Добірка наукової літератури з теми "Immunohistochemistry"

Immunohistochemistry (IHC) or immunocytochemistry is a method of localizing specific antigen in tissue or cells based on antigen antibody reaction. IHC is the way of validating morphological findings. It helps in tumor diagnosis and classification, identify prognostic and predictive markers. IHC has a long history that dates back more than 70 years, when Coons1 first developed immunofluroscence technique to detect corresponding antigen in frozen tissue section. At oxford, Taylor and Burns2 developed the first successful demonstration of antigens in routinely processed formalin fixed paraffin-embedded sections. Since the early 1990s, IHC has been applied in routine formalin fixed paraffin embedded tissue. 3-4 Validation of reagents, protocols, controls and staining results are vital steps of IHC. The basic principles and protocols for fresh-frozen tissue sections are the same as those for paraffin sections, except that the antigen retrieval and dewaxing procedures are not required for frozen tissue sections. Titrations may also differ and must be separately optimized. Basic principles: Antigen-antibody recognition is based on the three-dimensional (3D) structure of protein or some other antigen, which may be compromised by formalin-induced modification of protein conformation (“masking”) but is restored in part by Antigen retrieval. Anti-A antibody binds specifically to antigen A in the tissue section. Antigen B (B) is depicted as a second antigenic determinant that is part of the anti-A molecule; anti-B antibody, made in a second species, will bind to this determinant. Thus anti-B, the so-called secondary antibody, can be used to locate the site of binding of anti-A, the primary antibody, in a tissue section. Basic IHC procedure Antigen retrieval (AR)-Enzymatic digestion (proteinase or trypsin), heat treatment (Microwave, water bath or autoclave) Blocking of non-specific background staining Incubation with primary antibody in humidity chamber Add avidin-biotin-peroxidase complex, which binds to secondary antibody Add 3, 3’ diaminobenzidine (DAB) as a chromagen (color changing reagent), with hematoxylin (Mayer's) counterstaining Theranostic Application IHC is becoming increasingly important for the evaluation of predictive markers that can help select patients who may respond to particular targeted therapies. Some of these CD117 for GI stromal tumors, Herceptin (Genentech, South San Francisco, CA) for HER2-positive breast cancers, and rituximab for CD20-positive lymphomas.

The aim of this project was to compare and contrast the results of three related methodologies which are of particular relevance or potential within our department -. a manually performed streptAvidin Biotin Complex (sABC/ HRP) method which for many years has served as our 'standard' immunocytochemical method; an integrated semi-automated systems from DAKO based on the TechMate 500 immunostainer and the ChemMate reagent package;. a manually performed tyramide amplification method regarded as amongst the most sensitive immunocytochemical method currently available. Antigen retrieval investigating differing durations of exposure to the proteolytic enzyme trypsin, and heat mediated antigen retrieval using citrate buffer pH6.0 or DAKO high pH antigen retrieval solution were also included as parameters for investigation. To this aim a panel of eleven specific antibodies were assessed, each of which had previously displayed individual qualities worthy of investigation -. PGP9.5, CD30, oestrogen receptor, CD3 polyclonal, CD2, CD3 monoclonal, CD4, cyclin D1, kappa immunoglobulin light chain, lambda immunoglobulin light chain and IgD immunoglobulin heavy chain. The results of this work demonstrate that each of the systems investigated has its individual merits -. The manual sABC/ HRP method is the least complicated and expensive of the methods to perform. There are significant disadvantages in its used however. Being a manual method there are many steps where individual variation in working practices within a laboratory can and do affect results. The TechMate/ ChemMate system is a more sensitive system than the manual sABC/ HRP method also providing a cleaner end result and allowing higher dilutions of primary antibody to be employed. Tyramide amplification is the most sensitive system evaluated, the antibodies in this work generally demonstrated immunostaining at higher dilutions than the other methods tested although some, in particular those directed against immunoglobulin light chains, operated better under the ChemMate/ TechMate system. On occasion specific positive results were obtained which none of the other methods tested could achieve with certain antibodies, CD3 monoclonal and to a lesser extent CD2.

This thesis focus on the analysis of digitized microscopic image, especially on IHC stained colour images. The corresponding contributions focused on the automatic detection of stain colour and glands, the segmentation and quantification of cell nuclei, the analysis of liver cirrhosis and the development of a semi-automatic toolbox. Colour is the most important feature in the analysis of immunostained images. We developed a statistical colour detection model for stain colour detection based on the histograms of collected colour pixels. This is acting on the approach "what you see is what you get" which outperforms the other methods on the detection of several kinds of stain colour. Verifying the presence of nuclei and quantifying positive nuclei is the foundation of cancer grading. We developed a novel seeded nuclei segmentation method which greatly improves the segmentation accuracy and reduces both over-segmentation and under-segmentation. This method has been demonstrated to be robust and accurate in both segmentation and quantification against manual labelling and counting in the evaluation process. The analysis of gland architecture, which reflects the cancer stage, has evolved into an important aspect of cancer detection. A novel morphology-based approach has been developed to segment gland structures in H-DAB stained images. This method locates the gland by focusing on its morphology and intensity characteristics, which covers variations in stain colours in different IHC images. The evaluation results have demonstrated the improvements of accuracy and efficiency. For the successive development of three methods, we put them in a semi-automatic toolbox for the aid of IHC image analysis. It can detect different kinds of stain colour and the basic components in an IHC image. The user created models and parameters can be saved and transferred to different users for the reproduction of detection results in different laboratories. To demonstrate the flexibility of our developed stained colour detection technique, the tool has been extended to the analysis of liver cirrhosis. It is a novel method based on our statistical colour detection model which greatly improves the analysis accuracy and reduces the time cost.

Although it has been estimated that protein histidine phosphorylation account for about 6 % of the protein phosphorylation in eukaryotic cells; the knowledge of histidine phosphorylation and dephosphorylation is still limited. Lately, studies have appeared of a mammalian 14-kDa phospho- histidine phosphatase, also named protein histidine phosphatase and molecular cloning have provided some information of its physiological role. The object of the present study was to detect the protein expression of protein histidine phosphatase, PHPT1, in mouse tissue, by using immunohistochemistry. Tissue samples from a 4-week-old mouse (heart, liver, kidney, lung, muscle, and spleen), 5-month-old mouse (testis and intestinal), 8-month-old mouse (uterus) and an embryo from 14.5 days old mouse were obtained and processed for light microscopic examination. An absorption test was also made to confirm the specificity of the antibody. The results reveal that PHPT1 is mainly expressed in epithelium, heart- and skeletal muscle. These results provide new evidences for the understanding of the function of eukaryotic histidine phosphorylation and dephosphorylation.

KEYWORDS

Phosphohistidine, dephosphorylation, protein histidine phosphatase, phosphohistidine phosphatase, protein phosphorylation

Although it has been estimated that protein histidine phosphorylation account for about 6 % of the protein phosphorylation in eukaryotic cells; the knowledge of histidine phosphorylation and dephosphorylation is still limited. Lately, studies have appeared of a mammalian 14-kDa phospho- histidine phosphatase, also named protein histidine phosphatase and molecular cloning have provided some information of its physiological role. The object of the present study was to detect the protein expression of protein histidine phosphatase, PHPT1, in mouse tissue, by using immunohistochemistry. Tissue samples from a 4-week-old mouse (heart, liver, kidney, lung, muscle, and spleen), 5-month-old mouse (testis and intestinal), 8-month-old mouse (uterus) and an embryo from 14.5 days old mouse were obtained and processed for light microscopic examination. An absorption test was also made to confirm the specificity of the antibody. The results reveal that PHPT1 is mainly expressed in epithelium, heart- and skeletal muscle. These results provide new evidences for the understanding of the function of eukaryotic histidine phosphorylation and dephosphorylation. KEYWORDS Phosphohistidine, dephosphorylation, protein histidine phosphatase, phosphohistidine phosphatase, protein phosphorylation

Background: The rare nature of male breast cancer (MBC) has led to its management being guided by the extensive research conducted in the field of female breast cancer (FBC). The aim of this study was to evaluate MBC at both protein and molecular level to improve understanding of its pathology. Methodology: Immunohistochemistry analysis was performed in MBC (n=428) TMAs for 18 biomarkers (ERα, ERβ1, ERβ2, ERβ5, Total PR, AR, CK5/6, CK14, CK18, CK19, p53, Bcl-2, Her2, E-cadherin, Ki67, Survivin, Prolactin and FOXA1). The manual scoring of ERα and Ki67 was correlated with a fully automated immunohistochemistry image analysis system (ImmunoRatio™). Finally gene expression profiling (GEP) was undertaken in matched MBC (n=15) and FBC (n=10) samples. Results: There was poor 5 year overall survival (OS) in CK18 and CK19 negative patients (p= 0.05; p= 0.003), as well as poor 10 year OS in CK19 negative patients (p= 0.002). Age (p= 0.001) and nodal status (p= 0.04) was found to be independent predictors of OS at 5 years. There was significant correlations between manual and ImmunoRatio™ ERα (ρ= 0.872; p= 0.000) and Ki67 (r= 0.675; p= 0.000) scores. However due to a low measure of agreement it was not possible to validate Ki67 scoring using ImmunoRatio™. The functional enrichment analysis of GEP data using less stringent criteria (p < 0.05) identified 735 differentially expressed genes. The data analysis showed up-regulation of genes involved in ECM synthesis, degradation and re-modelling in MBC. The end product of one of the up-regulated genes (Fibronectin (FN1)) was validated in the MBC cohort with high fibronectin expression (60%) being positively associated with nodal status and showed a trend towards poor 5 year OS (p= 0.06). Conclusion: In MBC, epithelial cytokeratins, especially CK19 was found to be of prognostic significance. The extracellular matrix remodelling associated genes were found to be up-regulated in MBC. Fibronectin, end product of one of the up-regulated gene was found to have prognostic significance in MBC.

Primary scalp alopecia is classically divided into cicatricial (scarring) and non-cicatricial (non-scarring). Challenging cases are assessed with a scalp biopsy. The use of both horizontal and vertical sections (HoVert sections) has dramatically improved the accuracy of histopathological diagnosis. In this work, we have used immunostaining to address diagnostic difficulties, which persist despite all currently available tools. We performed an immunostain panel (CD3, CD4, CD8 and CD20) in order to distinguish pattern hair loss from alopecia aerate in cases which do not have the usual peribulbar lymphocytic infiltrate and showed that CD3+ T-lymphocytes within the empty fibrous follicular tracts favor a diagnosis of alopecia areata. We performed CD123 in order to distinguish lichen planopilaris from alopecia lupus erythematosus in cases with only a superficial lymphocytic infiltrate and an uninvolved interfollicular epidermis and showed that clusters of CD123+ plasmacytoid dendritic cells favor a diagnosis of lupus erythematosus. We performed cytokeratin 15 in order to assess whether the loss of the follicular bulge stem cells has diagnostic value in cicatricial alopecia and demonstrated that the loss of cytokeratin 15+ bulge stem cells is identified in lichen planopilaris, frontal fibrosing alopecia, and lupus erythematous, so cytokeratin 15 has no diagnostic value. We have attempted to integrate the new concepts and our findings into the traditional classifications of alopecia and proposed a new diagnostic algorithm. In conclusion, immunostaining combined with HoVert grossing advances the accuracy of histopathological diagnosis of primary scalp alopecia.
L’alopécie primitive du cuir chevelu est habituellement classée en cicatricielle et non-cicatricielle. Dans les cas difficiles, la biopsie du cuir chevelu peut aider au diagnostic. L’utilisation de coupes, à la fois verticales et horizontales sur le même spécimen (technique HoVert), a radicalement amélioré le diagnostic histopathologique. Dans ce travail, nous avons utilisé l’immunohistochimie pour évaluer les difficultés diagnostiques qui persistent malgré tous les outils actuels. Nous avons utilisé les CD3, CD4, CD8 et CD20 pour différencier l’alopécie androgénique de la pelade dépourvue de l’infiltrat lymphocytaire péribulbaire habituel et nous avons démontré que la présence de lymphocytes CD3+ dans les travées folliculaires fibreuses est en faveur de la pelade. Nous avons utilisé le CD123 pour différencier le lichen plan pilaire du lupus érythémateux alopécie avec infiltrat lymphocytaire superficiel et sans atteinte de l’épiderme interfolliculaire et nous avons démontré que la présence d’amas de cellules dendritiques plasmacytoïdes CD123+ est en faveur du lupus érythémateux. Nous avons utilisé la cytokératine 15 pour évaluer si la perte des cellules souches du bulge a une valeur diagnostique dans l’alopécie cicatricielle et nous avons démontré que cette perte s’observait de manière identique dans le lichen plan pilaire, l’alopécie frontale fibrosante comme dans le lupus érythémateux et n’avait donc aucune valeur diagnostique. Nous avons tenté d’intégrer les nouveaux concepts et nos données dans les classifications traditionnelles des alopécies et nous avons élaboré un nouvel algorithme diagnostique. L’association des immunomarquages avec la technique HoVert ouvre de nouvelles perspectives dans le diagnostic histopathologique des alopécies primaires du cuir chevelu.
Doctorat en Sciences médicales (Médecine)
info:eu-repo/semantics/nonPublished