1. 1. 1 Presented by Dr. Shrikant Sonune Guided by Dr Ashok Patil, Dr Shilpa Kandalgaonkar, Dr Mayur Chaudhary, Dr Suyog Tupsakhare, Dr Mahesh Gabhane. Immunohistochemical technique (part II)
2. 2. Historical aspect Fixation for IHC Antigen retrieval Labels Control
3. 3. 1941: Coons et all. have demonsrated antigens on tissue sections by using an antibody which was linked to an florescent label. 1966: Nakane and Pierce as well as Avrameas and Uriel reported the use of secondary antibody which was conjugated with peroxidase enzyme. 1970 : Sternberger et al described the peroxidase- antiperoxidase technique. 1971: Envall & Perlman reported the use of alkaline phosphatase labeling
4. 4. 1975: Kohler & Milstein described a revolutionary method for production of monoclonal antibodies 1976 : Huang et al described the use of trypsin digestion on paraffin sections as a means of reveling some antigens otherwise masked by formalin fixation & paraffin processing. 1977:Heggness & Ash proposed the use of avidin biotin for immunofluorescence.
5. 5. 1979: Sternberger described the peroxidase-antiperoxidase method. 1981: Hsu et all. have defined the method of avidin-biotin- peroxidase complex which is abbreviated as ABC method. 1984 : Alkaline phosphatase anti alkaline phosphatase technique using a red reaction product. 1993: Advent of one step system, based on a two layer dextran polymer. Reported by Pluzek et al at UK pathological society meeting.
6. 6. 1994 : Norton reported that the stainless steel pressure cooker was more efficient than the microwave oven for retrieving some antigens & this was conformed by Miller et al. 1998: Dako launched a new labeling system based on the dextran polymer technology.
7. 7. Intercellular antigens, Cell surface antigens, Tissue antigen for diagnosing autoimmune diseases Protein hormones in histopathological diagnosis Soluble antigens of the cell Diagnosis of the endocrine tumors Small amounts of peptides in endocrine or neuroendocrine cells Immunodeposits Tumoral markers Tumor typing
8. 8. 1. Fixation Fresh unfixed, fixed, or formalin fixation and paraffin embedding 2. Sectioning Tissue preparation
9. 9. 1. Antigen retrieval Proteolytic enzyme method and Heat-induced method 2. Inhibition of endogenous tissue components 3% H2O2 Pretreatment
10. 10. Make a selection based on the type of specimen, the primary antibody, the degree of sensitivity and the processing time required. Staining
11. 11. The requirements of a fixative vary according to the different techniques employed
12. 12. Blood smears are preserved by air-drying. The subsequent fixation method used depends upon the staining technique. Routine air-drying for 12 hours does not have a deleterious effect on most antigens studied immunocytochemically. Routine air-drying for 12 hours does not have a deleterious effect on most antigens studied immunocytochemically.
13. 13. Most cytology smears are immediately fixed in 95% ethanol or are spray-fixed with a carbowax containing alcoholic fluid. Ethanol fixation prevents staining for most leukocyte markers, such as T and B cell antigens.
14. 14. Two preparations can be made, one wet-fixed one air-dried. With wet-fixed smears, one of the main problems is the loss of cells, particularly clumps, during the Immunostaning incubations.
15. 15. Cryostat sections give much better antigen preservation than paraffin sections. Additionally, fixative can be used with cryostat sections, Different and optimal fixative for each antigen,
16. 16. The most popular fixatives contain Formalin (40% w/v formaldehyde in water), A neutral salt to maintain tonicity A buffering system to maintain pH. Well tolerated by tissues Have good penetration. Generally formalin-based fixatives are excellent for most Immunostaning procedure.
17. 17. Formaldehyde fixes tissue by reacting primarily with basic amino acids to form cross-linking methylene bridges. Relatively low permeability to macromolecules and that the structures of intracytoplasmic proteins are not significantly altered. The great variation in time and conditions for fixation that cause majority of problems in immunochemistry.
18. 18. Many antigens can be demonstrated after the use of appropriate pretreatment methods, Such as proteolytic enzyme digestion and/or antigen retrieval, particularly if polyclonal antisera are used.
19. 19. If monoclonal antibodies are to be utilized on formalin-fixed, paraffin-embedded tissue sections, then Does formaldehyde react with the epitope under investigation? Does it react with adjacent amino acids causing conformational changes? Does paraffin processing destroy the epitope under investigation?
20. 20. If there are conformational changes resulting from the reaction of formaldehyde with amino acids adjacent to the epitope, These can often be reversed using proteolytic enzyme digestion or antigen retrieval.
21. 21. If there are conformational changes in the epitope due to tissue processing, these are irreversible. Many epitopes are sensitive to heat, and during the paraffin-embedding step, tissues are heated to the melting point of wax, usually between 5060C.
22. 22. Saturated (1.2% w/v) aqueous picric acid 75 mL Formalin (40% w/v formaldehyde) 25 mL Glacial acetic acid 5 mL A saturated solution (w/v) contains 1.17 g/100 mL distilled water.
23. 23. This fixative penetrates rapidly and fixes all tissues very well, Fixation time is 112 hours Lipid-containing antigens may be affected. Tissues fixed in Bouins solution must be washed in 70% ethanol to precipitate soluble picrates prior to aqueous washes. After cutting sections, the yellow color in the tissue can be removed by treatment with 5% (w/v) sodium thiosulfate, followed by a water wash.
24. 24. To improve cytological preservation and minimize distortion associated with formaldehyde-based fixatives, mercuric chloride-based fixatives are used. These fluids are generally poor penetrators and are not well tolerated by the tissues.
25. 25. Frequently, mercuric chloride-based fixatives are used secondarily to formalin. Tissues are initially fixed in formal saline or neutral buffered formalin. Tissues are then immersed in the mercuric chloride- containing fluid for a further period of fixation.
26. 26. Mercuric chloride-containing fixatives are additive and coagulative. Suitable for the demonstration of intracytoplasmic antigens.
27. 27. The permeability of the tissue is greater Coagulant fixatives, Antibody penetration is better, Resulting in a more intense Immunostaning. These fixatives are also having additive effect.
28. 28. B5 is widely advocated for the fixation of lymph node Biopsies B5 STOCK SOLUTION Mercuric chloride 2 g Sodium acetate 2.5 g Distilled water 200 mL B5 WORKING SOLUTION B5 Stock solution 20 mL Formalin (40% w/v formaldehyde) 2 mL
29. 29. ZENKERS STOCK SOLUTION Mercuric chloride 50 g Potassium dichromate 25 g Sodium sulfate 10 g Distilled water to 1 liter ZENKERS WORKING SOLUTION Zenkers stock solution 100 mL Glacial acetic acid 5 mL CLEARING SOLUTION A Iodine 0.5 g 70% ethanol 100 mL CLEARING SOLUTION B Sodium thiosulfate 5% (w/v) in distilled water
30. 30. Morphological detail is generally well preserved, Penetration is poor Fixation time is 215 hours at room temperature, Sections are washed in running water for at least 1 hour to remove potassium dichromate deposits.
31. 31. The tissue is then dehydrated using clearing agent A, helps to remove mercuric deposits prior to sectioning. After cutting sections and collecting on clean glass slides, The tissue again incubated in clearing solution A for 12 minutes. Sections are rinsed in water, and placed in clearing solution for 12 minutes.
32. 32. This fixative is becoming increasingly popular, partly because it preserves membrane proteins. Zinc chloride 500 g Formalin (40% w/v formaldehyde) 3 L Glacial acetic acid 19 mL Distilled water 20 L
33. 33. WORKING SOLUTION 3% (w/v) Paraformaldehyde 50 mL M Disodium hydrogen orthophosphate 100 mL Lysine 0.9 g Sodium periodate 0.15 g Adjust to pH 7.4.
34. 34. First described by McLean and Nakane Preserve the micro anatomical relationship of cells and cytological details. Since the periodate oxidizes sugars to produce aldehydes which are cross linked with lysine. The paraformaldehyde stabilizes proteins.
35. 35. A recent modification to this solution involves the addition of potassium dichromate (PLDP). This chemical is added to preserve lipids and the fixative should therefore preserve all protein, carbohydrate and lipid antigenic determinants.
36. 36. It must be noted, however, that since additive compounds are formed, immunoreactivity may be blocked. Furthermore, cytological detail is not as good as with other fluids.
37. 37. Sainte-Marie first described the use of alcohol fixation and paraffin processing for immunocytochemistry. Small pieces of tissue are fixed rapidly and show good cytological preservation.
38. 38. They are in some ways ideal fixatives for immunocytochemistry. Since alcohols are coagulant fluids and do not form additive compounds, They permit good antibody penetration and do not block immunoreactive determinants.
39. 39. Conformational changes can occur. Alcohols precipitate carbohydrates Hence, useful for surface membrane antigens, which often display carbohydrate-containing epitopes.
40. 40. Applied to frozen sections or smears. Proteolytic digestion or antigen retrieval is of no use following alcohol fixation Ethanol fixation results in destruction of the tissue section or smear.
41. 41. Acetone is an excellent preservative of immunoreactive sites, leaving most sites intact. But it is a very poor penetrator. It is used only for smears and cryostat sections. Fixation is not complete, and extended incubation in buffers may result in chromatolysis and loss of membranes.
42. 42. The dilemma of trying to obtain good morphological preservation while maintaining immunoreactivity is even more apparent in ultra structural immunochemical studies. For routine electron microscopy, it is usual to employ glutaraldehyde primary fixation followed by post fixation in osmium tetra oxide.
43. 43. This combination produces excellent ultra structural detail with good preservation of membranes. With immunochemistry, however, the combination of glutaraldehyde and osmium tetra oxide is not generally useful.
44. 44. Pre treatment of ultrathin sections with hydrogen peroxide or sodium metaperiodate to counteract the deleterious effects of osmium. The glutaraldehyde primary fixation is not suitable for many antigens, at least when employing the concentrations of glutaraldehyde used for routine electron microscopy.
45. 45. WORKING SOLUTION 0.2 M Phosphate buffer, pH 7.4 50 mL 8% (w/v) Paraformaldehyde 25 mL 25% (w/v) Glutaraldehyde 0.8 mL Distilled water 24.2 mL
46. 46. If the tissue is frozen The sections may need to be used in immunohistochemistry Acetone fixed: - precipitates proteins onto cell surface---may extract lipids - is needed for many of the CD antibodies Unfixed: Advantage: antigens are unaltered Disadvantage: sections may fall off slide during staining Paraformaldehyde fixed: - needs to be freshly made, or frozen soon after
47. 47. - Deparaffinize ( remove the infiltrated paraffin wax, by using organic solvents) - The section then needs to be rehydrated, by sequential immersion in graded alcohols (100%, 70% , 50% and then PBS) - The deparaffinized section may need to be treated to expose buried antigenic epitopes with either proteases or by heating in low pH citrate buffer , or high pH EDTA buffer (Antigen Retrieval) If the tissue is paraffin embedded
48. 48. The loss of immunoreactivity by many antigens caused as a result of fixation in formalin. Hence many challenges for doing IHC in such cases
49. 49. Epitopes 2 types Formalin resistant- even after routine formalin fixation this type of epitopes remains unchanged Formalin sensitive- after routine formalin fixation this type of epitopes shows substantial changes. It can be divided into Irreversible Reversible or Masked Rescued by antigen retrieval
50. 50. The first attempt to improve the immunoreactivity of formalin-fixed tissue antigens was by use of tryptic digestion prior to immunofluorescent staining. Proteolytic digestion compensates for the impermeable nature of cross linked fixatives and allowing hidden determinants to be exposed.
51. 51. The concept of recovering lost immunoreactivity Through exposure to heat near the boiling point of water But ,went against the tenet of protecting proteins from the denaturing effect of heat.
52. 52. The principle of antigen retrieval relies on the application of heat for varying lengths of time to formalin fixed paraffin embedded (FFPE) tissue sections in an aqueous medium. After deparaffinizing and rehydrating the tissue sections, the slides are immersed in an aqueous solution commonly referred to as a retrieval solution.
53. 53. The following techniques can retrieve many masked antigens in routinely processed tissue: Proteolytic enzyme digestion Microwave antigen retrieval Microwave and trypsin antigen retrieval Pressure cooker antigen retrieval.
54. 54. Pre- treating formalin-fixed routinely processed paraffin sections with proteolytic enzymes to unmask certain antigenic determinants was described by Huange et al. (1976), Curran & Gregory (1977). and Mepham et al. (1979). The most popular enzymes-Trypsin -Protease
55. 55. It is generally accepted that the digestion somehow breaks down formalin cross linking. Hence the antigenic sites for number of antibodies are uncovered. Pure trypsin has a very limited digestion effect on formalin fixed paraffin section Essential for the demonstration of Cytokeratin & CD3.
56. 56. Pitfalls of proteolytic digestion . Under digestion results in very little staining. Over digestion can result in very sever tissue damage. Batches of trypsin varies even from same source.
57. 57. Pitfalls of proteolytic digestion . Many antigens do not retrieved . Some even destroyed by proteolytic enzyme. The specificity of some primary antigens altered. Allergy to trypsin
58. 58. Remains largely unknown Heat is obviously of great importance in reversing the damages caused by the fixation with formalin and embedding in paraffin. Some cross-links are reversible (Schiff bases), thus restoring the immunochemical integrity of the protein, while others are not (methylene bridges). Heat-induced AR has been more successful than the use of proteolytic enzymes
59. 59. Microwave oven heating retrieves many antigens though previously lost or destroyed by routine histopathological method. Dewaxed section of tissue is boiled with the various solutions like m-citrate buffer pH 6. The prerequisite for this technique is use of strong adhesive such as Vectabond or amino propyltriethoxysilane(APES).
60. 60. Replacing microwave oven with pressure cooker. Optimized microwave oven heating not produce consistent result. Comparatively this method is consistent & less time consuming. Also require the strong adhesive.
61. 61. Reported by Sandison et al in 1994 Heat pretreatment increases the sensitivity of sections to the subsequent proteolytic digestion. Drastically reduces the trypsin time
62. 62. Advantages Dilution factor of primary antibodies are greatly increase. Labeling of other antigens Disadvantages Proteolytic enzymes can destroy the sections. Staining distribution can be altered.
63. 63. Enzyme labels Colloidal metal labels Fluorescent labels Radiolabels
64. 64. Enzymes are most widely used labels. Chromogen using standard histochemical method produce a stable colored reaction end product suitable for light microscopy. Horseradish peroxidase is most widely used enzyme.
65. 65. There are also several chromogens for demonstration of peroxidase. Diaminobenzedine(DAB)tertrahydrochloride- brown 3-amino-9-ethylcarbazole- red 4chloro-l-naphthol-blue Hanker-Yates reagent-dark blue Alpha-naphthol pyronin redish purple.
66. 66. Colloidal gold is most common metal tracer in use. Pink in color when observed in transmitted light. Silver participation reaction can be used to amplify the visibility of gold conjugate thereby allowing the use of smaller, more stable gold conjugate. Colloidal gold has much wider usage with the electron microscope.
67. 67. Tissue/cells Need a good positive and negative Reagent When trying new technique
68. 68. Indicate that proper staining technique was achieved Use the same control over time Follow staining consistency over time Familiarity with pattern
69. 69. Maintenances Stain first and last longer Monitor antigenicity over time
70. 70. 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
71. 71. Internal control examples: S-100 protein in both melanoma and normal tissue Desmin present in blood vessel musculature
72. 72. Used to determine antibody specificity Use tissue without antigen expression May need to identify negative cells within tissue
73. 73. The ideal negative control is omission of primary antibody. In absorption control the primary antibodies are used but not in pure form. Pre-absorption of primary antibody with purified antigen. This type of control is used in characterization & evaluation of new antibodies.
74. 74. 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.
75. 75. Textbook of microbiology Anathnarayan.6th edition Theory & practices of histologycal thecnique bancroff 5th edition Immunohistochemical Staining Methods Fourth Edition Dako. Science of lab diagnosis by john croker . 2nd edition.