Immunohistochemistry & Immunocytochemistry Techniques PDF

Immunohistochemistry & immunocytochemistry techniques  Introduction The bonds between chemical compounds that are characterized by highest specificity are those that involve spatial matching (complementarity) of bonding moleculues. They are ca...

Immunohistochemistry & immunocytochemistry techniques  Introduction The bonds between chemical compounds that are characterized by highest specificity are those that involve spatial matching (complementarity) of bonding moleculues. They are called stereospecific interaction in result of which covalent bonds are not created, but instead molecules are bonding via weak but multiple bonds that are a result of vicinity of atoms. In this way bonding between antibody and antygen occurs, or between purine and pirymidine alkali in nucleic acids. ANTIGEN-ANTIBODY Hydrogen bonding Electrostatic interaction van der Waals Forces ▪ Histochemical techniques using this type of bonding are called affinity histochemistry and are characterized by the highest affinity among all histochemistry methods. Affinity histochemistry includes (among others): immunohistochemistry and hybrid cytochemistry.  Immuno… reactions - Methods based on detection and localization cells and tissues elements on the antigen-antibody reaction principle. - Allow detection of substances possessing antigenic traits with marked antibodies in tissues, - Routinely used techniques, allow precise identification of cell types and cancer markers. Zastosowanie badań IHC: Cancer type/Useful markers diagnostyka chorób nowotworowych Large intesitne cancer/ CEA, p53 Pancreatic określenie charakteru cancer/ CA 19-9, CK7, CK19, nowotworu p53 – łagodny czy Breast cancer/ mammoglobuline, ER, PR, HER2 złośliwy Prostate gland cancer/ PSA, AMACR, CKHMW, p63, BCC Primary określenie immunofenotypu hepatocellular cancer/ AFP nowotworu Cervical cancer/ p16 Endometrial określenie cancer/stopnia ER, PR, p53 zaawansowania nowotworu Gastric cancer/ CEA wskazanie na miejsce pochodzenia przerzutów, Lung cancer/ NSE, ALK tzn. Germ cellokreślenie pierwotnego tumors/ b-HCG, AFP, PLAP miejsca guza Ovary tumors/ Ca 125, b-HCG, AFP, CEA określenie wrażliwości ogniska chorobowego na Thyroid cancer/ Tyreoglobulina, kalcytonina, galektyna 3, stosowany lek  Antigen ▪ Antigen is a multicomponent substance that is identified by competent immune system cells as foreign, resulting in an initiation of the immunological response in form of production of specific antibodies and creating specific cell response. It is a molecule that reacts specifically with antibody or allergic cell. ANTIGEN TRAITS IMMUNOGENICITY ▪ Ability to stimulate specific immunological response/ response to create specific antibody against itself ANTIGENICITY ▪ Ability to specifically bind with antibodies (free ones and those that are lymphocytes B-receptors) and T-receptors In light of above traits, 2 types of antigenes are distinguished: immunogenes – exhibiting both above mentioned traits, haptens – exhibiting only antigeniticy  Antigen cont. ▪ Epitope, also known as antigenic determinant, is the part of an antigen that is recognized by the immune system, specifically by antibodies, B cells, or T cells. Epitopes Antigen Antibody ▪ Antigenes may be divided according to number of epitopes recognized by given antibody on single antigen: monovalent antigens – containing only one epitope and binding with Antibody single paratope. Haptens are always monovalent. polivalent antigens - binding with several paratopes simultaneously.  Antibody ▪ Antibodies are immunoglobulines produced by fully differentiated B lymphocytes– plasmocytes, capable of specific binding with an antigen. ▪ A/b are built from 4 polypetide chains – 2 light and 2 heavy, binded via disulfide bond. Variable regions (V) and constant regions (C) are in both types of chains. Variable region of each chain consists of 3 hypervariable (HV) and 4 framework regions (FR). HV create antygen binding sites, determining specificiyt of given a/b. ▪ 5 classes are distinguished: IgA IgD IgE IgG IgM ▪ a/b used in immunocyto/histo/chemistry are mostly IgG Paratope = antygen binding site an antibody’s fragment binding an epitope  Types of antibodies used in IHC Monoclonal – highly specific – recognize one antigenic determinant; monoclonal a/b are produced by undifferentiating, composing one clone, B lymphocytes; Advantages of monoclonal a/b: ▪ Monospecifity – all a/b molecules react with the same antigenic determinant, expressing the same affinity to antigen ▪ Enable distinguishing minimally different antigenes ▪ Smaller background in immunochemistry techniques Disadvantages of monoclonal a/b: ▪ Too high specificity may be a disadvantage: eg. a) examining multi-epitope antygen with only one kind of monoclonal antibodies that are able to bond only with one epitope may give inadequate ▪ b) no reaction, when eg. Working conditions cause partial denaturation of antygen (monoclonal a/b are sensitive to epitope structure changes) opu) ▪ Expensive and time consuming production process  How to obtain monoclonal a/b? Monoclonal a/b are obtained through so called somatic hybridization, ie. in result of a two cells fusion, in which one cell synthesizes and releases a/b of given specificity, and the second one is a multiple myeloma cell (cancer originating in lymphocytes B) with unlimited proliferating abilities. The result is a hybridoma – antibodies releasing cancer. Several factors influnece the proces of obtaining monoclonal : ▪ immunization, ▪ A fusion of B cells with multiple myeloma cells, ▪ cloning (series of dillution or cloning in agar), ▪ Selection of clones producing wanted a/b, ▪ propagation (multiplication) of desired clones..  Types of markers used in IHC Polyclonal a/b – less specific – a group of antibodies recognizing only one antigen, but reacting with its different epitopes; produced by various populations (clones) of plasmatic cells Advantages of polyclonal a/b: ▪ Recognize many epitopes within the same antygen , ▪ Enable using of higher dillutions, making false positve reactions less possible ▪ High bonding force, ▪ Bigger tolerance to changes in epitope’s conformation (less risk of falsely negative reaction because hiding one antigenic determinant does not have to influence other types of a/b binding with antigen), ▪ Frequently give strongher signals in immunochemistry techniques ▪ Cheap and quick in preparation and production Disadvantages of polyclonal a/b: ▪ Large quantity of unspecific antibodies, bigger background possible ▪ Uniqueness of production lines,  Markers used in IHC Posses features enabling observing them directly (fluorochromes) or indirect (enzymes) Must create permanent complexes Cannot change the characteristic of marked substance Show binding sites of antigen with antibody Antibodies may be marked with: Fluorochromes – fluorescent dyes, slide watched under fluorescent microscope Enzymes – slide watched under light microsope or electron microscope Metals – slide watched under electron microscope mikroskopie elektronowym  Markers used in immuno… methods 1. Fluorochromes (immunofluorescence) – substances that exhibit the ability to emit light after radiation ❑ Most ofte used: ▪ Fluorescein isothiocyanate (FITC) ▪ Fluorescein isocyanate (FIC) ▪ Tetramethylrhodamine(TRITC) ▪ Texas Red (TR)  Markers used in IHC 2. Enzymes ▪ An example of indirect marking ▪ A complex of antibody with enzyme recquires detection of the enzyme activity, resulting in colourful or high elctron density product of reaction in site of antigen ▪ Obsevation– light or electron microscope ▪ Sensitive method ❑ Most often used enzymatic markers: ▪ Horseradish peroxidase ▪ Alkaline phospatase ▪ Glucose oxidase  Markers used in IHC 3. Heavy metals In immunohistochemical testing on an electron microscope level: - ferritin - Colloid gold– gives very good contrast in electron microscope Colloid gold on a light electron microscope level: 1. Binding tissue with gold 2. Reduction of silver salt to metallic silver – colourful precipitate  Markers used in IHC Colloidal gold  Preparing histopathological material for tests 1. Preserving material for tests - fixation 2. Tissue proccessor stages 3. Embedding in paraffin 4. Slicing on microtome 5. Deparaffinization 6. Rehydration 7. Staining IHC reactions 8. Dehydration 9. Clearing 10. Closing What’s important during IHC reaction? Fixing Control reactions Embedding Direct method Cutting parafin blocks Indirect method Antigen retrieval Reaction type : Background busting Immunoenzymatic Selecting antibody Examination All stages preceding IHC reaction have future influence on the reaction result. What kind of material is used to perform IHC reaction? FFPE– most often, sections 3-4 µm thick on an increased adhesiveness glass slide Smears I. 1. Paraffin section preparation Deparaffinization of tissue – xylene – 56-600 C, subsequently in range of xylenes in room temp. Rehydration – alcohol 99,8% alc. 50% H2O  1. Utrwalenie materiału do IHC a) Used fixatives: Routinely: 10% buffered formalin pH 7,2 ALDEHYDES: ▪ Formaldehyde (FA) Most often ▪ Paraformaldehyde (PFA) 2-4% ▪ Glutaraldehyde (GA) – used mostly in microscope immunocytochemistry WORKING PRINCIPLE: during fixation they bind different functional groups of proteins, eg. amic, sulfhydryl, guanidyne, creating cross links  2. Antigen sites retrieval Fixing and embedding very often cause changes in antygen molecules, which inhibit or even prohibit antigenic deteminant recognition by specific antibodies (eg. Cross links). It results in weak intensity or even lack of IHC reaction. In such cases, after cutting the material and possible deparaffinization, methods reinstating original characteristic of antygen should be used, which is called antigenic determinant retrieval or unmasking. Most often used methods are: controlled digestion of tissue sections using proteolitic enzymes and microwaves  (enzyme-induced epitope retrieval) EIER controlled digestion of sections with proteolitic enzymes : - K proteinase - E proteinase - pronase - trypsin/chemotrypsin Enzymes dillution in PBS buffor pH 7,2; incubation – room temp. or 370 C  (heat-induced epitope retrieval) HIER boilng in cooker or water bath incubation in citrate buffer pH 6,0; EDTA pH 9,0; boil in 95֯C for 15-20 min., cool slowly rinse in PBS  Endogenous peroxidase activity quenching. A. Tissue section without previous incubation (falsly positive reaction – brown colour). B. B. Endogenous peroxidase activity quenching 3% H2O2 https://www.novusbio.com/blocking-non-specific-binding 3. Inhibiting endogenous enzymatic activity a) Inhibiting endogenous peroxidase activity Incubation of sections in 3% H2O2 water solution 3. Inhibiting endogenous enzymatic activity b) Inhibiting alkaline phosphatase activity Administation of specific inhibitor – levamisole to the substrate (incubating fluid detecting alkaline phosphatase activity)  4. Immunoglobulines’ unspecific binding sites blocking Biologial material is often able to unspecifcally bind proteins, thus also immunoglobulines. In result of immuno reaction, the sites unspecifically binding proteins would be equally visible as the sites of the searched antygen. Short term incubation of sections before immunoenzymatic reaction, with protein derived from different animal, than the one from which antibody was obtained This protein blocks sites in material, that binds immunoglobulines in unspecific way, however does not prevent from creating strong, specific binds between antygen and antibody used in IHC Bovine serum albumin (BSA) or not immunized animal A. B.  IHC reactions Direct reaction Indirect reaction – most common  Direct reaction Based on incubation of tisuue containing antygen A with marked antibody anti-A. Marked antibody creates a complex with antigen in tissue and enables its microscopicall localization. Rarely used because chemically marking may influence antiody’s ability to recognize specific antigen  Indirect reaction I. Stage Incubation of tested tissue with specific, unmarked antibody (Antigen + Unmarked specific antibody) II. Stage Incubation of tested tissue withe second marked antibody directed anti immunoglobuline from animal, from which 1st antibody was obtained (Unmarked specific antibody + marked antiglobuline A method much more sensitive than direct method. Direct vs indirect reaction  Detection of marking enzymes Last stage of immunoenzymatic procedure is a reaction detecting marking enzyme activity The result of this reaction, colourful, insoluble product marks the site of antigen-antibody complex.  Detection of marking enzymes 1. Detection of peroxidase activity Peroxidase activity causes (in presence of H2O2) oxidation of cytochemical substrates (electron donors) to colourfull products The method with 3,3’-diaminobenzidine (DAB) Horseradish peroxidase – a protein containing ferroporfirine – enable to reveal antygen catalizing reaction of transferring electrons from chromogen (substrate giving colourfull product) to H2O2 Leads to creating brown polimer – insoluble in water, alcohol or organic solvents (has the ability to bind heavy metals) Adding copper salt, cobalt, nickel or gold to incubation liquid with DAB causes enhancing the intensity of reaction and changes the colour from brown product to blue or black, what enhances the sharpness of raction  Detection of marking enzymes 2. Detection of alkaline phosphatase activity Alkaline phosphatase activity is routed out by method of concatenation with naphthol derivatives An enzyme catalizes reaction of transforming substrate: BCIP, what is acompaniated by creation of blue dye - indigo; these reactions donate H+ ions for reduction of another substrate – NBT with creation of purple salt – formazan.  Staining basophilic structures Hematoxylin acc. to Mayer is used in purpose to rout out cell nuclei and dyeing the whole structure of tissue to make it visible in light microscope (immunohistochemistry). Final stages of preparing slides Dehydration (alcohols of increasing concentrations) Clearing (xylen I-IV) Application of suitable medium and covering with cover glass  Closing glass slides in medium Crucial in case of archiving and improving microscopic image quality Protecting the staining from physical factors, improving brightness and controst of microscopic image  Control reactions Guarantee of results credibility Confirmation of used method specificity Detecting all artefacts Positive – in which we are expecting the occurence of immunopositve (stained structures) Negative – where colourful reaction should not occur  Control reaction 1. Positive control reaction - Conveyed on a selcted, model tissue containing detected antigen - Reaction points to correctness of used protocol: no colouring indicates methodology errors or inadequacy of used antibodies 2. Negative control reactions - With omiting specific antibody - With omiting specific antibody and secondary antibody - Reaction with substituing specific antibody with unspecific serum  Stages of IHC reaction Stage of IHC reaction Reagents Deparaffinization and hydration Xylen I, II, III Alcohol 99,8% 80%, redestilled water Antigen reveal Citric buffer / EDTA / K proteinase pH 6,0 /pH 9,0/; temp. 95 °C Redestilled water Blocking endogenous peroxidase 3% hydrogen peroxide activity Redestilled water Rinsing in PBS Eliminating background BSA Rinsing in PBS Incubation with I antibody Primary antibody Rinsing in PBS Incubation with II antibody II antibody + marker (horseradish peroxidase/ alkaline phosphatase) Detecting markers DAB (for peroxidase) brown polymer NBT/BCIP + levamisole (for alcalic phosphatase) red polimer Running water Nuclei staining Hematoxylin acc. Mayer Running water Dehydration Alcohol 80% 99,8% Clearing Xylene I, II, III, IV Closing glass slides DPX resin -> cover glass Localisaton of the Ag - Nuclear - ER/PR, S-100, TTF1 - Cytoplaxm S-100, SYN - Membrane – CD20, HER2 Nuclear – KI67 Membrane – HER2 Cytoplasmic -SYN Panels IHC 50 51 Metastasis 52 53  Why is IHC testing done? IHC tests are helpful in many diûerent circumstances, but these tests aren’t often part of the standard diagnostic process for most cancer types. For the most part, IHC tests are only ordered after a pathologist has reviewed the results of more routine tests. Pathologists diagnose most cancers just by looking at a biopsy sample under a microscope and using stains. However, if the analysis is more complex and requires checking for more speciüc characteristics to make a diagnosis, the pathologist may turn to IHC tests. The doctor and a pathologist typically determine whether or not immunohistochemistry is necessary on a case-by-case basis. However, IHC tests are routinely used to help diagnose most types of breast cancer, including: Invasive breast cancer (when the cancer has spread within breast tissue) Metastatic breast cancer (when the cancer has spread to distant parts of the body) Recurrent breast cancer (when the cancer has come back after treatment)  In patients with breast cancer, immunohistochemistry is used to test for the conditions listed below. Hormone receptor status: ER and PR - IHC tests may detect the presence or absence of hormone receptors on breast cancer cells. This knowledge informs how the cancer may be treated, as breast cancers that carry these receptors can be treated with hormone therapy drugs. HER2 status: IHC tests may check for HER2 receptors to determine whether breast cancer is HER2-positive or HER2-negative. If these receptors are found, the cancer is HER2-positive, which indicates that it’s likely to be fast-growing and may be treated by targeted therapy drugs that block the eûects of the HER2 proteins responsible for fueling the cancer’s growth. Another routine use of IHC testing is for Lynch syndrome (MSH2, MSH6, PMS2, MLH1), an inherited condition that may cause cancer or increase one’s risk of developing cancer. Lynch syndrome is most often linked to colorectal cancer, but it may also raise the risk for developing cancer in the uterus, stomach, liver, kidney and brain. Doctors may recommend using IHC to check for markers of Lynch syndrome in women who develop endometrial cancer, or anyone diagnosed with colorectal cancer before age 70.  Prostate cancer: Some prostate cancers are diþcult to diagnose using standard tests. Numerous antibodies may be used in IHC tests to learn more about the cancer, although performing these tests isn’t always necessary. IHC tests are more likely to be used if standard tests reveal mixed ündings. (AMACR, CKHMW, p63, PSA)  Gastrointestinal cancer: Some gastric cancers are linked to autoimmune diseases or bacterial strains, such as H. pylori. IHC tests may help inform treatment by determining whether these or other factors may be responsible for causing the cancer. IHC tests can also help diûerentiate between types of gastrointestinal cancer.  Lung cancer: After other tests conürm a lung cancer diagnosis, IHC tests may be necessary to deüne the type of lung cancer. For example, a pathologist will often need to use IHC to check for speciüc antigens to establish a case of non-small cell lung cancer. (TTF1, Napsin A, ROS1, ALK, p40, Ck5/6) https://diagnostics.roche.com/global/en/prod ucts/tests/lung-cancer-ihc-ish-portfolio.html  Lymphoma: It may be diþcult for a pathologist to recognize whether swollen lymph nodes are caused by an infection or cancer such as lymphoma. With IHC tests, the pathologist is able to test the white blood cells causing the swelling to see whether they carry cancer-indicating antigens. IHC tests may also help distinguish between diûerent lymphoma types. Diagnostic worküow for the diagnosis of aggressive mature B cell lymphomas. The worküow applies to DLBCLs that don’t fulûll the inclusion criteria for the speciûc DLBCL entities (i.e. primary mediastinal B cell lymphoma (PMBCL), intravascular DLBCL, EBV+ DLBCL, T cell rich histiocyte rich B cell lymphoma (TCRHRBCL), etc (*) and to blastoid lymphomas excluding lymphoblastic lymphomas and mantle cell lymphomas (**). In DLBCL discussion with the referring hematologist is highly recommended before proceeding with FISH analyses (***). https://link.springer.com/article/10.1007/s00428-019-02637-2  Understanding the IHC pathology report Once the analysis is complete, a pathology report will be prepared to summarize the diagnosis and other relevant information. It may take about two to 10 days for results to be ünalized and presented to the patient after a biopsy procedure. The diagnostic process may be longer or shorter, depending on the number of tests performed and their complexity. IHC tests typically only take one day longer than routine tests. More complicated analyses, such as ýow cytometry, tend to delay the process further. Once the patient receives his or her pathology report, it's important to look it over and take note of any questions or concerns. The doctor may go through the report in detail with the patient.  Benefits and risks of immunohistochemistry The beneüt of IHC testing is that, when necessary, it allows the care team to gain a more precise understanding of the cancer and how best to treat it. However, these tests aren’t 100 percent accurate. Misdiagnosis of any sort is harmful, with false-positives resulting in unnecessary treatment and trauma, and false-negatives leading to potentially detrimental treatment delays. The rate of false-positives and false-negatives vary signiücantly with an IHC. Numerous factors may inýuence the accuracy of these tests, including: Technology, equipment and materials used Storage and handling of the sample Errors made throughout the process  IHC test kits are regulated by the U.S. Food and Drug Administration. All test manufacturers must present data on their accuracy in repeated attempts and against other methods. Pathologists are also encouraged to regularly validate their own IHC systems to ensure that they are reporting accurate results. While a cancer misdiagnosis can occur, systems in place are designed to help avoid this, such as requiring a second pathologist to review ündings (in most accredited labs) and having a team of doctors and pathologists work together to interpret the results.  More information: https://www.slideshare.net/vbiogenex/basics- of-immunohistochemistryihc https://www.slideshare.net/appyakshay/imm unohistochemistry-75139545 Immunohistochemistry Preparing tissue sections according to standard protocol Primary antibody Secondary antibody Visualization and staining of basophilic structures Thank you for your attention