Single and Multiplex Immunohistochemistry Steve Westra, BS Reagent Product Specialist Leica Biosystems IHC Theory Polyclonal vs Monoclonal Polyclonal reagents Detect a multitude of epitopes Batch to batch variability Restricted to serum volume Less specific and problems with background Rabbit origin Monoclonal reagents Epitope specific Reproducible batches Development of standardized assays Unlimited volumes More specific and less background staining Generated in mice more recently developed in Rabbits IHC Theory Immunohistochemistry Uses antibodies developed in another species to visualize proteins by depositing a dye at the site of the antigen through a multi-step chemical reaction 1
IHC Theory Protocol Steps in Compact Polymer Polymer DAB Peroxidase/Protein Linker Primary mab Epitope RT Analytic: Detection Compact Polymer Compact polymer technology The most sensitive detection Leads to lower costs by using high primary a antibody dilutions Biotin free Clean background & IVD compliant Specific and intense staining DAB visualization Can be used for IHC and ISH Multi-link: detects both mouse and rabbit primary antibodies Available with alkaline phosphatase visualization Compact Polymer Refine Detection With Cytokeratin 8/18 Compact Polymer AP Red Detection With Melan A Analytic IHC Staining Quality Primary Antibody validation Clone, dilution, AR buffer, time and temperature Equipment calibration, GLP, Competency & training Use of controls Visualization Sensitivity and specificity Development Sensitivity and localization 2
Analytic IHC: Do it right the first time Overview Before you start Identify antibodies that will work for IHC Search for specific antibodies/conditions/protocols from:» Literature» Vendors» Peers Antibody selection Before Optimizing Antibodies Determine appropriate control tissue - Is the antigen present? Read the specification sheet for that antibody Call the manufacturer for information about antibody Look up Journal articles Vendor technical consultant Optimization: Titrations & Controls Tissue Control 4 multi-tissue control blocks for approximately 180/220 markers Block 1: Appendix, hepar, tonsil, pancreas Block 2: Brain, striated muscle, skin, melanoma Block 3: Lung, prostate, placenta, thyroid Block 4: Thymus, bone marrow, Hodgkin lymphoma, tonsil http://www.nordiqc.org/techniques/recommended_control_tissue.htm 3
Value Proposition Points Effective use of tissue Multiple antibodies on the same slide Fewer slides required for diagnosis Tissue safety gentle processing Effective staining for small foci Quality of Staining Minimal background staining Clear color differentiation Consistent staining Opportunity for Same Day Diagnosis Improved TAT One-step process How Does Multiplex Work The specimen is quenched with Hydrogen Peroxide to quench endogenous peroxidase activity A user supplied primary mouse and rabbit cocktail is applied Poly-HRP IgG reagent localizes mouse antibodies Poly-AP IgG reagent localizes rabbit antibodies The first substrate chromogen, 3,3 -Diaminobenizidine tetrahydrochloride (DAB), visualizes mouse antibodies via a brown precipitate The second substrate chromogen (Fast Red) visualizes rabbit antibodies via a red precipitate Hematoxylin (blue) counterstaining allows the visualization of cell nuclei New High Performance Chromoplex Multiple staining can be defined as the detection of two or more targets on one slide, thus increasing the information obtained from each slide and reducing turn around time compared to single staining or sequential staining. IHC Staining Methods 5 th Edition Nanna K. Christensen PhD and Lars Winther PhD 4
Why Multiplex PIN4 PIN4 Key Multiplex Stains PIN-4 Prostate Cancer P504s, CK5, P63 Benign vs Invasive Biocare/Chromoplex ADH-5 Breast Cancer CK5,7,14,18 & P63 Non-Invasive vs Invasive Biocare/Chromoplex CD34/CD71 double stain Differentiates Erythroid vs Myeloid CD34 (red): myeloblasts & 75% AML blasts: Often confused with erythroblasts CD71 (brown) only picks up erythroid Developed for use in bone marrow specimens to clearly separate the two Lineages (blasts vs. immature erythroid cells) 5
CD61(megakerocyte) -CD71(erythroid) ADH5 (CK5/14 + p63 + CK7/18) Used to differentiate 5 phenotypes of breast cancer Invasive vs. non-invasive breast lesions are easily distinguished. Hyperplasia of the usual type: p63 staining basal myoepithelium (DAB), CK5/14 (DAB) and CK7/18 staining luminal epithelium. ADH5 6
CD3-Ki67 (Large Cell Lymphoma) CD3-Ki67 PHH3 (Mitotic) Ki67 (Proliferative) 7
CD30/PAX5 double stain Identifies clearly Classical Hodkins Dual expression CD30 (red) cytoplasmic PAX5 (brown) weak nuclear In Hodgkins cells CD20 (B Cell) -CD3 (T Cell) 8
TTF1-Napsin A Adenocarcinoma vs. Squamous Cell Carcinoma Studies have shown NapsinA to be more sensitive and specific than TTF1 in lung andenocarcinomas and negative in all squamous carcinomas Other studies have shown NapsinA to stain more tumor cells and higher percentage of adenocarcinomas than TTF1 When TTF1 and NapsinA are used together, a higher sensitivity and specificity is achieved Often used in a panel with p63 and CK5 TTF1-Napsin A P63-CK5 Designed for squamous cell carcinomas P63-CK5 combination helps differentiate adenocarcinoma from squamous cell carcinoma Therefore when used in a panel with TTF-1 + Napsin A, this unique antibody cocktail of p63 + CK5 should prove useful for immunohistochemical analysis of poorly differentiated lung adenocarcinomas versus squamous cell carcinomas 9
p63-ck5 MITF-MART1 (Melanocytic Lesions) MITF-MART1 10
E-Cad (Ductal) p120 (Lobular) Kappa-Lambda (Monoclonality) Also can be done as dual ISH if desired. CK20-p53 + CD44 (Urothelial Dysplasia) 11
Experience Colorful Chromogens Diagnostic Biosystems Applications in Cancer Diagnostics Breast Colon Lung Prostate 12
4 Color IHC (Diagnostic Biosystems) Multiplex 5 Part Multiplexing 13
Thank you 14