Er Pr

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Er Pr

  1. 1. <ul><li>Evaluation </li></ul><ul><li>of ER/PR </li></ul><ul><li>Through </li></ul><ul><li>Immunohistochemistry </li></ul>NUDRAT JAWED BSMT 3 RD YEAR, 5 TH SEMESTER
  2. 2. Contents <ul><li>Hormone Receptors </li></ul><ul><li>Why ER/PR Testing? </li></ul><ul><li>Estrogen Receptors </li></ul><ul><li>Progesterone Receptors </li></ul><ul><li>Immunohistochemistry </li></ul><ul><li>Procedure Of IHC </li></ul><ul><li>Factors Affecting The Technique </li></ul><ul><li>Conclusion </li></ul>
  3. 3. HORMONE RECEPTORS <ul><li>It is a membrane receptor protein or in its interior that binds to a specific hormone. </li></ul><ul><li>The hormone causes many changes takes place in the cell. </li></ul><ul><li>Binding of hormone to its receptor often triggers the start of a biophysical signal that can lead to a further signal transduction pathways or triggers the activation or inhibition of genes. </li></ul>
  4. 4. Why ER/PR testing ? <ul><li>Prognostic Factor </li></ul><ul><li>Predictive Factor </li></ul><ul><li>ER protein expressed in 70-80% invasive breast carcinomas. </li></ul><ul><li>Rate of expression increase with patient age. </li></ul><ul><li>Less tumor tissue is required for ER/PR evaluation. </li></ul><ul><li>Assays can be performed on archival materials. </li></ul><ul><li>Exact location of ER/PR positive cells can be determined (i.e. tumor Vs normal breast tissue). </li></ul><ul><li>ER/PR positivity is associated with increased response to antiestrogen therapies. </li></ul>
  5. 5. Estrogen Receptors <ul><li>Estrogen regulates growth kinetics of epithelial linings (Breast and Endometrium). </li></ul><ul><li>It binds to ER (Cellular Proteins) directly stimulating proliferation and differentiation. </li></ul><ul><li>ER translocates the nucleus, where it can bind to promoter sites and thus regulates the expression of many other genes . </li></ul><ul><li>ER may be the best example of a tumor bio-marker. </li></ul>
  6. 6. Types <ul><ul><li>ER α </li></ul></ul><ul><ul><li>ER β </li></ul></ul><ul><li>Both ER α & ER β have a highly homologous structure. </li></ul><ul><li>The Co-expression ER α , ER β and PR as well as its association with indicators of low biological aggressiveness suggest that ER β positive tumors are likely to respond to hormonal therapy. </li></ul>
  7. 7. Progesterone Receptor <ul><li>Estrogen mediates part of its proliferative action of normal breast through transactivation of PR. </li></ul><ul><li>Progesterone is also a mitogenic stimulus for mammary epithelium. </li></ul><ul><li>PRs are ligand activated transcription factor members of the steroid hormone family of nuclear receptors. </li></ul><ul><li>In breast cancer, total PR levels are routinely measured as a guide to hormone therapy and as marker of disease prognosis together with ER. </li></ul><ul><li>It has two isoforms </li></ul><ul><li>PR-A PR-B </li></ul>
  8. 8. <ul><li>IHC is a technique, used to identify cellular or tissue Ag with the help of Ab. </li></ul><ul><li>This technique is based on immuno-reactivity of Ab and chemical properties and enzyme complexes which reacts with colorless substrate (Chromogen) to produce colored end product. </li></ul><ul><li>Site of Ag-Ab complex is identified by direct labeling or secondary labeling method. </li></ul><ul><li>Now this technique is used for a wide variety of Ag. </li></ul><ul><li>Staining not only makes sophisticated the diagnosis but also provide valuable information about pathogenesis of particular disease. </li></ul><ul><li>IHC made diagnosis easier and less expensive as compare to other technique. It has become an essential part of surgical pathology. </li></ul>
  9. 9. Envision System <ul><li>It is two step IHC technique. </li></ul><ul><li>Based on advance Labeled Streptavidin-biotin (LSAB) method. </li></ul><ul><li>HRP-labeled polymer conjugate with secondary Ab. </li></ul><ul><li>Extremely sensitive than other IHC techniques. </li></ul><ul><li>It can easily detect Ag present in low concentration. </li></ul>
  10. 10. Introduction <ul><li>This section shows the step by step Envision (DAKO) staining procedure using Estrogen Receptor (ER) antibody </li></ul><ul><li>on Breast carcinoma . </li></ul>
  11. 11. Step by Step Envision Staining Method <ul><li>Remove paraffin wax and hydrate tissue section. </li></ul>paraffin wax coated slide Deparaffinization and Rehydration SPECIMEN LNH 2009-013 ER/PR
  12. 12. Step by Step Envision Staining Method <ul><li>Remove paraffin wax and hydrate tissue section. </li></ul>tissue section Deparaffinization and Rehydration SPECIMEN LNH 2009-013 ER/PR
  13. 13. Step by Step Envision Staining Method <ul><li>Boiling for 50 minutes using citrate buffer solution pH 6.0. </li></ul>Rinse Deparaffinization and Rehydration Antigen Retrieval SPECIMEN LNH 2009-013 ER/PR SPECIMEN LNH 2009-013 ER/PR
  14. 14. Step by Step Envision Staining Method <ul><li>Rinse in distilled water and wash 2 times in PBS buffer. </li></ul>PBS Buffer Deparaffinization and Rehydration Antigen Retrieval Rinse SPECIMEN LNH 2009-013 ER/PR
  15. 15. Step by Step Envision Staining Method <ul><li>3% hydrogen peroxide solution for 10 minutes to inactivate endogenous peroxidase activity. </li></ul>3% Hydrogen Peroxide SPECIMEN LNH 2009-013 ER/PR Rinse Deparaffinization and Rehydration Antigen Retrieval Block Endogenous Peroxidase
  16. 16. Step by Step Envision Staining Method <ul><li>Thorough rinse in distilled water and wash 2 times in PBS buffer. </li></ul>PBS Buffer Rinse Deparaffinization and Rehydration Antigen Retrieval Block Endogenous Peroxidase Rinse SPECIMEN LNH 2009-013 ER/PR
  17. 17. Step by Step Envision Staining Method <ul><li>Optimally diluted ER antibody for 50 minutes. </li></ul>ER Antibody Deparaffinization and Rehydration Antigen Retrieval Block Endogenous Peroxidase Primary Antibody Rinse Rinse SPECIMEN LNH 2009-013 ER/PR
  18. 18. Step by Step Envision Staining Method <ul><li>Wash 2 times in PBS buffer. </li></ul>Deparaffinization and Rehydration Antigen Retrieval Block Endogenous Peroxidase Primary Antibody Rinse Rinse PBS Buffer Rinse SPECIMEN LNH 2009-013 ER/PR
  19. 19. Step by Step Envision Staining Method <ul><li>Biotinylated link antibody for 50 minutes. </li></ul>Link Antibody Deparaffinization and Rehydration Antigen Retrieval Block Endogenous Peroxidase Primary Antibody Rinse Rinse Rinse Biotinylated Secondary Antibody Rinse SPECIMEN LNH 2009-013 ER/PR
  20. 20. Step by Step Envision Staining Method <ul><li>Wash 2 times in PBS buffer. </li></ul>Deparaffinization and Rehydration Antigen Retrieval Block Endogenous Peroxidase Primary Antibody Biotinylated Secondary Antibody Rinse Rinse Rinse Rinse PBS Buffer SPECIMEN LNH 2009-013 ER/PR
  21. 21. Step by Step Envision Staining Method <ul><li>HRP conjugated streptavidin for 50 minutes. </li></ul>Deparaffinization and Rehydration Streptavidin HRP Antigen Retrieval Block Endogenous Peroxidase Primary Antibody Biotinylated Secondary Antibody Rinse Rinse Rinse Rinse HRP Streptavidin SPECIMEN LNH 2009-013 ER/PR
  22. 22. Step by Step Envision Staining Method <ul><li>Wash 2 times in PBS buffer. </li></ul>PBS Buffer Deparaffinization and Rehydration Streptavidin HRP Antigen Retrieval Block Endogenous Peroxidase Primary Antibody Biotinylated Secondary Antibody Rinse Rinse Rinse Rinse Rinse SPECIMEN LNH 2009-013 ER/PR
  23. 23. Step by Step Envision Staining Method <ul><li>DAB chromogen for 5 minutes. </li></ul>DAB Chromogen Deparaffinization and Rehydration Antigen Retrieval Block Endogenous Peroxidase Primary Antibody Biotinylated Secondary Antibody Streptavidin HRP Rinse Rinse Rinse Rinse Rinse DAB Chromogen SPECIMEN LNH 2009-013 ER/PR
  24. 24. Step by Step Envision Staining Method <ul><li>Wash 2 times in PBS buffer and rinse in distilled water. </li></ul>Deparaffinization and Rehydration Antigen Retrieval Block Endogenous Peroxidase Primary Antibody Biotinylated Secondary Antibody Streptavidin HRP DAB Chromogen Rinse Rinse Rinse Rinse Rinse Distilled Water Rinse SPECIMEN LNH 2009-013 ER/PR
  25. 25. Step by Step Envision Staining Method <ul><li>Counterstain in Harris’s Hematoxylin for 1 minute. </li></ul>Hematoxylin Counterstain Hematoxylin Harris’s Hematoxylin Deparaffinization and Rehydration Antigen Retrieval Block Endogenous Peroxidase Primary Antibody Biotinylated Secondary Antibody Streptavidin HRP DAB Chromogen Rinse Rinse Rinse Rinse Rinse Rinse SPECIMEN LNH 2009-013 ER/PR
  26. 26. Step by Step Envision Staining Method <ul><li>Thorough wash, 1 Dip in Ammonia water again washing in tap water to “blue” the nuclei. </li></ul>Hematoxylin Counterstain Deparaffinization and Rehydration Antigen Retrieval Block Endogenous Peroxidase Primary Antibody Biotinylated Secondary Antibody Streptavidin HRP DAB Chromogen Rinse Rinse Rinse Rinse Rinse Rinse Tap Water Rinse SPECIMEN LNH 2009-013 ER/PR
  27. 27. Step by Step Envision Staining Method <ul><li>Dehydrate and Coverslip with aqueous resins. </li></ul>Deparaffinization and Rehydration Antigen Retrieval Block Endogenous Peroxidase Primary Antibody Biotinylated Secondary Antibody Streptavidin HRP DAB Chromogen Rinse Rinse Rinse Rinse Rinse Rinse Hematoxylin Counterstain Rinse DPX Aqueous Mount SPECIMEN LNH 2009-013 ER/PR
  28. 28. FACTORS AFFECTING THE TECHNIQUE <ul><li>Pre-Analytical Variables </li></ul><ul><li>Fixation Time </li></ul><ul><ul><li>Min. 6-8 hrs Max. 48 hrs </li></ul></ul><ul><ul><li>Goldstein et al (Am J Clin Pathol 120:86-92, 2003) </li></ul></ul><ul><ul><li>ASCO-CAP Guidlines </li></ul></ul><ul><ul><li>Fixation should start < 30 min after surgical removal. </li></ul></ul><ul><li>Fixative Type </li></ul><ul><ul><li>10% Neutral Buffered Formalin is Highly Recommended </li></ul></ul><ul><ul><li>Ab reagents and IHC methodologies have been optimised for formalin fixed tissue. </li></ul></ul>
  29. 29. Contd. <ul><li>Analytical Variables </li></ul><ul><li>Automated Vs Manual Procedure </li></ul><ul><li>Antibody and Titre </li></ul><ul><ul><li>Primary Ab Clones </li></ul></ul><ul><ul><ul><li>Mouse Monolonal 1D11, 1D5 </li></ul></ul></ul><ul><ul><ul><li>Rabbit Polyclonal </li></ul></ul></ul><ul><ul><ul><li>Rabbit Monoclonal SP1 </li></ul></ul></ul><ul><ul><li>Concentrate Vs RTU </li></ul></ul><ul><li>Ag Retrieval </li></ul><ul><ul><li>HIER(Heat Induce Epitope Retrieval) </li></ul></ul><ul><ul><li>Enzyme Methodology </li></ul></ul><ul><li>Detection Kit (Secondary Ab) </li></ul>
  30. 30. INTERPRETATION <ul><li>Assess the invasive tumor only </li></ul><ul><li>Staining Pattern </li></ul><ul><li>Intensity, % of Positive cells </li></ul><ul><li>Internal Negative Control </li></ul><ul><li>Positive Control </li></ul><ul><li>Pit-falls: Edge Effect, Retraction Artifact. </li></ul>
  31. 31. QC/QA Measures <ul><li>Controls are essential, use with each run. </li></ul><ul><li>Use internal control (Non-neoplastic epithelium). </li></ul><ul><li>Use External control (Positive Cancer with non-neoplastic epithelium, ideally with high and low levels of ER) </li></ul><ul><li>Commercial controls, Cell lines available. </li></ul><ul><li>Routine audits of results </li></ul>
  32. 32. Conclusion <ul><li>ER/PR through IHC is widely validated as a predictive factor in breast cancer. </li></ul><ul><li>IHC reaction is influenced by tissue fixation and processing. </li></ul><ul><li>Retrieval may improve IHC but it does not recover tissue from autolysis or overfixation. </li></ul><ul><li>The choice of primary Ab, New rabbit monoclonal antibodies are commercially available. </li></ul><ul><li>New Detection system available with accurate and nonspecfic staining. </li></ul><ul><li>A quality control of pre-analytical, analytical and post analytical phases of IHC is recommended in order to optimize results. </li></ul>

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