Marylène Lejeune

Validation of various adaptive threshold methods of segmentation applied to follicular lymphoma digital images stained with 3,3’-Diaminobenzidine&Haematoxylin

AbstractThe comparative study of the results of various segmentation methods for the digital images of the follicular lymphoma cancer tissue section is described in this paper. The sensitivity and specificity and some other parameters of the following adaptive threshold methods of segmentation: the Niblack method, the Sauvola method, the White method, the Bernsen method, the Yasuda method and the Palumbo method, are calculated. Methods are applied to three types of images constructed by extraction of the brown colour information from the artificial images synthesized based on counterpart experimentally captured images. This paper presents usefulness of the microscopic image synthesis method in evaluation as well as comparison of the image processing results. The results of thoughtful analysis of broad range of adaptive threshold methods applied to: (1) the blue channel of RGB, (2) the brown colour extracted by deconvolution and (3) the ’brown component’ extracted from RGB allows to select some pairs: method and type of image for which this method is most efficient considering various criteria e.g. accuracy and precision in area detection or accuracy in number of objects detection and so on. The comparison shows that the White, the Bernsen and the Sauvola methods results are better than the results of the rest of the methods for all types of monochromatic images. All three methods segments the immunopositive nuclei with the mean accuracy of 0.9952, 0.9942 and 0.9944 respectively, when treated totally. However the best results are achieved for monochromatic image in which intensity shows brown colour map constructed by colour deconvolution algorithm. The specificity in the cases of the Bernsen and the White methods is 1 and sensitivities are: 0.74 for White and 0.91 for Bernsen methods while the Sauvola method achieves sensitivity value of 0.74 and the specificity value of 0.99. According to Bland-Altman plot the Sauvola method selected objects are segmented without undercutting the area for true positive objects but with extra false positive objects. The Sauvola and the Bernsen methods gives complementary results what will be exploited when the new method of virtual tissue slides segmentation be develop.Virtual SlidesThe virtual slides for this article can be found here: slide 1: http://diagnosticpathology.slidepath.com/dih/webViewer.php?snapshotId=13617947952577 and slide 2: http://diagnosticpathology.slidepath.com/dih/webViewer.php?snapshotId=13617948230017.

Role of Immune Escape Mechanisms in Hodgkin's Lymphoma Development and Progression: A Whole New World with Therapeutic Implications

Hodgkins lymphoma represents one of the most frequent lymphoproliferative syndromes, especially in young population. Although HL is considered one of the most curable tumors, a sizeable fraction of patients recur after successful upfront treatment or, less commonly, are primarily resistant. This work tries to summarize the data on clinical, histological, pathological, and biological factors in HL, with special emphasis on the improvement of prognosis and their impact on therapeutical strategies. The recent advances in our understanding of HL biology and immunology show that infiltrated immune cells and cytokines in the tumoral microenvironment may play different functions that seem tightly related with clinical outcomes. Strategies aimed at interfering with the crosstalk between tumoral Reed-Sternberg cells and their cellular partners have been taken into account in the development of new immunotherapies that target different cell components of HL microenvironment. This new knowledge will probably translate into a change in the antineoplastic treatments in HL in the next future and hopefully will increase the curability rates of this disease.

Segmentation of Stained Lymphoma Tissue Section Images

In order to obtain supporting tool for the pathologists who are investigating prognostic factors in folicular lymphoma a new method of color images segmentation is proposed. The method works on images acquired from immunohistochemically stained thin tissue sections of lymph nodes coming from patients with folicular lymphoma diagnosis. The proposed method of segmentation consists of: pre-processing, adaptive threshold, watershed segmentation and post-processing. The method is tested on a set of 50 images. Its results are compared with results of manual counting. It has been found that difference between the traditional method results and the proposed method is small for images with up to 100 nuclei while in more complicated images with more then 100 nuclei and with nuclei clusters this difference increases.

Equalisation of archival microscopic images from immunohistochemically stained tissue sections

A method of image equalisation that reduces non-uniformity of light distribution caused by optical devices and dust on camera sensors is presented. The method explores non-uniformity which occurs in archival images captured by a typical optical set which consists of a light microscope and a digital camera. A sufficient number of images with low density of foreground objects has been used to extract a global map of non-uniformity of the particular microscope and camera. The proposed method consists of two steps: – (1) extraction of the map of non-uniformity based upon a set of chosen images and – (2) correction of images acquired by the optical set. The global map is created based upon a modified value layer, the third layer of HSV colour space. The proposed method has been tested on images of immunohistochemically (IHC) stained samples of a biopsy tissue, and it has been validated using an image segmentation method developed earlier. The results of the light distribution equalization, as well as the equalized images segmentation turn out to be more similar to the reference method results (namely the manual counting results), than the results of the original images segmentation. The equalization method can be used for other types of images, but all of them should be acquired by the same optical set.

Performance of the digene LQ, RH and PS HPVs genotyping systems on clinical samples and comparison with HC2 and PCR-based Linear Array

BackgroundCertain Human Papillomaviruses (HPVs) are the infectious agents involved in cervical cancer development. Detection of HPVs DNA is part of the cervical cancer screening protocols and HPVs genotyping has been proposed for its inclusion in these preventive programs. The aim of this study was to evaluate three novel genotyping tests, namely Qiagen LQ, RH and PS, in clinical samples with and without abnormalities. For this, 305 cervical samples were processed and the results of the evaluated techniques were compared with those obtained in the HPVs diagnostic process in our lab, by using HC2 and Linear Array (LA) technologies.ResultsThe concordances and kappa statistics (k) for each technique compared with HC2 were 98.69% (k = 0.94) for LQ, 98.03% (k = 0.91) for RH and 91.80% (k = 0.82) for PS. There was a very good agreement in HPVs type-specific concordance for the most prevalent types HPV16 (kappa range = 0.83-0.90), HPV18 (k.r.= 0.74-0.80) and HPV45 (k.r.= 0.82-0.90).ConclusionsThe three tests showed an overall good concordance for HPVs detection when compared with HR-HC2 system. LQ and RH rendered lower detection rate for multiple infections than LA genotyping. However, our understanding of the clinical significance of multiple HPVs infections is still incomplete and therefore the relevance of the lower ability to detect multiple infections needs to be evaluated.

A multistep image analysis method to increase automated identification efficiency in immunohistochemical nuclear markers with a high background level

The “removal” of background from digital images (DIs) to identify only the objects of interest is difficult due to the overlapping color values of pixels of the nuclei and background. We outline a new automated procedure to quantify only immunohistochemically stained nuclear markers, despite the similar color of the background. This procedure was compared with the gold standard manual method and a previous method developed for low-background DIs.

Acute liver failure as the first manifestation of very late relapsing of Hodgkin’s disease

Hodgkins disease is, in general, a lymph node-based disease. It usually starts in an area within the lymphatic system and spreads, in an orderly manner, along the lymphatic chain to contiguous lymph node areas. There have been sporadic case reports of acute liver failure caused by hematological malignancies. Generally, liver failure is a feature of stage IV end-stage disease, when it occurs in lymphoma. Thus, hepatic involvement usually occurs late in the course of Hodgkins disease or with advanced-stage disease, and primary presentation in the liver with acute liver failure is extremely rare. In most cases, the diagnosis was made at autopsy. We describe a patient with Hodgkins disease presenting with acute liver failure. This is a very unusual Hodgkins disease form of presentation, because the acute liver failure was the presenting feature of the disease. Furthermore, the lymphoma occurred as a very late relapse, twenty years after the first diagnosis. To the best of our knowledge, such a case has not been described to date.

Roundness variation in JPEG images affects the automated process of nuclear immunohistochemical quantification: correction with a linear regression model

The volume of digital image (DI) storage continues to be an important problem in computer-assisted pathology. DI compression enables the size of files to be reduced but with the disadvantage of loss of quality. Previous results indicated that the efficiency of computer-assisted quantification of immunohistochemically stained cell nuclei may be significantly reduced when compressed DIs are used. This study attempts to show, with respect to immunohistochemically stained nuclei, which morphometric parameters may be altered by the different levels of JPEG compression, and the implications of these alterations for automated nuclear counts, and further, develops a method for correcting this discrepancy in the nuclear count. For this purpose, 47 DIs from different tissues were captured in uncompressed TIFF format and converted to 1:3, 1:23 and 1:46 compression JPEG images. Sixty-five positive objects were selected from these images, and six morphological parameters were measured and compared for each object in TIFF images and those of the different compression levels using a set of previously developed and tested macros. Roundness proved to be the only morphological parameter that was significantly affected by image compression. Factors to correct the discrepancy in the roundness estimate were derived from linear regression models for each compression level, thereby eliminating the statistically significant differences between measurements in the equivalent images. These correction factors were incorporated in the automated macros, where they reduced the nuclear quantification differences arising from image compression. Our results demonstrate that it is possible to carry out unbiased automated immunohistochemical nuclear quantification in compressed DIs with a methodology that could be easily incorporated in different systems of digital image analysis.

Improvements to Segmentation Method of Stained Lymphoma Tissue Section Images

We present the METINUS (METhod of Immunohistochemical NUclei Segmentation) , which is a improved and modified version of supporting tool for pathologists from 2010. The method supports examination of immunohistochemically stained thin tissue sections from biopsy of follicular lymphoma patients. The software localizes and counts FOXP3 expression in the cells’ nuclei supporting standard procedure of diagnosis and prognosis. The algorithm performs colour separation followed by object extraction and validation. Objects with statistical parameters not in specified range are disqualified from further assessment. To calculate the statistics we use the following: three channels of RGB, three channels of Lab colour space, brown channel and three layers completed with colour deconvolution. Division of the objects is done with support of watershed and colour deconvolution algorithm. Evaluation was performed on arbitrarily chosen 20 images with moderate quality of most typical tissues. We compared results of improved method with the previous version in the context of semiautomatic, pathologist controlled, computer-aided result of quantification as reference. Comparison is based on quantity of nuclei located per image using Kendall’s tau-b correlation coefficient. It shows concordance of 0.91 between results of proposed method and reference, while with previous version it is only 0.71.

JPEG2000 for automated quantification of immunohistochemically stained cell nuclei: a comparative study with standard JPEG format.

The Joint Photographic Experts Group (JPEG) standard format is one of the most widely used in image compression technologies. More recently, JPEG2000 format has emerged as a state-of-the-art technology that provides substantial improvements in picture quality at higher compression ratios. However, there has been no attempt to date to determine which of the two compression formats produces less variability in the automated evaluation of immunohistochemically stained digital images in agreement with their compression rates and complexity degrees. The evaluation of Ki67 and FOXP3 immunohistochemical nuclear markers was performed in a total of 329 digital images: 47 were captured in uncompressed Tagged Image File Format (TIFF), 141 were converted to three JPEG compressed formats (47 each with 1:3, 1:23 and 1:46 compression) and 141 were converted to three JPEG2000 compressed formats (47 each with 1:3, 1:23 and 1:46 compression). The count differences between images in TIFF versus JPEG formats were compared with those obtained between images in TIFF versus JPEG2000 formats at the three levels of compression. It was found that, using JPEG2000 compression, the results of the stained nuclei count are close enough to the results obtained with uncompressed images, especially in highly complex images at minimum and medium compression. Otherwise, in images of low complexity, JPEG and JPEG2000 had similar count efficiency to that of the original TIFF images at all compression levels. These data suggest that JPEG2000 could give rise to an efficient means of storage, reducing file size and storage capacity, without compromise on the immunohistochemical analytical quality.