Basics of Immunohistochemistry (IHC) Presented by Robert A Brunner, BA, HT(ASCP) Reagent Product Specialist Leica Biosystems Outline Use of IHC in Diagnostic Pathology Cell Structure Immunology and Primary Antibodies Sample Handling (Pre Analytic Workflow) Epitope Retrieval Detection/Visualization Systems Staining Patterns Importance of the IHC Tech Role Multi Step complex assay Errors- False Positive/ False Negative Accuracy/validity are crucial IHC Tech- First line of QC and QA Competent on Eval of Control tissues Know what you’re looking for! What Is Immunohistochemistry? Detects antigens in tissue sections with the use of Antibodies Enzyme/chromogen chemistry Augments/supports diagnosis- H&E is required Used for Diagnosis of Cancer Bacterial and Viral infections Neurogenitive diseases Cancer Abnormal Uncontrolled Cell growth and mitosis Can be invasive or in-situ Disruption of cell cycle can be due to: Damage to the DNA Over stimulation of the cell A lack of inhibition on the cell Viral infection IHC Is used to detect abnormal protein levels in tissue sections How Is Cancer Diagnosed? Cellular changes are seen microscopically Visible with the standard H&E Cell morphology changes from normal Nuclear dysmorphism - Nuclei often appear irregular Dissociation – The structure of the organ breaks down IHC Is often used to obtain a more specific diagnosis How Does IHC Help In Cancer Diagnosis? Classification of poorly differentiated tumors Stage and grade of tumor Identify Drug the origin of tumor therapy eligibility Prognosis CD3 CD20 Outline Use of IHC in Diagnostic Pathology Cell Structure Immunology and Primary Antibodies Sample Handling (Pre Analytic Workflow) Epitope Retrieval Detection/Visualization Systems Staining Patterns Cell Structure Cells The basic unit of living organisms Cells specialize and group together to form tissue Tissues Tissue is a grouping of similar cells from the same origin that together carry out a specific function Organs The collection of different tissues that together serve a common function Cell Anatomy Nucleus Cytoplasm DNA and nucleolus organelles -excluding the nucleus Cell membrane holds the cell’s contents and protects it DNA & Protein The Function of DNA DNA carries genetic information - used to create proteins What is Protein? String of amino acids folded into a 3-D structure Function as enzymes, as structural components (such as collagen, cartilage, and keratin), and as part of the cell signaling pathway Protein is often what we’re staining in IHC. Where is protein found? Everywhere in the body With IHC, we are demonstrating protein in the cell cytoplasm, nucleus, membrane Outline Use of IHC in Diagnostic Pathology Cell Structure Immunology and Primary Antibodies Sample Handling (Pre Analytic Workflow) Epitope Retrieval Detection/Visualization Systems Staining Patterns Immunology and Primary Antibodies Immune System Body’s defense system protect against disease comprised of many biological structures and processes Glossary Antibody Naturally occurring protein in the human body, which acts to identify and remove infection and disease Antigen A foreign substance present in the body which stimulates the immune response, and provide a site to where antibodies bind Epitope Region of an antigen to which the antibody binds Glossary Sensitivity Sensitive to disease How often is a positive result a true positive for disease AND How often is a Negative results a true negative for a disease Specificity Specific to a disease Does a positive result ONLY happen in that specific disease Positive result can happen with other diseases or processes Affinity The strength of interaction between an epitope and an antibody's antigen binding site. Immune System Organs Primary Organs Thymus & Bone Marrow Secondary Organs Spleen Tonsils & Adenoids Lymph Nodes & Vessels Peyer’s Patches Skin & Liver Immune System Cells B Cells Produce Antibodies What Is an Antibody? • Comprised of protein • Example: Immunoglobulin (Ig), • Has binding sites that link to foreign substancesAntigens • The exact binding site on the antigen where the antibody binds is referred to as the epitope • Each antibody only binds to a single epitope (or antigenic determinant) Antibody Isotypes IgG is most commonly used in IHC Antibody Structure Light Chain Heavy Chain • Variable Region different amino acid sequences unique antigen binding Area of epitope Location of antibody attachment to tissue • Constant Domain same amino acid sequence preserved amongst species Antibody Function with IHC Target antigen sites of human tissue Antibody selected based on affinity for a target or marker of interest Once bound, other reagents can be used to visualize it IHC Antibodies Monoclonal (Host Species) Mouse Rabbit Other Species (Rat, Camel) RARE Polyclonal (Host Species) Rabbit Other Species (Goat, Swine etc) RARE Monoclonal Antibodies Produced in Mice or Rabbits Homogeneous Population Directed at a single epitope Generated by B Cell clone from ONE animal Continued production of identical antibody by use of Hybridoma culture No lot to lot variability Mouse Monoclonal Rabbit Monoclonal - Easier to produce higher yield of Immunoglobulin - More stable cell lines over Rabbit - Most Monoclonal ab are mouse - More diverse Epitope recognition over mice - Improved immuno-response to small epitopes - Tend to be higher affinity Monoclonal (Mouse) Antibody Production Rabbit Monoclonal Antibody Production Complements of Spring Biosciences Inc Polyclonal Antibodies Most often generated in Rabbits Hetergeneous mixture of antibodies Reacts with many epitopes of the same antigen Generated by different B cell clones Variation from lot to lot Highly Sensitive Low Specificity Can produce background reaction Monoclonal vs Polyclonal Antibody Images courtesy of Dako IHC Education Guide 5th Edition Antibody Types and Formats Monoclonal vs Polyclonal Host Species Concentrated vs Ready to Use Titer Clone Used Diluent Used Incubation time and Temperature Examples are CD3, CD20, S-100, Ki67 etc Outline Use of IHC in Diagnostic Pathology Cell Structure Immunology and Primary Antibodies Sample Handling (Pre Analytic Workflow) Epitope Retrieval Detection/Visualization Systems Staining Patterns Pre-analytical Workflow/Sample Handling Preanalytical Processes Tissue Collection & Fixation Tissue Processing Microtomy Deparaffinization Tissue Collection • Placed in formalin as soon as possible • Delayed fixation allows • autolysis to occur • cause damage or loss of antigens Tissue Fixation • Formalin is the most commonly used • Formalin fixation creates crosslinks (methylene bridges) in proteins Fixation can influence IHC results – both positively and negatively! IHC Protocols need to be validated with EACH fixative used. Purpose of Fixation • Prevents autolysis and putrefaction. • Preserves cells and tissue components/tissue morphology. • Stabilizes the Antigen Formalin Fixation and IHC 10% Neutral Buffered formalin is most common fixative used 6-48 hours of fixation time is recommended Breast Tumors have specific CAP Guidelines for fixation Formalin forms hydrogen bonds with the antigen sites (Methylene Bridges) Requires Antigen Retrieval to open up binding sites for Antibodies to react with antigens Vimentin Antibody is used to determine over fixation Progressive fixation loss of reaction with extended formalin CAP Guidelines for Breast Markers Decalcification Fix tissue well before decalcification Do not allow prolonged exposure of tissue to decal solution Avoid harsh decals, such as those made with HCl Formic acid or EDTA decals are preferred Decalcification can cause loss of antigenicity if your processes aren’t designed with IHC in mind. If IHC is used on Decalcified tissues, Validation should be performed to assure results are not compromised by the decal procedure!! Tissue Processing • Samples must be fixed adequately before exposure to tissue processing reagents! • Removes water from tissue and infiltrates with paraffin wax. • Paraffin-infiltrated tissue is easier to cut into thin sections. • Poor Processing results in poor section quality and compromised IHC results Uneven Fixation Microtomy 3-5 um sections Free of wrinkles, tears and folds etc Clean water bath with Deionized water Mount Sections near center of the slide Avoid edges of the slide Avoid mounting too high or low on the slide Nitrile gloves can be worn to prevent Squamous cell contamination Microtomy Surface decal should be avoided when cutting IHC’s decal solutions can affect antigens and cause false negatives Positive Charged slides HIGHLY Recommended Not all are created equal Do not add adhesives to water bath Avoid using expired microscope slides positive charge diminishes over time Exposure to heat and humidity effects slide chemistry integrity Slide Drying Baking recommendations vary, but the primary goal is the same to remove the excess water from the slide and assist with section adhesion. Optimal Drying is overnight at room temp with fan (Carson/Hladik Cappellano) A safe procedure to use is baking slides at 55-60 degrees for 30 minutes to an hour. Convection ovens with blowers can decrease drying time Avoid excessive heat in the oven Nuclear and Cell Membrane are sensitive to dry heat and high temps False negative or weak reactions may happen Deparaffinization Complete Deparaffinization is essential IHC assays are performed in an aqueous solution, so paraffin must be completely removed Automated IHC instrumentation often deparaffinize on the instrument Follow your lab’s standard deparaffinization procedures to prepare slides for staining Outline Use of IHC in Diagnostic Pathology Cell Structure Immunology and Primary Antibodies Sample Handling (Pre Analytic Workflow) Epitope Retrieval Detection/Visualization Systems Staining Patterns Epitope Retrieval IHC Staining Steps Epitope Retrieval Blocking Primary Antibody Secondary Antibody(ies) Chromogen Counterstain Why Epitope Retrieval? • Formalin fixation blocks epitope sites by cross linking proteins • Epitope Retrieval reverses the obstructing effects caused by the crosslinking of formalin - Breaks Methylene bridges caused by hydrogen bonding by the formalin fixation • Two major methods are: • Enzyme-Induced Epitope Retrieval (EIER) • Heat-Induced Epitope Retrieval (HIER) Epitope Retrieval -Proteolytic Enzyme (EIER) Pronase, Protease, Pepsin, Proteinase K, Trypsin, etc Proteinase K is a commonly used Enzyme for EIER There are three main variables to consider with EIER: Concentration - Follow manufacturer’s instructions for working concentration Incubation time - Could be 1 to 60 minutes and 10-15 minutes is commonly used Incubation temperature - Is usually at 37 °C Epitope Retrieval with Proteolytic Enzymes Proteolytic Enzyme Disadvantages Many antigens can’t be retrieved via proteolytic epitope retrieval Over Digestion- Concentration too high or exposure time exceeded Distorted tissue morphology Loss of cellular detail Non specific staining Diminished staining and or false negative results Loss of tissue from the slide Heat-Induced Epitope Retrieval (HIER) • Originated in late 1980s for commercial use • The use of heat coupled with specific buffered solutions to recover antigens in formalin fixed paraffin embedded tissue • Citrate buffer 6.0 pH and EDTA buffer 9.0 pH are commonly used • Can be done manually or automated • Most fully automated IHC Platforms perform this function on the instrument with the use of heaters or baths • Manual methods use vegetable steamers, microwaves, water baths, and pressure cookers Heat-Induced Epitope Retrieval (HIER) There are three main variables to consider with HIER: Temperature of retrieval solution: should be around 95-100 °C Incubation time: varies. Commonly 10-90 minutes pH value of retrieval solution: depending on which solution is used Advantages of HIER Ability to dilute antibodies further Exposure of epitopes that were previously not available More intense reactions with decreased incubation times More uniform reaction Decreased background Day to day consistency of results Improved standardization (Lear 1995) Carson and Hladik/Cappellano 4th Edition Disadvantages of HIER Can cause tissue to fold and come off the slide Over-retrieved tissues can cause non-specific staining and background High pH (8.0 to 9.5) tends to be more harsh Tissue Lift off Morphology damage to tissue More intense reaction but can cause some background blush Low pH (6.0) tends to be more gentile May not be robust enough for some antigens Blocking Function- Prevents non-specific staining and false positives • • • Peroxide Block • Reduces the effects of endogenous peroxidase in found in tissue • Used in ALL Horse Radish Peroxidase Detection kits (HRP) Biotin Block • Blocks the biotin within tissue sections, which can react with avidin-biotin systems • Only used in Avidin-Biotin Detection Systems Protein Block • Reduces the potential for antibodies to non-specifically bind in tissue Outline Use of IHC in Diagnostic Pathology Cell Structure Immunology and Primary Antibodies Sample Handling (Pre Analytic Workflow) Epitope Retrieval Detection/Visualization Systems Staining Patterns Detection / Visualization Systems IHC Detection (Visualization) Systems Detects the presence of antigen in the tissue section Allows the visualization of the antigen reaction on the tissue section via light microscopy Uses antibody antigen reactions as well as enzyme reactions to produce a color precipitation Types of Chemistry Avidin Biotin Complex (ABC) Labeled Strep avidin biotin complex (LSAB) Polymer / monomer Primary Antibody An antibody specific for the marker of interest is applied after the Deparffinization, antigen retrieval and blocking steps IE, CD3, CD20, ER, PR ……… Polymer Detection System • Additional antibodies are bound to the primary antibody to allow for visualization. Chromogen Polymer enzyme (eg Peroxidase) Secondary antibody – Post Primary antibody Primary Antibody Antigen Secondary Antibody /Polymer Binds to the primary antibody. May be conjugated with biotin or polymer-enzyme complex. If the primary antibody is a mouse antibody, the secondary antibody must be of a different species, such as goat or rabbit, and be specific for mouse antibodies. Polymer Detection System • Secondary Antibody attached to Primary antibody attached to antigen in tissue section Secondary antibody – Post Primary antibody Primary Antibody Antigen Polymer Based IHC • Additional antibodies are bound to the primary antibody to allow for visualization. Chromogen Polymer enzyme (eg Peroxidase) Secondary antibody – Post Primary antibody Primary Antibody Antigen Enzymes & Chromogens Enzymes react with chromogens to create brown or red color Two common enzymes horseradish peroxidase (HRP) alkaline phosphatase (AP) HRP AP DAB Fast Red brown precipitate red precipitate Many other colors available Green, Blue, Yellow Horseradish Peroxidase Alkaline Phosphatase Converts chromogen into a visible precipitate in the presence of H2 O 2 Remove phosphate group from chromogen, leaving a visible precipitate Preferred in tissues with high endogenous phosphatase Preferred in tissues with high endogenous peroxidase and/or melanin concentrations Chromogens HRP + DAB = Brown AP + Fast Red = Red Red Chromogen vs. Melanin Red Chromogen, HMB-45 Brown is Melanin Pigment Useful- Pigmented tissues, Dual stains, Different contrasts (Microorganisms) PIN4 Double Stain P63, HMW Keratin and p504s P63 – Nuclear stain Basal Cells in glands of normal prostate CK903- Cytoplasmic Basal Cells in glands of normal prostate P504s- Cytoplasmic (granular) Prostatic Adenocarcinoma Cells Outline Use of IHC in Diagnostic Pathology Cell Structure Immunology and Primary Antibodies Sample Handling (Pre Analytic Workflow) Epitope Retrieval Detection/Visualization Systems Staining Patterns Staining Patterns Staining Patterns Nuclear Membranous Cytoplasmic Nuclear Staining Membranous Staining Cytoplasmic Staining IHC Resources http://pathmd.com CAP Publication (College of American Pathologists) http://www.asco.org/ (American Society of Clinical Oncology - ASCO) http://www.nordiqc.org http://www.immunoportal.com/ http://lists.utsouthwestern.edu/mailman/listinfo/histonet www.ihcworld.com http://web.ncifcrf.gov/rtp/lasp/phl/immuno/ www.biocompare.com Vendor Web Sites and Representatives (Histonet)
