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1.

Background

Currently available microscope slide scanners produce whole slide images at various resolutions from histological sections. Nevertheless, acquisition area and so visualization of large tissue samples are limited by the standardized size of glass slides, used daily in pathology departments. The proposed solution has been developed to build composite virtual slides from images of large tumor fragments.

Materials and methods

Images of HES or immunostained histological sections of carefully labeled fragments from a representative slice of breast carcinoma were acquired with a digital slide scanner at a magnification of 20×. The tiling program involves three steps: the straightening of tissue fragment images using polynomial interpolation method, and the building and assembling of strips of contiguous tissue sample whole slide images in × and y directions. The final image is saved in a pyramidal BigTiff file format. The program has been tested on several tumor slices. A correlation quality control has been done on five images artificially cut.

Results

Sixty tumor slices from twenty surgical specimens, cut into two to twenty six pieces, were reconstructed. A median of 98.71% is obtained by computing the correlation coefficients between native and reconstructed images for quality control.

Conclusions

The proposed method is efficient and able to adapt itself to daily work conditions of classical pathology laboratories.
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2.

Background

Eosinophilic oesophagitis (EoE) is characterized by the presence of eosinophils in oesophageal mucosa. Other inflammatory cells, mainly lymphocytes, dendritic cells, and mast cells may also play an important role in this disease. The aim of this study is to compare the inflammatory pattern of the mucosa between EoE and gastro-oesophageal reflux disease (GERD), using automatic image analysis in digital slides, and to assess treatment response after elimination diet and food challenge test.

Methods

From 2010 to 2013, 35 oesophageal biopsies from EoE and GERD patients were randomly selected. In six EoE biopsies, patients had been treated with selective food exclusion diet. Immunohistochemical study with CD3, CD20, CD4, and CD8 for lymphocyte populations, CD1a for dendritic cells, and CD117/c-kit for mast cells was performed. Slides were scanned using Leica Aperio Scanscope XT with 40× magnification. Immunohistochemical expression was quantified in 245 immunohistochemistry digital slides with Leica Aperio positive pixel count algorithm using two different approaches: whole slide analysis versus selection of a 2 mm2 hot spot area.

Results

Average eosinophil cell count was significantly higher (p < 0.001) in the first biopsy of EoE patients before treatment (30.75 eosinophils per high power field - HPF) than in GERD patients (0.85 eosinophils/HPF) or in EoE patients after treatment with elimination diet (1.60 eosinophils/HPF). In the immunohistochemical study, manual count and automatic image analysis showed a significant increase in the number of CD3 and CD8 cells in EoE patients, compared with GERD patients. However, the increase of CD117/c-kit was only statistically significant when manual counting procedures were used. CD20 positive cell count also showed a non-statistically significant tendency to reduce after elimination diet treatment.Manual eosinophil count correlated much better with CD3 and CD8 count using hot spot approach than with a whole slide approach.

Conclusions

Positive pixel count algorithm can be a useful tool to quantify the immunohistochemical expression of inflammatory cells in the diagnosis and follow up of eosinophilic oesophagitis.
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3.

Background

There are many scanners of glass slides on the market now. Quality of digital images produced by them may be different and pathologists who examine virtual slides on a monitor may subjectively evaluate it. However, objective comparison of quality of digital slides captured by various devices requires assessment algorithms, which will be automatically executed.

Methods

In this work such an algorithm is proposed and implemented. It is dedicated for comparing quality of virtual slides which show the same glass slide captured by two or more scanners. In the first step this method looks for the largest corresponding areas in the slides. This task is realized by defining boundaries of tissues and providing the relative scale factor. Then, a certain number of smaller areas, which show the same fragments of both slides, is selected. The chosen fragments are analyzed using Gray Level Co-occurrence Matrix (GLCM). For GLCM matrices some of the Haralick features are calculated, like contrast or entropy. Basing on results for some sample images, features appropriate for quality assessment are chosen. Aggregation of values from all selected fragments allows to compare the quality of images captured by tested devices.

Results

Described method was tested on two sets of ten virtual slides, acquired by scanning the same set of ten glass slides by two different devices. First set was scanned and digitized using the robotic microscope Axioscope2 (Zeiss) equipped with AxioCam Hrc CCD camera. Second set was scanned by DeskScan (Zeiss) with standard equipment. Before analyzing captured virtual slides, images were stitched and converted using software which utilizes advances in aerial and satellite imaging.The results of the experiment show that calculated quality factors are higher for virtual slides acquired using first mentioned device (Axioscope2 with AxioCam).

Conclusions

Results of the tests are consistent with opinion of the pathologists who assessed quality of virtual slides captured by these devices. This shows that the method has potential in automatic evaluation of virtual slides’ quality.
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4.

Introduction

Standardization and validation of the color displayed by digital slides is an important aspect of digital pathology implementation. While the most common reason for color variation is the variance in the protocols and practices in the histology lab, the color displayed can also be affected by variation in capture parameters (for example, illumination and filters), image processing and display factors in the digital systems themselves.

Method

We have been developing techniques for color validation and optimization along two paths. The first was based on two standard slides that are scanned and displayed by the imaging system in question. In this approach, one slide is embedded with nine filters with colors selected especially for H&;E stained slides (looking like tiny Macbeth color chart); the specific color of the nine filters were determined in our previous study and modified for whole slide imaging (WSI). The other slide is an H&;E stained mouse embryo. Both of these slides were scanned and the displayed images were compared to a standard. The second approach was based on our previous multispectral imaging research.

Discussion

As a first step, the two slide method (above) was used to identify inaccurate display of color and its cause, and to understand the importance of accurate color in digital pathology. We have also improved the multispectral-based algorithm for more consistent results in stain standardization. In near future, the results of the two slide and multispectral techniques can be combined and will be widely available.We have been conducting a series of researches and developing projects to improve image quality to establish Image Quality Standardization. This paper discusses one of most important aspects of image quality – color.
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5.

Background

Since microscopic slides can now be automatically digitized and integrated in the clinical workflow, quality assessment of Whole Slide Images (WSI) has become a crucial issue. We present a no-reference quality assessment method that has been thoroughly tested since 2010 and is under implementation in multiple sites, both public university-hospitals and private entities. It is part of the FlexMIm R&D project which aims to improve the global workflow of digital pathology. For these uses, we have developed two programming libraries, in Java and Python, which can be integrated in various types of WSI acquisition systems, viewers and image analysis tools.

Methods

Development and testing have been carried out on a MacBook Pro i7 and on a bi-Xeon 2.7GHz server. Libraries implementing the blur assessment method have been developed in Java, Python, PHP5 and MySQL5. For web applications, JavaScript, Ajax, JSON and Sockets were also used, as well as the Google Maps API. Aperio SVS files were converted into the Google Maps format using VIPS and Openslide libraries.

Results

We designed the Java library as a Service Provider Interface (SPI), extendable by third parties. Analysis is computed in real-time (3 billion pixels per minute). Tests were made on 5000 single images, 200 NDPI WSI, 100 Aperio SVS WSI converted to the Google Maps format.

Conclusions

Applications based on our method and libraries can be used upstream, as calibration and quality control tool for the WSI acquisition systems, or as tools to reacquire tiles while the WSI is being scanned. They can also be used downstream to reacquire the complete slides that are below the quality threshold for surgical pathology analysis. WSI may also be displayed in a smarter way by sending and displaying the regions of highest quality before other regions. Such quality assessment scores could be integrated as WSI's metadata shared in clinical, research or teaching contexts, for a more efficient medical informatics workflow.
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6.

Background

Ki67 labeling index (Ki67 LI), the percentage Ki67 immunoreactive cells, is a measure of tumor proliferation, with important clinical relevance in breast cancer, and it is extremely important to standardize its evaluation.

Aim

To test the efficacy of computer assisted image analysis (CAIA) applied to completely digitized slides and to assess its feasibility in routine practice and compare the results obtained using two different Ki67 monoclonal antibodies.

Materials and methods

315 consecutive breast cancer routinely immunostained for Ki-67 (223 with SP6 and 92 with MM1 antibodies previously examined by an experienced pathologist, have been re-evaluated using Aperio Scanscope Xs.

Results

Mean human Ki67 LI values were 36%± 14.% and 28% ± 18% respectively for SP6 and MM1 antibodies; mean CAM Ki67 LI values were 31%± 19% and 22% ± 18% respectively for SP6 and MM1. Human and CAIA evaluation are statistically highly correlated (Pearson: 0.859, p<0.0001), although human LI are systematically higher. An interobserver variation study on CAIA performed on 84 cases showed that the correlation between the two evaluations was linear to an excellent degree.

Discussion

Our study shows that a) CAIA can be easily adopted in routine practice, b) human and CAIA Ki67 LI are highly correlated, although human LI are systematically higher, c) Ki67 LI using different evaluation methods and different antibodies shows important differences in cut-off values.
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7.

Background

Digital immunohistochemistry (IHC) is one of the most promising applications brought by new generation image analysis (IA). While conventional IHC staining quality is monitored by semi-quantitative visual evaluation of tissue controls, IA may require more sensitive measurement. We designed an automated system to digitally monitor IHC multi-tissue controls, based on SQL-level integration of laboratory information system with image and statistical analysis tools.

Methods

Consecutive sections of TMA containing 10 cores of breast cancer tissue were used as tissue controls in routine Ki67 IHC testing. Ventana slide label barcode ID was sent to the LIS to register the serial section sequence. The slides were stained and scanned (Aperio ScanScope XT), IA was performed by the Aperio/Leica Colocalization and Genie Classifier/Nuclear algorithms. SQL-based integration ensured automated statistical analysis of the IA data by the SAS Enterprise Guide project. Factor analysis and plot visualizations were performed to explore slide-to-slide variation of the Ki67 IHC staining results in the control tissue.

Results

Slide-to-slide intra-core IHC staining analysis revealed rather significant variation of the variables reflecting the sample size, while Brown and Blue Intensity were relatively stable. To further investigate this variation, the IA results from the 10 cores were aggregated to minimize tissue-related variance. Factor analysis revealed association between the variables reflecting the sample size detected by IA and Blue Intensity. Since the main feature to be extracted from the tissue controls was staining intensity, we further explored the variation of the intensity variables in the individual cores. MeanBrownBlue Intensity ((Brown+Blue)/2) and DiffBrownBlue Intensity (Brown-Blue) were introduced to better contrast the absolute intensity and the colour balance variation in each core; relevant factor scores were extracted. Finally, tissue-related factors of IHC staining variance were explored in the individual tissue cores.

Conclusions

Our solution enabled to monitor staining of IHC multi-tissue controls by the means of IA, followed by automated statistical analysis, integrated into the laboratory workflow. We found that, even in consecutive serial tissue sections, tissue-related factors affected the IHC IA results; meanwhile, less intense blue counterstain was associated with less amount of tissue, detected by the IA tools.
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8.

Background

Automated image analysis on virtual slides is evolving rapidly and will play an important role in the future of digital pathology. Due to the image size, the computational cost of processing whole slide images (WSIs) in full resolution is immense. Moreover, image analysis requires well focused images in high magnification.

Methods

We present a system that merges virtual microscopy techniques, open source image analysis software, and distributed parallel processing. We have integrated the parallel processing framework JPPF, so batch processing can be performed distributed and in parallel. All resulting meta data and image data are collected and merged. As an example the system is applied to the specific task of image sharpness assessment. ImageJ is an open source image editing and processing framework developed at the NIH having a large user community that contributes image processing algorithms wrapped as plug-ins in a wide field of life science applications. We developed an ImageJ plug-in that supports both basic interactive virtual microscope and batch processing functionality. For the application of sharpness inspection we employ an approach with non-overlapping tiles. Compute nodes retrieve image tiles of moderate size from the streaming server and compute the focus measure. Each tile is divided into small sub images to calculate an edge based sharpness criterion which is used for classification. The results are aggregated in a sharpness map.

Results

Based on the system we calculate a sharpness measure and classify virtual slides into one of the following categories - excellent, okay, review and defective. Generating a scaled sharpness map enables the user to evaluate sharpness of WSIs and shows overall quality at a glance thus reducing tedious assessment work.

Conclusions

Using sharpness assessment as an example, the introduced system can be used to process, analyze and parallelize analysis of whole slide images based on open source software.
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9.
10.

Background

Digital pathology, i.e., applications of digital information technologies to pathology practice, has been expanding in the recent decades and the mode of pathology diagnostic practice is changing with enhanced precision. In the present study the changing processes of digital pathology in Japan were investigated and trends to future were discussed.

Methods

The changing status of digital pathology was investigated through reviewing the records of annual meetings of the Japanese Research Society of Telepathology and Pathology Informatics (JRST-PI) and of the Japanese pathology related medical and informatics journals. The results of the Japanese questionnaire survey conducted in 2008-2009 on telepathology and virtual slide were also reviewed. In addition effectiveness of an experimental automatic pathology diagnostic aid system using computer artificial intelligence was investigated by checking its rate of correct diagnosis for given prostate carcinoma digital images.

Results

Telepathology played a central role in the development of digital pathology in Japan. Both macroscopic and microscopic pathology digital images were routinely generated and used for diagnostic purposes in major hospitals. Virtual slide (VS) digital images were used first for education then for conference, consultation and also gradually for routine diagnosis and telepathology. The experimental automatic diagnostic aid system achieved the rate of correct diagnosis around 95% for prostate carcinoma and its use for automatic mapping of cancerous areas in a given tissue image was successful.

Conclusions

Advance in the digital information technologies gave revolutionary impacts on pathology education, conference, consultation, diagnosis, telepathology and also on pathology diagnostic procedures in Japan. The future will be bright for pathologists by the advanced digital pathology but we should pay attention to make the technologies and their effects under our control.
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11.

Background

Automated image analysis, measurements of virtual slides, and open access electronic measurement user systems require standardized image quality assessment in tissue-based diagnosis.

Aims

To describe the theoretical background and the practical experiences in automated image quality estimation of colour images acquired from histological slides.

Theory, material and measurements

Digital images acquired from histological slides should present with textures and objects that permit automated image information analysis. The quality of digitized images can be estimated by spatial independent and local filter operations that investigate in homogenous brightness, low peak to noise ratio (full range of available grey values), maximum gradients, equalized grey value distribution, and existence of grey value thresholds. Transformation of the red-green-blue (RGB) space into the hue-saturation-intensity (HSI) space permits the detection of colour and intensity maxima/minima. The feature distance of the original image to its standardized counterpart is an appropriate measure to quantify the actual image quality. These measures have been applied to a series of H&;E stained, fluorescent (DAPI, Texas Red, FITC), and immunohistochemically stained (PAP, DAB) slides. More than 5,000 slides have been measured and partly analyzed in a time series.

Results

Analysis of H&;E stained slides revealed low shading corrections (10%) and moderate grey value standardization (10 – 20%) in the majority of cases. Immunohistochemically stained slides displayed greater shading and grey value correction. Fluorescent stained slides are often revealed to high brightness. Images requiring only low standardization corrections possess at least 5 different statistically significant thresholds, which are useful for object segmentation. Fluorescent images of good quality only posses one singular intensity maximum in contrast to good images obtained from H&;E stained slides that present with 2 – 3 intensity maxima.

Conclusion

Evaluation of image quality and creation of formally standardized images should be performed prior to automatic analysis of digital images acquired from histological slides. Spatial dependent and local filter operations as well as analysis of the RGB and HSI spaces are appropriate methods to reproduce evaluated formal image quality.
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12.

Introduction

Since their introduction in 1999, fully automated, high speed, high-resolution whole slide imaging devices have become increasing more reliable, fast and capable. While by no means perfect, these devices have evolved to a point where one can consider placing them in a pre-diagnostic role in a clinical histology lab.

Methods

At the Massachusetts General Hospital, we are running a pilot study placing high end WSI devices in our main clinical histology lab (after the cover slipper and before slides are sent to the pathologist) to examine the requirement for both the machine and the laboratory.

Results

Placing WSI systems in the clinical lab stresses the system in terms of reliability and throughput. Significantly however, success requires significant modification to the lab workflow. It is likely laboratories need to move from manual, large batch processes to increasingly automated, continuous flow (or mini-batch) processes orchestrated by the LIS using bar coding to track and direct slides, and incorporating the decision to image into the specimen type and the histology orders. Furthermore, image quality, capture speed and reliability are functions of the quality of the histology presented to the WSI devices.

Conclusion

Imaging in pathology does not begin in a WSI robot but in the grossing room and in the histology lab. As more and more imaging devices are placed in histology lab, the inter-relationships histology and pathology imaging will become increasing understood.
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13.

Background

Application of virtual slides (VS), the digitalization of complete glass slides, is in its infancy to be implemented in routine diagnostic surgical pathology and to issues that are related to tissue-based diagnosis, such as education and scientific publication.

Approach

Electronic publication in Pathology offers new features of scientific communication in pathology that cannot be obtained by conventional paper based journals. Most of these features are based upon completely open or partly directed interaction between the reader and the system that distributes the article. One of these interactions can be applied to microscopic images allowing the reader to navigate and magnify the presented images. VS and interactive Virtual Microscopy (VM) are a tool to increase the scientific value of microscopic images.

Technology and Performance

The open access journal Diagnostic Pathology http://www.diagnosticpathology.org has existed for about five years. It is a peer reviewed journal that publishes all types of scientific contributions, including original scientific work, case reports and review articles. In addition to digitized still images the authors of appropriate articles are requested to submit the underlying glass slides to an institution (DiagnomX.eu, and Leica.com) for digitalization and documentation. The images are stored in a separate image data bank which is adequately linked to the article. The normal review process is not involved. Both processes (peer review and VS acquisition) are performed contemporaneously in order to minimize a potential publication delay. VS are not provided with a DOI index (digital object identifier). The first articles that include VS were published in March 2011.

Results and Perspectives

Several logistic constraints had to be overcome until the first articles including VS could be published. Step by step an automated acquisition and distribution system had to be implemented to the corresponding article. The acceptance of VS by the reader is high as well as by the authors. Of specific value are the increased confidence to and reputation of authors as well as the presented information to the reader. Additional associated functions such as access to author-owned related image collections, reader-controlled automated image measurements and image transformations are in preparation.

Virtual Slides

The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/1232133347629819.
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14.

Background

Experimental autoimmune neuritis (EAN) is a well-known animal model of human demyelinating polyneuropathies and is characterized by inflammation and demyelination in the peripheral nervous system. Fascin is an evolutionarily highly conserved cytoskeletal protein of 55 kDa containing two actin binding domains that cross-link filamentous actin to hexagonal bundles.

Methods

Here we have studied by immunohistochemistry the spatiotemporal accumulation of Fascin?+?cells in sciatic nerves of EAN rats.

Results

A robust accumulation of Fascin?+?cell was observed in the peripheral nervous system of EAN which was correlated with the severity of neurological signs in EAN.

Conclusion

Our results suggest a pathological role of Fascin in EAN.

Virtual slides

The virtual slides for this article can be found here: http://www.diagnosticphatology.diagnomx.eu/vs/6734593451114811
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15.

Background

This paper examines how the adoption of a subject-specific library service has changed the way in which its users interact with a digital library. The LitMiner text-analysis application was developed to enable biologists to explore gene relationships in the published literature. The application features a suite of interfaces that enable users to search PubMed as well as local databases, to view document abstracts, to filter terms, to select gene name aliases, and to visualize the co-occurrences of genes in the literature. At each of these stages, LitMiner offers the functionality of a digital library. Documents that are accessible online are identified by an icon. Users can also order documents from their institution's library collection from within the application. In so doing, LitMiner aims to integrate digital library services into the research process of its users.

Methods

Case study

Results

This integration of digital library services into the research process of biologists results in increased access to the published literature.

Conclusion

In order to make better use of their collections, digital libraries should customize their services to suit the research needs of their patrons.
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16.

Introduction

Collecting feces is easy. It offers direct outcome to endogenous and microbial metabolites.

Objectives

In a context of lack of consensus about fecal sample preparation, especially in animal species, we developed a robust protocol allowing untargeted LC-HRMS fingerprinting.

Methods

The conditions of extraction (quantity, preparation, solvents, dilutions) were investigated in bovine feces.

Results

A rapid and simple protocol involving feces extraction with methanol (1/3, M/V) followed by centrifugation and a step filtration (10 kDa) was developed.

Conclusion

The workflow generated repeatable and informative fingerprints for robust metabolome characterization.
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17.

Background

In recent years the visualization of biomagnetic measurement data by so-called pseudo current density maps or Hosaka-Cohen (HC) transformations became popular.

Methods

The physical basis of these intuitive maps is clarified by means of analytically solvable problems.

Results

Examples in magnetocardiography, magnetoencephalography and magnetoneurography demonstrate the usefulness of this method.

Conclusion

Hardware realizations of the HC-transformation and some similar transformations are discussed which could advantageously support cross-platform comparability of biomagnetic measurements.
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18.

Background

Immune cell infiltrates (ICI) of tumors are scored by pathologists around tumor glands. To obtain a better understanding of the immune infiltrate, individual immune cell types, their activation states and location relative to tumor cells need to be determined. This process requires precise identification of the tumor area and enumeration of immune cell subtypes separately in the stroma and inside tumor nests. Such measurements can be accomplished by a multiplex format using immunohistochemistry (IHC).

Method

We developed a pipeline that combines immunohistochemistry (IHC) and digital image analysis. One slide was stained with pan-cytokeratin and CD45 and the other slide with CD8, CD4 and CD68. The tumor mask generated through pan-cytokeratin staining was transferred from one slide to the other using affine image co-registration. Bland-Altman plots and Pearson correlation were used to investigate differences between densities and counts of immune cell underneath the transferred versus manually annotated tumor masks. One-way ANOVA was used to compare the mask transfer error for tissues with solid and glandular tumor architecture.

Results

The overlap between manual and transferred tumor masks ranged from 20%–90% across all cases. The error of transferring the mask was 2- to 4-fold greater in tumor regions with glandular compared to solid growth pattern (p < 10?6). Analyzing data from a single slide, the Pearson correlation coefficients of cell type densities outside and inside tumor regions were highest for CD4 + T-cells (r = 0.8), CD8 + T-cells (r = 0.68) or CD68+ macrophages (r = 0.79). The correlation coefficient for CD45+ T- and B-cells was only 0.45. The transfer of the mask generated an error in the measurement of intra- and extra- tumoral CD68+, CD8+ or CD4+ counts (p < 10?10).

Conclusions

In summary, we developed a general method to integrate data from IHC stained slides into a single dataset. Because of the transfer error between slides, we recommend applying the antibody for demarcation of the tumor on the same slide as the ICI antibodies.
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19.

Introduction

Developments in technology, web-based teaching and whole slide imaging have broadened the teaching horizon in anatomic pathology. Creating online learning material including many types of media such as radiologic images, whole slides, videos, clinical and macroscopic photographs, is now accessible to most universities. Unfortunately, a major limiting factor to maintain and update the learning material is the amount of resources needed. In this perspective, a French-national university network was initiated in 2011 to build joint online teaching modules consisting of clinical cases and tests. The network has since expanded internationally to Québec, Switzerland and Ivory Coast.

Method

One of the first steps of the project was to build a learning module on inflammatory skin pathology for interns and residents in pathology and dermatology. A pathology resident from Québec spent 6 weeks in France and Switzerland to develop the contents and build the module on an e-learning Moodle platform under the supervision of two dermatopathologists. The learning module contains text, interactive clinical cases, tests with feedback, virtual slides, images and clinical photographs. For that module, the virtual slides are decentralized in 2 universities (Bordeaux and Paris 7). Each university is responsible of its own slide scanning, image storage and online display with virtual slide viewers.

Results

The module on inflammatory skin pathology includes more than 50 web pages with French original content, tests and clinical cases, links to over 45 virtual images and more than 50 microscopic and clinical photographs. The whole learning module is being revised by four dermatopathologists and two senior pathologists. It will be accessible to interns and residents in the spring of 2014. The experience and knowledge gained from that work will be transferred to the next international resident whose work will be aimed at creating lung and breast pathology learning modules.

Conclusion

The challenges of sustaining a project of this scope are numerous. The technical aspect of whole-slide imaging and storage needs to be developed by each university or group. The content needs to be regularly updated and its accuracy reviewed by experts in each individual domain. The learning modules also need to be promoted within the academic community to ensure maximal benefit for trainees. A collateral benefit of the project was the establishment of international partnerships between French-speaking universities and pathologists with the common goal of promoting pathology education through the use of multi-media technology including whole slide imaging.
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20.

Background

The objective of this study was to evaluate angiogenesis according to CD34 antigen expression in estrogen receptor (ER)-positive and negative breast carcinomas.

Methods

This study comprised 64 cases of infiltrating ductal carcinoma in postmenopausal women divided into two groups: Group A: ER-positive, n = 35; and Group B: ER-negative, n = 29. The anti-CD34 monoclonal antibody was used as a marker for endothelial cells. Microvessel count was carried out in 10 fields per slide using a 40× objective lens (magnification 400×). Statistical analysis of the data was performed using Student's t-test (p < 0.05).

Results

The mean number of vessels stained with the anti-CD34 antibody in the estrogen receptor-positive and negative tumors was 23.51 ± 1.15 and 40.24 ± 0.42, respectively. The number of microvessels was significantly greater in the estrogen receptor-negative tumors (p < 0.001).

Conclusion

ER-negative tumors have significantly greater CD34 antigen expression compared to ER-positive tumors.
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