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Diagnostic of immunohistochemistry in pathology
HRDINOVÁ, Andrea
The bachelor thesis deals with the use of immunohistochemical methods in the diagnosis of neoplasms in pathology. Describes the use of antibody spectrum at the Department of Pathology, Český Krumlov Hospital, a.s. Immunohistochemical methods are nowadays an important method used in pathology for tumor typing and metastasis for the determination of primary tumors. They complement the standard staining methods for basic diagnostics such as Hematoxylin-Eosin and Van-Gieson, which are less specific, these methods mainly serve to differentiate the basic cellular components and tissue components of the preparation. Immunohistochemistry methods are based on the special antibody affinity principle and are divided into direct and indirect methods. For a direct method, an antibody labeled with, for example, an enzyme is used to detect the antigen and, upon binding, induces a color change observed under the microscope. Indirect method (two-stage, three-stage) is a more sensitive method, based on the principle of an unlabeled primary antibody to which a conjugated secondary antibody binds to an enzyme or biotin. In the three-stage, we use the affinity of avidin and biotin to form the avidin-biotin-enzyme complex (eg. peroxidase), which is shown and highlighted. In more detail, the thesis deals with the diagnosis of breast and sentinel lymph node tumors, both in the theoretical and practical part, and the incidence of these tumors since the introduction of the immunohistochemical method at the Department of Pathology of the Český Krumlov Hospital. Last but not least, a case report of individual interesting cases is described.
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Breast cancer immunohistochemistry
HYŽÍKOVÁ, Sabina
In my bachelor thesis I deal with imunohistochemical examination of breast cancer. I performed the research at the Department of pathology at the hospital in Písek. The thesis is divided into theoretical and practical part In the theoretical part I deal with anatomical structure of the breast, clinical symptoms, diagnostics together with imaging methods. Next risk factors, including genetic and hormonal aspects of breast cancer. Prognosis and also by prevention of tumor development and subsequent treatment of this disease. I summarized the general classification of tumors representing staging and grading. I also described pathological structures of the breast as benign and malignant lesions. In the practical part I deal with the description of the immunohistochemical method, I focused on how the method works and especially how the whole histological process looks like. I worked with a total amount of 218 biopsy samples of breast tissue, which were subsequently stained with this method and I determined 5 markers (oestrogen and progesterone receptors, HER-2/Neu, Ki67 and E-cadherin), which were used in their diagnosis. Based on the negativity of ER, PR and HER-2/Neu, I evaluated fourteen triple-negative lesions. Hormone receptors showed numerous positive results more than negative ones, while HER-2/Neu expression showed only slightly higher positivity in hundred and twenty two cases compared to the remaining ninety six negative cases. The results of the practical part of my bachelor thesis was finding, that the most prevalent histologic subtype of breast cancer is the invasive ductal carcinoma, which was represented in one hundred and ninety two cases, another type was invasive lobular carcinoma represented in twenty two cases and six ductal carcinoma in situ. The aim of my bachelor thesis was to present a brief description of the issue of breast cancer and point out the representation of the most frequently diagnosed subtypes as well as to describe the execution of a method that is typical for detecting breast cancer-associated receptors.
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Immunohistochemical and histological correlation of melanoma lesions
ČAPKOVÁ, Denisa
The bachelor thesis deals with the problematic of melanoma lesions. This theme is actual because melanoma is the most serious type of skin cancer and its incidence is moving to the younger generation. Because melanomas occur primary due to ultraviolet light exposure, one of the best possible ways to reduce the risk of developing melanoma is to protect yourself from ultraviolet radiation by using the sunscreen, wearing clothing that covers arms and legs and avoid using tanning beds. The prognosis of early melanoma is usually very good, so the detection and treatement is very important. In the theoretical part the current knowledge of melanoma, the characteristic, the occurence, the clasification, the prevention and the current possibilities of treatment were summarized. In the practical part the Hematoxylin eosin staining and the immunohistochemical method was described. There is also comparation of positive HMB-45 marker between each histological groups. The HMB-45 marker is absolutly specific for melanocytic tumors and has great utility fot the pathologist in differentiation of tumors with uncertain origin. From 2012 to 2018 were imunohistologicaly examined at the Departement of Pathology Nemocnice Písek a.s. 61 samples. Out of total 61 samples were 58 HMB-45 positive. The thesis has been focused on the imunohistological profile of each histological group. The imunohistologichemical profile was proven in 92%.
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Localization and quantification of mRNA coding digestive peptidases of Fascioloides magna
Peterková, Kristýna ; Kašný, Martin (advisor) ; Sojka, Daniel (referee)
Trematode peptidases are important molecules responsible for biocatalysis in many basal biological processes and are crucial in host-parasite interactions. Therefore, these enzymes are intensively studied in order to characterize their biological functions and to use them as potential diagnostic or therapeutic targets. Lately, investigation of transcriptome and secretome revealed, that adult Fascioloides magna (giant liver fluke) expresses and secretes a variety of peptidases. Thus, this thesis focuses on three newly identified enzymes: cathepsin L (FmCL), cathepsin B (FmCB) and cathepsin D (FmCD). In other trematode species, these cathepsins are being linked mainly with the digestion of host blood. We applied quantitative PCR (qPCR) to investigate relative expression levels of the three enzymes among three developmental stages - egg, miracidium and adult. It was revealed that all cathepsins have the highest expression in adult flukes in comparison to eggs and miracidia. We also localized the place of transcription of FmCL, FmCB and FmCD in adult fluke using RNA in situ hybridization. All of the peptidases were detected in gastrodermis, and in addition, they were localized in the reproductive system. The latter surprising finding is suggesting that these enzymes might have multiple functions in adult F....
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The use of immunohistochemistry for the examination of mammary gland biopsy.
MACHÁČOVÁ, Dominika
Background: Tumours of the breast are the most common tumours in the female population of the Czech Republic. Improvements in early diagnosis and therapy have led to gradual reduction of mortality with an increasing incidence of this disease. Aims: The aim of this study was to find out the significance of imunohistochemistry in diagnostics of tumours of the breast. Materials and methods: A total of 22 cases of malignant neoplasm of the breast were included in our study. Twenty two core-cut biopsies and twenty one resected specimens were eximined. Imunohistochemical staining with a total of 5 markers was performed - oestrogen and progesterone receptors, HER2, Ki-67, E-cadherin. In 9 cases, imunohistochemical examination in the resection specimens was also performed. The results of manual imunohistochemical staining and staining performed on autostainer Ventana Benchmark were compared. Results: The most frequent histologic subtype was invasive carcinoma of no special type in fourteen of cases, followed by four cases of invasive lobular carcinoma, two cases of atypical medullary carcinoma, one case of mixed (invasive carcinoma of no special type and invasive lobular carcinoma) carcinoma and one case of metaplastic carcinoma. In one case, a different diagnosis was made - invasive carcinoma of no special type was diagnosed in the core-cut biopsy while metaplastic carcinoma was diagnosed in the resected specimen. The expression of both oestrogen and progesterone receptors was variable. It ranged between 10-100 % (on average 81,9 % and 38,8 % respectively). Oestrogen receptors were found in 18 of cases while progesterone receptors were found in 16 of cases. The Ki-67 expression ranged between 2 and 40 % (on average 16,5 %); higher Ki-67 values ( 20 %) were found in tumours with loss of oestrogen and/or progesterone receptor expression and in poorly differentiated tumours. None of the cases was determinated as HER2 positive; a score 0 was used in 12 of the cases and a score 1+ in 10 of the cases. Conclusion: When comparing manual imunohistochemical staining staining with staining performed on the autostainer Ventana Benchmark very similar results were obtained.
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Diagnosis of Lynch syndrome based on pathologic examination
KRAUSOVÁ, Lenka
Lynch syndrome is an autosomal dominant disease predisposing to cancer development. Up to 5 % of colorectal cancers may be associated with Lynch syndrome. Due to its familial occurrence the diagnosis is important for family screening. Currently it is based on methods of modern pathology. Theoretical part describes structure of gastrointestinal tract, definition and history of Lynch syndrome, and methods of its laboratory diagnostics. The diagnosis can be based on evaluation of tumor microscopical features (Semi PREDICT score) in tissue sections, imunohistochemical investigation of MMR (mismatch repair) proteins, or molecular genetic MSI (microsatellite instability) testing. Practical part focuses mainly on correlation of immunohistochemistry and MSI testing. Randomly selected cases of colorectal cancer were organized into 2 study groups. The first group consisted of 25 cases with intact MMR immunoexpression, the second group comprised 25 cases with at least one MMR protein being deficient. For further verification molecular genetic MSI testing, along with BRAF gene analysis and MLH1 promoter methylation status to discriminate Lynch syndrome from sporadic cases, were performed. Germline analysis of MMR genes proved the diagnosis of Lynch syndrome in 5 cases of the second group. Semi PREDICT score sensitivity for MMR-deficiency prediction (and indirectly for Lynch syndrome detection) was 84 %, specificity 48 %. Sensitivity of MSI testing was 87 %, but only 50 % in Lynch syndrome subset of cases, specificity was 100 %. The results show major role of MMR immunohistochemistry in the diagnostics of MSI-H cancers, which is the cornerstone of Lynch syndrome screening.
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Use of Imunohistochemical Methods in Diagnostics of Neoplasms
VELKOVÁ, Michala
This thesis deals with the application of immunohistochemical methods in the diagnosis of neoplasms. Indications of immunohistochemical methods of particular departments in Hospital Písek, a.s. are outlined there. It describes the origins and development of this method in the Pathological Anatomy Department, Hospital Písek, a.s. In the theoretical part, I describe knowledge of single methods. As for immunohistochemistry, I focused on the determination of antigens and the use of suitable primary antibodies for diagnosing diseases, mostly neoplasms. There are 20 primary antibodies available at the Department of Pathological Anatomy. In the practical part, I compare conventional staining and immunohistochemical methods with conventional staining and special staining methods in histology for the diagnosis of tumours. Hematoxylin eosin serves for distinguishing of essential cellular components. Selective staining enables colour differentiation of various tissue components. Using immunohistochemistry, the presence of certain tissue antigens by antibody reaction with the immobilized chemical compounds is detected. This comparison I was applied at the differential diagnosis of malignant melanoma and myogenic tumours at the Pathological Anatomy Department, Hospital Písek, a.s. For the determination of malignant melanomas, initially the sample was histologically processed and stained with Hematoxylin eosin staining base. Secondarily, the argentaffin reaction was used to detect melanin pigment and immunohistochemical staining of antigen HMB 45 using a mouse monoclonal antibody against HMB 45 by Dako Company and S 100 protein using a polyclonal rabbit antibody against S 100 by Dako Company. 14 samples of patients with malignant melanoma were examined this way. HMB 45 was stained positively in 12 samples out of 14 in total. Monoclonal antibody against HMB 45 is for malignant melanoma quite specific. S 100 protein was detected in all tissue samples. Polyclonal antibody against S 100 shows higher sensitivity, however it is not very specific. It is more used to distinguish neuroectodermal and nonneuroektodermálních lesions. Melanin was detected in 11 tissue samples. Detection of melanin is also non-specific and complicated. Argentaffin reaction shows colouring of other substances contained in the cytoplasm, such as lipofuscin therefore it is necessary to use other methods for the melanoma diagnosis, e.g. immunohistochemistry. In the diagnosis of myogenic neoplasms, Hematoxylin eosin basic staining was used and subsequently immunohistochemical examination followed to detect smooth muscle actin and desmin using monoclonal mouse antibodies by Dako Company and transparent Hematoxylin van Gieson staining, which is used less than in the past. A total of 94 myogenic neoplasms (leiomyoms uterus) were examined. 72 neoplasms stained with Hematoxylin van Gieson showed a positive colour reaction of muscle mass and 22 neoplasms weak colour reaction. Smooth muscle actin and Desmin was detected by immunohistochemistry in 92 samples. The main problem of Hematoxylin van Gieson staining remains less specificity and sometimes ambiguous differentiability of colour tones in their own preparation impeding and preventing to set a reliable diagnosis in some cases. For this reason, immunohistochemical detection of Smooth muscle actin and Desmin (amongst others) is preferred as key parameters. Preferably, you can then use positive antibody reaction with Smooth muscle actinof myoepiteliels in the search for possible invasiveness mainly to differentiate in situ and microinvasive tumous in glandular organs. The differential diagnosis of myogenic origin tumours currently prefers immunohistochemical methods to special staining for high sensitivity and specificity, as well as in the differential diagnosis of malignant melanoma. When combined, both approaches complement each other well and provide relatively high degree of certainty in determining cells origin.
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