IMMUNOHISTOCHEMISTRY IN PATHOLOGY-1-2.pptx

immunohistochemistry in histopathology.

1. IMMUNOHISTOCHEMISTRY IN PATHOLOGY: A COMPENDIUM 1

2. Definition Principles Of Immunohistochemistry Procedures Of Immunohistochemistry Steps To Better Immunohistochemistry Application Of Immunohistochemistry Markers In Immunohistochemistry And Application Conclusion References 2 OUTLINE

3. Immunohistochemistry (IHC) is an important application of monoclonal as well as polyclonal antibodies to determine the tissue distribution of an antigen of interest in health and disease The antibody-antigen interaction is visualized using either chromogenic detection with a colored enzyme substrate such as Horseradish Peroxidase (HRP), Alkaline Phosphatase (AP), or fluorescent detection with a fluorescent dye ( Duraiyan et al., 2012). 3 WHAT IS IMMUNOHISTOCHEMISTRY

4. The principle of IHC has existed since the 1930s, but it was not until 1941 that the first IHC study was reported. Coons and his colleagues used Fluorescein isothiocyanate (FITC)-labeled antibodies with a fluorescent dye to localize pneumococcal antigens in infected tissues. Immunohistochemistry is carried out by exploiting the principle that antibodies bind specifically to an antigen in biological tissues, for example in the jaw, gonads or heart ( Duraiyan et al., 2012). 4 PRINCIPLES OF IMMUNOHISTOCHEMISTRY

5. TISSUE PROCESSING, FIXATION AND SECTIONING Tissue fixation preserves antigens and prevents autolysis and necrosis of harvested tissues. Paraffin embedding is the most common method, but frozen and floating sections are also available. Section tissue at 4-7 μm thickness, and mount on adhesion-treated slides ( Abcam , 2022). 5 PROCEDURES OF IMMUNOHISTOCHEMISTRY

6. 6 Fig 3.0: Diagrammatic representation of the key steps involved in IHC ( Verma , 2022).

7. ANTIGEN RETRIEVAL Antigen retrieval methods include heat induced antigen retrieval HIAR and enzymatic (protease-induced) In a microwave oven, deparaffinized and rehydrated slides are placed in 3% hydrogen peroxide for 5 minutes, then washed with deionized water. The slides are then placed in retrieval buffer and heated in the microwave at 100°C for 5-10 minutes. The slides are then cooled and washed twice ( Magaki et al., 2019) The antigen retrieval reagent requires citrate buffer solution, PBS buffer, EDTA, Tris -EDTA, and Tris-HCl ( Abcam , 2021). 7 PROCEDURES OF IMMUNOHISTOCHEMISTRY

8. BLOCKING PROTEINS Protein blocking step is required to reduce unwanted background staining. An ideal agent for the protein blocking is 5%–10% normal serum from the same species of secondary antibody. Other agents include protein buffers such as 0.1%–0.5% bovine serum albumin, gelatin, or non-fat dry milk (Kim et al., 2016). 8 PROCEDURES OF IMMUNOHISTOCHEMISTRY

9. ANTIBODY SELECTION AND VALIDATION Polyclonal and monoclonal antibodies are used for specific detection, with polyclonal antibodies created by injecting a protein or peptide fragment into animals, isolating antibodies from serum, and recognizing multiple epitopes , respectively, monoclonal antibodies react to a single epitope in an antigen (Kim et al., 2016). 9 PROCEDURES OF IMMUNOHISTOCHEMISTRY

10. TARGET ANTIGEN DETECTION METHODS The direct method is a one-step staining method and involves a labeled antibody reacting directly with the antigen in tissue sections. The indirect method involves an unlabeled primary antibody (first layer) that binds to the target antigen in the tissue and a labeled secondary antibody (second layer) that reacts with the primary antibody (Kim et al., 2016). 10 PROCEDURES OF IMMUNOHISTOCHEMISTRY

11. 11 Fig 1.0: Illustration of polymeric amplification system. DAB, diaminobenzidine ; HRP, horseradish peroxidase (Kim et al., 2016).

12. ANTIBODY APPLICATION Use serial dilutions of a concentrated antibody to determine the optimal concentration of an antibody . Apply 200μl of diluted primary antibody to slides and incubate at room temperature for 80 minutes. Wash in three changes of 0.1% TBS- Tween using different staining dishes for each wash. Apply species-specific secondary antibody and let sit for 15 minutes. Wash with 0.1% TBS- Tween , add orange horseradish peroxidase , and wash in three changes of 0.1% TBS- Tween for each wash ( Magaki et al., 2019). 12 PROCEDURES OF IMMUNOHISTOCHEMISTRY

13. 13 Fig 2.0: Chromogenic immunohistochemistry ( Leica , 2020).

14. COUNTERSTAINING, DEHYDRATION, CLEARING, AND MOUNTING Slides are placed in hematoxylin , rinsed in distilled water, and incubated in Bluing solution. After drying, they are immersed in three washes of Xylene , each lasting 5 minutes. The slides are then mounted in a permount and coverslip (Kim et al., 2016). 14 PROCEDURES OF IMMUNOHISTOCHEMISTRY

15. 15 Fig 4.0:   Immunohistochemical staining for tryptophan hydroxylase 2 ( Verma , 2022).

16. 16 Fig 5.0: Main staining patterns on chromogenic immunohistochemistry (Wikipedia, 2018).

17. U se high-quality sections E nsure proper fixation A void section adhesion issues C hoose appropriate antibodies Optimize retrieval methods C onsider cross-reactivity B lock endogenous peroxidase U se appropriate detection systems C arefully evaluate results ( Leica , 2020) 17 STEPS TO BETTER IMMUNOHISTOCHEMISTRY

18. Prognostic markers in cancers Tumors of uncertain histogenesis Prediction of response to therapy Infections In genetics Neurodegenerative disorders In muscle diseases Research application ( Duraiyan et al., 2012) 18 APPLICATION OF IMMUNOHISTOCHEMISTRY

19. Kiel (ki-67) is a prognostic indicator for tumors Cytokeratin identifies epithelial cells Cluster of differentiation (CD20) detect B-cells, especially in lymphomas Estrogen Receptors evaluate hormonal status in breast cancer ( Umphress , 2023) 19 MARKERS IN IMMUNOHISTOCHEMISTRY AND APPLICATION

20. Human epidermal growth factor receptor 2 (HER2) status for breast and gastric cancers Glial fibrillary acidic protein (GFAP) for astrocytic tumors CD4 for T-helper cells CD8 for cytotoxic T-cells CD15 for granulocytes ( Umphress , 2023). 20 MARKERS IN IMMUNOHISTOCHEMISTRY AND APPLICATION

21. IHC is a crucial tool for pathologists to understand disease pathophysiology and validate biomarker discovery, leading to personalized medicine. Despite recent automation and standardization, optimization and interpretation are essential for newly discovered molecules or antibodies. Validating specificity and sensitivity is crucial, and a full literature review is recommended before starting experiments. Careful planning and stabilizing inter observer consistency are also essential for objectifying IHC results. 21 CONCLUSION

22. Buza , N., & Hui , P. (2017). Immunohistochemistry in Gynecologic Pathology: An Example-Based Practical Update. Arch Pathol Lab Med, 141(8), 1052–1071. Duraiyan , J., Govindarajan , R., Kaliyappan , K., and Palanisamy , M. (2012). Applications of immunohistochemistry . Journal of Pharmacy & Bioallied Sciences, 4( 2), S307–S309. Hawes, D., Shi, S.-R., Dabbs , D. J., Taylor, C. R., & Cote, R. J. (2009). Modern Surgical Pathology (Second Edition), Volume 1, 48–70.   Kim, S.-W., Roh , J., and Park, C.-S. (2016). Immunohistochemistry for Pathologists: Protocols, Pitfalls, and Tips. Journal of Pathology and Translational Medicine, 50(6), 411–418. Magaki , S., Hojat , S. A., Wei, B., So, A., and Yong, W. H. (2019). An Introduction to the Performance of Immunohistochemistry . Methods Mol Biol , 1897, 289–298. Masuda, S., & Nakanishi, Y. (2023). Application of Immunohistochemistry in Clinical Practices as a Standardized Assay for Breast Cancer. Acta Histochem Cytochem , 56(1), 1–8. Sheffield, B. S. (2016). Immunohistochemistry as a Practical Tool in Molecular Pathology. Arch Pathol Lab Med, 140(8), 766–769. 22 Selected References