Descriptive Immunohistochemistry. Agenda. Agenda. What is Immunohistochemistry? 6/3/2014. Tissue from a dog

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1 Descriptive Immunohistochemistry Tissue from a dog Brian W. Smith, DVM, DACVP JPC, Veterinary Pathology Service Silver Spring, Maryland Descriptive Veterinary Pathology C.L. Davis DVM Foundation Agenda Shannon Lacy Jeremy Bearss JoLynne Raymond Overview What Why How Interpretation Description Common Antigens Case examples Agenda Overview What Why How Interpretation Description Common Antigens Case examples What is Immunohistochemistry? The application of enzyme conjugated antibodies to demonstrate the presence of a specific antigen in a tissue section Human papillomavirus (pink, ISH) within keratinocytes (green, anticytokeratin) 1

2 Indications Why do IHC? Indications After evaluating the H&E After developing a reasonable differential diagnosis Add corroborating evidence for a suspected diagnosis Better classify tissue origin (neoplasm) Identify markers of cellular events (e.g., proliferation, apoptosis) Identify an etiology IHC Targets Contraindications Cell structural components Enzymes and hormones Extracellular components Infectious agents Cytokines, chemokines and receptors Before carefully evaluating the H&E Before developing a reasonable differential diagnosis As a stab in the dark hoping to find the answer To stimulate the economy by charging the client lots of money for a complete immuno panel consisting of every stain available Methods How is IHC done? Example of an IHC Technique 1. Deparaffinize and rehydrate tissue 2. Antigen retrieval 3. Block endogenous peroxidase 4. Serum/protein block 5. Add primary antibody 6. Add secondary antibody (labeled polymer) 7. Add substrate chromogen solution 8. Counterstain 9. Dehydrate tissue and coverslip 2

3 Example of an IHC Technique 1. Deparaffinize and rehydrate tissue 2. Antigen retrieval 3. Block endogenous peroxidase 4. Serum/protein block 5. Add primary antibody 6. Add secondary antibody (labeled polymer) 7. Add substrate chromogen solution 8. Counterstain 9. Dehydrate tissue and coverslip Specimens Tissue sections, formalin fixed or frozen Cell smears, suspensions, cytospins Cells or tissue sections for electron microscopy Immunoelectron microscopy: Ebola virus Example of an IHC Technique An Antigen Retriever 1. Deparaffinize and rehydrate tissue 2. Antigen retrieval 3. Block endogenous peroxidase 4. Serum/protein block 5. Add primary antibody 6. Add secondary antibody (labeled polymer) 7. Add substrate chromogen solution 8. Counterstain 9. Dehydrate tissue and coverslip Antigen Retrieval Unmasks antigen epitopes for binding with antibody Protein cross links formed by formalin fixation Methods: Heat induced retrieval Enzymatic (proteolytic) digestion Too much can lead to destruction of the antigen of interest Example of an IHC Technique 1. Deparaffinize and rehydrate tissue 2. Antigen retrieval 3. Block endogenous peroxidase, ALP, biotin 4. Serum/protein block 5. Add primary antibody 6. Add secondary antibody (labeled polymer) 7. Add substrate chromogen solution 8. Counterstain 9. Dehydrate tissue and coverslip 3

4 Endogenous Peroxidases Erythrocytes Neutrophils Eosinophils Hepatocytes Endogenous Alkaline Phosphatase Intestine Kidney Liver Bone Neutralize with hydrogen peroxide Neutralize with heat or levamisole Example of an IHC Technique 1. Deparaffinize and rehydrate tissue 2. Antigen retrieval 3. Block endogenous peroxidase 4. Serum/protein block 5. Add primary antibody 6. Add secondary antibody (labeled polymer) 7. Add substrate chromogen solution 8. Counterstain 9. Dehydrate tissue and coverslip Antibodies Most are IgG Monoclonal or polyclonal High specificity High affinity Antibodies Most are IgG Monoclonal or polyclonal High specificity High affinity Mono vs. Poly Monoclonal antibody: Produced by a clone population from a single plasma cell which will only react with a single antigenic epitope Have high specificity and affinity for their antigen Polyclonal antibody: Produced by a mixed population of plasma cells which will react with multiple antigenic epitopes Used when optimal antigen retrieval is not possible 4

5 Simple & rapid Provides little signal amplification Requires large quantities of antibody Antigen Direct vs. Indirect Multistep detections Greater signal amplification Requires less antibody Only need a few off the shelf conjugated antibodies (e.g. antirabbit IgG) Primary antibody Avidin Biotin One form of signal amplification Secondary antibody is conjugated to biotin Biotin can recruit complexes of avidin, streptavidin, or neutravidin protein bound enzyme (e.g., peroxidase, ALKP) Thus the need to block endogenous biotin and enzymes Results in large numbers of branching enzyme molecules around a single secondary antibody Linker (biotin) or reporter molecule (enzyme or fluorophore) Secondary antibody Other Methods Polymer based techniques Bridge techniques Polyvalent techniques (secondary antibody binds both primary antibody and enzyme/chromogen system) Example of an IHC Technique 1. Deparaffinize and rehydrate tissue 2. Antigen retrieval 3. Block endogenous peroxidase 4. Serum/protein block 5. Add primary antibody 6. Add secondary antibody (labeled polymer) 7. Add substrate chromogen solution 8. Counterstain 9. Dehydrate tissue and coverslip Enzyme / Chromogen Systems Example of an IHC Technique Enzyme Chromogen Color Horseradish peroxidase 3,3 diaminobenzidine tetrachloride (DAB) Brown (PAP) Horseradish peroxidase 3 amino 9 ethycarbazole (AEC) Red Alkaline phosphatase (APAAP) Alkaline phosphatase 5 bromo 4 chloro 3 indolyphosphate/nickel blue tetrazoliumchloride (BCIP/NBT) BCIP/NBT with iodonitrotetrazolium violet (BCIP/NBT/INT) Dark purple Dark Brown Alkaline phosphatase Fast red Magenta Alkaline phosphatase New fuchsin Fuchsia 1. Deparaffinize and rehydrate tissue 2. Antigen retrieval 3. Block endogenous peroxidase 4. Serum/protein block 5. Add primary antibody 6. Add secondary antibody (labeled polymer) 7. Add substrate chromogen solution 8. Counterstain 9. Dehydrate tissue and coverslip 5

6 Agenda Overview What Why How Interpretation Description Common Antigens Case examples Trouble Spots IHC results are subject to more interpretation than other assays Antibody reactivity differs among species Fixation plays an important role: Different susceptibility of antigens Variable antigen retrieval Time of fixation and fixative variable Geographic differences, esp. altitude Importance of Controls Controls are critical for checking: Immunohistochemical technique Antigen retrieval effectiveness Appropriate secondary antibody function Antibody avidity in species of interest Antigen cross reactivity Control Types Internal vs. external Should be from same species Specific neoplasm / infectious agent control Individual organ controls Control panels (GI, lymph node, brain, adrenal, skeletal muscle, haired skin, thyroid, lung) Control Checklist Positive Internal Control Internal or external? Positive control stains positive Negative control stains negative Which antibody? Factor VIII-Ag 6

7 Common Complicating Factors Common Complicating Factors Generalized background staining Serum staining Common Complicating Factors Common Complicating Factors Melanin Hemosiderin Common Complicating Factors Agenda Overview What Why How Interpretation Description Common Antigens Case examples Tattoo pigment 7

8 IHC Description Tissue A single, concise description of the H&E For each stain: Evaluation of external control Evaluation of internal control Distribution, intensity, location of staining in tissue Interpretation Morphologic diagnosis Answer any questions Don t Do not describe normal Do not regurgitate information already provided on the panel Do not write separate descriptions for each magnification of the same stain Do not assume every IHC panel is a neoplasm! Do! H&E: Urinary bladder: Expanding the muscular tunics and infiltrating the serosa is a well circumscribed, moderately cellular neoplasm composed of spindle cells arranged in streams and bundles. Neoplastic cells have Vimentin: There is a positive internal control (vascular smooth muscle), and neoplastic cells have diffuse, intense, cytoplasmic immunopositivity (mesenchymal origin). Myoglobin:. Cytokeratin:. No!! Zero points for all these words: This immunohistochemical panel is composed of three H&E photomicrographs (one at 20X magnification, one at 200X magnification, and one at 400X magnification), as well as one photomicrograph at 200X magnification of a vimentin stain, one photomicrograph at 200X magnification of myoglobin, and one photomicrograph at 200X of cytokeratin. The 20X H&E demonstrates. The 200X H&E demonstrates. The 400 X H&E demonstrates. Vimentin and myoglobin are positive while cytokeratin is negative. Tangent High numbers of = many, numerous, myriad Low numbers of = few Small amounts of = scant Abundant amounts of Within the section Mitotic rate in the population Agenda Overview What Why How Interpretation Description Common Antigens Case examples 8

9 Common Target Antigens When Bruce was a Resident Intermediate filaments: keratin, vimentin, desmin, neurofilament protein (NFP), glial fibrillary acidic protein (GFAP) Enzymes: nuclear transcription factors, cytoplasmic / lysozomal enzymes Infectious agents: bacteria, viruses, protozoa Cell surface receptors: clusters of differentiation (CDs), TCRs Must know where to expect staining within the cell (membrane, cytoplasm, nucleus) Keratin + Vimentin + Carcinoma Sarcoma Neoplasm The End! Keratin + AND Vimentin + Mesothelioma Keratin AND Vimentin Neoplasm, NOS Vimentin The most ubiquitous intermediate filament in the body Embryonically expressed by all cells Retained expression by mesenchymal cells and many neoplastic cells Best use for this stain is to check for appropriate tissue preservation in a sample nnc4 Splenic lymphoma, dog Cytokeratin Complex mammary gland adenoma, dog No longer good enough to just know about pancytokeratin Type II (basic) Distribution Type I (acidic) CK1 Keratinized squamous epithlium CK10 CK3 Cornea CK12 CK4 Nonkeratinized epithelia CK13 CK5 Basal cells of glandular and squamous epithlium, CK14, CK17 myoepithelium, mesothelium CK6 Squamous epithelia CK16 CK7, CK8, Simple epithelia, superficial squamous epithelia CK18, CK19 Simple epithelia of GI tract, Merkel cells, urothelium CK20 Adapted from Dabbs, D: Diagnostic Immunohistochemistry, 2 nd ed., p. 67 9

10 Respiratory epithelium, horse, pancytokeratin Complex adenoma, cat CK5/6 Colonic adenocarcinoma, dog, CK 20 Ciliary body adenocarcinoma, pancytokeratin Keratin + and Vimentin + Traditionally these are co expressed by: Mesothelioma Synovial cell sarcoma Anaplastic carcinoma Carcinosarcoma Melanoma In reality, ANY carcinoma can co express these, as well as myometrium, vascular smooth muscle, plasma cells, PNETs, PNST, and other mesenchymal neoplasms Biliary epithelium and ductular reaction, CK7 10

11 Vimentin Tibiotarsal joint, dog Keratin Synovial sarcoma Hepatic capsule, cat Vimentin Keratin Mesothelioma 11

12 Miscellaneous Antigens Uroplakin III: prostatic and urothelial neoplasia HMB 45, PNL2, tyrosinase related antibodies: melanocytic neoplasia p63: myoepithelium and basal cells of stratified epithelium Calponin: myofibroblasts and myoepithelium ckit: mast cell tumors, GIST TTF 1: ANY thyroid tissue or carcinoma, respiratory epithelium, Clara cells, neuroendocrine, prostate, bladder, cervix, GI tract, breast Mammary adenoma, calponin and p63 Miscellaneous Antigens WT 1: in nephroblastoma, mesothelioma NFP: neuroblastoma, ganglioblastoma, pheochromocytoma NSE: virtually any type of neoplasm (even more ubiquitous than vimentin) CD56: natural killer cells GFAP + S Neural crest origin Spindle Cell Neoplasm MelanA, HMB 45, PNL2 + Spindle cell neoplasm Actin, Desmin + Muscle origin SMA + Myoglobin+ Factor VIII + CD31 (PECAM) + PNST Melanoma Smooth muscle Skeletal muscle (or cardiac) Vessel origin Desmin Primarily used to determine a possible myogenic origin for sarcomas Expressed in embryonic and adult skeletal, smooth and cardiac muscle Is not expressed in myoepithelium Glial Fibrillary Acidic Protein (GFAP) The major protein of astrocytes Also present in developing and neoplastic ependymal cells and oligodendrocytes Poorly differentiated (high grade) astrocytomas usually do not express GFAP 12

13 Factor VIII Related Antigen 99% von Willebrand factor Expressed in endothelial cells, megakaryocytes and platelets Useful in staining poorly differentiated hemangiosarcomas (sometimes) Cutaneous neoplasm, dog Factor VIII Antigen Platelet Endothelial Cell Adhesion Molecule (PECAM) (CD31) Expressed in endothelial cells, platelets and by some macrophages May be used in conjunction with Factor VIII to identify vascular origin Hemangiosarcoma S 100 Protein Primarily expressed by glial cells, but also expressed by: Adipocytes Chondrocytes Melanocytes Interdigitating reticular cells in LN Subcutaneous mass, cat 13

14 S-100 Protein Peripheral nerve sheath tumor Neoplasm thoracic wall, rat Desmin Actin Myoglobin Rhabdomyosarcoma Gastric submucosa, dog 14

15 Vimentin Desmin Actin Smooth Muscle Actin CD117 GFAP Leiomyoma 15

16 Melan A Oral neoplasm, dog Malignant Melanoma Epithelial Neoplasm Epithelial neoplasm Cytokeratin analysis Synaptophysin, ChromA + Tumor specific stains Multiple Neuroendocrine Insulin + Thyroglobin + Calcitonin + Parathyroid + Islet cell Thyroid follicular Thyroid parafollicular Parathyroid Multiple Neoplasm, neck, dog Thyroglobulin Thyroid follicular carcinoma Thyroid mass, baboon 16

17 Calcitonin Synaptophysin & Chromogranin Used to demonstrate neuroendocrine origin Very sensitive to formalin fixation C cell carcinoma Synaptophysin Chromogranin Pancreatic islet, dog Adrenal medulla, dog Synaptophysin Pharyngeal neoplasm, dog Neuroendocrine carcinoma 17

18 Synaptophysin Pancreas, Baboon Insulin Glucagon Somatostatin Round Cell Neoplasm Round cell neoplasm CD3 + CD79a + PAX 5 MUM1 + κ,λ + CD68+ Lysozyme + ckit + T cell B cell Plasma cell Histiocytic Mast cell tumor 18

19 Lysozyme Used as a macrophage marker In dogs it is expressed in: Monocytes, neutrophils Serous cells in mucosa associated exocrine glands Renal proximal tubular epithelium Kidney, dog Vimentin Lysozyme Histiocytic sarcoma CD3 Cutaneous neoplasm, dog Epitheliotropic lymphoma 19

20 CD3 Mass on the lip, dog Interpretation? CD79a Infectious Disease Diagnosis IHC can be used retrospectively to identify bacteria, fungi, viruses, protozoa, algae. Some cross reactivity with some of the antibodies for bacteria and protozoa Provides permanent preparation with the organism B-cell Lymphoma Marburg Virus, liver Coxiella burnetti, placenta 20

21 Yersinia pestis, lung Pneumocystis carinii, lung Agenda Overview What Why How Interpretation Description Common Antigens Case examples Self Evaluation Haired skin, dog Haired skin, dog 21

22 Vimentin GFAP S-100 Diagnosis? Diagnosis? Self exam Tissue from a dog Peripheral Nerve Sheath Tumor H&E Tissue from a dog 22

23 Vimentin Melan-A Vimentin Melan A S-100 Desmin S 100 protein Desmin Myoglobin Self evaluation #2 Tissue from a dog Morphologic Diagnosis: Myoglobin 23

24 Self evaluation #2 Tissue from a dog Tissue from a dog Morphologic Diagnosis: Mucous membrane: Rhabdomyosarcoma Tissue from a dog Tissue from a dog 24