Wojciech Kozlowski

New automated image analysis method for the assessment of Ki-67 labeling index in meningiomas.

Many studies have emphasised the importance of Ki-67 labeling index (LI) as the proliferation marker in meningiomas. Several authors confirmed, that Ki-67 LI has prognostic significance and correlates with likelihood of tumour recurrences. These observations were widely accepted by pathologists, but up till now no standard method for Ki-67 LI assessment was developed and introduced for the diagnostic pathology. In this paper we present a new computerised system for automated Ki-67 LI estimation in meningiomas as an aid for histological grading of meningiomas and potential standard method of Ki-67 LI assessment. We also discuss the concordance of Ki-67 LI results obtained by presented computerized system and expert pathologist, as well as possible pitfalls and mistakes in automated counting of immunopositive or negative cells. For the quantitative evaluation of digital images of meningiomas the designed software uses an algorithm based on mathematical description of cell morphology. This solution acts together with the Support Vector Machine (SVM) used in the classification mode for the recognition of immunoreactivity of cells. The applied sequential thresholding simulated well the human process of cell recognition. The same digital images of randomly selected tumour areas were parallelly analysed by computer and blindly by two expert pathologists. Ki-67 labeling indices were estimated and the results compared. The mean relative discrepancy between the levels of Ki-67 LI by our system and by the human expert did not exceed 14% in all investigated cases. These preliminary results suggest that the designed software could be an useful tool supporting the diagnostic digital pathology. However, more extended studies are needed for approval of this suggestion.

Accuracy of a remote quantitative image analysis in the whole slide images

The rationale for choosing a remote quantitative method supporting a diagnostic decision requires some empirical studies and knowledge on scenarios including valid telepathology standards. The tumours of the central nervous system [CNS] are graded on the base of the morphological features and the Ki-67 labelling Index [Ki-67 LI]. Various methods have been applied for Ki-67 LI estimation. Recently we have introduced the Computerized Analysis of Medical Images [CAMI] software for an automated Ki-67 LI counting in the digital images.Aims of our study was to explore the accuracy and reliability of a remote assessment of Ki-67 LI with CAMI software applied to the whole slide images [WSI].The WSI representing CNS tumours: 18 meningiomas and 10 oligodendrogliomas were stored on the server of the Warsaw University of Technology. The digital copies of entire glass slides were created automatically by the Aperio ScanScope CS with objective 20x or 40x. Aperios Image Scope software provided functionality for a remote viewing of WSI. The Ki-67 LI assessment was carried on within 2 out of 20 selected fields of view (objective 40x) representing the highest labelling areas in each WSI. The Ki-67 LI counting was performed by 3 various methods: 1) the manual reading in the light microscope - LM, 2) the automated counting with CAMI software on the digital images – DI , and 3) the remote quantitation on the WSIs – as WSI method. The quality of WSIs and technical efficiency of the on-line system were analysed. The comparative statistical analysis was performed for the results obtained by 3 methods of Ki-67 LI counting. The preliminary analysis showed that in 18% of WSI the results of Ki-67 LI differed from those obtained in other 2 methods of counting when the quality of the glass slides was below the standard range. The results of our investigations indicate that the remote automated Ki-67 LI analysis performed with the CAMI algorithm on the whole slide images of meningiomas and oligodendrogliomas could be successfully used as an alternative method to the manual reading as well as to the digital images quantitation with CAMI software. According to our observation a need of a remote supervision/consultation and training for the effective use of remote quantitative analysis of WSI is necessary.

Automatic Evaluation System of FISH Images in Breast Cancer

The paper presents the algorithm of an automatic evaluation of the fluorescent in situ hybridization (FISH) images in order to determine HER2 status of the breast cancer samples. The algorithm is based on the accurate measurement of the red/green spot ratio (the ratio of HER2/CEN17) per cell nucleus. The main points of this algorithm is an accurate detection of the nuclei of cells and application of the color map to detect the red and green spots localized in these cells. The results of the numerical experiments concerning these two problems, as well as the assessment of the positivity or negativity of the considered cases are presented and discussed in the paper. They confirm the high efficiency of the proposed solution.

The influence of the microscope lamp filament colour temperature on the process of digital images of histological slides acquisition standardization

BackgroundThe aim of this study is to compare the digital images of the tissue biopsy captured with optical microscope using bright field technique under various light conditions. The range of colours variation in immunohistochemically stained with 3,3-Diaminobenzidine and Haematoxylin tissue samples is immense and coming from various sources. One of them is inadequate setting of cameras white balance to microscopes light colour temperature. Although this type of error can be easily handled during the stage of image acquisition, it can be eliminated with use of colour adjustment algorithms. The examination of the dependence of colour variation from microscopes light temperature and settings of the camera is done as an introductory research to the process of automatic colour standardization.MethodsSix fields of view with empty space among the tissue samples have been selected for analysis. Each field of view has been acquired 225 times with various microscope light temperature and camera white balance settings. The fourteen randomly chosen images have been corrected and compared, with the reference image, by the following methods: Mean Square Error, Structural SIMilarity and visual assessment of viewer.ResultsFor two types of backgrounds and two types of objects, the statistical image descriptors: range, median, mean and its standard deviation of chromaticity on a and b channels from CIELab colour space, and luminance L, and local colour variability for objects specific area have been calculated. The results have been averaged for 6 images acquired in the same light conditions and camera settings for each sample.ConclusionsThe analysis of the results leads to the following conclusions: (1) the images collected with white balance setting adjusted to light colour temperature clusters in certain area of chromatic space, (2) the process of white balance correction for images collected with white balance camera settings not matched to the light temperature moves image descriptors into proper chromatic space but simultaneously the value of luminance changes. So the process of the image unification in a sense of colour fidelity can be solved in separate introductory stage before the automatic image analysis.

Increased cyclooxygenase expression and thymine dimer formation after repeated exposures of humans to low doses of solar simulated radiation.

Abstract:u2002 The impact of repeated doses of solar simulated radiation (SSR) has not been evaluated, particularly to determine if photoadaptation and photoprotection develop over time. In this study, erythema, pigmentation, cyclooxygenase (COX)‐1 and 2 expression and thymine dimer (dTT) formation were evaluated in the skin of irradiated subjects of phototype II or III. Groups of 7–10 volunteers were whole‐body irradiated with a low dose of SSR on each of 10 consecutive days followed by a single erythemal ultraviolet B (UVB) dose on a small body area, or irradiated only with the single erythemal UVB dose on a small body area, or irradiated with the low dose of SSR on each of 30 consecutive days, or were unirradiated. Erythema and pigmentation were measured 24u2003h after the final SSR or UVB, and skin biopsies collected for the assessment of COX+ cells and dTT+ nuclei. The repeated SSR exposures induced a small increase in pigmentation without erythema, and were slightly protective against the erythemal effects of the subsequent high UVB dose. The number of COX‐1+ and 2+ cells increased as a result of 10‐days SSR and rose still further after 30‐days SSR, indicating that photoadaptation had not developed. The SSR exposures did not result in any protection against the further increase in COX‐1 and 2 expression caused by the erythemal UVB dose. In contrast, for dTT formation, the repeated SSR exposures led to a limited degree of both photoadaptation and photoprotection.

Immunohistochemical evaluation of human epidermal growth factor receptor 2 and estrogen and progesterone receptors in invasive breast cancer in women

Introduction Estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER-2) expression are crucial in the biology of breast carcinoma. HER-2/neu gene is amplified and overexpressed in 15-30% of invasive breast cancers. HER-2-positive breast cancers have worse prognosis than HER-2 negative tumors and possess distinctive clinical features. The aim of this study was to assess the expression of HER2 in cancer tissue of patients with invasive breast cancer in correlation with tumor type, histological grade, tumor size, lymph node status, and expression of estrogen receptor and progesterone receptor. Material and methods Formalin-fixed, paraffin-embedded tissues from 40 patients with invasive HER-2-positive breast cancer and from 191 patients with HER-2-negative breast cancer were used in this study. HER2 expression was determined using the test HerceptTest™ DAKO. Results Among 231 cases of breast cancer, 18 invasive lobular carcinomas and 213 invasive ductal carcinomas were diagnosed. Sixty percent of HER-2-positive breast cancers were ER-positive compared with 77% in the HER-2-negative group (p = 0.002). The expression of PR was observed in 43% of HER-2-positive breast cancers and in 72% of HER2-negative tumors (p = 0.003). Excessive expression of HER2 protein was detected in 60% of patients positive for estrogen receptors, which may worsen prognosis in these patients. Conclusions Determination of HER2 overexpression in breast cancer patients, allows for a determination of a group of patients with a worse prognosis.

Localization of spots in FISH images of breast cancer using 3‐D shape analysis

The fluorescence in situ (FISH) belongs to the most often used molecular cytogenetic techniques, applied in many areas of diagnosis and research. The analysis of FISH images relies on localization and counting the red and green spots in order to determine HER2 status of the breast cancer samples. The algorithm of spot localization presented in the paper is based on 3‐D shape analysis of the image objects. The subsequent regions of the image are matched to the reference pattern and the results of this matching influence localization of spots. The paper compares different shapes of the reference pattern and their efficiency in spot localization. The numerical experiments have been performed on the basis of 12 cases (patients), each represented by three images. Few thousands of cells have been analysed. The quantitative analyses comparing different versions of algorithm are presented and compared to the expert results. The best version of the procedure provides the absolute relative difference to the expert results smaller than 3%. These results confirm high efficiency of the proposed approach to the spot identification. The proposed method of FISH image analysis improves the efficiency of detecting fluorescent signals in FISH images. The evaluation results are encouraging for further testing of the developed automatic system directed to application in medical practice.

Automatic cell recognition in immunohistochemical gastritis stains using sequential thresholding and SVM network

The paper presents program for automatic cell recognition and counting in selected immunohistochemical stains in the gastritis diseases. It is applied to cytoplasm reactivity markers, such as chromogranin A, serotonin and somatostatin antibodies. The program uses the sequential thresholding algorithm in combination with artificial neural network of support vector machine (SVM) type, to recognize the nuclei of the separated cells. The constructed algorithm imitates the human view of the image. The support vector machine is used for recognition of the immunoreactivity of the separated cell. The results corresponding to the exemplary images, confirm good accuracy, comparable to the human expert.

Strain of experimental animals and modulation of nitric oxide pathway: their influence on development of renal failure in an experimental model of hepatorenal syndrome

Introduction Pathomechanism of HRS is still poorly understood. The aim of our study was: (1) to test whether different strains of rats could develop typical HRS, and (2) to estimate the influence of activation and inhibition of nitric oxide for development of renal failure in course of HRS. Material and methods First, we used 16 of Wistar and 16 of Sprague-Dawley rats in galactosamine model of HRS. Next, we used 48 of SDR rats, which received saline, N-nitro-L-arginine or L-arginine before and after liver damage. Twenty four hours urine and blood samples were collected 48 h after saline or Ga1N injection. Biochemical parameters were determined in serum or urine and then creatinine clearance and osmolality clearance were calculated. Liver and kidney tissues were collected for histopathological examination. Results Liver failure developed in all tested groups with significant increase of bilirubin (p < 0.001), ALT (p < 0.001) and ammonia (p < 0.001). Nevertheless we did not achieve any evidence of renal failure in Wistar, but we found typical renal failure in Sprague-Dawley group with significant decrease in creatinine clearance (p < 0.0012) and increase in concentration of creatinine and urea (p < 0.001) and (p < 0.001) respectively. Inhibition of NOS prevented development of renal failure with significant improvement of GFR both before (p < 0.0017) and after (p < 0.003) Ga1N injection. Injection of L-arginine after Ga1N injection did not caused significant improvement of GFR. Conclusions Our study showed, that genetic factors might be responsible for development of renal failure in course of HRS and nitric oxide play important role in acute model of this syndrome.

Novel segmentation algorithm for identification of cell membrane staining in HER2 images

We presented new approach to membrane staining determination in HER2 images.Our solution using predefined linear patterns is resistive to noise and artefacts.The system has shown good performance for HER2 breast cancer images. The paper presents new approach to the problem of a membrane staining determination in HER2 images of breast cancer. It is based on representation of the small membrane sections by the defined linear patterns of different shapes, which combined together allow representing the complex shape of the membrane staining. The discovered linear structures existing in the image are localized and recognized applying the specially devised masks sliding over the whole analyzed image. The method is resistant to the noise inherent in the image and to the varying detail complexity of the image. Its usefulness in HER2 image processing was verified on examples of the HER2 images of the breast cancer representing different grades of membrane staining.