IMMUNOHISTOCHEMISTRY AND IMMUNOPATHOLOGY PRACTICE IN PULMONARY PATHOLOGY Dr. Kemal BAKIR Gaziantep University, Medical F

1. IMMUNOHISTOCHEMISTRY AND IMMUNOPATHOLOGY PRACTICE IN PULMONARY PATHOLOGY Dr. Kemal BAKIR Gaziantep University, Medical Faculty, Department of Pathology TTS 10th Annual Congress April 25th-29th 2007 - ANTALYA

2. PRESENTATION • History • Aim • Methods • IHC in Pulmonary Pathology • Pitfalls in IHC • Conclusion

3. HISTORY: Diagnostic immunochemistry was first introduced in 1940s by Coons et al who defined bacterial antigens and fluorescein-binded antibodies in infected tissues. Being used for the last 30 years, improvement of immunohistochemistry (IHC) occured at the end of 1980s and the beginning of 1990s. Although a few antibodies were included in the panel at the beginning, thousands of antibodies are now used in routine practice.

4. Immunopathology: • immunohistochemistry, • immunofluorescein, • in situ hybridisation.

5. Immunopathology can be applied to • Tissue sections, • Aspiration materials, • Fluids such as bronchoalveolar lavage

8. Direct method

9. Two-step indirect method

10. Three-step indirect method

11. Soluble Enzyme Immune Complex Technıques

16. In situ hybridisation (ISH) is a system depending on a labelled riboprobe, that is complementary to a unique nucleic acid sequence (which is tried to be evaluated or to be shown) in target cell or organisms Probes which are used in (ISH) may also be labelled with digoxigenin. The probe can be detected using enzyme-labeled anti-digoxigenin antibody. This technique also allows precise localization and can be quantitatively assessed.

17. History • Aim • Methods • IHC in Pulmonary Pathology • Pitfalls in IHC • Conclusion

18. When IHC used? • confirm the diagnosis with HE • contribute to etiopathogenesis • establish various cell types and areas of accumulations in inflammatory processes • manage treatments and to investigate new treatment modalities • evaluate prognostic parameters • get information about mechanisms of rejection and damage in transplantations

19. History • Aim • Methods • IHC in Pulmonary Pathology • Pitfalls in IHC • Conclusion

20. There are many steps in IHC : • Routin Tissue Processing • Practicing of IHC • Interpretation of IHC

21. Routin Tissue Processing • Tissue fixation ( Type and duration of fixation) • Tissue processing • Deparaffinisation

22. Practicing of IHC • Antigen retrieval • Staining method and protocol • Validity of solutions used in the staining procedure • Controls

23. Interpretation of IHC • Working of the technician and pathologist in harmony • To choose the antibody panel • Sensitivity of the antibody panel • Clone of the antibody • Right interpretation, scoring and evaluation of the positivities.

27. Detection Systems: PAP APAAP (Strept)avıdın-bıotın Polymer-based Technology Staınıng Of Multıple Tıssue Markers At The Same Tıme

28. Although IHC is generally used with avidin-biotin based detection systems, polymer-based detection systems are now being used because of false results due to binding of biotin with cells especially in kidneys and liver.

29. It’s supposed that tyramin-based amplification systems will be used more efficiently in the future

30. Immunohistochemical reactivity is observed in cells and in stroma. The reactivity in cells may be membranous, cytoplasmic and/or nuclear . The reactivity is assesed considering either the staining extent or intensity (or a combination of both), and the results are evaluatedin a wide spectrum, with ‘positive’ at one end, and ‘negative’ at the other (e.g. Weak, intermediate, strong reactivity; or +,++,+++…)

33. History • Aim • Methods • IHC in Pulmonary Pathology • Pitfalls in IHC • Conclusion

34. When pulmonary pathology is considered; IHC may be helpful in: • Diffuse parenchymal pulmonary disorders, • Diseases such as COPD and asthma, • To assess the number and types of inflammatory cells • Infective diseases • Benign and malignant pulmonary neoplasia • Mesothelial lesions • Experimental studies cocerning pathogenesis and treatment modalities.

35. In Asthmatic Patients; Chronic T-cell mediated immunologic reaction on the bronchial wall of asthmatic patients was investigated immunohistochemically. T-cells (CD2, CD5, CD7 and CD8 monoclonal antibodies), B cells ( CD19, CD20 monoclonal antibodies), Monocytes and macrophages (CD 68), Eosinophils (EG1) and Activated eosinophils(EG2) were tried to be shown and assessed quantitatively.

36. In addition, CD4:CD8 ratio was determined by a double staining immunopathology procedure to assess T-cell subtypes.

37. The number and ratio of T-cells, eosinophils and macrophages can be established in biopsies from proximal airways and bronchioloalveolar lavage (BAL) from distal airways. In sudden deaths due to asthma, T-cell lymphocytosis is increased in BAL. This increase was not observed in asthmatic patients and it was relatively preserved as normal. CD4:CD8 ratio is reversed with an increase in CD8+ T-cells.

38. Eosinophilia and immunohistochemical nitric oxide positivity in sputum, lavage and BALspecimens are helpful in differential diagnosis of eosinophilic bronchitis and asthma which have similar clinical presentations.

39. CD8+ T-cells were investigated in patients with chronic obstructive pulmonary disease (COPD). When compared with non-smokers, an increase in CD8 was observed in smokers. The role of smoking in pathogenesis of COPD was demonstrated by this way.

40. Immunopathology, especially IHC also helps in the diagnosis and follow-up of diffuse parenchymal pulmonary diseases

41. IHC is also used in experimental studies which investigate pulmonary pathologies in humans and try to developnew therapeutic approaches. • One of them is to determine the accumulating cell type and localisation in pulmonary hypersensitivity. • Dendritic cells tend to accumulate arround bronchioles • Macrophages preferarteries • CD4+ T cells tend to accumulate at the same locations withdendritic cells.

42. Lung cancer is the first leading cause of death due to cancer worldwide. Non-small cell lung carcinoma (NSCLC) accounts for more than 80% of all lung carcinomas. For this reason, it’s essential to make the differential diagnosis of these tumors and predicting the prognosis.

43. β-catenin is used in differential diagnosis of adenocarcinomas. Positivity of c-kit is helpful in the differential diagnosis of adenocarcinomas, on the other hand it’s also related with prognosis.

44. Β-katenin

45. IHC is also used to determine prognostic factors in NSCLC. • These molecular prognostic factors are classified as: • Growth factors and receptors, oncogenes • Programmed cell death • Regulation of cell cyclus • Angiogenesis and tumor progression

48. RNP

49. Another field of IHC is the differential diagnosis of mesothelioma from adenocarcinoma. For this purpose; calretinin, cytokeratin5/6, podoplanin, WT1 and also MOC-31, Ber-EP4, B72.3, CEA, BG-8 and TTF-1 may be used.

50. . MOC-31, Ber-EP4, estrogen receptors and calretinin are known to be useful markersin differantial diagnosis of peritoneal mesothelioma and serous carcinoma.