germinal center area of secondary follicles in a consistent and unbiased manner using image analysis. Tonsil specimens were obtained from children with clinical evidence of recurrent tonsillitis and/or idiopathic tonsillar hypertrophy. Normal control biopsy specimens were obtained from children with no history of ear, nose, or throat infections. The results show a significant increase in the germinal center area in tonsils exhibiting hypertrophy when compared to normal controls (p < .002). Image analysis is an important tool for making quantitative size comparisons in lymphoid tissues.

Acute Disease↗

DNA image analysis in childhood acute lymphoblastic leukemia.

DNA index (DI) and percentages of cells in S and G2/M phase were determined in Feulgen stained nuclei of blasts from 31 cases of childhood ALL at diagnosis. In 6 cases the results of DNA analysis and cytogenetics were concordant showing hyperdiploidy. Two other cases with normal karyotype were revealed as DNA aneuploid with image analysis. Cases with cytogenetic abnormalities like translocation, deletion or presence of single or double supernumerary chromosomes had DI within normal ranges. Nine ALL cases (29%) were found to be DNA aneuploid--8 hyperdiploid and 1 hypodiploid. The percentages of cells in S and G2/M phase for blasts from bone marrow (mean 17.6%) were significantly higher than those estimated in the peripheral blood (mean 1.57%). We conclude that analysis by image cytometry can detect aneuploid DNA content even in cases, which showed a normal karyotype and provides new information concerning the biological aspects of leukemic blasts.

Adolescent↗

Automated cell differentiation of bronchoalveolar lavage samples with two-step image analysis.

OBJECTIVE: To establish an easy method for performing an automated differential cell count on bronchoalveolar lavage (BAL) cytocentrifuge samples using morphometric parameters acquired by a digital image analyzer. STUDY DESIGN: The study population comprised 20 satisfactory cytocentrifuge preparations routinely processed in our laboratory. Images of at least 40 fields of interest were digitized and stored. The images were analyzed to assess the planimetric parameters. These were used for cell identification. Basic morphometric parameters were acquired by an automated image analysis procedure. The results obtained by this method are compared to those of the manual counting procedure. RESULTS: Following sequential analysis, computerized identification corresponded, in > 97% of the cells, to the results of manual cell typing. After data analysis, < 1% of cells remained unidentifiable. CONCLUSION: Although the number of specimens evaluated is not yet large enough to allow a definitive statement, the use of image analysis systems seems a promising approach in automated differential cell counting in BAL preparations.

Bronchoalveolar Lavage Fluid↗

Quantitative comparison and evaluation of two commercially available, two-dimensional electrophoresis image analysis software packages, Z3 and Melanie.

While a variety of software packages are available for analyzing two-dimensional electrophoresis (2-DE) gel images, no comparisons between these packages have been published, making it difficult for end users to determine which package would best meet their needs. The goal here was to develop a set of tests to quantitatively evaluate and then compare two software packages, Melanie 3.0 and Z3, in three of the fundamental steps involved in 2-DE image analysis: (i) spot detection, (ii) gel matching, and (iii) spot quantitation. To test spot detection capability, automatically detected protein spots were compared to manually counted, "real" protein spots. Spot matching efficiency was determined by comparing distorted (both geometrically and nongeometrically) gel images with undistorted original images, and quantitation tests were performed on artificial gels with spots of varying Gaussian volumes. In spot detection tests, Z3 performed better than Melanie 3.0 and required minimal user intervention to detect approximately 89% of the actual protein spots and relatively few extraneous spots. Results from gel matching tests depended on the type of image distortion used. For geometric distortions, Z3 performed better than Melanie 3.0, matching 99% of the spots, even for extreme distortions. For nongeometrical distortions, both Z3 and Melanie 3.0 required user intervention and performed comparably, matching 95% of the spots. In spot quantitation tests, both Z3 and Melanie 3.0 predicted spot volumes relatively well for spot ratios less than 1:6. For higher ratios, Melanie 3.0 did much better. In summary, results suggest Z3 requires less user intervention than Melanie 3.0, thus simplifying differential comparison of 2-DE gel images. Melanie 3.0, however, offers many more optional tools for image editing, spot detection, data reporting and statistical analysis than Z3. All image files used for these tests and updated information on the software are available on the internet (http://www.umbc.edu/proteome), allowing similar testing of other 2-DE image analysis software packages.

Electronic Data Processing↗

Image analysis and data management of ELISPOT assay results.

The recent renaissance of enzyme-linked immunospot (ELISPOT) assays largely is the result of advances in image analysis. Information on the frequency of antigen-specific T-cells and also on the secretion rate of the individual cells is captured in spots generated using this technique. Although the overall assessment of ELISPOT results can be conducted visually, this is inevitably subjective, inaccurate, and cumbersome. In contrast, objective, and accurate measurements are fundamental to good science. Validated image analysis algorithms and procedures, therefore, have become critical for elevating the quality of ELISPOT assays results. As cytokine and granzyme B ELISPOT assays become the gold standard for monitoring antigen-specific T-cell immunity in clinical trials, the pressure increases to make ELISPOT analysis transparent, reproducible and tamperproof, complying with Good Laboratory Practice and Code for Federal Regulations Part 11 guidelines. In addition, ELISPOT assays in clinical and basic science settings frequently require high degrees of throughput, thus further raising the need for advanced data management and statistical analysis. The ImmunoSpot software portfolio has been specifically designed to meet all these needs, using the techniques described in this chapter.

Cytokines↗

Image analysis system for assessment of immunohistochemically stained proliferative marker (MIB-1) in oesophageal squamous cell carcinoma.

The prognosis of oesophageal cancer patients is related to the portion of MIB-1 positively stained tumour nuclei. In this study, an image analysis system was developed based on LEICA Image Processing and Analysis System to reduce the subjective, tedious and inaccurate manual counting of nuclei staining. Representative oesophageal cancer tissues were collected and immunohistochemical preparations of MIB-1 were made. The MIB-1 positive nuclei in these tumours were assessed by quantitative counting, semi-quantitative counting, and three computer assessment methods using LEICA QWIN PRO. Our results showed that computer assessment methods were reliable and consistent. The procedure using the system could be accomplished within 15 min. Overlapped or missed counting of nuclei by the observer were eliminated. The image analysis system can really assist experts in obtaining reliable data for the prognosis of oesophageal cancer patients quickly.

Biomarkers↗

A digital SLR or intra-oral camera: preference for acquisition within an image analysis system for measurement of disclosed dental plaque area within clinical trials.

OBJECTIVES: The aim of this investigation was to compare the accuracy and reproducibility of images acquired from two types of camera (digital SLR camera, Kodak DCS410, KJP, UK and an intra-oral camera, Schick Technologies, UK) for application within an imaging system used for the quantification of disclosed dental plaque. METHODS: Subjects refrained from brushing their teeth for 24 h. Their teeth were then disclosed with Erythrosin FDC Red 3 and duplicate sets of images were obtained by each examiner of the upper central and lateral incisors. Images were then saved and measured for dental plaque area. These data were used to calculate reliability. RESULTS: No statistically significant bias in the measurements of plaque area was found. The reliability results showed the method was reliable. However, the image analysis system incorporating the 35 mm SRL camera was more reliable for both operators, as demonstrated by their intra-operator results. This was also collaborated by the inter-operator results. CONCLUSION: In this investigation the digital SLR camera combined with the image analysis system and frame permitted greater reliability of dental plaque surface area measurements than the digital intra-oral camera. This conclusion was derived both from the reliability data and from the perceived ease of use and flexibility of both camera types. When combined with the use of the custom-made frame, the images using the digital SLR camera were highly reproducible, confirming the systems application within clinical trials.

Adult↗

Distribution of lymphoid cells in tonsillar compartments in relation to infection and age. A quantitative study using image analysis.

The distribution of lymphoid cells in the mantle zone, germinal center, interfollicular area, and subepithelial area of the tonsil was evaluated quantitatively by image analysis in 66 subjects aged 3 to 66 years. The number of Ig-positive cells in the tonsil decreased with advancing years in all compartments. This inverse correlation to age was statistically significant for IgD-, IgM-, and IgG-positive cells. For T-cells, overall change of each T-cell subset with age was smaller than those of Ig-positive cells. An age-related marked decline was seen for CD4-positive cells only in the subepithelial area and for CD8-positive cells only in the interfollicular area. Ki-67-positive cells, cells undergoing active division, were mainly found in the germinal centers and also diminished with advancing years. Patients with frequent episodes of tonsillitis demonstrated a significant increase of IgD-positive cells and IgG-positive cells in interfollicular and subepithelial compartments and a decrease of CD4-positive T-cells in the germinal centers and subepithelial areas. These results suggest that the tonsillar involution with age is immunologically associated in all compartments with the decrease of Ig-positive cells and Ki-67-positive activated cells resulting in a relative increase of T-cell subsets. The method of image analysis provides a novel and unique approach for quantitative immunohistological study of the tonsil.

Adolescent↗

Image analysis studies of the degree of irreversible deformation of sickle cells in relation to cell density and Hb F level.

We analyzed, quantitatively by image analysis, the degree of irreversible deformation of red cells (SS cells) from patients with homozygous sickle cell disease, and studied the relationships among the degree of irreversible cell deformation, cell density, and Hb F level. SS cells from 25 patients (aged 1-36 years) whose Hb F levels ranged from 2.5% to 40.0%, were fully oxygenated and then were separated into four fractions by density centrifugation. Every fraction was studied for morphology and Hb F content. We found that the irreversible deformation of SS cells from the circulation occurred mainly by elongation and that the degree of elongation was extremely variable. We also found that in the cells of patients with Hb F levels < 20% the degree of irreversible elongation increases as cell density increases, suggesting that dehydration occurs concomitantly with irreversible elongation. Statistical analysis (Student t test) indicated that there were significant differences (P = 0.008 or < 0.001) in the degree of elongation among density-fractionated SS cells from patients with Hb F < 20%, although there was no significant difference (P > 0.1) among those from patients with Hb F > or = 20%. We also found that cell density increased as Hb F level of the density fraction decreased in all patients with Hb F < 20% but not always in those with Hb F > or = 20%. This suggests that cells with lower Hb F levels are selectively susceptible to dehydration. Furthermore, we found that the mean degree of irreversible elongation decreases linearly with increasing levels of Hb F and reaches the normal range at 21-24%. Since the degree of irreversible deformation of SS cells quantified by image analysis is directly related to cell density, and inversely to Hb F levels, mechanical stress or membrane damage caused by Hb S polymerization may be an important factor in the formation of dense cells in vivo.

Adolescent↗

Zonal image analysis of tumour vascular perfusion, hypoxia, and necrosis.

A number of laboratories are utilising both hypoxia and perfusion markers to spatially quantify tumour oxygenation and vascular distributions, and scientists are increasingly turning to automated image analysis methods to quantify such interrelationships. In these studies, the presence of regions of necrosis in the immunohistochemical sections remains a potentially significant source of error. In the present work, frozen MCa-4 mammary tumour sections were used to obtain a series of corresponding image montages. Total vessels were identified using CD31 staining, perfused vessels by DiOC(7) staining, hypoxia by EF5/Cy3 uptake, and necrosis by haematoxylin and eosin staining. Our goal was to utilise image analysis techniques to spatially quantitate hypoxic marker binding as a function of distance from the nearest blood vessel. Several refinements to previous imaging methods are described: (1) hypoxia marker images are quantified in terms of their intensity levels, thus providing an analysis of the gradients in hypoxia with increasing distances from blood vessels, (2) zonal imaging masks are derived, which permit spatial sampling of images at precisely defined distances from blood vessels, as well as the omission of necrotic artifacts, (3) thresholding techniques are applied to omit holes in the tissue sections, and (4) distance mapping is utilised to define vascular spacing.

Animals↗

Double in situ hybridization in combination with digital image analysis: a new approach to study interphase chromosome topography.

Double in situ hybridization with mercurated and biotinylated chromosome specific DNA probes in combination with digital image analysis provides a new approach to compare the distribution of homologous and nonhomologous chromosome targets within individual interphase nuclei. Here we have used two DNA probes representing tandemly repeated sequences specific for the constitutive heterochromatin of the human chromosomes 1 and 15, respectively, and studied the relative arrangements of these chromosome targets in interphase nuclei of human lymphocytes, amniotic fluid cells, and fibroblasts, cultivated in vitro. We have developed a 2D-image analysis approach which allows the rapid evaluation of large numbers of interphase nuclei. Models to test for a random versus nonrandom distribution of chromosome segments are discussed taking into account the three-dimensional origin of the evaluated 2D-distribution. In all three human diploid cell types the measurements of target-target and target-center distances in the 2D-nuclear image revealed that the labeled segments of the two chromosomes 15 were distributed both significantly closer to each other and closer to the center of the nuclear image than the labeled chromosome 1 segments. This result can be explained by the association of nucleolus organizer regions on the short arm of chromosome 15 with nucleoli located more centrally in these nuclei and does not provide evidence for a homologous association per se. In contrast, evaluation of the interphase positioning of the two chromosome 1 segments fits the random expectation in amniotic fluid and fibroblast cells, while in experiments using lymphocytes a slight excess of larger distances between these homologous targets was occasionally observed. 2D-distances between the labeled chromosome 1 and 15 segments showed a large variability in their relative positioning. In conclusion our data do not support the idea of a strict and permanent association of these homologous and nonhomologous targets in the cell types studied so far.

Cell Nucleolus↗

Location of subunits within the acetylcholine receptor by electron image analysis of tubular crystals from Torpedo marmorata.

The binding sites on the nicotinic acetylcholine receptor of labels specific for the alpha-, beta-, and delta-subunits were determined by electron image analysis, using tubular crystals of receptors grown from the postsynaptic membranes of Torpedo marmorata electric organ. The labels were alpha-bungarotoxin (which attaches to the acetylcholine binding sites on the pair of alpha-subunits), Fab35 (a monoclonal antibody Fab fragment directed against the main immunogenic region of the alpha-subunit), Fab111 (a monoclonal antibody Fab fragment directed against a cytoplasmic site on the beta-subunit), and wheat germ agglutinin (which binds to N-acetylglucosamine residues on the delta-subunit). These labels, bound to receptors in the crystals, were located by comparing labeled with native structures, averaged in each case over more than 5,000 molecules. From the assignments made, we find that the clockwise arrangement of subunits around the receptor, viewed from the synaptic face, is: alpha, beta, alpha, gamma, and delta; that the main immunogenic region is at (or close to) the side of the alpha-subunit; and that the two acetylcholine binding sites are at the synaptic end of the alpha-subunits, 27-28 A from the central axis and approximately 53 A apart. In the crystal lattice, neighboring molecules are paired so that their delta- and alpha-subunits are juxtaposed, an organization that appears to relate closely to the grouping of receptors in vivo.

Animals↗

Digital image analysis in dental research applied for treatment of fissures on occlusal surfaces of premolars.

The aim of this paper was to quantitatively assess caries changes of teeth by using digital image analysis. Digital images of stained sections of crowns of teeth were acquired with a computer-assisted light microscope. In each image, spots representing the main and total demineralization of enamel were segmented to determine their area. The area of total demineralization was significantly different between premolars with sealed fissures and unprotected premolars as indicated by the Mann-Whitney test. Fissures on occlusal surfaces of premolars were characterized by their width, height, and distance from the bottom of the fissure to the enamel-dentin junction. This distance was also significantly different between protected and unprotected teeth. The results of our in vitro studies of enamel lesions allow us to plan an effective diagnosis, prophylaxis, and treatment of early caries changes in in vivo conditions.

Adolescent↗

Rapid and sensitive neurotoxicity test based on the morphological changes of PC12 cells with simple computer-assisted image analysis.

In order to develop a rapid and sensitive bioassay for the screening of chemicals with possible neurotoxicity, a computer-assisted simple image-analysis system was developed to quantify small changes in the specific morphology of the cultured pheochromocytoma cell line, PC12. This cell forms a neuron-like microfibril network (neurites) in response to a nerve growth factor (NGF) stimulation in vitro. Dichrolvos (DDVP) and methylmercury chloride (MMC) were employed as model neurotoxicants. In DDVP treatment, there was no large difference in the ED50s (effective dose that reduces the morphological index by 50%) among the toxicities determined from various morphological indices, but they were significantly lower than those observed by whole-cell-area-based toxicity assay using the hepatoblastoma cell line, Hep G2. In contrast, in MMC treatment, neurite-length-based toxicity was observed as early as 2 h, and at 48 h this was lower by over three orders of magnitude compared with whole-cell-area-based one (2.06 x 10(-7) mM vs. 6.42 x 10(-4) mM). These results demonstrate that the developed bioassay using image analysis of nerve-tissue-derived cell morphology allows us to screen possible neurotoxic chemicals very rapidly with highly enhanced sensitivity, particularly for some chemicals that preferentially act on nerve fibers.

Journal Article↗

An image analysis study of DNA content in early colorectal cancer.

The DNA content of 168 consecutive T3,N0,M0 (Dukes' B, Astler-Coller B2) colorectal cancers was studied using image analysis on formalin-fixed paraffin-embedded tissues. 72 cases (43%) were classified as diploid and the remaining 96 (57%) as non-diploid. After a median follow-up period of 6.7 years, a significant survival advantage was found for diploid compared with non-diploid cases (logrank test; P = 0.008). The long-term (8 year) survival rate was 70% for diploid and 46% for non-diploid tumours. Subgroup analysis showed that the survival advantage conferred by tumour diploidy was greatest in large (> or = 5 cm) cancers and was found both in colonic and rectal cancer cases. These data indicate that tumour ploidy status measured by image analysis might be useful in determining risk of colorectal cancer recurrence and death in patients following resection of early colorectal cancer.

Adult↗

Automatic extraction and measurement of leukocyte motion in microvessels using spatiotemporal image analysis.

This paper describes a computer vision system for the automatic extraction and velocity measurement of moving leukocytes that adhere to microvessel walls from a sequence of images. The motion of these leukocytes can be visualized as motion along the wall contours. We use the constraint that the leukocytes move along the vessel wall contours to generate a spatiotemporal image, and the leukocyte motion is then extracted using the methods of spatiotemporal image analysis. The generated spatiotemporal image is processed by a special-purpose orientation-selective filter and a subsequent grouping process newly developed for this application. The orientation-selective filter is designed by considering the particular properties of the spatiotemporal image in this application in order to enhance only the traces of leukocytes. In the subsequent grouping process, leukocyte trace segments are selected and grouped among all the segments obtained by simple thresholding and skeletonizing operations. We show experimentally that the proposed method can stably extract leukocyte motion.

Algorithms↗

Improvement of the quantification of estrogen and progesterone receptors in paraffin-embedded tumors by image analysis.

It has been shown previously that estrogen receptors (ER) detected by immunohistochemical examination of paraffin-embedded tissue sections could be quantified by computerized image analysis. Several factors were identified that were, in part, responsible for the modest correlation obtained with biochemical assay results. In the present study, 45 formalin-fixed, paraffin-embedded breast carcinomas from a previous study were reevaluated to determine if current methods could provide a better correlation and more consistent results. Sections of the tumors were made to react with estrogen and progesterone receptor antibodies (ER-ICA and PgR-ICA) and the intensity of the stain was quantified using an image analysis system. Vimentin immunostain was used to assess the degree of antigenic loss. Quantitation was performed only on the areas with nuclear staining. The correlation of the estrogen and progesterone receptor values obtained by the dextran charcoal-coated method with the percentages of stained areas and with the intensity of the stain was excellent. The agreement between both methods was 91.1% for estrogen and 86.7% for progesterone receptor values. These results represent a significant improvement compared with those found in a previous study (87% agreement for estrogen receptor). The current approach to estrogen and progesterone receptor quantitation is simplified and eliminates subjectiveness in the selection of the fields for evaluation. The studies are reproducible because discrepancies due to sampling techniques are excluded. Finally, the method validates the technique as a substitute for cytosol-based methods. The results of the two vastly dissimilar assays, the pitfalls of the "gold standard" dextran charcaol-coated assay, and the need for retrospective studies to acquire a range of quantified ER-ICA and PgR-ICA values with clinical significance are discussed.

Breast Neoplasms↗