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Megakaryocyte ploidy in thrombocytosis: improved microdensitometric measurements with a new image analysis system.

Megakaryocyte (MK) nuclear deoxyribonucleic acid (DNA) content was measured on a new image analysis system. Feulgen-stained bone marrow aspirate smears were analysed from 9 patients with thrombocytosis. Average optical density (OD) of stained nuclei was evaluated by pixel discrimination over 128 grey levels. Projected nuclear area was delineated manually. The product of these two parameters gave an index of nuclear DNA content. Neutrophils were used as 2N reference cells. Reproducibility of OD, nuclear area and their product was excellent for individual cells (CV less than 2.0% for MKs, less than 4.0% for neutrophils). The average CV for DNA content of 18 groups of 10 neutrophils was 5.5% (range 3.0-9.7%). A significant linear regression existed between MK nuclear area and DNA content (p much less than 0.001) for 627 MKs in essential thrombocythaemia (ET) and 346 MKs in reactive thrombocytosis (RT). Compared with RT, more 8N and 64N MKs were seen in ET (p less than 0.05). 128N MKs were unique to ET. Image analysis of MK ploidy may assist the clinical discrimination between primary and secondary thrombocytosis.

DNA↗

DNA ploidy measurements in prostate cancer: differences between image analysis and flow cytometry and clinical implications.

The relationship between DNA ploidy and prognostic parameters has been established in prostate cancer. However, comparison of various techniques used for DNA ploidy analysis has not been sufficiently addressed in prostate carcinoma. In the present study, grossly identified discrete carcinoma foci from 48 consecutive radical prostatectomy specimens were analyzed by flow cytometry and image analysis using both imprints and tissue sections. Correlations with other prognostically important pathological findings such as grade, size, extracapsular extension, positive surgical margin, and seminal vesicle and lymph node involvement were done. Image analysis detected 26 (54.2%) nondiploid tumors compared to 15 (31.3%) nondiploid tumors by flow cytometry. No significant differences were demonstrated between imprints and tissue sections. DNA ploidy status showed a good correlation with most other pathological findings listed above. In conclusion, DNA ploidy status determination gives clinically useful information which correlates well with the other useful pathological parameters of prognostic importance. Image analysis may be more sensitive than flow cytometry in detecting nondiploid populations. Tissue sections appear to be as reliable as imprints when properly controlled.

Adenocarcinoma↗

Computer image analysis of variance between human chromosome replication sequences and G-bands.

A computer image analysis system was applied to the quantitative study of chromosomal early- and late-replication patterns from the leukocytes of several normal human donors, and these patterns were compared with the chromosomal G-banding patterns. The first and last few hours of replication were discriminated by selective bromodeoxyuridine vs. thymidine incorporation in DNA and a Hoechst-blacklight-Giemsa stain technique. Image analysis with Tufts Piquant system involved automatic determination of chromosome boundaries, centromeres and telomeres, linear chromatid axes, chromatid density measurements along each axis, and comparative length normalized density profiles for each chromatid and the chromosome. Consistent complementary early- and late-replication patterns were determined for autosomes 1-6 and the X chromosomes. Limited intracellular or interindividual variability occurred in the intensity of a few active replication peaks but not in their location. However, there were very distinct regions of noncorrespondence between the late-replication patterns and the G-band patterns, in contrast with previous observations, although many similarities were also evident. These differences are interpreted with reference to a general model of replication sequence control of cell differentiation.

Analysis of Variance↗

Multiparameter digital image analysis of biliary, ampullary, and pancreatic adenocarcinomas.

Periampullary adenocarcinomas arise from pancreatic, biliary, or ampullary ductal epithelium. Their origin is difficult or impossible to discern by routine light microscopy of cytologic or small biopsy specimens. We used image analysis to describe the morphologic features of Feulgen-stained nuclei of biliary and ampullary adenocarcinomas and to compare these findings with those of pancreatic ductal adenocarcinomas. Surgically resected cases of ampullary adenocarcinoma (n = 7) and cholangiocarcinoma (n = 26) were selected on the basis of available formalin-fixed, paraffin-embedded tissue and diagnostic clinical data. Disaggregated nuclei were stained by the Feulgen reaction and analyzed using a digital image processor. Data from 15 morphonuclear parameters were assessed and compared with the results from the morphonuclear analysis of 22 pancreatic ductal adenocarcinomas. Multivariate analysis with canonical transformation of the data defined the ampullary adenocarcinomas and cholangiocarcinomas as occupying similar factorial distributions, whereas the pancreatic carcinomas were separate and distinct. Monovariate analysis identified seven parameters distinguishing pancreatic carcinoma from both ampullary carcinoma and cholangiocarcinoma, with P values < or = 0.001 and two others having P values < or = 0.01. Adenocarcinomas of ampullary or bile duct origin possess similar morphonuclear features described by image analysis. Image analysis provides a mechanism to discriminate adenocarcinomas arising from the bile ducts or ampulla from those arising in the pancreas.

Adenocarcinoma↗

Use of computer-assisted image analysis for noninvasive evaluation of oral lichenoid reactions and oral leukoplakia.

The objective of the present study was to elaborate and to evaluate a method in which the clinical appearance of a mucosal lesion could be expressed by means of color and morphologic features extracted from digitized images. The ability to express mucosal changes with image features was evaluated, as was the accuracy of a classification of the lesions on the basis of image feature analysis. Computer-assisted image analysis was performed on digitized color slides from 76 patients with lichenoid reactions that included both oral lichen planus and contact lesions and 20 patients with homogenous oral leukoplakia. Color features were measured according to the intensity-hue-saturation color system, and morphologic features were estimated by tracing the hyperkeratotic area. Hyperkeratinizing and inflammatory tissue reactions could be demonstrated and defined by image analysis. Image features indicating inflammatory tissue reactions were more pronounced in cases of oral lichenoid reactions. Morphologic parameters could express the anatomic appearance of the lesions. The comparison between histopathologic diagnosis and a classification through computerized analysis demonstrated that 90.5% of oral lichenoid reactions could be separated from homogenous oral leukoplakia by linear discriminant analysis.

Analog-Digital Conversion↗

Integrated image analysis solutions for PET datasets in damaged brain.

OBJECTIVE: To identify and discuss the problems inherent in the processing of multiparametric functional imaging datasets from patients with acute brain injury, using "triple oxygen" positron emission tomography (PET) as an example. To present an integrated imaging solution for analysis of such datasets and report on its use in practice. METHODS: Structured analysis of the steps in image analysis for triple oxygen studies in patients with acute brain injury, subarachnoid haemorrhage and carotid artery disease was undertaken. Systematic identification of the drawbacks of conventional manual processing techniques, which make use of modules from different commercially available software packages was carried out. This analysis informed the construction of an image analysis gateway that addressed these issues. RESULTS: We describe an integrated image analysis gateway (PETAn) that provides for the automated integration of all processing steps, along with outputs that detail analysis stages, error logs and results. Implementation of PETAn allowed batch processing of clinical PET data sets, reduced the level of technical expertise required for analysis, improved the quality of results and achieved significant reductions in operator analysis time. CONCLUSIONS: While modules from several imaging software suites can be usefully combined to allow analysis of multiparametric PET data sets from patients with acute brain injury, sequential use of these modules for various steps in image analysis is fraught with difficulty. Careful automation and integration of these steps greatly facilitates the interrogation of information-rich data sets and increases research productivity.

Algorithms↗

Proliferative activity in non-Hodgkin's lymphomas. A comparison of the bromodeoxyuridine labeling index with PCNA immunostaining and quantitative image analysis.

Cellular proliferation is being evaluated as an independent prognostic indicator in a variety of tumors, including non-Hodgkin's lymphomas (NHL). Although the labeling index (LI), using either tritiated thymidine or monoclonal antibodies to bromodeoxyuridine, is considered to be the gold standard of cell kinetic measurements, recent advances in the use of antibodies specific to cellular proliferating antigens and computer-based image analysis have potentially established a new technology for evaluating the growth fraction of NHL. Proliferating cell nuclear antigen (PCNA), a protein associated with DNA polymerase that is expressed only in the nuclei of cells actively synthesizing DNA, was quantitated in 46 formalin-fixed, paraffin-embedded samples of various types of NHL using the Cell Analysis Systems 200 image analyzer. The results were expressed as the percentage of nuclear area (%NA) positive for PCNA. These results were compared with the bromodeoxyuridine LI, and a correlation coefficient of 0.78 was calculated using first-order linear regression. An average positive NA of 8.2% for low-grade NHL, 32% for intermediate-grade, and 43% for high-grade NHL was observed. The potential advantages PCNA quantitation by image analysis may offer over LI are discussed.

Antibodies↗

[Automatic image analysis of argentophil protein granule quantitative measurement on nucleolus area of limbus epithelial neoplasms].

OBJECTIVE: To study the pathological diagnostic significance of argentophil protein (AgNOR) quantitative analysis at nucleolus area of limbus epithelial neoplasms. METHODS: Automatic image analysis system was used for argentophil protein granule measurement in the pathologic diagnosis of 45 cases with limbus epithelial neoplasms, including benign hyperplasia, atypical hyperplasia, carcinoma in situ and squamous cell carcinoma. RESULTS: In various lesions, there were the differences in the distributive pattern, size and area of AgNOR granules, which were closely related to the nature of the neoplasms (benign or malignant) and its cell differentiation. CONCLUSION: Automatic image analysis of AgNOR quantitative measurement on limbus epithelial neoplasms may indicate the proliferative activity and cell differentiation in various lesions, help to differentiate benign or malignant neoplasms and effectively enhance the level of pathologic diagnosis.

Carcinoma in Situ↗

Comparison of VHS video recording system with apple Macintosh-based image analysis and modified CODA-3 systems in equine motion analysis.

A VHS video--computer-based image analysis combination is described as a low sampling rate motion analysis system. Video recordings were taken indoor without any artificial illumination at 25 fps sampling rate. The horse studied was running on a high-speed treadmill and observed at 1.6, 4 and 7 m/s velocities at walk, trot and canter, respectively. Left forelimb and hindlimb were recorded separately from lateral view. For comparison, parallel CODA-3 recordings were taken at the same time from the same position. Joint angles were expressed and compared in angle-time diagrams. Sampling of both systems has been synchronised by a timer device at +/- 1/300 s error level. Results obtained with the two different recording systems were comparable in all joints measured with the exception of the fetlock. Inaccuracies in fetlock recordings are thought to be eliminated by measuring at controlled illumination. As a conclusion, the VHS-Macintosh setup appears to be promising as a simplified system for gait analysis.

Animals↗

Automated image analysis of live/dead staining of the fungus Aureobasidium pullulans on microscope slides and leaf surfaces.

An image analysis program and protocol for the identification and enumeration of live versus dead cells of the yeast-like fungus Aureobasidium pullulans was developed for both populations on microscope slides and leaf surfaces. Live cells took up CellTracker Blue, while nonviable cells stained with DEAD Red. Image analysis macro programs running under Optimas software were used to acquire images and to differentiate and enumerate viable from nonviable cells. The software was capable of discriminating green as a third parameter for identification and quantification of green fluorescent protein-expressing cells in a wild-type population.

Green Fluorescent Proteins↗

Computerized image-analysis microspectroscopy of tissue sections.

A simple technique using monochromatic light was applied to computer-assisted image analysis of tissue sections. The method consists of the determination of the spectral characteristics of different elements of interest and selective monochromatic illumination of a microscopic field followed by image analysis. Results show a dramatic increase in resolution and contrast. By means of an image subtraction technique at two wavelengths, it was possible to achieve specific optical isolation and quantification of image components.

Animals↗

[DNA content in colorectal carcinomas: application of image analysis to archival material].

The most studied biological parameter of human tumours is the DNA nuclear content. In the present study we assessed the DNA content of 197 colorectal cancers from 180 patients using archival material (tissues routinely processed and paraffin-embedded) for image analysis. The metastatic lymph nodes were also included in the study in 54 Dukes "C" stage cases and 19/25 Dukes "D" stage cases. A total of 452 sections, 5 mu thick, were submitted to image analysis in static cytometry. DNA diploid pattern was found in 72/452 samples (15.9%) and the DNA-aneuploid pattern in 380/452 (84.1%). Two different areas for each tumour were examined but we found a concordance of diploid pattern in only 23 cases. DNA-aneuploidy, regarded as presence of at least one sample with an aneuploid peak in the same tumour, reached 88% in our study. In conclusion the image analysis of several samples obtained from different areas of the same tumour clearly revealed: a pronounced heterogeneity of ploidy patterns in colon cancer; an overall prevalence of DNA-aneuploidy in colon cancer; a prevalence of diploid pattern in tumours localized in right portion of the colon and in younger patients.

Adenocarcinoma↗

Image analysis of hepatocyte nuclei in assessing di(2-ethylhexyl)phthalate effects eluding detection by conventional microscopy.

The effect of di(2-ethylhexyl)phthalate (DEHP) administered in single intraperitoneal doses of 30, 300 and 3000 mg/kg in Syrian golden hamsters was studied by means of routine pathologic investigations, electron microscopy and image analysis. The morphological evaluations did not show apparent differences between the control and treated animals. Such differences, however, were recognized by using image analysis. They concerned morphology of the hepatocyte nuclei and were defined by quantitative parameters reflecting geometrical, optical and structural properties. Of importance for differentiating dose/effect relationships were features of chromatin structure. In order to describe those features we developed special algorithms capable of identifying and characterizing regions of condensed chromatin as subimages. These were distinguished by their size, shape and optical density and showed typical distributions within the nucleus. As our results demonstrate, image analysis methods permit detection of DEHP related pathology in animals which, as far as is evident from routine morphologic evaluations, belong to the no-effect experimental group.

Animals↗

Detection of subgroups from flow cytometry measurements of heterotrophic bacterioplankton by image analysis.

BACKGROUND: Flow cytometry is an invaluable tool for the analysis of large series of samples in aquatic microbial ecology. However, analysis of the resulting data is often inefficient or does not reflect the complexity of natural communities. Because bacterioplankton assemblages frequently fall into several clusters with respect to their cellular properties, these subgroups seem to be a promising level of abstraction. Image analysis was used to detect clusters from flow cytometry data. The method was tested on a bacterial community under heavy protozoan grazing pressure. METHODS: A bivariate histogram of flow cytometry data was transformed into a gray-scale image for image analysis. After low-pass filtration, regional maxima were delimited by a watershed algorithm. The resulting areas were then used as gates on the original measurements. RESULTS: Three clusters could be detected from the bacterial assemblage. Protozoan grazing had a strong impact on the bacterial community, which could be analyzed in detail at the level of individual subgroups. CONCLUSIONS: Investigation at the level of bacterial subgroups allowed a more detailed analysis than whole-community statistics and delivered essential and ecologically meaningful information. Image analysis proved to be an adequate tool to detect the subgroups without a priori knowledge.

Animals↗

Quantitative immunohistological analysis of the microvasculature in untreated human glioblastoma multiforme. Computer-assisted image analysis of whole-tumor sections.

Because histologically prominent microvascular proliferation is frequently present in glioblastoma multiforme, it has been hypothesized that this neoplasm is particularly dependent on neovascularization for its continued growth and that antiangiogenic therapy might be especially useful. To quantify the histological aspects of microvascular proliferation in glioma, a feasible and reproducible method was developed for computer-assisted image analysis of the visualized microvasculature in glial tissue. This method was used to compare several vascular parameters in histological whole-tumor sections of untreated human glioblastoma multiforme with those in histologically normal cerebral cortex and white matter. There was a significant increase in mean number, area, and perimeter of blood vessels per microscopic field in glioblastoma multiforme compared to normal cerebral white matter. In a substantial number of tumor fields, however, the vascular density was in the same range as that of normal cerebral white matter. The striking heterogeneity of the microvasculature within glioblastoma multiforme was illustrated by the significantly higher standard deviation for the vascular parameters in tumor tissue. The results of this study suggest that many regions of glioblastomas multiforme are not overtly angiogenesis dependent and may be difficult to treat by antiangiogenic therapy alone.

Adult↗

Quantitative DNA determinations by image analysis. I. Application to human pulmonary cytology.

A description is made of the application of image analysis to quantitative deoxyribonucleic acid (DNA) determinations in a variety of cells. Optical density of the video signal from Feulgen-stained material was integrated across the area of the nucleus and values obtained related to values derived from known diploid cells. Analysis was performed on three classes of material: (1) biological material with known haploid, diploid and tetraploid nuclear DNA (standards), (2) metaplasias of the bronchial epithelium and (3) bronchogenic carcinomas. Results obtained with image analysis corresponded with expected data for the biologic standards and with microspectrophotometric data obtained for metaplasia and epidermoid carcinoma of the bronchial system. Application of this and other quantitative methods to cancer screening and biology is discussed.

Carcinoma, Bronchogenic↗

3-D portal image analysis in clinical practice: an evaluation of 2-D and 3-D analysis techniques as applied to 30 prostate cancer patients.

PURPOSE: To investigate the clinical importance and feasibility of a 3-D portal image analysis method in comparison with a standard 2-D portal image analysis method for pelvic irradiation techniques. METHODS AND MATERIALS: In this study, images of 30 patients who were treated for prostate cancer were used. A total of 837 imaged fields were analyzed by a single technologist, using automatic 2-D and 3-D techniques independently. Standard deviations (SDs) of the random, systematic, and overall variations, and the overall mean were calculated for the resulting data sets (2-D and 3-D), in the three principal directions (left-right [L-R], cranial-caudal [C-C], anterior-posterior [A-P]). The 3-D analysis included rotations as well. For the translational differences between the three data sets, the overall SD and overall mean were computed. The influence of out-of-plane rotations on the 2-D registration accuracy was determined by analyzing the difference between the 2-D and 3-D translation data as function of rotations. To assess the reliability of the 2-D and 3-D methods, the number of times the automatic match was manually adjusted was counted. Finally, an estimate of the workload was made. RESULTS: The SDs of the random and systematic components of the rotations around the three orthogonal axes were 1. 1 (L-R), 0.6 (C-C), 0.5 (A-P) and 0.9 (L-R), 0.6 (C-C), 0.8 (A-P) degrees, respectively. The overall mean rotation around the L-R axis was 0.7 degrees, which deviated significantly from zero. Translational setup errors were comparable for 2-D and 3-D analysis (ranging from 1.4 to 2.2 mm SD and from 1.5 to 2.5 mm SD, respectively). The variation of the difference between the 2-D and 3-D translation data increased from 1.1 mm (SD) for zero rotations to 2.7 mm (SD) for out-of-plane rotations of 3 degrees, due to a reduced 2-D registration accuracy for large rotations. The number of times the analysis was not considered acceptable and was manually adjusted was 44% for the 2-D analysis, and 6% for the 3-D analysis. CONCLUSION: True 3-D analysis of setup errors for a group of 30 patients with prostate cancer demonstrated that setup rotations are rather small. The deformation of the projected anatomy in portal images caused by out-of-plane rotations leads to a reduced 2-D registration accuracy. For rotations larger than 3 degrees this effect can be quite pronounced, making 3-D registration the preferred method. Furthermore, the automatic 3-D registration has a higher success rate, most likely because this technique uses more information compared to the 2-D method.

Feasibility Studies↗

Quantitation of megakaryocytopoiesis by computerized automatic in culture image analysis.

A computer-assisted automatic image procedure was karyocytopoiesis in culture. This analysis system was based on acetylcholinesterase staining, a specific staining for murine bone marrow megakaryocytes, and an image capturing instrument with a computer program. Two kinds of routine megakaryocyte culture methods were used, the plasma clot and the serum-free agar systems. A comparison between manual counting and the instrument was made. The image analysis software was able to distinguish between megakaryocytes (MK) at different stages of maturation. The results show that this analysis system can simultaneously detect not only the number of megakaryocytes and their colonies in each dish, but also the surface area of individual megakaryocytes. In addition, this analysis system functions automatically 24 hours a day and the results obtained are reproducible. Using this system, we have confirmed previous observations that thrombopoietin (TPO) and heparin stimulate both proliferation and maturation of megakaryocytes. In addition, we found that platelet factor 4 (PF-4) significantly reduced the number of megakaryocytes but not their cell surface area, whereas TGFbeta1 decreased both number and surface area of megakaryocytes, suggesting that PF4 and TGFbeta1 negatively regulate megakaryocytopoiesis by different mechanisms. We noticed that megakaryocytes grown under agar culture conditions regularly had an increased size in comparison with those grown in a plasma clot system, which may be an indication that the plasma clot culture media contains an inhibitor(s) of megakaryocyte maturation. Our data indicate that this image analysis system, in addition to its automatic and reproducible features, is more efficient and allows detection of more parameters than routine manual microscopic detection.

Journal Article↗