IHC interpretation- and its quality control .pptx

Immunohistochemistry history, antigen antibody interaction, quality control

1. IMMUNOHISTOCHEMISTRY PRINCIPLES & TECHNIQUES

2. Introduction Immuno + Histo + chemistry : technique for identifying antigens in tissue ( histo ) by means of antigen-antibody interactions (immunochemistry)

3. History Coons, creech , jones in 1942 developed indirect immunofluorescence method to demonstrate pneumococcal antigen in tissue Graham & karnovsky in 1966, developed enzyme tagging method (horseradish peroxidase) Nakane & pierce in 1967, developed enzyme labelled antibody technique Taylor et al. in 1974, introduced IHC in routine formalin-fixed paraffin embedded section (FFPE)

4. PRINCIPLE OF IMMUNOHISTOCHEMISTRY Localization of antigens in tissues sections by the use of labeled antibodies as specific reagents through antigen-antibody interactions that are visualized by a marker (label) such as fluorescent dye, enzyme, radioactive element or colloidal gold. In simple words study of tissue sections (detecting antigens/protein) stained with labelled antibodies.

5. Fundamentals of immunohistochemistry Existence and distribution of an antigen detected micro chemically is made manifest macro chemically Sharp localization of target component in cell and tissue is based on satisfactory signal (given by the labelled secondary antibody) to background staining ratio.

6. Antigen Antigen is any molecule that induces the formation of an antibody Usually bears one or more bonding sites Epitopes/antigenic determinant site – composed of amino acids or monosaccharide units forms a highly specific topographic sites for antibody binding Antigenic determinant sites maybe divided into continuous or discontinuous This issue reflects the variable influence of formalin fixation on “antigenicity” and the variations in antigen retrieval

7. Antibody Antibody molecules are immunoglobulins made of four polypeptides : two identical light chains (kappa/lambda) and two identical heavy chains (gamma, delta, alpha,mu , epsilon) Five major classes : IgG, IgM, IgA, IgE , IgD MC antibody used in IHC is IgG

8. Antigen-Antibody binding Binding Amino acid side chains of Abs forms a cavity complementary to an specific antigen epitope and the bonds that are formed are made of hydrogen bonds, electrostatic forces and van der Waals forces.

9. Factors in an Ag-Ab reaction Affinity : 3D fit of the epitope with the corresponding site of antibody Avidity : multivalency of the antibody along with affinity & structural arrangement Specificity : precise detection of specific epitope of the antigen by antibody Sensitivity : detection of the relative amount of antigen in particular technique

10. Primary antibodies Monoclonal antibodies Hybridoma technique by kohler & Milstein (1975) Recognize only a single type of epitope practical specificity reflected by IHC is excellent with monoclonal antibodies

11. Hybridoma technique This technique is utilized to produce abundant amount of pure homogenous immunoglobulin Antibody producing B lymphocyte is fused with a malignant immortal plasma cell The resultant hybrid cell shows unlimited proliferation and produces specific antibodies (monoclonal antibodies)

12. Primary antibodies Mixture of antibodies to different epitopes Sensitive but less specific – more background staining

13. Labels Enzyme labels – enzyme incubated with chromogen produces a stable coloured reaction end product Most commonly used is Horse Radish Peroxidase with chromogen DAB (diamino_) Fluorescent labels Radiolabels Colloidal metal (gold or gold with silver precipitation) labels

14. Enzyme label – Horse radish peroxidase It is most used because of its smaller size, does not hinder binding of antibody to adjacent sites Endogenous peroxidase activity is present in cells of myeloid lineage, which can be removed by blocking procedures (most popular is hydrogen peroxide-methanol) Most common chromogen used is diaminobenzidine tetrahydrochloride – dark brown reaction product

15. Endogenous peroxidase blocking After primary antibody incubation, incubation in absolute methanol containing 0.5% hydrogen peroxide for 10 mins in room temperature Removes the endogenous peroxidase activity without interfering with immunoreactivity

16. Direct method Primary antibody is directly tagged with an enzyme or fluorescence Advantage : rapid & simple method Disadvantage : Different primary antibody should be labelled differently for the antigen Hardly used except in skin & kidney (direct immunofluorescence)

17. Enhanced polymer one step staining (new direct technique) A large number of primary antibody molecules and peroxidase enzymes are attached to the dextran polymer backbone Signal implication is increased with more sensitivity

18. Indirect method Unconjugated primary antibody is added to the section An enzyme labeled secondary antigen binds to the primary antigen A substrate chromogen solution is added producing a colored end product Advantages : Signal amplification- Secondary antibody binds to Different antigenic sites in primary Antibody 2. Higher working dilutions

19. Polymer chain two step technique Unconjugated primary antibody followed by a secondary antibody conjugated to a labeled polymer dextran chain 70 molecules of the label (HRP enzyme) and 10 molecules of secondary antibody . Polymer chain may contain anti mouse & anti rabbit IgG and therefore can be used for both mouse & rabbit primary antibodies. Biotin free. Quick, reliable & reproducible method.

20. Unlabeled antibody-enzyme complex techniques (PAP & APAAP) PAP (peroxidase antiperoxidase ) Alkaline phosphatase technique (APAAP) Same principle as PAP These techniques have been replaced by ABC &Polymer methods

21. (strep)avidin –biotin technique (ABC) Most widely used along with indirect polymer techniques Three step technique This method relies on the marked affinity of basic glycoprotein avidin (streptavidin produced from streptomyces avidinii ) for the water soluble vitamin biotin

22. (strep)avidin –biotin technique (ABC) Tissues rich in endogenous biotin such as liver & kidney require a avidin/biotin block before primary antibody incubation

23. Avidin vs Streptavidin Avidin High isoelectric ph of 10 , it has a + charge at neutral ph leading to non specific binding with – ve charged structures Being a glycoprotein it reacts with carbohydrate moiety of lectin Streptavidin Shows more affinity for biotin Has no carbohydrate, no non specific reactions Isoelectric point is close to neutrality High stable reagent

24. Steps in IHC Followed by Counterstain with hemotoxylin Mounting

25. Steps in IHC Tissue sections Antigen retrieval Blocking endogenous enzyme Blocking of background staining Primary antibody incubation Secondary antibody incubation Chromogen substrate Counterstain with hematoxylin Mounting

26. Step 1 : Deparaffinization Polylysine (0.1% solution) coated slides are used to ensure adherence This step removes paraffin still on the slide, which would otherwise interfere in immune reactivity

27. Step 2 : Antigen retrieval Fixation modifies the tertiary structure of antigens making them undetected by specific antibodies Cross linking fixative like formalin leads to formation of methylene bridges which cross link proteins and therefore mask antigenic sites Antigen retrieval dissociates irrelevant proteins and restors immunoreactivity by denaturing the secondary and tertiary structures of irrelevant proteins

28. Heat induced epitope retrieval Two factors are important Ph value of AR soluton Most antigens show no significant variation in ph variation, usually ranges from 1-10.0 0.01 M citrate buffer of ph 6.0 is the most frequently used retrieval solution The TRIS EDTA of ph 9.0 and 0.1 M EDTA of ph 8.0 is the second most commonlu used 0.01 M TRIS-HCL ph of 1 or 10 was little superior to the citrate buffer Heating time and heating time : there is an inverse correlation between time & temperature, a low temperature (around 90c) helps in preservation of tissue morphology

29. Blocking – endogenous enzyme & background staining Major causes of background staining are hydrophobic & ionic interactions Hydrophobic bonds are the result of cross linking of amino acids both within and between adjacent protein molecules due to aldehyde fixation Collagen, epithelium and adipocytes give rise to background staining due to hydrophobic interactions Non specific staining is most commonly produced because the primary antibody is attracted non immunologically to charged groups in connective tissue elements

30. Blocking – endogenous enzyme & background staining Blocking of nonspecific sites/ background staining is achieved by serum block Apply 3% bovine serum albumin directly to smear for 30 mins Endogenous enzyme block Peroxidase block Biotin block

31. Washing Almost after every step except serum blocking a wash should be performed This will remove unwanted proteins or chemicals Wash is done in TRIS buffered saline

32. Primary antibody incubation For optimum staining primary antibody is to be used at proper dilution To be incubated in a moist chamber Time for incubation depends on the individual antibody varying from 1 hour to overnight Wash after in TRIS buffered saline

33. Secondary antibody incubation In a standard 3 step ABC protocol Incubate with secondary reagent (biotinylated immunoglobulin) followed by buffer rinse Incubate slides with ABC complex conjugated with HRP followed by buffer rinse In a two step HRP polymer method HRP polymer secondary antibody incubated in one step

34. Detection of immune reaction by chromogen Addition of DAB reagent or other chromogen for 5-15 minutes Stop reaction by bringing section to deionized water

35. Counter staining Counterstaining with Meyers hematoxylin Followed by dehydration & mounting

36. What do we do here Take 3-4 micron sections on poly-L-Lysine coated slides Deparaffinize (incubation 60 to 70 degree Celsius for 20 mins) Xylene wash 1 & 2 – each 10 mins 100% alcohol 1&2, 80%, 70% 50% alcohol each 5 mins During this process to pre heat the ART (TRIS buffer) solution, while maintaining the ph of 7 Wash the slides in water for 2 mins Wash the slides in distilled water for 2 mins & put the slides in a coupling jar & keep in the heated pressure cooker and wait for 2-3 whistles, then cool it Mark the smear in PAP pen Add hydrogen peroxide for blocking – 10 mins

37. What do we do here Wash buffer 1&2 – each 5 mins Primary antibody – 20 mins Wash buffer 1&2 – each 5 mins Secondary antibody (HRP/ANTIBODY with the dextran polymer) 20 mins Wash buffer 1&2 – 5 mins each Add DAB (diaminobenzidine tetrahydrochloride) – 10 mins After 10 mins buffer wash – several times Counterstain with hematoxylin 30 seconds 50%,70%,80%,100%,100% alcohol – each 1 dip Wash buffer preparation 475 ml distilled water + 25 ml wash buffer DAB chromogen preparation 1ml subtraction buffer + 10 microliter DAB

38. Quality control Quality control is an essential part of immunostaining and is divided into three phases Preanalytical : Proper fixation : proper fixative with optimal time duration Slide preparation : drying of slide before staining Insufficient fixation & variable tissue processing are two crucial factors responsible for unreliable & inconsistent IHC results.

39. Quality control Analytical : Primary antibody – proper selection of primary antibody, optimal dilution, volume Antigen retrieval Validation of antibody – primary antibody to be validated with the adequate number of positive (specificity & sensitivity) and negative specificity) controls Internal controls – tissue being tested contains the antigen used under question in adjacent normal tissue. optimum positive internal control indicates appropriate immunoreactivity.

40. Controls Positive & negative tissue control should subjected to the same protocol in order to for accurate control One positive tissue control is indicated for each set of tests To be done on a separate slide or on the same slide Internal positive control like CD31 & CD34 expressed by the endothelial cells If positive tissue control is unsatisfactory – invalid protocol If the positive control block/slide is old & cut previously-stored : stability of antigens are questionable

41. Troubleshooting False positive Poor fixation Variation in ph of retrieval solutions Endogenous enzyme activity due to inadequate blocking Tissue drying Chromogen precipitation & streaking Automation error usually incomplete rinsing False negative Incomplete deparaffinization Incorrect retrieval solution Inadequate heat induced epitope retrieval temperature Wrong antibody concentration Wrong positive control Human error Incorrect chromogen

42. Troubleshooting Problems Inadequate fixation Antibody too diluted Drying out of tissue during processing Chromogen not working Solutions Fixation (at least 2hours up to 48 hours) Increase concentration, lengthen the incubation/heating time Use humidity chamber Add chromogen to labelling solution and monitor for colour change

43. Common applications of IHC Characterization of poorly differentiated malignancy Differentiating in situ lesion from invasive malignancy Metastatic tumor workup Determination of viral etiology Prognostic and therapeutic indication

44. Interpretation of immunohistochemistry Four broad categories : Membranous pattern : characteristic for antigens within the the cell membrane, examples are most of the CD markers & HER-2 Nuclear pattern : characteristic for antigens located within the nuclei or nuclear membrane, examples are transcription factors & ER/PR Cytoplasmic pattern : characteristic for antigens located in the cytoplasm, examples are vimentin, desmin & cytokeratin Extra cellular staining pattern : tissue matrix antigens

45. PRIMARY SCREENING ANTIBODY PANEL

46. Estrogen Receptor & progesterone receptor

47. Estrogen Receptor & progesterone receptor

48. HER-2 neu receptor

49. Tissue Microarray: A rapidly evolving diagnostic and research tool A microarray contains many small representative tissue samples from hundreds of different cases assembled on a single histologic slide, and therefore allows high throughput analysis of multiple specimens at the same time. Tissue microarrays are paraffin blocks produced by extracting cylindrical tissue cores from different paraffin donor blocks and re-embedding these into a single recipient (microarray) block at defined array coordinates.

50. Tissue Microarray: A rapidly evolving diagnostic and research tool . Using this technique, up to 1000 or more tissue samples can be arrayed into a single paraffin block. It can permit simultaneous analysis of molecular targets at the DNA, mRNA, and protein levels under identical, standardized conditions on a single glass slide, and also provide maximal preservation and use of limited and irreplaceable archival tissue samples. This versatile technique, in which data analysis is automated facilitates retrospective and prospective human tissue studies.

51. Advantages and applications of tissue microarrays Amplification of a scarce resource Simultaneous analysis of very large numbers of specimens Experimental uniformity Decreased assay volume, time and cost. Does not destroy original block for diagnosis and thus conserves valuable tissue

52. References Diagnostic immunohistochemistry , theranostic and genomic applications by David.j.Dabbs Basic & advanced laboratory techniques in histopathology and cytology by Pranab dev Immunohistochemistry in tumor diagnostics by muin s.s.tuffaha Quick reference handbook for surgical handbook by natasha rekhtman