immunohistochemistry Flashcards
What is immunohistochemistry routinely used for?
Identification of specific or highly selective cellular epitopes or antigens in frozen or paraffin-embedded tissues.
What does immunocytochemistry detect?
Organism in cytologic preparations (fluids, sputum samples, and fine needle aspirates).
What is the principle of immunohistochemistry and immunocytochemistry?
Antigen-antibody interactions.
What are the applications of immunohistochemistry?
Disease diagnosis, drug development, biological research.
What are polyclonal antibodies?
Antibodies produced by several clones of plasma cells using laboratory animals such as rabbits, goats, pigs, sheep, horses, guinea pigs.
How are polyclonal antibodies produced?
Lab animals are immunized with a purified specific immunogen having the antigen of interest, which responds by producing humoral antibodies against the antigen. Immunoglobulin-rich serum is collected from the lab animal.
What are monoclonal antibodies?
Antibodies produced by a single clone of plasma cells using hybridoma and cloning techniques, typically involving mice.
What is proteolytic enzyme digestion used for in sample preparation?
To break down formalin cross-links to unmask and allow certain antigenic sites to be exposed, useful for heavy chain immunoglobulins, complement, and specific antigens.
What enzymes are commonly used in proteolytic enzyme digestion?
Trypsin (0.1% trypsin in 0.1% CaCl) and protease (0.05 to 0.1% protease) adjusted to pH 7.8 with NaOH and preheated to 37°C.
What is microwave antigen retrieval?
Boiling formalin-fixed deparaffinized sections in buffers such as 0.01 M citrate buffer (pH 6.0), EDTA (pH 8.0), Tris EDTA (pH 8 or 10.0) with optimal exposure to heat for 10 to 60 minutes, most satisfactory at 20 minutes.
What is pressure cooker antigen retrieval?
A less time-consuming method with more consistent antigen recovery.
What is keratin a marker for?
Epithelial cells, including epithelial tumors (carcinomas) and non-epithelial tumors (mesotheliomas and non-seminomatous germ cell tumors).
What carcinomas are positive for CK7?
Lung, breast, uterus, and ovaries carcinomas.
What carcinomas are positive for CK20?
Colon and stomach carcinomas.
What is epithelial membrane antigen (EMA)?
A high molecular weight protein aiding in determining the site of the tumor, positive in adenocarcinomas of the breast, lung, and kidneys.
What is carcinoembryonic antigen (CEA)?
An oncofetal antigen present in GIT, pancreas, lung, breast, ovary, uterus, and cervix carcinomas, differentiates adenocarcinoma from mesothelioma.
What is thyroid transcription factor-1 (TTF-1)?
Distinguishes lung adenocarcinoma from mesothelioma, positive in thyroid, lung, and neuroendocrine tumors.
What is prostate-specific antigen (PSA)?
Extremely useful in the diagnosis of prostatic adenocarcinoma, positive in certain pancreatic and salivary gland tumors.
What is actin a marker for?
Muscle differentiation, identifying tumors derived from smooth, skeletal, and cardiac muscle.
What is vimentin a marker for?
A 57 kD intermediate filament in normal mesenchymal cells and their neoplastic counterparts, always positive in melanomas and Schwanommas.
What is desmin a marker for?
A 53 kD intermediate filament in smooth and striated muscles, highly specific for myogenic tumors.
What is GFAP (glial fibrillary acidic protein)?
A 51 kD intermediate filament protein expressed by CNS glial cells, particularly astrocytes, used to confirm astrocytoma diagnosis.
What is neurofilament (NF)?
Expressed by cells of neural origin, particularly neurons, peripheral nerves, and neuroendocrine cells, positive in tumors with neuronal or neuroendocrine differentiation.
What is S-100 protein?
A low molecular weight calcium-binding protein expressed by CNS glial cells, Schwann cells, melanocytes, and other cell types.
What is neuron-specific enolase (NSE)?
An isoenzyme marker providing strong evidence of neural or neuroendocrine differentiation.
What is chromogranin?
Found in neural secretory granules of endocrine tissues, a marker for neuroendocrine differentiation.
What is synaptophysin?
A 38 kD transmembrane protein associated with presynaptic vesicles of neurons, identified in normal neurons and neuroendocrine cells.
What is human chorionic gonadotropin (HCG)?
Synthesized by placental syncytiotrophoblasts, a marker for choriocarcinoma.
What is alpha fetoprotein (AFP)?
Synthesized by normal hepatocytes, a marker for endodermal sinus tumors and hepatocellular carcinomas.
What is placenta-like alkaline phosphatase (PLAP)?
Produced by placental syncytiotrophoblasts in late pregnancy, a marker for germ cell tumors.
What markers are positive for myogenic tumors?
Actin, desmin, myo-D1, myoglobin, myogenin.
What markers are positive for fibrohistiocytic tumors?
CD68 or FAM 56 with alpha-1-antitrypsin and alpha-1-antichymotrypsin for malignant, vimentin for undifferentiated.
What markers are positive for vascular tumors?
Factor VII-related antigen, CD31, Ulex europaeus 1.
What markers are positive for melanomas?
S100 protein, melanosome (HMB-45), Melan-A (MART-1).
What markers are positive for lymphomas?
Leukocyte common antigen (LCA), CD3, CD4, CD5 for T cells, CD19, CD20, CD23 for B cells, CD15, CD30, Ig LC, and HC for Reed-Sternberg cells.
What are cell proliferation markers?
Ki-67 (MIB-1), PCNA (proliferating cell nuclear antigen).
What are the types of labels used in immunohistochemistry?
Enzyme labels, colloidal metal labels, fluorescent labels, radiolabels.
What is the traditional direct technique?
The primary antibody is conjugated directly to the label, with direct interaction between the labeled antibody and antigen in the histological or cytological preparation.
What is the two-step indirect technique?
An unconjugated primary antibody binds to the antigen, followed by a labeled-secondary antibody directed against the primary antibody.
What is the three-step indirect technique?
Another labeled-antibody is used directed against the labeled-secondary antibody.
