Immunohistochemistry PDF

IMMUNOHISTOCHEMISTRY Dr Temba Mudariki  Learning Outcomes Understand the Principles of Immunohistochemistry Interpret Immunohistochemical Staining Identify Suitable Antibodies Perform Immunohistochemical Staining Analyse and Interpret Results Understand Clinical Applica...

IMMUNOHISTOCHEMISTRY Dr Temba Mudariki  Learning Outcomes Understand the Principles of Immunohistochemistry Interpret Immunohistochemical Staining Identify Suitable Antibodies Perform Immunohistochemical Staining Analyse and Interpret Results Understand Clinical Applications Quality Assurance and Troubleshooting Ethical and Professional Considerations Communication and Reporting Critical Evaluation of Research Literature  Introduction IHC identifies proteins using antigen-antibody binding & signal amplification Developed in 1940s by Coons establishing immunofluorescence technique Advancements in antibodies, staining, microscopy have revolutionized applications 1940s: Coons develops immunofluorescence using fluorophore-labelled antibodies 1960s: PAP method introduces enzyme signal amplification 1990s: Polymer-based detection systems improve sensitivity 2000s: Digital analysis incorporation advances quantification Importance in Molecular Diagnostics Visualizes protein expression levels and localization within tissues Identifies predictive biomarkers like HR, HER2 guiding therapy in breast cancer Determines prognostic markers such as Ki-67, p53 correlating with outcomes Provides spatial context of protein presence within tissue microenvironment Advances precision medicine via characterization of molecular signatures  Antigen-Antibody Interactions Antigen retrieval methods expose masked epitopes via heat, enzymes, pH Primary antibodies specifically bind target antigens Secondary antibodies coupled to enzymes/polymers amplify detection  Signal Amplification Methods Enzyme-Based Amplification Polymer-Based Amplification Tyramide Signal Amplification (TSA)  Visualization Techniques Chromogenic detection uses DAB, FastRed yielding precipitate under microscope Fluorescent detection employs fluorophore-labelled probes for enhanced sensitivity Digital analysis software conducts quantitative image assessments objectively  Differentiation of Staining Patterns Cytoplasmic staining indicates intracellular proteins’ localization and function Nuclear staining reveals gene/protein expression and transcription factors Membranous staining suggests cell surface receptors and adhesion molecules Controls confirm specificity while positive/negative staining guides interpretation  Factors Influencing Antibody Selection Evaluating target antigen specificity and antibody validation are paramount Reviewing cross-reactivity with other antigens ensures assay specificity Assessing sensitivity enables detection of low-abundance proteins Confirming tissue compatibility optimizes consistent staining across samples Antibody Validation and Quality Control Incorporating controls validates staining patterns and antibody performance Following guidelines like IWGP enhances credibility for clinical/research use Sourcing antibodies from reputable manufacturers provides documentation Ensuring specificity, sensitivity and reliability through testing new antibodies  Tissue Preparation & Antigen Retrieval Techniques Formalin fixation and paraffin-embedding preserve antigens and morphology Sectioning tissues into thin slices 3-5 μm for slide preparation Optimized processing is foundational for successful and accurate staining results HIER administers heat treatment in water bath or pressure cooker undoing crosslinks Enzymatic digestion uses proteases to enzymatically expose antigens pH-mediated retrieval involves adjusting pH to restore acid/base antigens Antibody Incubation and Signal Detection Antibody Incubation and Signal Detection –crucial for specific visualisation Primary Antibody Incubation Secondary Antibody and Signal Amplification Signal Detection Correlation of Staining Patterns with Pathological Conditions & Molecular Diagnosis and Prognostic Implications Distinguishing protein expression patterns in cancer subtypes guides classification Assessing tumour grade correlates proliferation/differentiation markers with aggressiveness Identifying immune cells and cytokines characterizes inflammatory conditions Biomarker expression reveals molecular diagnosis and targeted therapy selection Correlating prognostic markers with outcomes indicates survival predictions Therapeutic predictive markers guide treatment selection and immunotherapy responses  Disease Diagnosis and Subtyping Disease Diagnosis and Subtyping IHC aids diagnosis and subtyping by identifying biomarkers: Cancer Diagnosis Identifies tissue-specific (cytokeratins) and subtype (HR, HER2) markers Infectious Diseases Detects microbial antigens aiding diagnosis from tissues Autoimmune Disorders Helps identify immune complexes and autoantibodies contributing to diagnosis Precisely characterizing biomarkers facilitates accurate disease classification.  Prognostic and Predictive Biomarkers Immunohistochemistry - Prognostic and Predictive Biomarkers IHC is instrumental in evaluating prognostic and predictive biomarkers: Tumour Prognosis Biomarkers like Ki-67, p53, BCL-2 determine aggressiveness and predict patient prognosis Therapeutic Predictive Markers HR, HER2 guide treatment decisions in breast cancer Immunotherapy Biomarkers PD-L1 evaluation assists in predicting immunotherapy responses  Treatment Selection IHC contributes to selecting appropriate treatments: Targeted Therapy Detecting targets like EGFR, ALK aids selecting patients for targeted drugs Hormone Therapy Assessing HR status guides hormone-based therapy in breast and prostate cancers Immunotherapy Selection PD-L1 expression helps identify patients benefiting from checkpoint inhibitors  Ensuring Accuracy and Reliability of Results Ensuring quality and accuracy of IHC results is crucial. Key measures include: Validated Antibodies Selecting validated antibodies with documented specificity/sensitivity confirms target detection reliability Standardized Protocols Following standardized procedures for retrieval, dilution, detection promotes consistency across experiments Positive and Negative Controls Including controls in each run validates assay performance and staining pattern specificity Quality Tissue Samples Working with properly fixed, processed high-quality samples underpins reliable, interpretable results  Common Issues and Troubleshooting Measures Common IHC issues arise but can be addressed through troubleshooting: High Background Staining Adjust blocking, optimize antibody dilution/secondary/detection to reduce non-specific binding Weak or No Signal Re-evaluate antigen retrieval, optimize conditions to enhance exposure and signal Uneven Staining or Artifacts Ensure uniform sectioning, address artifacts from cutting or age of reagents Non-Specific Binding Review primary/secondary antibody choice and cross-reactivity, add blocking steps  Professional Conduct and Guidelines Compliance Professional Integrity Upholding honesty in conducting and reporting IHC findings ensuring accurate representation Compliance with Guidelines Adhering to regulatory and organizational guidelines ensures ethical and responsible practices Communication and Reporting of Immunohistochemical Findings Written Reports and Visual Representations Comprehensive Reporting Providing detailed methodology, results, interpretation and limitation reports conveys significance Visual Representations Clear photomicrographs and graphics effectively communicate staining patterns and results Professional Standards and Guidelines Adherence Professional Standards and Guidelines Adherence Quality Assurance Ensuring validation, quality controls and reporting standards maintains integrity and reliability Transparency and Disclosure Providing transparent methods, results and conflict disclosures adheres to standards of scientific communication ethics Current Advancements and Emerging Technologies Multiplex IHC Simultaneous visualization of multiple proteins provides comprehensive spatial and molecular information Digital Pathology Integration Integration with digital platforms facilitates quantitative analysis, spatial profiling and digital biomarkers Spatial Omics Technologies Technologies like spatial transcriptomics and proteomics provide deeper microenvironment insights  Potential Impact on Molecular Diagnostics Potential Impact on Molecular Diagnostics Precision Medicine Advanced techniques enhance characterization of molecular signatures, contributing to tailored therapies Biomarker Discovery Facilitating discovery of novel tissue-based biomarkers for prognostic/predictive applications Therapeutic Target Identification Insights aid target identification and validation, guiding targeted/immunotherapy development Future Directions in Immunohistochemistry and Molecular Diagnostics Integration of Multi-Omics Combining IHC with genomics, transcriptomics and proteomics for comprehensive disease understanding Standardization and Quality Assurance Continued efforts to ensure reproducibility and reliability of IHC-derived molecular data Clinical Translation Bridging research and practice by implementing advances in routine diagnostics and therapeutics