Understanding Immunohistochemistry (IHC) and Novocastra Products

2. Introduction to IHC & Novocastra products 95.7936 Rev B

3. IHC • Immunohistochemistry(IHC) is the technique used to visualize, identify/classify tumour or viral cell types in tissue sections in the context of the tissue morphology. • Used in conjunction with routine staining tests (Hematoxylin and Eosin) to confirm or assist in a diagnosis • Used primarily to detect cancer but also used to detect certain infectious diseases

4. IHC • Step 1. Formation of Immune Complexes • Step 2. Visualization of Immune complexes using enzymes

5. Standard terminology • Antigen • A foreign protein present in the body which stimulates the immune system to produce antibodies. • Antigenicity • The ability of an antigen to trigger an immune response • Epitope • Region of an antigen to which the antibody binds

6. Standard terminology • Antibody • Naturally occurring protein in the human body, acts to identify and remove infection and disease • Produced by B cells in response to an immunologic challenge • Antibodies belong to a specific protein group called immunoglobulins (Ig) • Classes of Ig: IgG, IgA, IgM, IgD and IgE (in order of abundance)

7. Standard terminology • Classes of Immunoglobulin (Ig) • IgA – First line of defense against microbes. Found in saliva, respiratory secretions, milk and intestinal secretions. • IgM – Primary response to immune challenge – short lasting • IgD – Coexpressed with IgM as a primary response • IgG – Secondary immune response – long lasting • IgE – Seen in response to allergies and parasites

8. Antibodies Antibody • Antibodies for IHC are manufactured to bind to a specific antigen or epitope • Two types of antibodies are manufactured • Monoclonal and polyclonal

9. Monoclonal Antibody • Produced by clones of plasma cells (commonly mice) • Immunochemically similar • Antibodies from a given clone are identical, hence reacting with the same epitope on the antigen • More specific than polyclonal antibodies but not more sensitive

10. Polyclonal Antibody • Produced by different cells and commonly in rabbit • Immunochemically dissimilar • Antibodies react with different epitopes on the antigen • Not as specific, but more sensitive, than monoclonals due to stronger signal • Cross reactivity with different antigens can be a problem

11. Detection systems Peroxidase Detection Systems: • Use biotinylated secondary antibodies to locate the bound primary antibody, followed by Streptavidin-horseradish peroxidase (HRP) conjugates Polymer Detection Systems: • Secondary antibodies are directly polymerized with HRP into compact polymers bearing a higher ratio of enzymes to antibodies Alkaline phosphatase systems: • The enzyme used in the conjugation is alkaline phosphatase instead of horseradish peroxidase and the chromogen is usually red.

12. Detection Systems Streptavidin-biotin • Primary antibody binds to antigenic site • Secondary antibody (biotinylated) binds to primary antibody • Enzyme substrate labelled streptavidin binds to biotin on secondary antibody • DAB chromogen becomes oxidised and forms a brown coloured precipitate • Other chromogens, such as Fast Red, are able to produce different coloured precipitate (red)

13. Detection Systems

14. Detection Systems Dextran Based Polymer • Primary antibody binds to antigenic site • Numerous peroxidase and secondary antibody molecules are attached to long dextran chain • Long dextran molecule diminishes tissue penetration • Less bonds of antibody/enzyme complexes per antigen • Not as sensitive as Compact Polymer

15. Detection Systems

16. Detection Systems Compact Polymer -Superior staining • Primary antibody binds to antigenic site • Numerous peroxidase molecules compacted together • Smaller molecular structure • Enhanced tissue penetration • More bonds of antibody/enzyme complexes per antigen are formed • Greater sensitivity

17. Compact Polymer Detection Systems

18. Biotin-free Higher ratio of enzymes to antibodies Smaller, more compact than dextran polymers Either mouse or rabbit secondary antibody – 2 systems required Multi-link secondary antibody mouse and rabbit in the one system Compact Polymer Systems

19. IHC protocol • All IHC protocols use similar structure, however they might be different: • Retrieval types and times • Detection systems used • Incubation times • Inclusion or exclusions of certain steps (e.g Blocking, DAB enhancing) • Choice of technique is based on laboratory needs, requirements, costs and funding

20. IHC protocol Sequential steps involved in IHC process • Tissue sections cut • Sections are usually 3-5 microns in thickness for IHC • Slides baked • Slides are placed in the 60°C oven 30mins to adhere tissue to the slides and prevent lifting or baked on board Bond for 20 minutes. • De-waxing of slides and bring to distilled water • Removes wax from tissues to enable reagent penetration and staining

21. IHC protocol • Blocking reagent application • A peroxide block is it blocks the endogenous peroxidase found in many tissues and stops it reacting with DAB later in the IHC process. • A protein block or a block for endogenous alkaline phosphatase may be used if using and alkaline phosphatase kit (red chromogen).

22. IHC protocol • Epitope retrieval • Enzyme Digestion / Proteolytic Digestion - achieved by applying enzyme solution over the tissue and incubating at 37°C (eg protease) or room temperature (eg proteinase K) • Or • HIER (Heat Induced Epitope Retrieval) • Manual process of heat retrieval usually employs the use of a microwave, pressure cooker or water bath as a source of heat energy. Slides are incubated in a buffer for a set period at a specific temperature (100°C).

23. IHC protocol • Application and incubation of primary antibody • Antibody is designed to bind to certain antigens or epitopes that may be present in the tissue. • Incubation with secondary antibody • Steptavidin method • Incubation with antibody raised to the host of the primary antibody, commonly mouse or rabbit. • Polymer method • Incubation with a post primary reagent to increase sensitivity and specificity.

24. IHC protocol • Peroxidase Detection Systems: • Use biotinylated secondary antibodies to locate the bound primary antibody, followed by Streptavidin-horseradish peroxidase (HRP) Conjugates Polymer Detection Systems: • Secondary antibodies are directly polymerized with HRP into compact polymers bearing a higher ratio of enzymes to antibodies

25. IHC protocol • Incubation with chromogen • The antigen:antibody complex is visualised most commonly with Diaminobenzidine (DAB) where the conjugated enzyme is horseradish peroxidase • The antigen:antibody complex is visualised most commonly with AEC or neutral red where the conjugated enzyme is alkaline phosphatase

26. Definitions • Substrate / chromogen • There are many chromogens available on the market but the most popular one is a compound commonly called DAB. Active DAB is a mixture of 3,3-diaminobenzidine and hydrogen peroxide in buffer. DAB can be oxidised by the enzyme Peroxidase, producing a permanent brown precipitate. • Antigen retrieval • A process which partially reverses the cross linking effects of formalin fixation, dramatically improving binding ability and staining results for some antibodies. Usually performed at high temperature in specific buffers.

27. Definitions • Blocking reagents • Reagents designed to stop background staining of non specific elements during the IHC process • Buffers • Buffered saline solution such as PBS (phosphate buffered saline) or TBS (TRIS buffered saline) - used during IHC washing steps • DAB enhancers • Solutions of heavy metals such as nickel or copper used to enhance DAB staining. They create the illusion of stronger staining by changing the colour of the DAB precipitate (to darker brown or black) to improve contrast.

28. Definitions • Counterstain • Stain applied after completion of IHC protocol to visualize structure of the tissue and provide contrast to the DAB staining. Usually haematoxylin. • Enzyme labelled antibodies • Antibodies which have peroxidase molecules attached to them in order to facilitate reaction with DAB. Used in direct detection methods.

29. How is Cancer Diagnosed Using IHC? • Nearly every tumour reacts with one or several antibodies available that will give away its identity • Panels of antibodies are often used to identify tumours Example of antibody panel: • The negative results are as important as the positives • Architecture of the tissue is also important in diagnosis

30. Staining Patterns Membrane

31. Staining Patterns Nuclear

32. Staining Patterns Cytoplasmic

33. Living up to Life Novocastra ProductsNovolink™ Detection SystemReference Range AntibodiesAncillary Reagents

34. NovolinkTM Detection System Volumes Novolink™ Polymer Detection Systems • RE7290-K, 50 tests • RE7140-K, 250 tests • RE7150-K, 500 tests • RE7280-K, 1250 tests

35. Reagents in the Novolink™ Detection SystemPeroxidase BlockProtein BlockPost Primary BlockNovolink™ PolymerDab ChromogenDab Substrate BufferHematoxylin

36. Benefits of NovolinkTM High Sensitivity leads to improved staining • Higher Sensitivity can be exploited to reduce Primary Antibody Incubation periods • eg 60min to 30min as per new protocol or make results more reproducible • Higher sensitivity can lead to lower costs per test

37. Nine Different Areas of Pathologies • Breast IHC • Melanoma IHC • Leukaemia IHC • Lymphoma IHC • Prostate IHC • Gastrointestinal IHC • Renal IHC • Lung IHC • Special IHC

38. Novocastra™ Reference Range http://www.leica-microsystems.com/products/total-histology/novocastra-reagents/reference-range/

39. www.leica-microsystems.com Reference Range- Breast Panel Estrogen Receptor- (ER 6F11) Progesterone Receptor- (PGR 16) Her2- C-erbB-2 (CB11) Ki67- (MM1) Akt - (Protein Kinase B) Cytokeratin 5 Cytokeratin 7 Cytokeratin 20 Epidermal Growth Factor- EGFR p53 p63

40. www.leica-microsystems.com Breast Markers • Estrogen /Progesterone receptors • Cerb B2 / Her2 • Determining the hormone receptor status in breast cancer for a prediction of response to hormone therapy.

41. www.leica-microsystems.com Estrogen Receptor • ER content of breast cancer tissue is an important parameter in the prediction of prognosis and response to endocrine therapy. • ER-6F11 is raised to N-terminal of ER protein and does not bind to C-terminal region. • Study found 6F11 5% more sensitive than 1D5. • SP1 binds towards C-terminal end of ER protein and may detect non-functional splice variants of the protein. SP1 may overestimate ER levels. • External evaluation demonstrated ER-6F11 outperforms SP1 when used optimally .

42. www.leica-microsystems.com Estrogen Receptor SP1 6F11 Functional N ERα 66kDa C SP1 Non-Functional N C ER 46 kDa isoform Page 42

43. www.leica-microsystems.com ER-6F11 Nuclear staining of breast carcinoma cells in breast glands. Weak to no cytoplasmic staining

44. www.leica-microsystems.com ER-6F11 Nuclear staining of normal breast ducts. Weak to no cytoplasmic staining.

45. www.leica-microsystems.com References • 1. Kaufmann O, Kother S, Dietel M. (1998) Use of antibodies against estrogen • and progesterone receptors to identify metastatic breast and ovarian • carcinomas by conventional immunohistochemical and tyramide signal • amplification methods.Mod Pathol. Apr11(4):357-63. • 2. Leake R, Barnes D, Pinder S, Ellis I, Anderson L, Anderson T, Adamson R, • Rhodes T, Miller K, Walker R. J. (2000). Immunohistochemical detection of • steroid receptors in breast cancer: a working protocol. Clin Pathol 53:634– • 635. • 3. Lei L., Haynes MP et al. (2003). Plasma membrane localisation and • function of estrogen receptor alpha variant ER a46. PNAS 100 4807-4812.

46. www.leica-microsystems.com Progesterone Receptor PR is expressed as two isoforms, PRA and PRB. PRA form is a truncated version of the PRB form. Clone 16, is specific for the A form of PR. PR (16) was compared to 636 and found to be superior

47. www.leica-microsystems.com Progesterone Receptor (16) Nuclear staining of breast carcinoma cells in breast glands Nuclear staining of normal breast ducts

48. www.leica-microsystems.com References • 1. Kaufmann O, Kother S, Dietel M. (1998) Use of antibodies against estrogen and progesterone receptors to identify metastatic breast and ovarian carcinomas by conventional immunohistochemical and tyramide signal amplification methods.Mod Pathol. Apr11(4):357-63.

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