Immunohistochemistry (IHC) is a technique used in pathology to determine the origins, prognosis, and treatment of tumors by detecting antigens in cells of a tissue section. It is important for accurate and consistent IHC results that laboratories follow rigorous quality control procedures and standards for tissue fixation, processing, sectioning, and staining. Key considerations include using the appropriate fixation time and temperature, optimizing antigen retrieval, selecting the proper primary antibody and detection system, and including appropriate controls. Delays in fixation or suboptimal fixation can compromise antigenicity and affect IHC results.
This document provides an overview of histology and microscopy. It discusses the four basic tissue types studied in histology, as well as techniques for tissue collection, processing, fixation, embedding and section cutting to prepare samples for microscopic examination. It also describes the principles of light microscopy and electron microscopy, and different types of microscopes used including bright field, dark field, phase contrast and fluorescence microscopes. The importance of histology for understanding the relationship between tissue structure and function is emphasized.
Practical Issues of FISH in Solid TumorsYosep Chong
This document discusses practical issues related to FISH analysis in solid tumors. It covers general procedures, sample adequacy assessment, quality control in test procedures and interpretation, and troubleshooting. Key points include the importance of proper tissue fixation and tumor cell adequacy. Quality control measures for baking, digestion, hybridization and imaging are outlined. Factors like probe type and cutoffs as well as using positive controls are emphasized for accurate interpretation. External quality assurance programs are also referenced.
In situ hybridization (ISH) is a technique that uses labeled probes to localize specific DNA or RNA sequences within cells in a tissue sample. It allows researchers to obtain information about gene expression and genetic loci in their cellular context. There are two main types of ISH - fluorescence in situ hybridization (FISH) and chromogenic in situ hybridization (CISH). ISH requires many optimization steps for each tissue and probe used but can provide insights into physiological processes and disease pathogenesis by identifying specific mRNA sequences within individual cells.
Practical Histopathology and cytopathology
Histopathological examination is used to provide diagnostic information that is important for timely diagnosis of disease to determine treatment plan. Fresh tissue is extremely fragile & subject to autolysis.
The document discusses fixation in histology and cytology. It describes the aims of fixation as preserving tissue structure and preventing autolysis and bacterial growth. Fixation causes some changes like tissue shrinkage and hardening. The types of fixation include immersion, coating, vapor and perfusion. Formalin and alcohol are common fixatives. Factors like pH, temperature, duration and osmolarity influence fixation quality. The choice of fixative depends on the tissue and technique used, for example glutaraldehyde for electron microscopy. Useful fixative formulas are also provided.
This document provides information on handling specimens for histopathology. It discusses what histopathology is, general rules for biopsies including avoiding necrosis and orienting specimens. It describes different types of biopsies like incisional, punch and excisional biopsies. It outlines how to take surgical specimens, send them to the pathology lab in proper containers and labels, and handle specimens including fixation. The document also discusses which information is important for the pathologist and rapid diagnosis techniques like cytopathology and frozen sections.
Frozen sections allow for rapid microscopic analysis of tissue during surgery. The procedure involves freezing tissue, sectioning it with a cryostat microtome, and staining with H&E. This enables real-time diagnosis and assessment of surgical margins. Accuracy depends on the pathologist's experience and tissue type, but diagnoses can often be provided to surgeons within 10-15 minutes to guide surgery. Regular audits help ensure the quality of individual frozen section services.
This document discusses quality control in microbiology. It covers various aspects of quality control including the pre-analytic, analytic and post-analytic phases of diagnostic testing. In the pre-analytic phase, it discusses proper specimen collection, transport and processing. The analytic phase covers microscopic examination, culture processing, identification and antimicrobial susceptibility testing. It also discusses quality control of equipment, reagents and culture media. The post-analytic phase involves reporting of results and interaction with epidemiologists. Maintaining accurate records and samples is also emphasized.
This document provides an overview of histology and microscopy. It discusses the four basic tissue types studied in histology, as well as techniques for tissue collection, processing, fixation, embedding and section cutting to prepare samples for microscopic examination. It also describes the principles of light microscopy and electron microscopy, and different types of microscopes used including bright field, dark field, phase contrast and fluorescence microscopes. The importance of histology for understanding the relationship between tissue structure and function is emphasized.
Practical Issues of FISH in Solid TumorsYosep Chong
This document discusses practical issues related to FISH analysis in solid tumors. It covers general procedures, sample adequacy assessment, quality control in test procedures and interpretation, and troubleshooting. Key points include the importance of proper tissue fixation and tumor cell adequacy. Quality control measures for baking, digestion, hybridization and imaging are outlined. Factors like probe type and cutoffs as well as using positive controls are emphasized for accurate interpretation. External quality assurance programs are also referenced.
In situ hybridization (ISH) is a technique that uses labeled probes to localize specific DNA or RNA sequences within cells in a tissue sample. It allows researchers to obtain information about gene expression and genetic loci in their cellular context. There are two main types of ISH - fluorescence in situ hybridization (FISH) and chromogenic in situ hybridization (CISH). ISH requires many optimization steps for each tissue and probe used but can provide insights into physiological processes and disease pathogenesis by identifying specific mRNA sequences within individual cells.
Practical Histopathology and cytopathology
Histopathological examination is used to provide diagnostic information that is important for timely diagnosis of disease to determine treatment plan. Fresh tissue is extremely fragile & subject to autolysis.
The document discusses fixation in histology and cytology. It describes the aims of fixation as preserving tissue structure and preventing autolysis and bacterial growth. Fixation causes some changes like tissue shrinkage and hardening. The types of fixation include immersion, coating, vapor and perfusion. Formalin and alcohol are common fixatives. Factors like pH, temperature, duration and osmolarity influence fixation quality. The choice of fixative depends on the tissue and technique used, for example glutaraldehyde for electron microscopy. Useful fixative formulas are also provided.
This document provides information on handling specimens for histopathology. It discusses what histopathology is, general rules for biopsies including avoiding necrosis and orienting specimens. It describes different types of biopsies like incisional, punch and excisional biopsies. It outlines how to take surgical specimens, send them to the pathology lab in proper containers and labels, and handle specimens including fixation. The document also discusses which information is important for the pathologist and rapid diagnosis techniques like cytopathology and frozen sections.
Frozen sections allow for rapid microscopic analysis of tissue during surgery. The procedure involves freezing tissue, sectioning it with a cryostat microtome, and staining with H&E. This enables real-time diagnosis and assessment of surgical margins. Accuracy depends on the pathologist's experience and tissue type, but diagnoses can often be provided to surgeons within 10-15 minutes to guide surgery. Regular audits help ensure the quality of individual frozen section services.
This document discusses quality control in microbiology. It covers various aspects of quality control including the pre-analytic, analytic and post-analytic phases of diagnostic testing. In the pre-analytic phase, it discusses proper specimen collection, transport and processing. The analytic phase covers microscopic examination, culture processing, identification and antimicrobial susceptibility testing. It also discusses quality control of equipment, reagents and culture media. The post-analytic phase involves reporting of results and interaction with epidemiologists. Maintaining accurate records and samples is also emphasized.
This document discusses the process of histopathology specimen processing. There are several key steps:
1) Tissue specimens are fixed, typically in formalin, to preserve their structure. 2) The tissues then undergo dehydration using increasing concentrations of alcohol, followed by clearing with xylene to remove the alcohol. 3) The tissues are then infiltrated with paraffin wax through impregnation to harden them. 4) The tissues are embedded in paraffin blocks. 5) Sections are cut from the paraffin blocks using a microtome and placed on slides. The slides are then stained to visualize the tissue structures under a microscope.
The document discusses common pitfalls in diagnostic immunohistochemistry (IHC), including issues that can occur in pre-analytic, analytic, and post-analytic phases. Some key pitfalls mentioned are insufficient tissue fixation time, improper epitope retrieval methods, suboptimal antibody choices, and lack of appropriate controls. The author emphasizes the importance of validation, controls, and following standardized protocols to avoid false positives and false negatives in IHC.
This document provides information on general histopathology techniques. It defines histology and histopathology, and describes the process of collecting biopsy specimens including fixation, storage, and labeling. It discusses different types of biopsies like core needle, fine needle, excisional, and incisional biopsies. Common histological techniques are also outlined, such as frozen section, fine needle aspiration, and exfoliative cytology. Immunohistochemistry is also briefly described.
This document discusses tissue preparation techniques for immunohistochemistry. It begins by listing names and identification numbers. It then outlines the goals of being able to identify fixatives, understand sectioning, and describe whole mount preparation. It discusses various fixation methods and fixatives used including formalin, paraformaldehyde, glutaraldehyde, and acetone. It describes sectioning techniques using paraffin embedding and vibratome sectioning. It concludes by discussing advantages and disadvantages of different preparation methods and stresses that no single method is ideal for all antigens.
The document summarizes the process of tissue processing which involves fixing, dehydrating, clearing, infiltrating with wax, embedding, sectioning, staining, and mounting tissue samples in order to examine them microscopically. Key steps include fixation to prevent decay, dehydration using alcohol to remove water, clearing with xylene to remove alcohol, infiltration and embedding in paraffin wax, sectioning thin slices with a microtome, staining typically with hematoxylin and eosin for examination, and mounting on slides. The goal is to prepare tissue for microscopic analysis while maintaining structure.
This document discusses practical approaches for diagnosing viral diseases in poultry, including clinical diagnosis, rapid field diagnostic tests, serological diagnosis, molecular diagnosis, and isolation/characterization. Clinical diagnosis is based on case history, clinical signs, examination of live/dead birds, and gross lesions. Rapid field tests can detect viruses but require high viral titers. Serological tests detect antibodies but have delays. Molecular diagnosis using PCR technologies can sensitively and specifically detect pathogens. The document emphasizes that clinical signs alone are not confirmatory and that multiple diagnostic approaches should be used to accurately diagnose poultry viral diseases.
This document discusses the process of tissue processing for histological examination. It involves several key steps: specimen identification and labelling, gross examination, fixation, dehydration, clearing, infiltration, embedding, sectioning, and staining. Fixation using formalin or other fixatives preserves tissue structure. Dehydration removes water and replaces it with alcohol or other solvents to allow for infiltration of paraffin wax. The wax infiltrates and impregnates the tissue, allowing it to be sectioned thinly for microscopic examination after staining. The document provides details on common fixation and processing methods and their purposes in preparing tissue for histological analysis.
The document discusses frozen section procedures, which allow rapid microscopic analysis of surgical specimens. The procedure involves freezing tissue samples and slicing thin sections that are immediately stained and examined. Frozen sections allow pathologists to provide intraoperative diagnoses to surgeons to guide surgical decisions. While quick, frozen sections provide lower quality slides than formal histology methods. Accuracy is generally high for clearly benign or malignant cases but lower for uncertain "borderline" diagnoses. The procedure was pioneered in 1905 and involves using a cryostat machine to freeze and slice tissue samples for immediate examination.
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The Embryology laboratory should been designed to provide an environment that is as close to optimum as possible for the growth of human embryos and to provide the best resulting pregnancy rates for patients undergoing IVF cycles.
Here, we discuss what is the components of IVF laboratory.
This document provides an introduction to histopathology and the process of tissue fixation. It defines histopathology as the microscopic examination of tissue to study disease. The key steps in tissue fixation are described as collection and transport of biopsy specimens, followed by processing including fixation, dehydration, clearing, embedding, cutting and staining of tissues. Critical factors that ensure proper fixation include freshness of tissues, penetration of fixatives, duration of fixation, and choice of fixative based on the tissue and intended analysis. Formalin is the most commonly used fixative but others including glutaraldehyde and osmium tetroxide are also described.
This presentation in mainly focused of understanding of automation and its utility in cytopathology. It will be very usefull for postgraduate in pathology, cytopathologist and cytotechnicians.
PREPARATION OF HISTOLOGICAL SPECIMENS.pptxAnthonyMatu1
This document provides information on the processes involved in preparing histological specimens, including tissue fixation, processing, sectioning, and staining. It describes the objectives of tissue fixation as preventing autolysis and bacterial attack while maintaining tissue structure. The main types of fixatives and factors affecting fixation are outlined. Tissue processing involves dehydration using graded alcohols, clearing with agents like xylene, and embedding in paraffin wax. Sections are cut with a microtome and stained, with hematoxylin and eosin being a common staining method that colors nuclei blue and cytoplasm pink. Other specialized staining techniques are also mentioned.
Histology is the microscopic study of tissues. Key steps in processing tissues for histological examination include fixation, dehydration, clearing, embedding in paraffin wax, sectioning, and staining. Tissues are first fixed in chemicals like formaldehyde to preserve their structure. They are then dehydrated using graded alcohols to remove water. Next, tissues are cleared using solvents like xylene to make them permeable to paraffin prior to embedding. The embedded tissues can then be thinly sectioned and stained for microscopic examination. Proper tissue processing is important for high quality histological analysis.
Immunohistochemistry (IHC) is a method that combines biochemical, histological and immunological techniques into a simple but powerful assay for protein detection. IHC provides valuable information as it visualizes the distribution and localization of specific cellular components within cells and in proper tissue context.
Histological Techniques: Perform Fixation and Tissue ProcessingLouisaChew
This document discusses fixation and tissue processing techniques in histology. It begins by describing the aims of fixation as preventing autolysis, putrefaction, and tissue distortion. An ideal fixative is said to preserve tissue structure and chemistry without shrinkage or swelling. Fixatives are classified based on their chemical properties, effects on cells and tissues, and whether they use single or multiple chemicals. Factors that affect fixation include temperature, tissue size, fixative concentration, duration of fixation, agitation, pH, and osmolality.
The document then discusses the aims of tissue processing as removing water from tissues and making them suitable for embedding in paraffin. The key steps are dehydration, clearing, and infiltration with par
Introduction to Histopathology and Lab organization.pptxsandeep singh
This document provides an introduction to histopathology and laboratory organization. It defines key terms like histology, histopathology, and histotechniques. It explains that histopathology involves examining tissues under a microscope to diagnose diseases. The organization of a histopathology laboratory has three parts: construction and equipment, staff, and protocols. Laboratories need specific rooms and equipment for receiving samples, processing, staining, and storage. Staff roles include pathologists, technicians, and attendants. Laboratories must establish standard operating procedures and safety measures to properly handle tissues and chemicals.
Histopathology techniques are used to demonstrate minute structural alterations in tissues caused by disease. Key techniques include fixing tissues in formalin to preserve structure, processing tissues through dehydration, clearing and infiltration steps, embedding in paraffin wax, sectioning with a microtome, staining, and mounting slides. Histopathology allows diagnosis of diseases through microscopic examination of tissue structures and any pathological changes present. It is a crucial technique when other testing may not be possible or provides definitive confirmation of diseases.
Central sterile supply department(cssd)anees fatima
The Central Sterile Supply Department (CSSD) is responsible for receiving, processing, sterilizing, storing and distributing sterile supplies and equipment for hospitals. Key functions of the CSSD include cleaning, packaging, sterilizing items using autoclaves or ethylene oxide, storing sterile supplies, and issuing items to different hospital units. CSSDs aim to provide safe sterile supplies efficiently while standardizing equipment and assisting in infection control.
This document discusses cytopreparatory techniques used in cytopathology. It covers different types of cytology samples like exfoliative cytology obtained from washing, smearing or brushing epithelial surfaces, as well as aspiration cytology using fine needle aspiration. The advantages of cytopathology are described as being non-invasive, allowing for faster reporting to guide clinicians, and being relatively inexpensive. The key steps in cytopreparatory techniques are outlined as specimen evaluation, smear preparation, fixation, and staining. Different fixation methods are also summarized.
This document discusses the process of histopathology specimen processing. There are several key steps:
1) Tissue specimens are fixed, typically in formalin, to preserve their structure. 2) The tissues then undergo dehydration using increasing concentrations of alcohol, followed by clearing with xylene to remove the alcohol. 3) The tissues are then infiltrated with paraffin wax through impregnation to harden them. 4) The tissues are embedded in paraffin blocks. 5) Sections are cut from the paraffin blocks using a microtome and placed on slides. The slides are then stained to visualize the tissue structures under a microscope.
The document discusses common pitfalls in diagnostic immunohistochemistry (IHC), including issues that can occur in pre-analytic, analytic, and post-analytic phases. Some key pitfalls mentioned are insufficient tissue fixation time, improper epitope retrieval methods, suboptimal antibody choices, and lack of appropriate controls. The author emphasizes the importance of validation, controls, and following standardized protocols to avoid false positives and false negatives in IHC.
This document provides information on general histopathology techniques. It defines histology and histopathology, and describes the process of collecting biopsy specimens including fixation, storage, and labeling. It discusses different types of biopsies like core needle, fine needle, excisional, and incisional biopsies. Common histological techniques are also outlined, such as frozen section, fine needle aspiration, and exfoliative cytology. Immunohistochemistry is also briefly described.
This document discusses tissue preparation techniques for immunohistochemistry. It begins by listing names and identification numbers. It then outlines the goals of being able to identify fixatives, understand sectioning, and describe whole mount preparation. It discusses various fixation methods and fixatives used including formalin, paraformaldehyde, glutaraldehyde, and acetone. It describes sectioning techniques using paraffin embedding and vibratome sectioning. It concludes by discussing advantages and disadvantages of different preparation methods and stresses that no single method is ideal for all antigens.
The document summarizes the process of tissue processing which involves fixing, dehydrating, clearing, infiltrating with wax, embedding, sectioning, staining, and mounting tissue samples in order to examine them microscopically. Key steps include fixation to prevent decay, dehydration using alcohol to remove water, clearing with xylene to remove alcohol, infiltration and embedding in paraffin wax, sectioning thin slices with a microtome, staining typically with hematoxylin and eosin for examination, and mounting on slides. The goal is to prepare tissue for microscopic analysis while maintaining structure.
This document discusses practical approaches for diagnosing viral diseases in poultry, including clinical diagnosis, rapid field diagnostic tests, serological diagnosis, molecular diagnosis, and isolation/characterization. Clinical diagnosis is based on case history, clinical signs, examination of live/dead birds, and gross lesions. Rapid field tests can detect viruses but require high viral titers. Serological tests detect antibodies but have delays. Molecular diagnosis using PCR technologies can sensitively and specifically detect pathogens. The document emphasizes that clinical signs alone are not confirmatory and that multiple diagnostic approaches should be used to accurately diagnose poultry viral diseases.
This document discusses the process of tissue processing for histological examination. It involves several key steps: specimen identification and labelling, gross examination, fixation, dehydration, clearing, infiltration, embedding, sectioning, and staining. Fixation using formalin or other fixatives preserves tissue structure. Dehydration removes water and replaces it with alcohol or other solvents to allow for infiltration of paraffin wax. The wax infiltrates and impregnates the tissue, allowing it to be sectioned thinly for microscopic examination after staining. The document provides details on common fixation and processing methods and their purposes in preparing tissue for histological analysis.
The document discusses frozen section procedures, which allow rapid microscopic analysis of surgical specimens. The procedure involves freezing tissue samples and slicing thin sections that are immediately stained and examined. Frozen sections allow pathologists to provide intraoperative diagnoses to surgeons to guide surgical decisions. While quick, frozen sections provide lower quality slides than formal histology methods. Accuracy is generally high for clearly benign or malignant cases but lower for uncertain "borderline" diagnoses. The procedure was pioneered in 1905 and involves using a cryostat machine to freeze and slice tissue samples for immediate examination.
Hóa mô miễn dịch và các câu hỏi liên quan. Hóa mô miễn dịch và các câu hỏi liên quan. Hóa mô miễn dịch và các câu hỏi liên quan. Hóa mô miễn dịch và các câu hỏi liên quan. Hóa mô miễn dịch và các câu hỏi liên quan. Hóa mô miễn dịch và các câu hỏi liên quan. Hóa mô miễn dịch và các câu hỏi liên quan. Hóa mô miễn dịch và các câu hỏi liên quan. Hóa mô miễn dịch và các câu hỏi liên quan. Hóa mô miễn dịch và các câu hỏi liên quan. Hóa mô miễn dịch và các câu hỏi liên quan. Hóa mô miễn dịch và các câu hỏi liên quan. Hóa mô miễn dịch và các câu hỏi liên quan. Hóa mô miễn dịch và các câu hỏi liên quan. Hóa mô miễn dịch và các câu hỏi liên quan. Hóa mô miễn dịch và các câu hỏi liên quan. Hóa mô miễn dịch và các câu hỏi liên quan. Hóa mô miễn dịch và các câu hỏi liên quan. Hóa mô miễn dịch và các câu hỏi liên quan. Hóa mô miễn dịch và các câu hỏi liên quan. Hóa mô miễn dịch và các câu hỏi liên quan. Hóa mô miễn dịch và các câu hỏi liên quan. Hóa mô miễn dịch và các câu hỏi liên quan. Hóa mô miễn dịch và các câu hỏi liên quan. Hóa mô miễn dịch và các câu hỏi liên quan. Hóa mô miễn dịch và các câu hỏi liên quan. Hóa mô miễn dịch và các câu hỏi liên quan. Hóa mô miễn dịch và các câu hỏi liên quan. Hóa mô miễn dịch và các câu hỏi liên quan. Hóa mô miễn dịch và các câu hỏi liên quan. Hóa mô miễn dịch và các câu hỏi liên quan. Hóa mô miễn dịch và các câu hỏi liên quan. Hóa mô miễn dịch và các câu hỏi liên quan. Hóa mô miễn dịch và các câu hỏi liên quan. Hóa mô miễn dịch và các câu hỏi liên quan. Hóa mô miễn dịch và các câu hỏi liên quan. Hóa mô miễn dịch và các câu hỏi liên quan. Hóa mô miễn dịch và các câu hỏi liên quan. Hóa mô miễn dịch và các câu hỏi liên quan. Hóa mô miễn dịch và các câu hỏi liên quan. Hóa mô miễn dịch và các câu hỏi liên quan. Hóa mô miễn dịch và các câu hỏi liên quan. Hóa mô miễn dịch và các câu hỏi liên quan. Hóa mô miễn dịch và các câu hỏi liên quan. Hóa mô miễn dịch và các câu hỏi liên quan. Hóa mô miễn dịch và các câu hỏi liên quan. Hóa mô miễn dịch và các câu hỏi liên quan. Hóa mô miễn dịch và các câu hỏi liên quan. Hóa mô miễn dịch và các câu hỏi liên quan. Hóa mô miễn dịch và các câu hỏi liên quan. Hóa mô miễn dịch và các câu hỏi liên quan. Hóa mô miễn dịch và các câu hỏi liên quan. Hóa mô miễn dịch và các câu hỏi liên quan. Hóa mô miễn dịch và các câu hỏi liên quan. Hóa mô miễn dịch và các câu hỏi liên quan. Hóa mô miễn dịch và các câu hỏi liên quan. Hóa mô miễn dịch và các câu hỏi liên quan. Hóa mô miễn dịch và các câu hỏi liên quan. Hóa mô miễn dịch và các câu hỏi liên quan. Hóa mô miễn dịch và các câu hỏi liên quan. Hóa mô miễn dịch và các câu hỏi liên quan. Hóa mô miễn dịch và các câu hỏi liên quan. Hóa mô miễn dịch và các câu hỏi liên quan. Hóa mô miễn dịch và các câu hỏi liên quan. Hóa mô miễn dịch và các câu hỏi liên quan. Hóa mô miễn dịch và các câu hỏi liên quan. Hóa mô miễn dịch và các câu hỏi liên quan. Hóa mô miễn dịch và các câu hỏi liên quan. Hóa mô miễn dịch và các câu hỏi liên quan.
The Embryology laboratory should been designed to provide an environment that is as close to optimum as possible for the growth of human embryos and to provide the best resulting pregnancy rates for patients undergoing IVF cycles.
Here, we discuss what is the components of IVF laboratory.
This document provides an introduction to histopathology and the process of tissue fixation. It defines histopathology as the microscopic examination of tissue to study disease. The key steps in tissue fixation are described as collection and transport of biopsy specimens, followed by processing including fixation, dehydration, clearing, embedding, cutting and staining of tissues. Critical factors that ensure proper fixation include freshness of tissues, penetration of fixatives, duration of fixation, and choice of fixative based on the tissue and intended analysis. Formalin is the most commonly used fixative but others including glutaraldehyde and osmium tetroxide are also described.
This presentation in mainly focused of understanding of automation and its utility in cytopathology. It will be very usefull for postgraduate in pathology, cytopathologist and cytotechnicians.
PREPARATION OF HISTOLOGICAL SPECIMENS.pptxAnthonyMatu1
This document provides information on the processes involved in preparing histological specimens, including tissue fixation, processing, sectioning, and staining. It describes the objectives of tissue fixation as preventing autolysis and bacterial attack while maintaining tissue structure. The main types of fixatives and factors affecting fixation are outlined. Tissue processing involves dehydration using graded alcohols, clearing with agents like xylene, and embedding in paraffin wax. Sections are cut with a microtome and stained, with hematoxylin and eosin being a common staining method that colors nuclei blue and cytoplasm pink. Other specialized staining techniques are also mentioned.
Histology is the microscopic study of tissues. Key steps in processing tissues for histological examination include fixation, dehydration, clearing, embedding in paraffin wax, sectioning, and staining. Tissues are first fixed in chemicals like formaldehyde to preserve their structure. They are then dehydrated using graded alcohols to remove water. Next, tissues are cleared using solvents like xylene to make them permeable to paraffin prior to embedding. The embedded tissues can then be thinly sectioned and stained for microscopic examination. Proper tissue processing is important for high quality histological analysis.
Immunohistochemistry (IHC) is a method that combines biochemical, histological and immunological techniques into a simple but powerful assay for protein detection. IHC provides valuable information as it visualizes the distribution and localization of specific cellular components within cells and in proper tissue context.
Histological Techniques: Perform Fixation and Tissue ProcessingLouisaChew
This document discusses fixation and tissue processing techniques in histology. It begins by describing the aims of fixation as preventing autolysis, putrefaction, and tissue distortion. An ideal fixative is said to preserve tissue structure and chemistry without shrinkage or swelling. Fixatives are classified based on their chemical properties, effects on cells and tissues, and whether they use single or multiple chemicals. Factors that affect fixation include temperature, tissue size, fixative concentration, duration of fixation, agitation, pH, and osmolality.
The document then discusses the aims of tissue processing as removing water from tissues and making them suitable for embedding in paraffin. The key steps are dehydration, clearing, and infiltration with par
Introduction to Histopathology and Lab organization.pptxsandeep singh
This document provides an introduction to histopathology and laboratory organization. It defines key terms like histology, histopathology, and histotechniques. It explains that histopathology involves examining tissues under a microscope to diagnose diseases. The organization of a histopathology laboratory has three parts: construction and equipment, staff, and protocols. Laboratories need specific rooms and equipment for receiving samples, processing, staining, and storage. Staff roles include pathologists, technicians, and attendants. Laboratories must establish standard operating procedures and safety measures to properly handle tissues and chemicals.
Histopathology techniques are used to demonstrate minute structural alterations in tissues caused by disease. Key techniques include fixing tissues in formalin to preserve structure, processing tissues through dehydration, clearing and infiltration steps, embedding in paraffin wax, sectioning with a microtome, staining, and mounting slides. Histopathology allows diagnosis of diseases through microscopic examination of tissue structures and any pathological changes present. It is a crucial technique when other testing may not be possible or provides definitive confirmation of diseases.
Central sterile supply department(cssd)anees fatima
The Central Sterile Supply Department (CSSD) is responsible for receiving, processing, sterilizing, storing and distributing sterile supplies and equipment for hospitals. Key functions of the CSSD include cleaning, packaging, sterilizing items using autoclaves or ethylene oxide, storing sterile supplies, and issuing items to different hospital units. CSSDs aim to provide safe sterile supplies efficiently while standardizing equipment and assisting in infection control.
This document discusses cytopreparatory techniques used in cytopathology. It covers different types of cytology samples like exfoliative cytology obtained from washing, smearing or brushing epithelial surfaces, as well as aspiration cytology using fine needle aspiration. The advantages of cytopathology are described as being non-invasive, allowing for faster reporting to guide clinicians, and being relatively inexpensive. The key steps in cytopreparatory techniques are outlined as specimen evaluation, smear preparation, fixation, and staining. Different fixation methods are also summarized.
Myocardial infarction, commonly known as a heart attack, results from abrupt reduction in coronary blood flow causing ischemic myocardial necrosis. In over 90% of cases, an acute thrombus forms in an artery previously partially obstructed by atherosclerosis, blocking blood flow. Release of cardiac enzymes like troponins and CK-MB are diagnostic of myocardial infarction. Symptoms include chest pain and ECG changes like ST elevation or new left bundle branch block. Treatment focuses on reperfusion through medications or angioplasty to restore blood flow.
Myocarditis is inflammation of the heart muscle that can lead to dilated cardiomyopathy. It has infectious, toxic, or autoimmune causes. Patients may be asymptomatic or experience cardiac symptoms
Intraepidermal Carcinoma.pptx in the childrenDinu85
1) Squamous cell carcinoma in situ (SCCIS) is a non-invasive form of skin cancer where cancerous cells are located in the epidermis.
2) SCCIS is most commonly caused by long-term exposure to ultraviolet radiation from sunlight, which damages skin cell DNA and leads to abnormal cell growth.
3) SCCIS typically appears as red, scaly patches or plaques on sun-exposed skin like the head and neck. If left untreated it could develop into invasive skin cancer.
Financing in Health care Sector in Sri Lanka - Suleiman.pptxDinu85
The document discusses healthcare financing in Sri Lanka. It notes that Sri Lanka has a centralized healthcare system managed by the Ministry of Health. It also has extensive primary care and focuses on preventative care. Hospitals provide secondary and tertiary care. Healthcare is financed through government budget allocations, taxes, international donors, private insurance, and out-of-pocket payments. While Sri Lanka has made progress in healthcare access and outcomes, challenges remain around equitable access, rising costs, and reliance on out-of-pocket payments.
This document discusses various pulmonary conditions that pediatric pulmonologists may examine via X-ray, including obstructive bronchitis, pneumonia, congestive heart failure, emphysema, atelectasis, and acute bronchitis.
This document provides an overview of faecal microbiota transplantation (FMT). It discusses the history and timeline of FMT, the gut microbiota, common indications for FMT like Clostridium difficile infection, the procedure for FMT including donor screening and administration methods, advantages and disadvantages of FMT, regulatory issues, and areas for future research.
This document discusses the role of heredity in pathology and genetic diseases. It defines important genetic terms like genotype, phenotype, karyotype, mutation and types of mutations. It then classifies genetic diseases and describes different types of chromosome diseases and genetic disorders resulting from mutations in chromosomes, genomes and genes. Specific examples of genetic conditions are explained like Down syndrome, Turner syndrome, and phenylketonuria. Methods used in genetic research like cytogenetic analysis and twin studies are also summarized. The concepts of phenocopy and diathesis, which are environmental influences on genetic traits, are defined.
Epilepsy is characterized by recurrent unprovoked seizures resulting from excessive neuronal cell discharges. Seizures are classified as generalized or partial. Anti-epileptic drugs work by potentiating GABA, blocking sodium or calcium ion channels, or antagonizing glutamate. Their clinical uses depend on the seizure type. Common anti-epileptic drugs include carbamazepine, phenytoin, valproic acid, ethosuximide, and clonazepam. Each drug has specific characteristics like side effects, interactions, and indications.
Rotaviruses are the most common cause of severe diarrhea in infants and young children worldwide. They contain 11 segments of double-stranded RNA and belong to the Reoviridae family. Nearly all children are infected by rotavirus by age 5. While first infections after 3 months of age are usually symptomatic, subsequent infections tend to be milder. Treatment focuses on rehydration and prevention of dehydration. Vaccines provide the most effective prevention.
1. Coronary artery disease (CAD) occurs when there is an imbalance between the oxygen supply and demand of the heart muscle, usually due to atherosclerosis of the coronary arteries.
2. CAD is the leading cause of death worldwide for those over age 20 and prevalence increases with age.
3. Risk factors for CAD include hypertension, diabetes, dyslipidemia, smoking, obesity, and sedentary lifestyle.
4. Treatment involves medication, percutaneous interventions such as stents, and coronary artery bypass grafting depending on the severity and location of blockages.
Blastomycosis is a fungal infection caused by inhaling spores of the Blastomyces dermatitidis fungus, which can be found in moist, wooded areas. Symptoms resemble the flu but may become more severe and prolonged, with skin lesions or organ involvement possible. Risk factors include immunosuppression and living in or visiting endemic regions. Diagnosis involves culturing the fungus from infected tissues or fluids. Treatment requires long-term antifungal drugs like itraconazole. Prognosis depends on a person's immune status, with immunosuppressed individuals facing higher mortality risks. Prevention focuses on avoiding high-risk environmental areas.
This slide is special for master students (MIBS & MIFB) in UUM. Also useful for readers who are interested in the topic of contemporary Islamic banking.
How to Add Chatter in the odoo 17 ERP ModuleCeline George
In Odoo, the chatter is like a chat tool that helps you work together on records. You can leave notes and track things, making it easier to talk with your team and partners. Inside chatter, all communication history, activity, and changes will be displayed.
Strategies for Effective Upskilling is a presentation by Chinwendu Peace in a Your Skill Boost Masterclass organisation by the Excellence Foundation for South Sudan on 08th and 09th June 2024 from 1 PM to 3 PM on each day.
Exploiting Artificial Intelligence for Empowering Researchers and Faculty, In...Dr. Vinod Kumar Kanvaria
Exploiting Artificial Intelligence for Empowering Researchers and Faculty,
International FDP on Fundamentals of Research in Social Sciences
at Integral University, Lucknow, 06.06.2024
By Dr. Vinod Kumar Kanvaria
Chapter wise All Notes of First year Basic Civil Engineering.pptxDenish Jangid
Chapter wise All Notes of First year Basic Civil Engineering
Syllabus
Chapter-1
Introduction to objective, scope and outcome the subject
Chapter 2
Introduction: Scope and Specialization of Civil Engineering, Role of civil Engineer in Society, Impact of infrastructural development on economy of country.
Chapter 3
Surveying: Object Principles & Types of Surveying; Site Plans, Plans & Maps; Scales & Unit of different Measurements.
Linear Measurements: Instruments used. Linear Measurement by Tape, Ranging out Survey Lines and overcoming Obstructions; Measurements on sloping ground; Tape corrections, conventional symbols. Angular Measurements: Instruments used; Introduction to Compass Surveying, Bearings and Longitude & Latitude of a Line, Introduction to total station.
Levelling: Instrument used Object of levelling, Methods of levelling in brief, and Contour maps.
Chapter 4
Buildings: Selection of site for Buildings, Layout of Building Plan, Types of buildings, Plinth area, carpet area, floor space index, Introduction to building byelaws, concept of sun light & ventilation. Components of Buildings & their functions, Basic concept of R.C.C., Introduction to types of foundation
Chapter 5
Transportation: Introduction to Transportation Engineering; Traffic and Road Safety: Types and Characteristics of Various Modes of Transportation; Various Road Traffic Signs, Causes of Accidents and Road Safety Measures.
Chapter 6
Environmental Engineering: Environmental Pollution, Environmental Acts and Regulations, Functional Concepts of Ecology, Basics of Species, Biodiversity, Ecosystem, Hydrological Cycle; Chemical Cycles: Carbon, Nitrogen & Phosphorus; Energy Flow in Ecosystems.
Water Pollution: Water Quality standards, Introduction to Treatment & Disposal of Waste Water. Reuse and Saving of Water, Rain Water Harvesting. Solid Waste Management: Classification of Solid Waste, Collection, Transportation and Disposal of Solid. Recycling of Solid Waste: Energy Recovery, Sanitary Landfill, On-Site Sanitation. Air & Noise Pollution: Primary and Secondary air pollutants, Harmful effects of Air Pollution, Control of Air Pollution. . Noise Pollution Harmful Effects of noise pollution, control of noise pollution, Global warming & Climate Change, Ozone depletion, Greenhouse effect
Text Books:
1. Palancharmy, Basic Civil Engineering, McGraw Hill publishers.
2. Satheesh Gopi, Basic Civil Engineering, Pearson Publishers.
3. Ketki Rangwala Dalal, Essentials of Civil Engineering, Charotar Publishing House.
4. BCP, Surveying volume 1
Main Java[All of the Base Concepts}.docxadhitya5119
This is part 1 of my Java Learning Journey. This Contains Custom methods, classes, constructors, packages, multithreading , try- catch block, finally block and more.
This presentation includes basic of PCOS their pathology and treatment and also Ayurveda correlation of PCOS and Ayurvedic line of treatment mentioned in classics.
How to Setup Warehouse & Location in Odoo 17 InventoryCeline George
In this slide, we'll explore how to set up warehouses and locations in Odoo 17 Inventory. This will help us manage our stock effectively, track inventory levels, and streamline warehouse operations.
This presentation was provided by Steph Pollock of The American Psychological Association’s Journals Program, and Damita Snow, of The American Society of Civil Engineers (ASCE), for the initial session of NISO's 2024 Training Series "DEIA in the Scholarly Landscape." Session One: 'Setting Expectations: a DEIA Primer,' was held June 6, 2024.
2. Source:
• Carson F., Hladik C. HISTOTECHNOLOGY 3rd Edition
• Diagnostic Immunohistochemistry NORDICQC Krakow,
Poland, 12th-13th October 2015 (seminars)
• Dako’s Guidebook to Immunohistochemical Staining
Methods
3. In the clinical laboratory:
a heavily used technique,
for assisting the pathologist in making diagnosis,
Used to determine:
the origins,
prognosis,
and treatment of a tumor
Rigorous quality control (QC)
Accurate and consistent results
Expected staining patterns
Trobbleshooting:
Fixation
Processing
Tissue types
5. • Fixation & tissue preparation
• Pre-treatment/epitope retrieval
(Appropriate and efficient epitope
retrieval)
• Primary antibody selection
(Appropriate choice & titre of
antibody/clone)
• Detection system s
pecific & sensitive detection system
• Appropriate choice of
control material
Important considerations
for IHC
6. START
IHC Images by Kornstein, MD, Medical College of Virginia
Abnormal 2+ Abnormal 3+
Normal 0
Normal
Normal 1+
Normal Abnormal low
amplification
Abnormal high
amplification
ALMOUST
THE END
Important considerations for IHC
8. LITTLE BIT OF HISTORY…
The term “antibody” was coined by Paul Ehrlich in 1891.
The principle of IHC has been known since the 1930s,
It was not until 1942 that the first IHC study was reported…(Coons et al. used FITC-labeled antibodies to identify Pneumococcal
antigens in infected tissue).
1974 Taylor and Burns developed IHC on routinely processed FFPE tissues
1975 Köhler and Milstein presented the hybridoma technique to produce monoclonal antibodies (mAbs) by fusing an antibody-
producing B cell with a myeloma cell that is selected for its ability to grow in tissue culture
1979 Peroxidase anti-peroxidase technique
early 1980’s avidin & biotin complex
Since then, improvements have been made in protein conjugation, tissue fixation methods, detection labels and microscopy,
making immunohistochemistry a routine and essential tool in diagnostic and research laboratories
12. • Fixation & tissue preparation
• Pre-treatment/epitope retrieval
(Appropriate and efficient epitope
retrieval)
• Primary antibody selection
(Appropriate choice & titre of
antibody/clone)
• Detection system s
pecific & sensitive detection system
• Appropriate choice of control
material
Important considerations
for IHC
13. Fixation & tissue preparation
Fixation is a complex series of chemical events that differ for the
different groups of substance found in tissues.
• A fixative is described as a chemical substance which will
preserve the shape, structure, relationship and chemical
constituents of tissues and cells after death.
The aim of fixation according to IHC:
1- during fixation, hydrogen bonds are formed – which must be reserved to have successful
antibody binding to the antigen site.
14. Fixation & tissue preparation
– preservation of tissues in its original condition.
Aldehyde based
• 10% NBF
• 4% formaldehyde with PBS buffer
• 2% formaldehyde with picric acid and PBS
• Immersion v. transcardial perfusion
15.
16. 16
Fixation & tissue preparation
- factors affecting fixation good quality of IHC
• Formalin is best at 10% pH 7.2 (7.0 -7.4)
• Too high a concentration may adversely affect the
tissues and produce artefact similar to excessive
heat.
40% formaldehyde 250 ml
- Na2HPO4 x H2O 6,5 g
- NaH2PO4 4,0 g
- H2O dest. 750 ml
17. General principles of fixation
Amount of fixing fluid should be approx. 20 times more than the volume of
tissue held in a container with a required fixation time.
Temperature has an important effect. ROOM TEMPARATURE: 21°C
A lower temperature retard fixation – reduce autolytic reaction.
A higher temperature will decrease the required for fixation but will
increase autolysis.
• Formalin is best at 10% pH 7.2 (7.0 -7.4)
18. Fixation & tissue preparation
FIXATIVES FOR PARAFFIN-PROCESSED TISSUE
- new regulation regarding predictive marker staining responsibility on the
clinical laboratory fo dokumenting the time of fixation
- Time: 6 - 48 h – standard: 24 h
The speed of fixation of most fixative is almost 1 mm/hour
(according to Medawar penetration index: 1 mm/ 1 godz.
d = k √ t (d = depth k = penetration index t = time (h)
19. Penetration time at K = 3.6( d = K x √t )
• 1 hour = 3.6 mm
• 4 hours = 7.2 mm (1.8 mm/hr)
• 16 hours = 14.4 mm (0.9 mm/hr)
• 64 hours = 28.8 mm (0.45 mm/hr)
• 256 hours = 57.6 mm (0.225 mm/hr)
(to double the depth takes 4x the time)
Fixation & tissue preparation
NORDICQC, seminars 20
20. 100 % binding of formaldehyde after 24 hours at 25°C
50 % binding of formaldehyde after 100 min. at 25°C room temp.37˚C
Helander, KG. Kinetic studies of formaldehyde binding in tissue.
Biotechnique and Histochemistry. 1994; 69, 177 -179 NORDICQC, seminars 20
21. Am J Surg Pathol, Vol. 24, No. 7, 2000
Reaction!!!!!
− Penetration is just a part of the problem
− The main clue !!!!! reaction!!!
− „GOLDEN HOUR” – first hour
is the most important during fixation
we can save or completly destroy the tissue
(IMMUNOREACTIVITY)
22. Appropriate tissue fixation and processing
• – Problem 1: Delayed fixation
• – Problem 2: Too short fixation in NBF
• – Problem 3: Other fixatives than NBF
• – Too long fixation in NBF is not a problem !!!??
Appropriate tissue fixation and processing
Fixation & tissue preparation
NORDICQC, seminars 20
23. • Because fixation varies markely from laboratory to laboratory
• Vimentin provides an excellent way of determinating when tissue has been
overfixed.
• Vimentin staining is usually excelent in paraffin sections of tissues that have
been optimally fixed in formalin but is progressively lost as the lenght of
time in the fixative increases.
• When the vimentin staining is completely negative the tissues are
overfixed and all antibodies should be interpreted with caution.
• When the preservation of vimentin is uneven, then all immunostains should
be read in the area of most intense vimentin staining
Fixation & tissue preparation
DAKO, seminars 2015
25. TissueSafe – urządzenie, w którym tkanki możemy
opakować w folię, w próżni co eliminuje konieczność
utrwalenia jej w miejscu pobrania (np. blok
operacyjny).
- Urządzenie to pozwala również na transport
tkanek z miejsca pobrania do laboratorium
histopatologicznego.
- Tkanki przewożone są w temperaturze 4 °C.
- Otrzymaną tkankę utrwalamy dopiero po wyjęciu z
worka próżniowego, w laboratorium.
- Od początku możemy nadzorować proces
utrwalania materiału.
Important considerations for IHC
Tissue preparation standards NEW ALTERNATIVE
26. Ventana: System 2 + 2
1. Fresh tissue material – on ice – department of clinical pathomorphology
2. Cutting the fresh material
3. Placing the fresh thin tissue sections into 10% buffer formalin for 2 h in 4°c
4. Last step of fixation: 2 h in 45°c
Chafin D, Theiss A, Roberts E, Borlee G, Otter M, et al. (2013) Rapid Two-Temperature Formalin Fixation.
PLoS ONE 8(1): e54138. doi:10.1371/journal.pone.0054138
Important considerations for IHC
Tissue preparation standards NEW ALTERNATIVE
30. Estrogen receptor expression at
different delayed formalin fixation
times (0 minutes, 4 and 8 hours,
and overnight).
Note the decreased
number/percentage of positive cells
and the intensity of the stain with
increased time of delayed fixation.
31.
32. Sectioning
• Paraffin
• Most commonly used
• Must heat and process through xylenes and alcohols – ruins some
antigens
• BEST if not stored more than two weeks – lose antigenicity after
that time
Fixation & tissue preparation
Sections usually 3-4µm
HER2 IHC = 4µm
Thinner sections 2-3µm required for:-
Bone marrow trephines
Lymph nodes
Renal biopsies
Thicker sections 6-20µm required for:-
Cases for amyloid (histochemistry)
(6-8µm)
33.
34.
35. Model A22
Zalecane do bardzo
cienkich sekcji w
krojeniu rutynowym i
badaniach
naukowych. Nadaje
się do tkanki
włóknistej, a także do
cięcia
wstążeczkowego.
Długość - 80mm;
Wysokość - 8 mm; Kąt
żyletki - 22°;
Model A35
Zalecane do bardzo
cienkich sekcji w
krojeniu rutynowym i
badaniach
naukowych. Nadaje
się zarówno do
tkanki miękkiej jak i
twardej .
Długość - 80mm;
Wysokość - 8 mm;
Kąt żyletki - 35°
Model C35
Zalecane wyłącznie
do skrawania w
kriostatach w celu
uzyskania bardzo
cienkich sekcji.
Jedyna żyletka
wykonana ze stali
węglowej.
Długość - 80mm;
Wysokość - 8 mm; Kąt
żyletki - 35°
Model R35
Zalecane do
skrawania
wstążeczkowego.
Nadaje się przede
wszystkim do tkanki
twardej, możliwe jest
jednak również
skrawanie materiału
miękkiego.
Długość - 80mm;
Wysokość - 8 mm; Kąt
żyletki - 35°
36. Model N35
Zalecane do
skrawania bardzo
twardych materiałów
w krojeniu
rutynowym.
Długość - 80mm;
Wysokość - 8 mm;
Kąt żyletki - 35°
Model N35HR
Zalecane do
skrawania bardzo
twardych i trudnych
materiałów zarówno
w skrawaniu
rutynowym jak i
wstążeczkowym.
Długość - 80mm;
Wysokość - 8 mm;
Kąt żyletki - 35°
Model S35L
Zalecane do
skrawania
rutynowego tkanek o
dużych wymiarach
zatopionych
w bloczku
parafinowym oraz
większych biopsji.
Długość - 120 mm;
Wysokość - 8 mm;
Kąt żyletki - 35°
Model S35LL
Zalecane do
skrawania
rutynowego tkanek o
bardzo dużych
wymiarach
zatopionych
w bloczku
parafinowym.
Długość - 180 mm;
Wysokość - 8 mm;
Kąt żyletki - 35°
39. H&E
Score lines visible on the
waterbath
David Muskett - SD/CP5 Microtomy
Tissue preparation - sectioning
40. This short ribbon of sections that was cut from a cold block shows considerable compression
(30–40%). In this case re-setting the knife tilt angle overcame the problem.
David Muskett - SD/CP5 Microtomy
Optimise knife tilt angle
Tissue preparation - sectioning
41. This block face has cracked because it was frozen to –15 °C in a freezer prior to cutting. The
cracks may make sectioning and flotation difficult because the wax is no longer bound to the
tissue. David Muskett - SD/CP5 Microtomy
Avoid freeze damaging
Tissue preparation - sectioning
42. David Muskett - SD/CP5 Microtomy
Carefully choose a section
Tissue preparation - sectioning
47. • Fixation & tissue preparation
• Pre-treatment/epitope retrieval
(Appropriate and efficient epitope
retrieval)
• Primary antibody selection
(Appropriate choice & titre of
antibody/clone)
• Detection system s
pecific & sensitive detection system
• Appropriate choice of
control material
Important considerations
for IHC
48. Pre-treatment/epitope retrieval
(Appropriate and efficient epitope retrieval)
two common categories of retrieval methods
1. HIER – HEAT-INDUCED EPITOPE RETRIEVAL
2. EIER – ENZYME-INDUCED EPITOPE ETRIEVAL
• Those methods are necessary to break down the hydrogen bonds,
formed during fixation
• Overfixation results in in loss of immuneactivity false negative
results
Important considerations
for IHC
49. Advantages in epitope retrieval:
- Ability to furthere dilute antibodies
- Exposure of epitopes sites not previously detectable
- More intense reactions with decreased incubation times
- More uniform staining
- Decreased background staining
- Possibility of better standarization
Pre-treatment/epitope retrieval
(Appropriate and efficient epitope retrieval)
50. Antigen retrieval Pre-treatment / Epitope retrieval:
Defined as an unmasking method
for ”re-storing” blocked antigens
in formaldehyde fixed tissue
The key to an optimal IHC reaction…
51. HIER Heat Induced Epitope Retrieval
Citrate 6.0//Tris-EDTA 9.0//EDTA 8.0 / 9.0
95°C 20 min.
52. IHC Procedure validation
Antibody dilution 1:25 1:50 1:100
Antigen retrieval 1. HIER low pH
2. HIER high pH
3. Enzyme
(protease)
1.HIER low pH
2.HIER high pH
3.Enzyme
(protease)
1.HIER low pH
2.HIER high pH
3. Enzyme
(protease)
Incubation 1. 30 min. 37°C
2. 1 h RT
3. 16 h 4°C
1. 30 min. 37°C
2. 1 h RT
3. 16 h 4°C
1. 30 min. 37°C
2. 1 h RT
3. 16 h 4°C
53.
54.
55. • Fixation & tissue preparation
• Pre-treatment/epitope retrieval
(Appropriate and efficient epitope
retrieval)
• Primary antibody selection
(Appropriate choice & titre of
antibody/clone)
• Detection system specific &
sensitive detection system
• Appropriate choice of control
material
Important considerations
for IHC
56. Monoclonal v. polyclonal
Antibodies monoclonals
• Mouse monoclonals - one clone
to one antigen determinant
• High specificity - low to high
sensitivity
• Mouse or rabbit hybridoma
Primary antibody selection
57. Rabbit polyclonals - more clones to more antigen determinants
• Low to high specificity - high sensitivity
• Polyclonal antibodies
• Many different species
• Tends to have more non-specific reactivity
• Can have very different
• avidity/affinity batch-to-batch
• Rabbit monoclonals -
• High specificity – high sensitivity
Polyclonal antibodies reacting with various epitopes
Each antibody is made by a different B-cell
Monoclonal v. polyclonal
Primary antibody selection
58. Antibody specifiacation sheet:
• The antibody specification sheet provided with a commercial antibody contains
valuable information regarding the antibody.
• The expiration date under recommended storage conditions
• Long-term//short-term storage temparatures (some antibodies require long-term
storage at -20°C. Measures to ensure that the antibody storage condition is optimal
will assist with antibody stability and maximum reactivity for the life of the antibody.
• The protein concentration and suggested working dilution ranges
• Pretreatment (antigen retrieval) solution; pH, and//or method if recommended
• Expected positive and negative tissue types INCLUDING NORMAL AND TUMOR
• Reference and publication list, and applications of antobody
• ANTIBODY TYPE: IN VITRO (IVD), RESEARCH USE ONLY (RUO), FOR RUTINE
DIAGNOSIS
59. Prediluted and concentrated antibodies
• Commercialy available – READY TO USE antibodies –
validated
• Concentrated antibodies – dilutions must be performed, to
find opitimal dilution for antigen detection
CONTROL REACTIONS – ANTIBODY VALIDATION
60. CONTROL REACTIONS – ANTIBODY VALIDATION
• EXAMPLE OF STANDARD LABORATORY PROTOCOL:
• Antigen retrieval - combination of heating method/
• - combination of time
• Peroxidase quenching – peroxidase block
• – combination of time
• Non-specific binding block – combination of time
• Detection system - universal polymer//HRP//AP
• Staining times: - primary antibody combination
• 30 minute//1 hour//16 hours
• - detection system//chromogen
• 20 minutes //1-10 minutes
69. 1:800
1:400
1:50 1:100 1:200
IHC staining results of serially diluted Chromogranin A
antibody on pancreas.
Primary antibody selection rules
70. At 1:50, the Islet cells stain strongly but there is also a
strong background staining.
1:50
background staining
Islet cells
Primary antibody selection rules
71. At 1:800, there is no background staining but the Islet
cells stain very weak.
1:800
Primary antibody selection rules
72. At 1:200, there is good contrast and no background
staining, it is therefore the optimal working dilution.
1:200
Primary antibody selection rules
74. Primary Antibody Incubation Time / Temparature
• Incubation time is inversely proportional to antibody concentration.
• Higher concentration of antibody allows shorter incubation time.
• 30 minutes 37°C // 1 hour RT // 16 hours 4°C
• Humidity chambers must be used when incubating at higher
temperature to prevent drying of tissue sections.
• 16 /4°C – allow sometines to better antibody penetration – used in
VALIDATION
75. IHC rules: Blocking
Background staining
• Specific
• Polyclonal antibodies – impure antigen used
• Inadequate fixation – diffusion of antigen – often worse in
center of large block
• Non-specific
• Endogenous enzymes peroxidases//AP
76. Structure of DAB
NH3
+
NH3
+
+H3
N
+H3
N 4Cl
-
DAB stained slides can be coverslipped with permanent organic
based mounting media.
In some IHC procedures, the dark brown reaction product can
be modified and intensified by adding metals (copper or
cobalt) to DAB solution.
Commonly used enzyme labels for IHC procedures include
horseradish peroxidase (HRP) HRP, from horseradish plant, is an enzyme that
catalyze the reduction of hydrogen peroxide (H2O2) to water and oxygen.
alkaline phophatase (AP)
Commonly used chromogens for HRP include
3-amino-9-ethylcarbazole (AEC)
3,3’-diaminobenzidine (DAB)
77. • Horse Radish Peroxidase (HRP)
• + high sensitivity
• + precise chromogenic reaction
• + can be amplified by metal (Cu/Ni)
•
• Alkaline Phosphatase (AP)
• + suitable for cryostate sections and cytology
(hematology)
• + double IHC staining
• - granular chromogenic reaction
• - relative low sensitivity
Enzyme
78. Control material can be categorized in different ways:
„
„
Reagent controls
– Positive reagent control is the actual reagent;
e.g. the primary antibody when tested on control material.
Testing is done during development and product validation to
ensure reagent specificity and sensitivity.
– Negative reagent control is a reagent substitute for the
primary antibody used to assess potential specificity
issues/false positive staining reaction
„
„
79. Tissue controls
– Positive tissue control is tissue with the specific antigen
at known, relevant and stable level.
Purpose is to docu-ment correct staining.
Control material can be categorized in different ways:
80. Negative tissue control
-is tissue without the specific antigen present – or not present in
specific regions.
- Purpose is to document specificity of the staining.
Tissue blocks/tissue microarrays,
- consisting of a few to several different
tissues that may serve as control material
for a range of antibodies
Control material can be categorized in different ways:
81. Internal tissue controls
– The presence of the target antigen (protein) within normal
elements of the tissue under investigation is an internal positive
control
„
„
Control material can be categorized in different ways:
82. „
„
Cell line controls
– Control material based on cultured cells with specific
antigenic characteristics. This type of control typically is made
for very specific purposes, in particular predictive assays (HER2)
Control material can be categorized in different ways:
83. Controls in Daily Routine Testing
On-slide positive tissue controls,
where specific controls are
placed on the same slide as the test
specimen are strongly preferred.
A) Illustration of the principle of
multi-core blocks. Tissue controls are
the cores in red, yellow, gray and
green. Patient sample is placed below
B) Example of on-slide tissue
controls. TMA
C) Example of cell line controls.
84. Controls in Daily Routine Testing
Tissue Controls
- both positive and negative tissue controls provide important information
and must be included in daily routine regardless of type of detection
system used.
- the tissues used for controls are carefully selected from normal or
cancer tissues previously analyzed in the laboratory.
- However, in some cases the actual patient tissue being tested can be
used as tissue control. HER2 - gastic
- For all tissue controls, if the staining does not perform as expected,
results from the respective test specimen should be considered invalid.
85. Controls in Daily Routine Testing
Positive tissue control
- The positive tissue control must contain the target
antigen at relevant, known and stable expression
level.
- It serves to document that proper staining has been
performed and confirms that the target retrieval procedure
has been carried out correctly.
- Thus, a positive tissue control assesses correct staining
protocol performance (temperature, time and correct
application of reagents).
86. Controls in Daily Routine Testing
Positive tissue controls
- are indicative of properly prepared tissue, they should
be as accurate as possible in the same manner as
the patient samples.
- Optimally, autopsy/biopsy/surgical specimens should be
fixed, processed and embedded as soon as
possible for best preservation of antigens.
- Generally, autopsy tissue is least preferred because of
inevitable delays before fixation (degradation of some
antigens)
87. Control material
On-slide positive tissue controls
-In the daily routine, positive tissue controls may be run on a
separate slide - as a batch control or daily control, or they
may be included on the same slides as the test specimens.
- on-slide positive controls, are
specific controls are placed
on the same slide as the test
specimen strongly preferred
88. Control material
More and more laboratories are using TMA cores as
on-slide controls.
-building a a few basic multi-tissue control blocks
- each containing a small number of control tissues to
cover most of the markers used in clinical
diagnostics.
89. Control material
The multi-tissue control blocks contains:
appendix, tonsil, pancreas and liver,
The reaction patterns are described for approximately 100 different
antibodies.
101. Thorough rinse in distilled water and
wash 2 times in PBS buffer.
Rinse
Deparaffinization and Rehydration
Block Endogenous Peroxidase
Antigen Retrieval
Rinse
SPECIMEN
Lab
Vision
Corp.
NM-123
Colon
carcinoma
MS-1375
CEA
Step by Step IHC Staining Method
102. 5% bovine serum albumin (BSA) to block nonspecific
staining.
Protein Block
Deparaffinization and Rehydration
Block Endogenous Peroxidase
Antigen Retrieval
Rinse
Rinse
SPECIMEN
Lab
Vision
Corp.
NM-123
Colon
carcinoma
MS-1375
CEA
Step by Step IHC Staining Method
103. Optimally diluted primary antibody for 20 minutes.
Primary Antibody
Deparaffinization and Rehydration
Block Endogenous Peroxidase
Antigen Retrieval
Protein Block
Rinse
Rinse
SPECIMEN
Lab
Vision
Corp.
NM-123
Colon
carcinoma
MS-1375
CEA
Step by Step IHC Staining Method
104. Wash 2 times in PBS buffer.
Deparaffinization and Rehydration
Block Endogenous Peroxidase
Antigen Retrieval
Protein Block
Primary Antibody
Rinse
Rinse
Rinse
SPECIMEN
Lab
Vision
Corp.
NM-123
Colon
carcinoma
MS-1375
CEA
Step by Step IHC Staining Method
105. Secondary antibody for 20 minutes.
Secondary Antibody
Rinse
Deparaffinization and Rehydration
Block Endogenous Peroxidase
Antigen Retrieval
Protein Block
Primary Antibody
Rinse
Rinse
SPECIMEN
Lab
Vision
Corp.
NM-123
Colon
carcinoma
MS-1375
CEA
Step by Step IHC Staining Method
106. Wash 2 times in PBS buffer.
Deparaffinization and Rehydration
Block Endogenous Peroxidase
Antigen Retrieval
Protein Block
Primary Antibody
Secondary Antibody
Rinse
Rinse
Rinse
Rinse
SPECIMEN
Lab
Vision
Corp.
NM-123
Colon
carcinoma
MS-1375
CEA
Step by Step IHC Staining Method
107. DAB chromogen for 1 minute.
DAB Chromogen
Deparaffinization and Rehydration
Block Endogenous Peroxidase
Antigen Retrieval
Protein Block
Primary Antibody
Secondary Antibody
Rinse
Rinse
Rinse
Rinse
Rinse
SPECIMEN
Lab
Vision
Corp.
NM-123
Colon
carcinoma
MS-1375
CEA
Step by Step IHC Staining Method
109. Counterstain in Mayer’s Hematoxylin
for 1 minute.
Deparaffinization and Rehydration
Block Endogenous Peroxidase
Antigen Retrieval
Protein Block
Primary Antibody
Secondary Antibody
DAB Chromogen
Rinse
Rinse
Rinse
Rinse
Rinse
Hematoxylin Counterstain
SPECIMEN
Lab
Vision
Corp.
NM-123
Colon
carcinoma
MS-1375
CEA
Step by Step IHC Staining Method
110. SPECIMEN
Lab
Vision
Corp.
NM-123
Colon
carcinoma
MS-1375
CEA
Thorough wash in tap water to “blue” the nuclei.
Hematoxylin Counterstain
Deparaffinization and Rehydration
Block Endogenous Peroxidase
Antigen Retrieval
Protein Block
Primary Antibody
Secondary Antibody
DABChromogen
Rinse
Rinse
Rinse
Rinse
Rinse
Rinse
Step by Step IHC Staining Method
111. Step by Step IHC Staining Method
Coverslip using mounting medium.
Deparaffinization and Rehydration
Block Endogenous Peroxidase
Antigen Retrieval
Protein Block
Primary Antibody
Secondary Antibody
DAB Chromogen
Rinse
Rinse
Rinse
Rinse
Rinse
Hematoxylin Counterstain
Rinse
Mount
SPECIMEN
Lab
Vision
Corp.
NM-123
Colon
carcinoma
MS-1375
CEA
112.
113.
114.
115.
116.
117.
118.
119.
120. “Stronty formalinowe", albo
"hematyna kwaśnej formaliny".
Pigment ten tworzy się w środowisku
kwaśnej formaliny.
Hematyna powstaje z hemoglobiny,
reagującej z formaliną w środowisku
kwaśnym, stąd największe
nagromadzenie tego barwnika
obserwowane jest w preparatach
zawierających dużo krwinek.
131. Improving antibody penetration
Need this for intracellular (cytoplasmic, nuclear) or
membrane components when epitope is inside cell
membrane
Detergents most popular
Triton-X
Tween
Also decreases surface tension – better coverage
Can’t use for membrane proteins
Acetone/Methanol
Precipitate proteins outside cell membranes- more accessible
Saponin
Punches holes in cell membrane – holes close up when
removed