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11. ihc IHC makes it possible to visualize the distribution and localization of specific cellular components with in a cell or tissue.pptx
- 3. Introduction • Immunohistochemistry (IHC) Combine s histological, immunological and biochemical techniques for the identification of specific tissue components by means of a specific antigen/antibody reaction tagged with a visible label • IHC makes it possible to visualize the distribution and localization of specific cellular components with in a cell or tissue.
- 4. Immunohistochemistry • Technique for identifying cellular or tissue constituents (antigens) by means of antigen- antibody interactions, the site of antibody binding being identified either by direct labeling of the antibody, or by use of a secondary labeling method. • Immunohistochemistry- using tissue sections. • Immunocytochemistry – cytological preparations
- 6. 1945 – Albert Coons 1st used an Ab labeled with a fluorescent dye to visualize tissues 1st fluorescent dye attached to an Ab was fluorescein isothiocyanate (FITC) – it emits green fluorescence A fluorescence microscope is required to visualize this fluorochrome and they have a tendency to fade . Also morphology is difficult to demonstrate Due to these limitations – IHC was developed Here enzymes are used as labels and visualized with an appropriate chromogen using light microscope
- 7. 1966 – 1st developed enzyme labeling instead of fluorescent label Used enzyme was – horse radish peroxidase (HRP) – hence also known as immunoperoxidase technique It was done on frozen tissues 1970 : Sternberger et al described the peroxidase antiperoxidasetechnique. 1971: Envall& Perlman reported the use of alkaline phosphatase labeling 1975: Kohler & Milstein described a revolutionary method for production of monoclonal antibodies
- 8. 1977:Heggness & Ash proposed the use of avidin– biotin for immunofluorescence 1981: Hsu et all. have defined the method of avidin- biotinperoxidase complex which is abbreviated as ABC method 1993: Advent of one step system, based on a two layer dextran polymer. Reported by Pluzek et al at UK pathological society meeting. 1998: Dako launched a new labeling system based on the dextran polymer technology.
- 9. TERMINOLOGIES • ANTIGENS : molecules that induces formation of an Ab and is foreign to the animal into which it is introduced. • Sites on Ag that are capable of inducing Ab formation are known as Epitopes/ Antigenic determinant – the exact site on the Ag with which the Ab combines. • Epitopes may be classified as -
- 11. ANTIBODIES • Antibody is also known as immunoglobulin. The antibody is produced by plasma cells in response to antigenic stimulation. • IgG is the most frequently used Ab for IHC. • The paratope of Ab binds to the epitope of Ag.
- 13. Primary antibodies • Polyclonal antibodies – Large complex antigens may have multiple epitopes and elicit several antibody types. Mixtures of different antibodies to a single antigen are called polyclonal antibodies. • Monoclonal antibodies – Antibodies specific for a single epitope and produced by a single clone are called monoclonal antibodies
- 16. amounts
- 17. Affinity • Three dimensional fit of Ab to its specific antigen • Measures the binding strength Avidity • Functional combining strength of an antibody with its antigen. Antibody specificity • characteristic of an antibody to bind selectively to a single epitope Sensitivity • Relative amount of antigen that an IHC technique is able to detect.
- 18. Principle : Ag-Ab Interaction • Immunohistochemistry is a method for localizing specific antigen in tissues or cells based on antigen - antibody reaction. • The site of antibody binding is identified either by tagging the antibody, directly or indirectly with a visible label. • Enzymes , Fluorescent dye, colloidal metal, radioactive marker.
- 19. LABELS Need To make the Ag-Ab reaction product easily visible Types of Labels • Enzyme Labels • Colloidal Metal Labels • Fluorescent Labels • Radiolabels
- 20. Enzyme Labels • Most widely used in IHC • Commonly used enzyme label is Horseradish Peroxidase. Along with the preferred chromogen DAB, gives dark brown colored reaction product. • Its small size does not hinder the binding of antibodies to adjacent sites. • Chance of contamination is minimized. • Stable enzyme. • Endogenous activity is easily quenched.
- 22. • Calf intestinal Alkaline phosphatase – APAAP technique. • Glucose oxidase • Bacterial β-D-Galactosidase
- 23. Colloidal metal labels • When used alone, colloidal gold conjugates appear pink when viewed using the light microscope. • A silver precipitation reaction can be used to amplify the visibility of the gold conjugates • Silver may also be used as a conjugate, and it gives a yellow color that is visible directly
- 24. Fluorescent Labels • Flouramine • Rhodamine
- 25. Radiolabels • Radioisotope tracers. • Requires autoradiographic facilities
- 26. Direct Technique Traditional New Immunohistochemical Methods Indirect Technique Polymer chain two step indirect New Unlabeled Ab Enzyme- Complex (PAP & APAAP) Immuno gold –Silver Staining Avidin-Biotin Hapten Labeling Tyramine signal amplification MICA
- 27. Direct method • Primary antibody is conjugated directly to the label • Little signal amplification and lower sensitivity Indirect method • Primary antibody is unlabelled • Secondary antibody directed against the immunoglobulin is conjugated with the label
- 28. Traditional direct technique • Primary antibody is conjugated directly to the label- a fluorochrome, horseradish peroxidase and alkaline phosphatase • Advantage - simple to use as they only require one application of reagent • Main disadvantage is that the sensitivity is low. • Risk of not detecting small amounts of antigen that sometimes could be crucial to the diagnosis.
- 30. New direct technique (enhanced polymer one step staining method) • A large number of primary antibody molecules and peroxidase enzymes are attached to a dextran polymer “backbone”. • It is possible to obtain sensitivity approaching that achieved by avidin-biotin technology but with fewer steps. • Advantages - rapid & sensitive; better reproducibility. • Disadvantages - numbers of primary antibodies available with this system are limited.
- 33. Indirect technique • The unconjugated primary antibody is applied, followed by a labelled antibody against the first antibody. • Horseradish peroxidase labeling is most commonly used with the appropriate chromogen substrate. • More sensitive, rapid and relatively inexpensive.
- 35. New indirect technique (Dextran polymer conjugate two-step visualization system) • Based on the dextran technology employed in the EPOS labeling system. • The primary antibody in the EPOS model is replaced with a secondary antibody. • Greater sensitivity, less time-consuming.
- 38. Unlabeled antibody enzyme-complex techniques • PAP uses a soluble peroxidase – antiperoxidase complex - used as a third layer - bound to the unconjugated primary antibody by a second layer of ‘bridging’ antibody • APAAP - uses the alkaline phosphatase-anti-alkaline phosphatase complexes- not so successful as excessive background staining can sometimes be serious drawback.
- 42. Immunogold silver staining technique (IGSS) • Introduced by Faulk and Taylor (1971). • In this method the gold particles are enhanced by the addition of metallic silver layers to produce a metallic silver precipitate which overlays the colloidal gold marker. • silver lactate as the ion supplier • hydroquinone as the reducing agent. • Disadvantage: Formation of fine silver deposits in the background
- 44. Avidin and Biotin Method • The biotin has the high affinity for avidin. In this method the secondary antibody is tagged with biotin. Now avidin conjugated with horseradish peroxidase is used. • The biotinylated secondary antibody is tightly bound with the peroxidase-conjugated avidin
- 45. Avidin and Biotin Conjugated Procedure • This is the modification of the above-mentioned procedure to further increase the sensitivity of the test. • Here a preformed complex of avidin-biotin and horseradish peroxidase is used • The presence of multiple molecules of horseradish peroxidase enhances the visualization reaction
- 47. Streptavidin-biotin method • Affinity of the glycoprotein avidin for biotin, a LMW vitamin. • A similar molecule, streptavidin -extracted from bact. streptomyces avidinii- lack oligosaccharide residues and neutral isoelectric point. • Variants - peroxidase and alkaline phosphatase, either directly bound or biotinylated
- 48. 3-step technique: • 1. unconjugated primary antibody as the first layer, • 2. followed by a biotinylated secondary antibody. • 3. The third layer is either a complex of enzyme-labelled biotin and streptavidin, or enzyme-labelled streptavidin
- 50. Hapten labeling technique • Uses haptens such as dinitrophenol and arsanilic acid. • Hapten is linked to the primary antibody and a complex is built up using an anti-hapten antibody and either hapten labeled enzyme or hapten-labeled PAP complex.
- 52. Biotinylated tyramide signal amplification • Bobrow et al. first described the use of biotinylated tyramide to enhance signal amplification, in 1989. • The technique is based around the streptavidin biotin technique. • Advantage: Enables many antigens which had previously been non-reactive in formalin-fixed paraffin-embedded tissue to be demonstrated. • Disadvantage: Excessive background staining
- 53. Procedure: Application of the primary antibody subsequent incubations in biotinylated secondary antibody horseradish peroxidase-labeled streptavidin subsequent treatment with the biotinylated tyramide amplification reagent. hydrogen peroxide free biotin radicals. These reactive biotin molecules bind covalently to proteins adjacent to the site of the reaction.
- 55. Tyramine signal amplification (TSA) method. In this technique biotinylated tyramine is used in the presence of HRP and hydrogen peroxide. The HRP converts the biotinylated tyramine to reactive biotinylated tyramide that further reacts with tyrosine in the amino acid of the tissue and deposits biotin. This biotin is deposited only in the antigen-antibody reaction site. This biotin is visualized by avidin-biotin technique
- 56. Mirror Image Complementary Antibody labeling technique (MICA) • Involves the sequential use of mutually attractive antibodies. • Raising antibodies against each antibody’s species, which enables each antibody to be both antigen and antibody in respect to each other. • 60 times more sensitive than conventional avidin-biotin systems. • Complex and time consuming procedure. • Cannot be used without automation.
- 58. IHC can be performed on.... • Formalin fixed paraffin embedded sections. • Frozen sections • Smears • Cytospins
- 59. Protocol in IHC Tissue section Antigen retrieval Blocking of endogenous enzyme Primary antibody Secondary antibody Chromogen substrate Counter stain Mounting Microscopic observation
- 60. Fixation Goals of fixation • Prevent autolysis by rapidly terminating enzymatic/metabolic activities • Prevent bacterial decomposition. • Preserve tissue structures while stabilizing and hardening the tissue for processing.
- 61. • Fixative can cause changes in the steric configuration of proteins, which may mask antigenic sites (epitopes) and adversely affect binding with antibody. • It is well recognized that cross-linking fixatives (formaldehyde) alter the IHC results for a significant number of antigens, whereas coagulant fixatives, especially ethanol, have been reported to produce fewer changes • A robust and optimized fixation protocol is a critical step in an immunohistochemistry protocol as an antigen that has been inappropriately fixed maynot be detected in downstream detection.
- 62. • The most popular choice of fixatives for routine histology are formalin based, either as a 10% solution or with the addition of different chemical constituents. • When formalin-based fixatives are used, intermolecular and intramolecular cross-linkages are formed with certain structural proteins. These are responsible for the masking of tissue antigens. • Methylene bridges forms between reactive sites on tissue proteins.
- 63. Advantages of formalin • good preservation of morphology, even after prolonged fixation and economical • sterilizes tissue specimens in a more reliable way than precipitating fixatives, particularly for viruses. • Carbohydrate antigens are well preserved • Cross-linking of protein in situ avoids leaching out of proteins that may diffuse in water or alcohol. • Many low–molecular-weight antigens (peptides) are well preserved in tissue by formalin.
- 64. Slide Preparation IHC-P: Paraffin-embedded Tissue • Prior to immunostaining, harvested and fixed in 10% neutral buffered formalin (NBF) to preserve cell morphology and target epitopes. IHC-F: Frozen Tissue • Frozen tissue should be stored at -80°C • When ready to stain, equilibrate tissue at -20°C for 15 minutes before attempting to section. Section the tissue to a 6-8 μm thickness using a microtome
- 65. Slide Storage • freshly cut slides- For best results • Slides may lose antigenic potential over time in storage. • If slides must be stored, store at 4°C.
- 66. Deparaffinization/ rehydration • Paraffin wax must be completely removed for staining • This is done through a series of sequential xylene/ethanol/water washes that remove the wax and rehydrate the tissue for subsequent antibody binding. • Insufficient paraffin removal can lead to spotty, uneven background staining.
- 67. Basic Steps in IHC Tissue section Antigen retrieval Blocking of endogenous enzyme Primary antibody Secondary antibody Chromogen substrate Counters tain Mounting Microscopic observation
- 68. Antigen Retrieval Need for antigen retrieval To unmask the antigenic sites usually blocked during Formalin fixation. Methods: 1. Proteolytic enzyme digestion 2. Heat Pre-treatments- a) Microwave Ag Retrieval b) Pressure Cooker Ag Retrieval c) Steamer d) autoclave e) Water bath 3. Combination of Microwave & Trypsin Method 4. Commercial Retrieval Solutions
- 69. Proteolytic enzyme digestion • Described by Huang et al. (1976), Curran and Gregory (1977), and Mepham et al. (1979). • The most popular enzymes employed today are trypsin and protease, but other proteolytic enzymes such as chymotrypsin, pronase, proteinase K, and pepsin may also be used. • Principle- Digestion breaks down formalin cross-linking and hence the antigenic sites for a number of antibodies are uncovered. • Under-digestion results in too little staining, because the antigens are not fully exposed. Over-digestion can produce false positive staining, high background levels, and tissue damage.
- 70. Heat-mediated antigen retrieval techniques • Heat-based antigen retrieval methods have brought a great improvement in the quality and reproducibility of immunohistochemistry. They have also widened its use as an important diagnostic tool in histopathology. • Theories: Heavy metal salts (as described by Shi et al. 1991) act as a protein precipitant, forming insoluble complexes with polypeptides, and that protein precipitating fixatives display better preservation of antigens than do cross-linking aldehyde fixatives
- 71. During formalin fixation intermolecular and methylene bridges and weak Schiff bases form intramolecular cross-linkages, which may prevent it from being recognized by a specific antibody. Heat-mediated antigen retrieval removes the weaker Schiff bases but does not affect the methylene bridges, so the resulting protein conformation is intermediate between fixed and unfixed. Morgan et al. (1997), who postulated that calcium coordination complexes formed during formalin fixation prevent antibodies from combining with epitopes on tissue-bound antigens. High temperature weakens or breaks some of the calcium coordinate bonds, but the effect is reversible on cooling
- 72. Microwave antigen retrieval • Shi et al. (1991) first established the use of microwave heating for antigen retrieval. • Gerdes et al. (1992) used microwave antigen retrieval with a non-toxic citrate buffer at pH 6.0 . • Cattoretti et al. (1993) established microwave oven heating as an alternative to proteolytic enzyme digestion. • Antigen retrieval solutions: 0.01 M citrate buffer at pH 6.0 and 0.1 mM EDTA at pH 8.0 • Uneven heating and the production of hot spots
- 73. Pressure cooker antigen retrieval • Norton et al. (1994) suggested the use of the pressure cooker as an alternative to the microwave oven. Batch variation and production of hot and cold spots in the microwave oven could be overcome. • Pressure cooking is said to be more uniform than other heating methods. • A pressure cooker at 15 psi (10.3 kPa) reaches a temperature of around 120°C at full pressure • It is preferable to use a stainless steel domestic pressure cooker, because aluminium pressure cookers are susceptible to corrosion from some of the antigen retrieval buffers
- 74. Water bath • Kawai et al. (1994) demonstrated that a water bath set at 90°C was adequate for antigen retrieval. • Increasing the temperature to 95–98°C, antigen retrieval was improved and the incubation times could be decreased. • Advantage - gentler on the tissue sections because the temperature is set below boiling point. • antigen retrieval buffer does not evaporate and • expensive commercial antigen retrieval solutions can be safely reused • Disadvantage -antigen retrieval times are increased compared to other methods.
- 75. Steamer • Steam heating appears to be less efficient than either microwave oven heating or pressure cooking • Advantage - less damaging to tissues than the other heating methods
- 76. Autoclave • This method offers an alternative form of heatmediated antigen retrieval, producing good results for nuclear antigens such as MIB1, p21, and p53
- 77. Combined microwave antigen retrieval and trypsin digestion • A combination of microwave antigen retrieval followed by 30 seconds trypsin digestion enables more reliable identification of light chain restricted plasma cells in myeloma compared with the use of trypsin alone Advantages • Light chains in plasma cells and AL amyloid are more easily demonstrated. • Labelling of other antigens, e.g. high molecular weight cytokeratin. • The dilution factors of primary antibodies are greatly increased
- 78. Disadvantages • As microwave oven pre-treatment causes the sections to be highly sensitive to proteolytic digestion, tissue can easily be damaged by excessive trypsinization. • The staining distribution with some antibodies can be severely altered
- 79. Advantages of heat pretreatment • Some antigens previously thought lost in routinely processed paraffin embedded sections are now recovered by heat pretreatment. • Many antigens are retrieved by uniform heating times, regardless of length of fixation.
- 80. Pitfalls of heat pretreatment • Care should be taken not to allow the sections to dry after heating, as this destroys antigenicity. • The boiling of poorly fixed material also damages nuclear detail. • Fibrous and fatty tissues tend to detach from the slide. • Prevention: Vectabond or APES-coated slides (3-Amino propylene triethoxy silane) can be dipped in 10% formal saline for 1–2 minutes and air dried before picking up the sections.
- 81. Commercial antigen retrieval solutions • There are numerous commercial antigen retrieval solutions available. • They can be either specialized high pH solutions (recommended for certain antibodies) or lower pH 6.0 for more general use. • Citrate buffer pH 6.0 • EDTA buffer pH 8.5 • Tris-HCL buffer pH-10
- 82. Basic Steps in IHC Tissue section Antigen retrieval Blocking of endogenous enzyme Primary antibody Secondary antibody Chromogen substrate Counters tain Mounting Microscopic observation
- 83. Blocking endogenous enzymes • In case of immunoperoxidase enzyme method, it is necessary to block the tissue endogenous peroxidase enzymes that are commonly present in RBCs, polymorphs and histiocytes. To block this endogenous peroxidase, the tissue should be kept in 0.5–1% hydrogen peroxide in absolute methanol for 10 min • Alkaline phosphatase (AP) is present in the WBC, liver, intestine, bone, etc. • When AP is used as a visualizing enzyme, it is necessary to block this enzyme. 1 mM levamisole in 0.5 M HCl solution is capable to block AP enzyme.
- 84. • Endogenous biotin may create problem in biotin-based visualization technique. Biotin is present in the liver, kidney, spleen, etc. Polymer-based method can effectively overcome this problem of endogenous biotin staining.
- 85. Blocking background staining The major causes of background staining in immunohistochemistry are • hydrophobic and ionic interactions and • endogenous enzyme activity. • Non-specific uptake of antigen, particularly the high affinity of collagen and reticulin for immunoglobulins, can cause high levels of background staining.
- 86. Hydrophobic interactions • Hydrophobic interactions are the result of the cross-linking of amino acids, both within and between adjacent protein molecules. Proteins are rendered more hydrophobic by aldehyde fixation and the extent of hydrophobic cross-linking of tissue proteins is primarily a function of fixation • Tissues that give background staining as a result of hydrophobic interactions include collagen and other connective tissues, epithelium, and adipocytes. Prevention: • Addition of a blocking protein • Addition of a detergent such as TritonX • Addition of a high salt concentration, 2.5%NaCl, to the buffer. • Addition of the blocking serum to the diluted primary antibody.
- 87. Basic Steps in IHC Tissue section Antigen retrieval Blocking of endogenous enzyme Primary antibody Secondary antibody Chromogen substrate Counters tain Mounting Microscopic observation
- 88. Application of Primary Antibody
- 89. Basic Steps in IHC Tissue section Antigen retrieval Blocking of endogenous enzyme Primary antibody Secondary antibody Chromogen substrate Counters tain Mounting Microscopic observation
- 90. CHROMOGEN SUBSTRATE Most often used substrate: • 3,3'-Diaminobenzidine Tetrahydrochloride (DAB) • DAB reacts with HRP to form a brown precipitate at the site of antibody binding. Peroxidase (HRP horse radish peroxidase)
- 91. Other Peroxidase Substrates • 4-chloro-1-naphthol = BLUE • Alpha napthol pyronin = RED • 3-amino-9-ethylcarbazole (AEC) = RED • 3,3,5,5-tetramethyl benzidine = blue • Hanker-Yates reagent - darkblue
- 92. • Alkaline phosphatase Fast blue BB = blue Fast red TR =red New fuchsin = red 5-bromo-4-chloro-3-indolyl phosphate/nitroblue tetrazolium (NBT) = blue • Glucose oxidase Tetrazolium = blue Tetranitroblue tetrazolium = black
- 93. Basic Steps in IHC Tissue section Antigen retrieval Blocking of endogenous enzyme Primary antibody Secondary antibody Chromogen substrate Counters tain Mounting Microscopic observation
- 94. Counterstain • The final step in the process is counterstaining and mounting slides. • Counterstains used are: Haematoxylin Hoechst stain and DAPI (4',6-diamidino-2-phenylindole) Hematoxylin is used as the nuclear counterstain for most routine IHC staining.
- 96. • Hoechst stains are part of a family of blue fluorescent dyes used to stain DNA. There are three related Hoechst stains: Hoechst 33258, Hoechst 33342, and Hoechst 34580. • DAPI (4',6-diamidino-2-phenylindole) is a fluorescent stain. It is used extensively in fluorescence microscopy.
- 97. Basic Steps in IHC Tissue section Antigen retrieval Blocking of endogenous enzyme Primary antibody Secondary antibody Chromogen substrate Counters tain Mounting Microscopic observation
- 98. Mounting and examination • Aqueous and non aqueous (permanent) mounting media are available. • The mounting media depends on the chromogen used during the detection step and its solubility in organic solvents or water • Water insoluble Chromogens should not be used with nonaqueous mounting media and water soluble chromogens should not be used with aqueous media • Nonaqueous mounting media is not compatible with water; therefore, the samples must be first dehydrated with a series of ethanol and xylene washes
- 99. CONTROLS • Controls validate immunohistochemical results. • It is essential that any method using immunohistochemistry principles include controls to test for the specificity of the antibodies involved
- 100. Positive control: • The use of a section with known positivity to the test Ab. • Indicate that proper staining technique was achieved • Use the same control over time • Follow staining consistency over time • Stain first and last longer • Monitor antigenicity over time
- 101. Internal control • Built-in or intrinsic controls • Target antigen within normal tissue elements in addition to tissue elements being evaluated • Can replace external positive control • S-100 protein in both melanoma and normal tissue • Desmin present in blood vessel musculature
- 102. Negative control • Omission of the primary Ab or replacement of the specific primary Ab by an Ig which is directed against an unrelated antigen. • Used to determine antibody specificity • No staining reaction occur
- 103. Absorption control • To demonstrate that an antibody is binding specifically to the antigen of interest, it is first pre-incubated with the immunogen. This should inactivate the antibody and the tissue should show little or no staining. • The staining pattern produced by the primary antibody can be compared to that produced by the pre-absorbed antibody. • Addition of the mixture of antibody plus protein may result in higher non-specific staining.
- 104. BLOCKING CONTROL • The bonding between the primary & secondary antibody is prevented in indirect technique by use of some control. • This type of control is know as blocking control.
- 105. PRACTICAL ASPECTS Dilution of immune serum antibodies • Use primary antibody at the correct dilution. • When applying an untested antibody, a broad dilution series be used to insure false -ve results do not occur • Dilutions that identify normal cells may be too dilute to demonstrate tumor cells that originate from them. • Most primary antibodies and labeling systems are provided with a recommended dilution range.
- 106. Washes • To prevent problems with interpretation and background staining, it is necessary to remove the unbound antibody before incubation in the next layer. • By washing the sections between antibody incubations in TRIS- buffered saline [TBS].
- 107. Troubleshooting • Inadequately fixed or processed tissue • Improper antibody dilution or reagents • Fluctuation in pH of buffer or retrieval soln • Proteolytic enzyme exposure • Temperature • Improper chromogen • Expiry date • Thickness, wrinkles, folds and tears – non specific staining • Improper storage of control tissue – false negative
- 108. False negative staining • Incomplete deparaffinization • Incorrect retrieval solution • Inadequate heat induced epitope retrieval • Temperature • Antibody concentration • Chromogen incompatibility • Positive control selection • Process failure
- 109. False positive staining • Poor quality of fixation • Pretreatment • Overdigestion • High temp oven exposure • Intrinsic tissue biotin • Antibody concentration • Detection system • Species cross reactivity • Tissue drying • Chromogen
- 110. Current applications of immunohistochemistry Tumor Pathology • Diagnosis of Malignancy • Prognostic Markers • Predicting response to treatment • Detection of metastases • Screening of inherited cancer syndromes
- 111. Non-Tumor Pathology • Neurodegenerative diseases • Brain trauma • Muscle diseases • Amyloidosis • Dementias
- 112. IHC Significance • While there are countless medical detection systems (x-ray, CT scan, MRI etc.); There are few identification tools available. • With new carcinogens being discovered and an ever increasing population; the demand for cancer diagnostics is at an all-time high. • Even with the emergence of genetic testing; in many cases, IHC remains the fastest and most cost-effective diagnostic tool from which accurate and often life-saving diagnosis and/or prognosis can be derived.
- 113. Primary Applications • Diagnosis: -- Identification of the cause of an illness or set of symptoms. • Prognosis: -- Predicting the likely outcome of an illness
- 114. Limitations of Immunohistochemistry • Experience : Experience is critical in standardizing the procedure including the selection and proper dilutions of necessary reagents and regular performance of all the appropriate controls. Interpretation too has its foundation in experience. • Availability of antibodies: The paucity of antibody with high degree of specificity for cellular and tissue antigens was serious limitation until recently. This has been remedied in part by using hybridoma technique for monoclonal antibodies. • Antigen loss: The specificity of an antibody for particular antigen and its ability to react with that antigen require the preservation of antigen configuration
- 115. CONCLUSION • Immunohistochemistry is a powerful adjunct to the normal H&E procedures. • Require a lot of skill & experience. • Basic principle and steps are simple and easily understood. • Strict protocol and an eye for details is necessary for consistent and reliable results.
- 116. REFERENCES •Theory and practice of histological techniques – Bancroft 7th edition •Basic and advanced laboratory techniques in histopathology and cytology- P.Dey