MLS Review - Immunohistochemistry

MEDICAL LABORATORY SCIENCE REVIEW IMMUNOHISTOCHEMISTRY OUTLINE • • • • • Immunohistochemistry Labeling of Antibodies Specimens for IHC Detection Tumor Markers IMMUNOHISTOCHEMISTRY IMMUNOHISTOCHEMISTRY • • • • • • Immunohistochemistry o Used in disease diagnosis ▪ Involved in staging of cancer o Not a routine procedure o Involves antigen-antibody reactions ▪ Antigens - found in cells ▪ Antibody - detectors to detect abs Purpose of IHC o For identification of tissue or cellular antigens or phenotypic markers o For detection of organisms (microbes or microorganisms that cause diseases) in cytologic preparations (sputum, body fluids, fine needle aspirates) Immunoglobulins (Ig) - antibody used in IHC; belonging to the class of serum proteins IgG - Most common form of antibody used in IHC Types of Antibodies used in IHC o Polyclonal ▪ Produced by different cells ▪ Can react with various epitopes • Epitope - ag attachment site reacting with ab ▪ From lab animal sources ▪ Main source: rabbit • Rabbits are immunized to produce antibodies ▪ Other sources: goat, pigs, sheep o Monoclonal ▪ More specific ▪ Produced from individual clone of plasma cell • Produced from one cell ▪ Reacts only with one specific type of epitope ▪ Animal source: mice IHC o Ag + Ab = End-Product (EP) o EP can be detected using: ▪ Light microscope (LM) - enzymes, colloidal metals ▪ Fluorescent microscope - use of fluorochrome dye o Enzymatic Method ▪ Antibodies are labeled with enzyme before reaction with antigen • Ab → label with enzyme → chromogen + substrate → linking with Ag (incubate) → counterstain (incubate) ▪ After reacting with targeted ag, an Ag-Ab complex is produced ▪ Enzyme complex catalyzes substrate to yield a colored product ▪ Colored product is analyzed using LM/EM LABELLING OF ANTIBODIES • • • • First step in IHC Antibodies must be labeled before use Labeling techniques 1. Use of enzymes 2. Use of fluorochrome dyes (immunofluorescence) 3. Use of colloidal metals 4. Use of radioactive isotopes Use of Enzymes o Enzymatic method of IHC - uses enzymes o Ab + Enzyme + chromogen + substrate o Most commonly used enzyme labels ▪ Horseradish peroxidase (HRP) - most widely used ▪ Alkaline Phosphatase (AP) - alternative for HRP o Use of enzymes will require chromogens (color developers) - to produce colored product ▪ 3,3’ diaminobenzidine (DAB) - brown color ▪ 3- amino-9-ethylcarbazole (AEC) - brick red o Chromogens will require use of substrate ▪ Substrate - one consumed by the enzyme ▪ For HRP - hydrogen peroxide (H2O2) ▪ For AP - phosphate o Chromogens for HRP ▪ DAB • Compatible Counterstains: hematoxylin (common), methyl green, methyl blue ▪ AEC • Compatible Counterstains: hematoxylin, methyl blue ▪ End-products observed using light microscope ▪ Counterstains - utilized after linking with antigen o Chromogens for AP ▪ BCIP (5-Bromo-4-Chloro-3-IndolylPhosphate)/NBT (Nitro Blue Tetrazolium) • Color: bluish violet or black violet • Suitable counterstains: nuclear fast red and brilliant green ▪ Fast Red TR salt • Color: rose color • Suitable counterstains: none o Hematoxylin as counterstain ▪ Traditional counterstain ▪ Nuclear stain ▪ Forms of hematoxylin used in IHC: • Mayer’s - reddish violet nucleus • Harris - red nucleus o Commonly used because tissue morphology are well-defined ▪ 30-60 mins at Room Temp. - Incubation time and temperature to link antibody with peroxidase o Advantages of Enzymatic method of IHC: ▪ Standard samples can be stored for a long time ▪ Hematoxylin can be used as counterstain ▪ End-product can easily be identified using LM/EM ▪ Will not require fluorescence microscope o Enzymatic Method of IHC Summary HRP AP Notes Most commonly used Substrate H2O2 Phosphate Chromogens DAB, AEC BCIP/NBT, Fast Red TR Salt 1 • • • Use of fluorochrome label/dyes o In the labelling, use of fluorochrome dye will render the technique as immunofluorescence ▪ Immunofluorescence (IF-IHC) is an IHC technique o Require fluorescence microscope to observe results o FITC – fluorescein isothiocyanate ▪ Most commonly used Use of Colloidal metals o Gold - most commonly used ▪ End product: pink o End-products can be noted under light microscope Use of Radioactive isotopes • • 1. 2. Used to detect antigens aside from antibodies From plant or animal sources Can bind with tissue carbohydrate Can also be labeled using enzymes similar to antibodies • SPECIMENS FOR IHC 1. 2. • • Frozen Section o Requested for rapid diagnosis (while px in OR) o Carried out intraoperatively o Microtomes used: freezing & cryostat (commonly used) o Tissues are prepared as cryostat section and must be fixed for a few sections using: methanol or acetone Processed Specimens o Types: ▪ Formalin-fixed ▪ Paraffin Embedded o These specimens are already placed on a slide o Requires antigen retrieval prior to detection Purpose of fixation in IHC: o To preserve position of antigens being detected o To prevent destruction of labile antigenic sites ▪ Destruction will remove attachment sites Formaldehyde - Preferred fixative for IHC • • • 2 general methods of Antigen Retrieval: o Heat-induced Epitope Retrieval (HIER) o Protease-induced epitope retrieval (PIER) Purpose of antigen retrieval: o To remove/expose antigen from tissues for detection o To increase accessibility of antibody to antigen Proteolytic Enzyme Retrieval/ Protease induced epitope retrieval (PIER) o Most common enzymes used are: ▪ Trypsin ▪ Protease o Enzymes can break formalin crosslinks ▪ Formalin crosslinks - formed during fixation to preserve the cells Heat-induced Epitope Retrieval (HIER) 1. Microwave antigen retrieval - 20 mins. 2. Pressure cooker antigen retrieval - less time consuming 3. Autoclave heating 4. Water bath heating 5. Steamer heating 6. Decloaker heating 7. Combination of microwave & enzyme digestion Use of Control as requirement in IHC o Purpose: ▪ To test for specificity of antibodies involved ▪ To prevent misinterpretation due to false (+) and false (-) results Controls o Positive Control ▪ Tissue section containing antigen being detected o Negative Control ▪ To prepare, omit the primary antibody from staining schedule or replace primary antibody by an Ig that is directed against an unrelated antigen o Internal Tissue control ▪ Also known as Built-in control ▪ Contains the target antigen not only in the tissue elements but also in adjacent normal tissue elements DETECTION DETECTION USING LABELED ENZYME ANTIBODY • ANTIGEN RETRIEVAL • To be done only for paraffin embedded & formalin fixed tissues (processed samples) Should not be done for frozen sections Peroxidase Block – to block endogenous peroxidases, uses 3% H2O2 Protein block – blocks all non-specific sites & reduces background, uses 10% normal serum o Preferred than peroxidase block CONTROLS IN IHC • LECTINS • • • • PRE-TREATMENT BLOCKING Direct IHC o Traditional method o Use of only one labeled antibody o Principle: 1. Use of only 1 ab that is labeled 2. Add the labeled ab to the ag being detected 3. End-product observed under LM o • Enhanced Polymer One-Step staining Method (EPOS) ▪ Improvement of traditional method (Direct IHC) ▪ A type of direct method ▪ Involves addition of dextran polymer ▪ More sensitive method Indirect IHC o 2-step method o Requires use of 2 antibodies o Primary antibody - specific to antigen being detected o Secondary antibody - attach/bond to the Fc portion of primary antibody thereby increasing sensitivity by increasing signal amplification 2 AUTOMATION IN IHC OTHER MARKERS • • • Presence of automated workstation in IHC Advantages: o Greater consistency of staining o Faster and more accurate o Less manpower o Decreased use of reagents TUMOR MARKERS • • • Found in tissues and are markers for malignancies Sarcoma - connective tissue in origin Carcinoma - epithelial cell in origin EPITHELIAL TUMOR MARKERS • • • • • • • • • hCG (Human Chorionic Gonadotropin) o Synthesized by placental syncitiotrophoblast o Marker for choriocarcinoma AFP (Alpha feto protein) o Marker for embryonal carcinomas & teratomas as well as hepatocellular carcinomas o Teratoma - usually benign monstrous tumor Lymphomas o Best screening marker for lymphoma o Also known as CD45 Ki & M1B-1 o Two most common markers to assess proliferation of tumor cells (cervical cancer) Keratin o Marker for epithelial tissue cells o For diagnosis of carcinoma o CK - cytokeratin o CK 7 - positive for CA of lungs, uterus, ovaries o CK 20 - positive for colon & stomach cancer o Not all types of carcinoma will be positive for CK EMA (Epithelial Membrane Antigen) o Determines the site of tumor o Useful in detecting adenocarcinoma o Positive for cancer (CA) of breast, lung, kidney o Most useful in breast adenocarcinoma CEA (Carcinoembryonic antigen) o An oncofetal antigen o Differentiates adenocarcinoma from mesothelioma ▪ Adenocarcinoma - cancer of epithelial tissues involving the glands ▪ Mesothelioma - cancer affecting mesothelial cells which cover most internal organs o Elevated in certain cancers of the GIT o Used in monitoring CA in colon, pancreas, cervix, breast TTF-1 (Thyroid Transcription Factor 1) o Differentiates lung adenocarcinoma from mesothelioma o Found in all thyroid neoplasms/cancer PSA (Prostate Specific Antigen) o Highly specific antigen for prostate o Elevated in cancer in prostate, pancreas, and with salivary tumors o Also elevated with prostate infection ER/PR (Estrogen-Progesterone Receptor) o Normally found in breast epithelial cells but not in myoepithelial cells o Useful in determining prognosis and treatment of breast carcinoma as well as for identifying of metastatic breast carcinoma o Positive ER/PR denotes good prognosis INTERMEDIATE FILAMENT MARKERS • • • • Actin - identify tumors derived from smooth, cardiac & skeletal muscles Vimentin - Present in normal mesenchymal cells and their neoplastic counterparts: sarcoma, melanoma, leukemia, seminoma, schwannomas o Schwannoma - cancers involving Schwann cells (PNS) Desmin - Highly specific for myogenic tumors Glial fibrillary acidic protein (GFAP) - Confirms diagnosis of astrocytoma 3