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An image analysis system that estimates conjunctival hyperemia is described. The system gives a good estimate of minor to moderate hyperemia in the conjunctiva, and is easy and fast to operate.
1. Among different etiological concepts in schizophrenia research is the disconnect on hypothesis involving distributed brain regions. Adequate empirical research requires correlational studies of multiple brain regions. In this pilot study, the authors therefore tested the applicability of an automated image analysis device as a scanning tool to detect cytoarchitectural abnormalities in Brodmann area (BA) 10. 2. The authors applied the gray level index (GLI) method as automated image analysis on 10 schizophrenic brains compared to 10 controls. The GLI as perikarya-neuropil-ratio is obtained as the ratio between the area covered by cellular cross sections and the area of the total measuring field in 101 continous measuring fields from pial surface to the cortical depth. Resulting data provide a specific cytoarchitectonic profile curve. An analysis was performed separately for mean GLI and GLI values in six compartments covering approximately the different cortical laminae. 3. A statistically significant reduction of the mean GLI was demonstrated in the schizophrenic group covering laminae III to VI, as detected by multivariate analysis and corroborated by univariate analyses and t-tests. 4. This result clearly underlines a cytoarchitectonic disturbance with a perikarya neuropil-ratio reduction in BA 10, that is associated with schizophrenia. This is suggestive either of an increased neuropil fraction or a decreased neuronal perikarya fraction. The latter could either be due to a volume or a total number reduction of neuronal perikarya. These data are compatible with previously published data on cell loss in schizophrenics in BA 10.
AIMS: To compare the results of breast cancer sections with HercepTesttrade mark immunohistochemistry (IHC) scores ranging from 0 to 3+ with fluorescence in situ hybridisation (FISH) for HER2 amplification. The HER2 digital scoring application of the Micrometastasis Detection System (MDS) was used, together with manual scoring of FISH and HercepTest, to determine whether this system provides an accurate alternative. METHODS: Paraffin wax embedded sections were stained using HercepTest and analysed by eye and automated quantitative image analysis. FISH was performed using the PathVysion fluorescent probe and scored by eye and automated quantitative image analysis using MDS. RESULTS: Of 114 cases, 26% were amplified by FISH, whereas only 18% scored 3+; 32% of IHC 2+ cases were amplified by FISH, and one showed borderline amplification. Six percent of IHC negative cases (0 or 1+) were amplified by FISH, and one showed borderline amplification. Of IHC 3+ cases, 10% were non-amplified by FISH. Classification discrepancies were seen in 18% of HercepTest cases scored by eye and using the MDS system. MDS was consistent with visual FISH scoring and correctly differentiated most ambiguous visual IHC scores. CONCLUSIONS: FISH provides a more accurate and consistent scoring system for determining HER2 amplification than HercepTest. The MDS system provides a reliable, consistent alternative to visual IHC and FISH scoring. IHC is still a valuable technique to aid in identification of isolated or heterogeneous tumour populations for subsequent FISH analysis, and a combined FISH and HercepTest approach to all breast cancer cases may be the most efficient strategy.
This paper examines temporal changes in the organisation of the cytoskeleton within isolated articular chondrocytes cultured for up to 7 days in agarose constructs. Fluorescent labelling and confocal microscopy were employed to visualise microtubules (MT), vimentin intermediate filaments (VIF) and actin microfilaments (AMF). To quantify the degree of cytoskeletal organisation within populations of cells, a novel image analysis technique has been developed and fully characterised. Organisation was quantified in terms of an Edge Index, which reflects the density of 'edges' present within the confocal images as defined by a Sobel digital filter. This parameter was shown to be independent of image intensity and, for all three cytoskeletal components, was validated statistically against a visual assessment of organisation. Both MT and VIF exhibited fibrous networks extending throughout the cytoplasm, while AMF appeared as punctate units associated with the cell membrane. The use of the Edge Index parameter revealed statistical significant temporal variation, in particular associated with VIF and AMF. These findings indicate the possibility of cytoskeletal mediated temporal variation in many aspects of cell behaviour following isolation from the intact tissue. Furthermore, the image analysis techniques are likely to be useful for future studies aiming to quantify changes in cytoskeletal organisation.
Argyrophil nucleolar organizer regions (AgNORs) were morphometrically evaluated in rat anterior pituitaries of control and oestrogenized rats using an automatized (LUCIA M--Laboratory Universal Computer Image Analysis Micro) and a non-automatized approaches to investigate not only the sensitivity and effectiveness of the automatized procedure but also the morphology of AgNORs in oestrogen-induced rat anterior pituitary hyperplasia. In the experimental oestrogen-induced rat anterior pituitary hyperplasia, the image analysis system LUCIA M was compared to a non-automatized morphometric procedure and proved to be very efficient and yielded analogical results. The AgNOR morphology in the oestrogenized pituitaries was characterized by an increase of the total AgNOR area and clustering of AgNORs in the large nucleoli.
BACKGROUND: Successful automated chromosome analysis requires the development of new techniques to increase and standardize chromosome length and improve banding patterns. METHODS: Human and plant cells were pretreated with the DNA intercalator 9-aminoacridine (9-AMA), and chromosomes were stained with GTG and aceto-orcein banding techniques and investigated by an image analysis system. RESULTS: The human optimal chromosome spreads with the 850 G-band resolution level, suitable for image analysis, were obtained by 9-AMA pretreatment for 1 h at a final concentration of 0.5-1 microg/ml, as compared with 600-700 bands after ethidium bromide treatment and about 400 bands without pretreatment. The best results for plant chromosomes were obtained after pretreatment with 1-2 microg/ml of 9-AMA for 12-24 h. The chromosomes elongated approximately 1.5-fold, and the resolution of chromosome banding patterns increased, reaching approximately 140 bands per haploid set in the case of camomile. CONCLUSIONS: 9-AMA is an efficient reagent for the standardization and increasing the resolution of chromosome banding patterns in human and plant chromosomes. It is extremely important for chromosome investigation in small plants.
It has recently been reported that atypical fibroxanthoma (AFX) is a predominantly diploid lesion in contrast to malignant fibrous hystiocytoma (MFH) which is usually aneuploid. To test this hypothesis, DNA content quantification was undertaken on Feulgen-stained cytology and tissue section preparations from 10 cases of AFX by image analysis. The large atypical cells which characterize AFX were aneuploid in each case. Smaller spindle-shaped cells found in this lesion were diploid. The results suggest that AFX is indistinguishable from MFH by DNA content estimation and highlight an advantage of image analysis over flow cytometry.
Human sperm heads were detected and tracked using semiautomatic image analysis. Measurements of size and shape on two specimens from each of 26 men showed that the major component of variability both within and between subjects was the number of small elongated sperm heads. Variability of the computed features between subjects was greater than that between samples from the same subject.
Methods for the quantitation of antigen or antibody specific cells within lymphoid structures rely on the laborious and often inaccurate counting of stained cells visually under the microscope. To circumvent these problems we used a combination of computer-linked image analysis, with the Quantimet 720 and specific staining for IgE by immunoperoxidase, to produce a relatively easy, accurate method for the quantitation of specifically stained cells within tonsils and lymph nodes. Frozen sections of bovine lymph nodes and tonsils were stained in an indirect immunoperoxidase technique with either rabbit anti-bovine IgE or normal rabbit serum as the primary reagent. Examination of the stained slides on the Quantimet 720 image analyzing computer revealed cells that were sufficiently dark to be considered above the 'threshold level' of background staining. These were counted as positive picture points. A quantitative evaluation of the relative number of IgE containing cells within the particular lymphoid tissue was arrived at by determining the difference between the percent positive picture in the control tissue incubated with normal rabbit serum (background) and in the sample treated with rabbit anti-IgE. Using this method, it was readily possible to quantitate the presence of IgE containing cells in the tonsils of calves; the highest percentage of positive tissue was present in the tonsils of calves exposed to ovalbumin. Image analysis is a useful technique for quantitative evaluation of any immunoperoxidase stained tissues.
Gastroesophageal reflux is a disorder with well-characterized histopathological features in the adult, but the incidence and pathogenesis of epithelial injury in children are poorly understood. Esophageal suction biopsies from 80 infants exhibiting symptoms of reflux were studied by computer-assisted image analysis and the results compared to routine histological scoring. The histological features evaluated were the number of intraepithelial lymphocytes and eosinophils, papillary height, interpapillary basal cell height, and a novel measure "integrated basal cell height." We quantitatively evaluated these histological criteria by computer-assisted image analysis and compared these results to four subjective grades of esophagitis: low, mild, moderate and high. In this report we now describe this quantitative histopathologic method for the evaluation of pediatric esophageal biopsies. Utilizing this method we demonstrate that both inter- and intra-observer variability were sufficiently low to stratify the mucosal changes reliably into at least four categories. The reliability of this objective analytic technique will permit studies into the disease progression and regression in pediatric reflux esophagitis.
BACKGROUND: The aim of the present study was to evaluate the prognostic significance of DNA ploidy and Ki67 expression in non-small cell lung carcinoma (NSCLC). METHODS: This prospective study included 96 patients with stages I-IIIA NSCLC who underwent surgical excision. DNA image analysis cytometry was applied on imprints. Calculation of the DNA index (DI) and the 5c exceeding rate (5cER) was performed and the histograms were classified as peridiploid, peritetraploid, and x-ploid-multiploid. The Ki67 immunoreactivity was determined according to the avidin-biotin complex immunoperoxidase method. RESULTS: DNA histogram classification disclosed 30 peridiploid cases, 15 peritetraploid and 51 x-ploid-multiploid. Forty-eight cases (50%) had 5cER > 5%. The Ki67 immunoreactivity was below 25% in 53 tumors (62.4%) and above 25% in 32 (32.6%). Our results revealed the existence of a statistically significant relationship of DNA ploidy with nodal status (p = 0.042) and grade (p = 0.005). Adenocarcinomas and large cell carcinomas were more frequently encountered in x-ploid-multiploid tumors as compared to squamous cell carcinomas, which were more frequently peridiploid (p = 0.003). 5cER showed statistically significant association with nodal status (p = 0.037). Univariate analysis with respect to survival revealed significant association with stage (p < 0.001), nodal status (p < 0.001), tumor status (p < 0.001), DNA ploidy (p = 0.008) and 5cER (p = 0.0124). Multivariate analysis revealed stage and ploidy status as independent factors: peridiploid tumors were associated with better survival as compared to x-ploid-multiploid tumors (p = 0.022). CONCLUSION: Our results suggest that DNA ploidy, as determined by image analysis, provides an independent prognostic parameter for patients with NSCLC and thus, could be used to identify a subset of patients with more aggressive tumors.
5-aminolevulinic acid mediated changes in tissue specific fluorescence were studied in bladder cancer. Bladders of normal patients and also patients diagnosed with cancer were instilled with 5-aminolevulinic acid and the resultant protoporphyrin IX mediated fluorescence intensity was imaged and quantified with confocal laser microscopy and fluorescence image analysis. Urothelial tumour cells were observed to fluoresce more intensely than normal urothelial cells. Submucosa and muscle tissues exhibited minimal fluorescence compared to urothelial cells of malignant origin and also normal urothelial cells. Degree of fluorescence intensity was in the order of malignant urothelium > normal urothelium > normal submucosa > normal muscles. Fluorescence intensity was also found to increase with duration of ALA instillation. Grade 3 malignant cells produced more fluorescence compared to grade 2 and grade 1. Similarly, T1 transitional cell carcinoma (TCC) showed increased fluorescence intensity than that of Ta TCC. Also, tumour blood vessels fluoresced more intensely compared to blood vessels found in normal bladder tissue. Tissue specific ALA mediated PpIX micro fluorescence can be used as a diagnostic technique for early detection of neoplasms and confocal laser microscopy and fluorescence image analysis are advantageous diagnostic tools for the photodynamic diagnosis of bladder neoplasms in vivo.
OBJECTIVE: For morphologic and functional investigations on the physiology and pathology of human blood platelets, their adhesion plays an important role. This feature is normally assessed concerning qualitative aspects, but there is a lack of quantitative investigations. STUDY DESIGN: We developed a model to quantify in vitro manipulations on thrombocytes by measuring their adhesion areas automatically. This was achieved by combining the VIDAS 2.5 image analysis system with reflection contrast microscopy (RCM). The evaluation was performed independent of the position of the grey value histogram by analyzing each digitized image corresponding to its individual mean grey value. RESULTS: The applicability and validity were verified from the example of a pharmacologic experiment administering the platelet stimulant oxidized low density lipoprotein (ox-LDL) to thrombocyte suspensions and confirming it by aggregometry. We assessed the temporal increase in adhesion area and mean total adhesion area of activated platelets, comparing unstimulated to stimulated ones. Ox-LDL accelerated the adhesion process and increased the adhesion area. CONCLUSION: The combination of RCM with consecutive image analysis provides useful parameters for in vitro examination on vital human platelets. Large amounts of data can be accumulated in a short time for an evaluation of influences of and on blood platelets.
The use of antibodies permits the study of oncogene product expression in cells and tissues. However, quantitation of the levels of expression in immunohistochemical preparations is beset by difficulties, and the available scoring system provide semiquantitative data at best. Here we describe the use of computer-assisted image analysis for determination of oncoprotein levels in a model system and compare the results with those generated by flow cytometric analysis. The oncogene products measured are located in the nucleus (c-myc p62 and c-fos p55), the inner surface of the membrane (c-ras p21), and both sides of the membrane (c-erbB-2 p185). In each instance, both analytic modalities yielded concordant results. Our data indicate that computer-assisted image analysis is a useful tool for quantitating cell components in immunohistochemical preparations.
The new technique of colour television image analysis has been used to produce seven-colour contour maps of the radiodensity of micro-radiographs of enamel caries. This method permits the detailed measurement of the mineral content of enamel carious lesions to a sensitivity of +/-3% of the level of complete mineralisation. By selection of appropriate aluminium step-wedges, exposed simultaneously with the tooth section, the whole range of mineral content can be assessed.
The analysis system for banding chromosomes (SYSTEM), an image analysis software, was programmed. By the SYSTEM the photos of Chinese G banding chromosomes were analyzed. It showed that the karyotype of the chromosomes of persons was recorded in high speed and efficiency, and that the chromosomes of alike ordinal number from different persons could be compared directly. That would presage, by this kind of the high effective operation, a favorable condition was created for the application of the polymorphism of the chromosomes to the parentage diagnosis.
Isolated rat lenses were exposed to oxidative stress generated by 100 microM H2O2, 2 mM ADP and 100 microM ferrous ammonium sulfate. Oxidation-induced cataract formation was followed by measuring loss of transmitted light intensity using quantitative digital image analysis, which offers distinct advantages over conventional photography. In the presence of oxidants, total and average light transmitted by the lens decreased exponentially as a function of time; the cortex showing a greater rate of decline in transmitted light intensity than the nucleus, which led to a change in the distribution pattern of light intensity. Lenses developing oxidative cataracts also showed a significant increase in diameter and an increase in the total wet weight. Maximal increase in lens diameter preceded maximal decrease in light intensity. These studies demonstrate the utility of quantitative image analysis in studying changes in lens geometry and transparency, and suggest that cataract formation is a single rate process.
Surfactant (Curosurf, diluted to 5 mg/ml) was suspended in normal saline with 3 mM CaCl2 and mixed with different concentrations of albumin or fibrinogen. The samples were vortexed to generate microbubbles, and the equivalent circle diameter of these bubbles was determined with computer-aided image analysis. Bubbles in the original surfactant suspension had an average diameter of 27 microns after 2 min, and their size increased only little during a 15-min period of observation. The diameter of bubbles generated in the presence of albumin (4 or 40 mg/ml) or fibrinogen (4 mg/ml) was 4-5 times larger, indicating surfactant inhibition. We conclude that evaluation of microbubble stability with image analysis provides an objective and rapid assessment of surfactant inhibition, and we speculate that the method could also be used for quantification of surfactant activity in airway samples.