C.L. Davis Foundation 2015 Descriptive Veterinary Pathology Course IHC Resources IHC Identification Targets Antibodies Antibodies 1
Antibodies Specimens Antigen Retrieval Unmasks antigen epitopes Methods Heat Enzymatic digestion Too much heat can destroy antigen Immunoelectron Microscopy-Ebola Virus IHC TECHNIQUES 2
Indirect Methods PAP and Streptavidin Endogenous Peroxidases Enzyme Horseradish Peroxidase Alkaline Phosphatase Substrate Diaminobenzidine 3 Amino 9 ethyl Carbozole(AEC) Histomark Red New Fuchsin BCIP/MBT Enzyme Chromogen Color HrP 3,3 diaminobenzidine (DAB) Brown HrP 3 amino 9 ethycarbazole (AEC) Red Alkaline phosphatase 5 bromo 4 chloro 3 indolyphospate/nickel blue Purple tetrazoliumchloride (BCIP/NBT) Alkaline phosphatase BCIP/NBT with iodonitrotetrazolium violet Dark brown Alkaline phosphatase Fast red magenta Alkaline phosphatase New fuchsin fuchsia 3
Controls Reagent controls Tissue controls 4
Trouble Spots Common complications Nonspecific Specific 5
Lymph node, nonhuman primate The Key to Description IHC Description Example 6
Intermediate Filaments Intermediate Filaments What are they? Limitations of Intermediate Filaments Cytokeratin Type II (basic) distribution Type I (acidic) CK1 Keratinized squamous epithelium CK10 CK3 Cornea CK12 CK5 Nonkeratinized epithelium CK13 CK6 Basal cells of glandular and squamous epithelium, myoepithelium, mesothelium CK14, CK17 CK7, CK8 Simple epithelia, superficial squamous epithelia CK16 Simple epithelia of GI track, Merkel cells, urothelium CK20 7
Cytokeratin Keratin Expression Keratin Expression Respiratory epithelium, horse Complex adenoma, cat Colonic Adenocarcinoma, dog 8
Ocular neoplasm, cat Ciliary body adenocarcinoma Vimentin Vimentin Expression Vimentin Expression Splenic lymphoma, dog 9
Dual Expression of Intermediate Filaments Complex mammary gland adenoma, dog Vimentin Tibiotarsal joint, dog Keratin DX: Synovial sarcoma Hepatic capsule, cat 10
Vimentin Keratin DX: Mesothelioma Desmin Desmin Expression Desmin Gingiva, dog 11
Neural Fibrillary Protein (NFP) Gingiva, dog NFP Expression Cerebral mass, dog Veterinary Pathology, 41:3, pp.282-84 NFP Expression Glial Fibrillar Acidic Protein (GFAP) Ganglioneuroblastoma 12
GFAP Mass, subcutis, cat Peripheral nerve sheath tumor Nestin Lysozyme Other Macrophage Markers Kidney, dog 13
Lysozyme Multifocal vimentin + with a positive internal control Histiocytic sarcoma Factor VIII Related Antigen Cutaneous neoplasm, dog Factor VIII Antigen Platelet Endothelial Cell Adhesion Molecule (PECAM) (CD31) Hemangiosarcoma 14
Thyroglobulin Thyroid Gland Thyroid transcription factor 1 (TTF 1) Calcitonin Neoplasm, neck, dog Thyroglobulin Thyroid follicular carcinoma Thyroid mass, baboon Calcitonin S 100 Protein Medullary carcinoma 15
Neoplasms that express S 100 Subcutaneous mass, cat S-100 Protein Neuron Specific Enolase (NSE) Peripheral nerve sheath tumor Synaptophysin and Chromogranin Synaptophysin Pancreatic islet cell, dog 16
Chromogranin Adrenal medulla, dog Pharyngeal neoplasm, dog Synaptophysin Muscle Markers Actin or muscle specific actin Smooth muscle actin Myoglobin Neuroendocrine carcinoma Actin Smooth Muscle Actin 17
Myoglobin S 100+ Neural crest origin GFAP + PNST MelanA, HMB45, PNL2 + Melanoma Spindle cell neoplasm Actin, desmin + Muscle origin SMA + Smooth muscle Myoglobin + Skeletal or cardiac muscle Factor VIII or CD31 + Vascular origin Actin Neoplasm thoracic wall, rat Desmin Myoglobin 18
Rhabdomyosarcoma Gastric submucosa, dog Diffuse vimentin immunoreactivity Diffuse desmin immunoreactivity Diffuse actin immunoreactivity 19
Diffuse smooth muscle actin immunoreactivity Negative staining for CD117 DIAGNOSIS? Negative staining for GFAP Myoepithelial Markers Mammary neoplasm, cat 20
Calponin Leukocyte and Platelet Markers CD 1a CD 3 CD 18 Complex adenoma CD 45RA CD 56 Leukocyte and Platelet Markers CD 3+ T cell lymphoma Round cell neoplasm CD79a, BLA 36, CD45RA, CD20 B cell lymphoma Kappa, lambda light chains Plasma cell tumor Lysozyme, CD68, MAC 387 Histiocytic Duodenal mass, dog Vet. Path., 39:5, pp.557-64 CD 117 21
Mast cell tryptase Dx: Mast Cell Tumor Cutaneous neoplasm, dog CD 3 DX: Epitheliotropic lymphoma Mass on the lip, dog CD 3 CD 79a Interpretation? DX: B-cell Lymphoma 22
Diagnosis of Melanomas Oral neoplasm, dog Melan A Infectious Disease Diagnosis DX: Malignant Melanoma MERS-Coronavirus; Lung Lassa, brain 23
MERS Coronavirus Ebola Zaire (Guinea strain), liver Morphometric analysis Vimentin Haired skin, dog 24
GFAP S-100 Self exam Tissue from a dog Haired skin: Peripheral Nerve Sheath tumor H&E Tissue from a dog Tissue from a dog Vimentin Melan-A 25
Tissue from a dog Tissue from a dog S-100 protein Myoglobin Tissue from a dog Self evaluation #2 Tissue from a dog rhabdomyosarcoma Desmin 26