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28 Diagnostic Immunohistochemistry Immunohistochemistry is a method to detect antigens in tissues that employs an enzyme-linked antibody specific for antigen. The enzyme degrades a colorless substrate to a colored insoluble substance that precipitates where the antibody and, therefore, the antigen are located. Identification of the site of the colored precipitate and the antigen in the tissue section is accomplished by light microscopy. Diagnostic pathology services routinely offer approximately 100 antigens, identified by immunoperoxidase technology, which are used in diagnosis. Immunoperoxidase method: Nakene and Pierce, in 1966, first proposed that enzymes be used in the place of fluorochromes as labels for antibodies. Horseradish peroxidase (HRP) is the enzyme label most widely employed. The immunoperoxidase technique permits the demonstration of antigens in various types of cells and fixed tissues. This method has certain advantages that include the following: (1) the use of conventional light microscopy; (2) the stained preparations may be kept permanently, (3) the method may be adapted for use with electron microscopy of tissues, and (4) counterstains may be employed. The disadvantages include the following: (1) the demonstration of relatively minute positively staining areas is limited by the resolution of the light microscope; (2) endogenous peroxidase may not have been completely eliminated from the tissue under investigation; and (3) diffusion of products results from the enzyme reaction away from the area where antigen is localized. The peroxidase–antiperoxidase (PAP) technique (Figure 28.1) employs unlabeled antibodies and a PAP reagent. This has proven highly successful for the demonstration of antigens in paraffin-embedded tissues as an aid in surgical pathologic diagnosis. Tissue sections preserved in paraffin are first treated with xylene, and after deparaffinization they are exposed to a hydrogen peroxide solution that destroys the endogenous peroxidase activity in tissue. The sections are next incubated with normal swine serum, which suppresses nonspecific binding of immunoglobulin molecules to tissues containing collagen. Thereafter, the primary rabbit antibody against the antigen to be identified is reacted with the tissue section. Primary antibody that is unbound is removed by rinsing the sections, which are then covered with swine antibody against rabbit immunoglobulin. This so-called linking antibody will combine with any primary rabbit antibody in the tissue. It is added in excess, which will result in one of

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its antigen-binding sites remaining free. After washing, the PAP reagent is placed on the section, and the antibody portion of this complex, which is raised in rabbits, will be bound to the free antigen-binding site of the linking antibody on the sections. The unbound PAP complex is then washed away by rinsing. To read the sections microscopically, it is necessary to add a substrate of hydrogen peroxide and aminoethylcarbazole (AEC), which permits the formation of a visible product that may be detected with the light microscope. The AEC is oxidized to produce a reddish-brown pigment that is not water-soluble. Peroxidase catalyzes the reaction. Because peroxidase occurs only at sites where the PAP is bound via linking antibody and primary antibody to antigen molecules, the antigen is identified by the reddish-brown pigment. The tissue sections can then be counterstained with hematoxylin or other suitable dye, covered with mounting medium and cover slips, and read by conventional light microscopy. The PAP technique has been replaced, in part, by the avidin–biotin complex (ABC) technique (Figure 28.2). Streptavidin is a protein isolated from streptomyces that binds biotin. This property makes streptavidin useful in the immunoperoxidase reaction that is employed extensively in antigen identification in histopathologic specimens, especially in surgical pathologic diagnosis. Immunodiagnosis involves the use of antibody assays, immunocytochemistry, the identification of lymphocyte markers, and other techniques to diagnose infectious diseases and malignant neoplasms. Decorate is a term used by immunologists to describe the reaction of tissue antigens with monoclonal antibodies, described as “staining,” in the immunoperoxidase reaction. Thus, a tissue antigen stained with a particular antibody is said to be decorated with that monoclonal antibody. Immunoperoxidase techniques give a reddish-brown color to the reaction product that is read by light microscopic observation. Antibroad-spectrum cytokeratin is a mouse monoclonal antibody that may be used to identify cells of normal and abnormal epithelial lineage and as an aid in the diagnosis of anaplastic tumors. The cytokeratins are a group of intermediate filament proteins that occur in normal and neoplastic cells of epithelial origin. The 19 known human cytokeratins are divided into acidic and basic subfamilies. They occur in pairs in epithelial tissues, the composition


Tissue Ag

ABC COMPLEX Tissue Ag Rabbit primary antibody

Sheep antirabbit immunoglobulin [linking antibody] [in excess]

Rabbit primary antibody

Biotinylated secondary antibody (e.g., goat antirabbit)

Peroxidase

Reagent A

Reagent B

Avidin DH (in excess)

Biotinylated Peroxidase H

Peroxidase

Peroxidase-antiperoxidase (PAP)

Development in chromogenic hydrogen donor and hydrogen peroxide. (The reaction product is seen as a reddish brown or brown granular deposit depending upon the chromogenic hydrogen donor used.)

AVIDIN (with four bindin sites for biotin)

FIGURE 28.1 The peroxidase–antiperoxidase (PAP) technique.

of pairs varying with the epithelial cell type, stage differentiation, cellular growth, environment, and disease state. The pankeratin cocktail recognizes most of the acidic and all of the basic cytokeratins, making it a useful general stain for nearly all epithelial tissues and their tumors. This antibody binds specifically to antigens located in the cytoplasmic region of normal simple and complex epithelial cells. The antibody is used to qualitatively stain cytokeratins in sections of formalin-fixed paraffin-embedded tissue. Antipankeratin primary antibody contains a mouse monoclonal antibody raised against an epitope found on human epidermal keratins. It reacts with 56.5-kDa, 50-kDa, 48kDa and 40-kDa cytokeratins of the acidic subfamily, and 65- to 67-kDa, 64-kDa, 59-kDa, 58-kDa, 56-kDa, and 52kDa cytokeratins of the basic subfamily. In anaplastic tumors, the percentage of tumor cells showing cytokeratin reactivity may be small (less than 5%). Unexpected antigen expression or loss of expression may occur, especially in neoplasms. Occasionally, stromal elements surrounding heavily stained tissue and or cells will show immunoreactivity. The clinical interpretation of any staining or its absence must be complemented by morphological studies and evaluation of proper controls. AE1/AE3 pan-cytokeratin monoclonal antibody (Figure 28.3) provides the broadest spectrum of keratin reactivity among

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BIOTIN PEROXIDASE

Development in chromogenic hydrogen donor and hydrogen peroxide. (The reaction product is seen as a reddish brown or brown granular deposit depending upon the chromogenic hydrogen donor used.)

FIGURE 28.2 The avidin–biotin complex (ABC) technique.

FIGURE 28.3 Cytokeratin cocktail — prostate.


the 19 catalogued human epidermal keratins and produces positive staining in virtually all epithelia. Anti-high molecular weight human cytokeratin antibodies are mouse monoclonal antibodies that identify keratins of approximately 66 kDa and 57 kDa in extracts of the stratum corneum. The antibody labels squamous, ductal, and other complex epithelia. It is reactive with both squamous and ductal neoplasms and variably with those derived from simple epithelium. Consistently positive are squamous cell carcinomas and ductal carcinomas, most notably those of the breast, pancreas, bile duct, and salivary gland; transitional cell carcinomas of the bladder and nasopharynx; and thymomas and epithelioid mesotheliomas. Adenocarcinomas are variably positive. The antibodies are largely unreactive with adenomas of endocrine organs, carcinomas of the liver (hepatocellular carcinoma), endometrium, and kidney. Mesenchymal tumors, lymphomas, melanomas, neural tumors, and neuroendocrine tumors are unreactive. Antihuman cytokeratin (CAM 5.2) (cytokeratin 8,18) (Figure 28.4) is a monoclonal antibody against cytokeratins which are polypeptide chains that form structural proteins within the epithelial cell cytoskeleton. Nineteen different molecular forms of cytokeratin have been identified in both normal and malignant epithelial cell lines. Because specific combinations of cytokeratin peptides are associated with different epithelial cells, these peptides are clinically important markers for classifying carcinomas (tumors of epithelial origin) and for distinguishing carcinomas from malignant tumors of nonepithelial origin such as lymphomas, melanomas, and sarcomas. The identification of cytokeratin has gained increasing importance in immunopathology. Cytokeratin 7 (K72), mouse: Anticytokeratin 7 (K72) mouse monoclonal antibody reacts with proteins that are

FIGURE 28.4 Cytokeratin 18 — salivary gland.

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FIGURE 28.5 Cytokeratin 7 — adenocarcinoma of the lung.

found in most ductal, glandular, and transitional epithelium of the urinary tract and bile duct epithelial cells. Cytokeratin 7 distinguishes between lung and breast epithelium that stain positive, and colon and prostate epithelial cells that are negative. This antibody also reacts with many benign and malignant epithelial lesions, e.g., adenocarcinomas of the ovary, breast, and lung. Transitional cell carcinomas are positive and prostate cancer is negative. This antibody does not recognize intermediate filament proteins. Antihuman cytokeratin 7 antibody (Figure 28.5) is a mouse monoclonal antibody directed against the 54-kDa cytokeratin intermediate filament protein identified as cytokeratin 7, a basic cytokeratin found in most glandular epithelia and in transitional epithelia. The antibody reacts with a large number of epithelial cell types including many ductal and glandular epithelia. In general, the antibody does not react with stratified squamous epithelia but is reactive with transitional epithelium of the urinary tract. The antibody reacts with many benign and malignant epithelial lesions. Keratin 7 is expressed in specific subtypes of adenocarcinomas from ovary, breast and lung, whereas carcinomas from the gastrointestinal tract remain negative. Transitional cell carcinomas express keratin 7 whereas prostate cancer is generally negative. The antibody does not react with squamous cell carcinomas, rendering it a rather specific marker for adenocarcinoma and transitional cell carcinoma. In cytological specimens, the antibody permits ovarian carcinoma to be distinguished from colon carcinoma. Antihuman cytokeratin-20 monoclonal antibody (Figure 28.6) reacts with the 46-kDa cytokeratin intermediate filament protein. It reacts with intestinal epithelium, gastric foveolar epithelium, a number of endocrine cells of the upper portions of the pyloric glands, as well as with the urothelium and Merkel’s cells in the epidermis. The antibody has been tested


human cytokeratins. It may be used to aid in the identification of cells of epithelial lineage. The antibodies are intended for qualitative staining in sections of formalinfixed paraffin-embedded tissue. Antikeratin primary antibody specifically binds to antigens located in the cytoplasmic regions of normal epithelial cells. Unexpected antigen expression or loss of expression may occur, especially in neoplasms. Occasionally, stromal elements surrounding heavily stained tissue and or cells will show apparent immunoreactivity. The clinical interpretation of any staining, or its absence, must be complemented by morphological studies and evaluation of proper controls.

FIGURE 28.6 Cytokeratin 20 — adenoma of the colon.

on a series of carcinomas including primary and metastatic lesions. There is a marked difference in expression of cytokeratin 20 among various carcinoma types. Neoplasia expressing cytokeratin 20 are derived from normal epithelia expressing cytokeratin 20. Colorectal carcinomas consistently express cytokeratin 20, whereas adenocarcinomas of the stomach express cytokeratin 20 to a lesser degree. Adenocarcinomas of the gall bladder and bile ducts, ductal cell adenocarcinomas of the pancreas, mucinous ovarian tumors, and transitional-cell carcinomas have been found to stain positively with the antibody. Most of the carcinomas from other sites were not positive using the antibody to cytokeratin 20, e.g., adenocarcinomas of the breast, lung and endometrium, and nonmucinous tumors of the ovary. Merkel cell carcinomas of the skin stain normally with the anticytokeratin 20 antibody. There was a lack of positivity in small-cell lung carcinomas and in intestinal and pancreatic neuroendocrine tumor cells.

Nonsquamous keratin (NSK) is a marker, demonstrable by immunoperoxidase staining, that is found in glandular epithelium and adenocarcinomas but not in stratified squamous epithelium. Epithelial membrane antigen (EMA) (Figure 28.7) is a marker that identifies, by immunoperoxidase staining, most epithelial cells and tumors derived from them, such as breast carcinomas. However, various nonepithelial neoplasms, such as selected lymphomas and sarcomas, may express EMA also. Thus, it must be used in conjunction with other markers in tumor identification and/or classification. Antiepithelial membrane antigen (EMA) antibody is a mouse monoclonal antibody directed against a mucin epitope present on most human epithelial cells. This antibody reacts with epithelial mucin, a heavily glycosylated molecule with a molecular weight of circa 400 kDa. Epithelial membrane antigen is widely distributed in epithelial tissues and tumors arising from them. Normal glandular epithelium and tissue from nonneoplastic diseases stain in lumen membranes and cytoplasm. Malignant neoplasms of glandular epithelium frequently show a change

Cytokeratin (34betaE12), mouse: Anticytokeratin (34betaE12) mouse monoclonal antibody detects cytokeratin 34betaE12, a high-molecular-weight cytokeratin that reacts with all squamous and ductal epithelium and stains carcinomas. This antibody recognizes cytokeratins 1,5,10, and 14 that are found in complex epithelia. Cytokeratin 34betaE12 shows no reactivity with hepatocytes, pancreatic acinar cells, proximal renal tubes, or endometrial glands; there has been no reactivity with cells derived from simple epithelia. Mesenchymal tumors, lymphomas, melanomas, neural tumors, and neuroendocrine tumors are unreactive with this antibody. Cytokeratin 34betaE12 has been shown to be useful in distinguishing prostatic adenocarcinoma from hyperplasia of the prostate. Anti-low molecular weight cytokeratin is a mouse monoclonal antibody directed against an epitope found on

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FIGURE 28.7 Epithelial membrane antigen — squamous carcinoma.


in pattern with the appearance of adjacent cell membrane staining. EMA is of value in distinguishing both large-cell anaplastic carcinoma from diffuse histiocytic lymphoma and small-cell anaplastic carcinoma from well and poorly differentiated lymphocytic lymphomas. An epithelial cell adhesion molecule (EpCAM) is considered a pan-carcinoma antigen. It is highly expressed on a variety of adenocarcinomas of different origin such as breast, ovary, colon, and lung, whereas its expression in normal tissue is very limited. Intermediate filaments are 7- to 11-nm diameter intracellular filaments observed by electron microscopy that are lineage specific. They are intermediate in size between actin microfilaments, which are 6 nm in diameter, and microtubules, which are 25 nm in diameter. They are detected in cell and tissue preparations by monoclonal antibodies specific for the filaments and are identified by the immunoperoxidase method. The detection of various types of intermediate filaments in tumors is of great assistance in determining the histogenetic origin of many types of neoplasms. Vimentin is a 55-kDa intermediate filament protein synthesized by mesenchymal cells such as vascular endothelial cells, smooth muscle cells, histiocytes, lymphocytes, fibroblasts, melanocytes, osteocytes, chondrocytes, astrocytes, and occasional ependymal and glomerular cells. Malignant cells may express more than one intermediate filament. For example, immunoperoxidase staining may reveal vimentin and cytokeratin in breast, lung, kidney, or endometrial adenocarcinomas. Antivimentin antibody is a mouse monoclonal antibody raised against purified bovine eye lens vimentin. This antibody reacts with the 57-kDa intermediate filament protein, vimentin. This reagent may be used to aid in the identification of cells of mesenchymal origin. The antibody is intended for qualitative staining in sections of formalinfixed, paraffin-embedded tissue. It binds specifically to antigens located in the cytoplasm of mesenchymal cells. The clinical interpretation of any staining, or its absence, must be complemented by morphological studies and evaluation of proper controls.

lines in striated muscle fibrils. In smooth muscle, desmin interconnects cytoplasmic dense bodies with membranebound dense plaques. Desmin antibody reacts with leiomyomas, rhabdomyomas, and perivascular cells of glomus tumors of the skin (if they are of myogenic nature). This antibody is basically used to demonstrate the myogenic components of carcinosarcomas and malignant mixed mesodermal tumors. Antidesmin antibody is a mouse monoclonal antibody (clone DE-R-11) raised against purified porcine desmin that reacts with the 53-kDa intermediate filament protein desmin. This reagent may be used to aid in the identification of cells of myocyte lineage. The antibody is intended for qualitative staining in sections of formalin-fixed, paraffin-embedded tissue. Antidesmin primary antibody specifically binds to antigens located in the cytoplasm of myocytic cells. The clinical interpretation of any staining, or its absence, must be complemented by morphological studies and evaluation of proper controls. Myoglobin is an oxygen-storing muscle protein that serves as a marker of muscle neoplasms, demonstrable by immunoperoxidase staining for surgical pathologic diagnosis. Myoglobin antibody is a reagent that stains normal striated muscle and striated muscle containing tumor. Using immunohistochemical procedures on formalin-fixed paraffin-embedded tissues, this antibody stains human skeletal and cardiac muscle. Actin, the principal muscle protein, which together with myosin causes muscle contraction, is used in surgical pathology as a marker for the identification of tumors of muscle origin. Actin is identified through immunoperoxidase staining of surgical pathology tissue specimens.

Desmin is a 55-kDa intermediate filament molecule found in mesenchymal cells that include both smooth and skeletal muscle, endothelial cells of the vessels, and probably myofibroblasts. In surgical pathologic diagnosis, monoclonal antibodies against desmin are useful in identifying muscle tumors.

Antimuscle actin primary antibody is a mouse monoclonal antibody (clone HUC1-1) directed against an actin epitope found on muscle actin isoforms. This reagent may be used to aid in the identification of cells of myocytic lineage. The antibody is intended for qualitative staining in sections of formalin-fixed, paraffin-embedded tissue. Antimuscle actin antibody specifically binds to antigens located in the cytoplasmic regions of normal muscle cells. Unexpected antigen expression or loss of expression may occur, especially in neoplasms. Occasionally, stromal elements surrounding heavily stained tissue and or cells will show immunoreactivity. Clinical interpretation must be complemented by morphological studies and the evaluation of appropriate controls.

Desmin (D33), mouse: Desmin antibody detects a protein that is expressed by cells of normal smooth, skeletal, and cardiac muscles. The light microscope has suggested that desmin is primarily located at or near the periphery of Z

Antihuman Îą-smooth muscle actin is a mouse monoclonal antibody that reacts with the Îą-smooth muscle isoform of actin. The antibody reacts with smooth muscle cells of vessels and different parenchyma without exception, but with

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FIGURE 28.8 S-100 — metastatic melanoma — lymph node.

FIGURE 28.9 Glial fibrillary acidic protein — brain.

different intensity, according to the amount of α-smooth muscle actin present in smooth muscle cells, myoepithelial cells, pericytes, and some stromal cells in the intestine, testes, breast, and ovary. The antibody also reacts with myofibroblasts in benign and reactive fibroblastic lesions and perisinusoidal cells of normal and diseased human livers.

Glialfibrillary acidic protein (GFAP) (Figure 28.9) is an intermediate filament protein constituent of astrocytes, which is also abundant in glial cell tumors. The immunoperoxidase technique employing monoclonal antibodies against the GFAP is used in surgical pathologic diagnosis to identify tumors based on their histogenetic origin.

S-100 (Figure 28.8) is a heterodimeric protein comprised of α and β chains. It is present in a variety of tissues and is especially prominent in nervous system tissue including brain, neural crest, and Schwann cells. It also is positive in breast ducts, sweat and salivary glands, bronchial glands and Schwann cells, serous acini, malignant melanomas, myoepithelium, and neurofibrosarcomas.

Antiglial fibrillary acidic protein (GFAP) antibody is a rabbit polyclonal antibody directed against glial fibrillary acidic protein present in the cytoplasm of most human astrocytes and ependymal cells. This reagent may be used to aid in the identification of cells of glial lineage. The antibody is intended for qualitative staining in sections of formalin-fixed, paraffin-embedded tissue. Anti-GFAP antibody specifically binds to the glial fibrillary acidic protein located in the cytoplasm of normal and neoplastic glial ells. Unexpected antigen expression or loss of expression may occur, especially in neoplasms. Occasionally, stromal elements surrounding heavily stained tissue and or cells will show immunoreactivity. The clinical interpretation of any staining, or its absence, must be complemented by morphological studies and evaluation of proper controls.

S-100 protein is a marker, demonstrable by immunoperoxidase staining, that is extensively distributed in both central and peripheral nervous systems and tumors arising from them, including astrocytomas, melanomas, Schwannomas, etc. Most melanomas express S-100 protein. Such nonneuronal cells as chondrocytes and histiocytes are also S-100 positive. S-100 protein antibody is a mouse monoclonal antibody specific for S-100 protein that is found in normal melanocytes, Langerhans cells, histiocytes, chrondrocytes, lipocytes, skeletal and cardiac muscle, Schwann cells, epthelial and myoepithelial cells of the breast, salivary and sweat glands, and glial cells. Neoplasms derived from these cells also express S-100 protein, albeit nonuniformly. A large number of well-differentiated tumors of the salivary gland, adipose and cartilaginous tissue, and Schwann cell derived tumors express S-100 protein. Almost all malignant melanomas and cases of histiocytosis X are positive for S-100 protein. Despite the fact that S-100 protein is a ubiquitous substance, its demonstration is of great value in the identification of several neoplasms, particularly melanomas.

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Synaptophysin (Figure 28.10) is a neuroendocrine differentiation marker that is detectable by the immunoperoxidase technique used in surgical pathologic diagnosis. Tumors in which it is produced include ganglioneuroblastoma, neuroblastoma, ganglioneuroma, paraganglioma, pheochromocytoma, medullary carcinoma of the thyroid, carcinoid, and tumors of the endocrine pancreas. Antihuman synaptophysin antibody is a rabbit antibody that reacts with a wide spectrum of neuroendocrine neoplasms of neural type including neuroblastomas, ganglioneuroblastomas, ganglioneuromas, pheochromocytomas, and chromaffin and nonchromaffin paragangliomas. The antibody also labels neuroendocrine neoplasms of epithelial


FIGURE 28.10 Synaptophysin — pancreas.

FIGURE 28.11 Neuron-specific enolase (NSE) — pancreas.

type including pituitary adenomas, islet cell neoplasms, medullary thyroid carcinomas, parathyroid adenomas, carcinoids of the bronchopulmonary and gastrointestinal tracts, neuroendocrine carcinomas of the bronchopulmonary and gastrointestinal tracts, and neuroendocrine carcinomas of the skin.

Anti-Ri antibody is an antibody found in serum and spinal fluid of patients with opsoclonus without myoclonus that occurs in conjunction with gait ataxia in women with breast cancer. The anti-Ri antibody reacts with 55-kDa and 80-kDa proteins present in the nuclei of CNS neurons and breast tumor cells. The condition may remit, manifest exacerbations and remissions, and occasionally respond to steroids or other immune interventions.

Anti-Purkinje cell antibody has been detected in the circulation of subacute cerebellar degeneration patients and in those with ovarian neoplasms and other gynecologic malignancies. Anti-GM1 antibodies are antibodies found in 2 to 40% of Guillain-Barré syndrome (GBS) patients. They are mainly IgG1, or IgA, rather than IgM, even though IgM anti-GM1 antibodies have been found in a few GBS cases. Anti-GM1 are more frequent in GBS patients who experienced C. jejuni infection (up to 50% of cases). Titers are highest initially and fall as the disease progresses. These antibodies are present in spinal fluid, apparently due to disruption of the blood–nerve barrier. Anti-GM1 antibodies recognize surface epitopes on Campylobacter bacteria, stains, and possibly a saccharide identical to the terminal tetrasaccharide of GM1 that has been found in Campylobacter lipopolysaccharide. IgG anti-GM1 has been postulated to selectively injure motor nerves. Anti-Ewing’s sarcoma marker (CD99) is a mouse monoclonal antihuman MIC2 gene product. (Ewing’s sarcoma marker) antibody reacts only with glioblastoma and ependymoma of the central nervous system and certain islet cell tumors of the pancreas. Because the MIC2 gene products are most strongly expressed on the cell membrane of Ewing’s sarcoma (ES) and primitive peripheral neuroectodermal tumors (pPNET), demonstration of the gene products allows for the differentiation of these tumors from other round-cell tumors of childhood and adolescence.

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Neuron-specific enolase (NSE) (Figure 28.11) is an enzyme of neurons and neuroendocrine cells, as well as their derived tumors, e.g., oat cell carcinoma of lung, demonstrable by immunoperoxidase staining. NSE occurs also in some neoplasms not derived from neurons or endocrine cells. Neuron-specific enolase (NSE) antibody is a murine monoclonal antibody directed against γ-γ enolase present on most human neurons, normal and neoplastic neuroendocrine cells, and some megakarocytes. This reagent may be used to aid in the identification of cells of neural or neuroendocrine lineage. The antibody is intended for qualitative staining in sections of formulin-fixed paraffin-embedded tissue. AntiNSE antibody specifically binds to the γ-γ enolase located in the cytoplasm of normal and neoplastic neuroendocrine cells. Unexpected antigen expression or loss expression may occur, especially neoplasms. Occasionally, stromal elements surrounding heavily stained tissue and/or cells will show immunoreactivity. The clinical interpretation of any staining, or its absence, must be complemented by morphological studies and evaluation of proper controls. A neurofilament is a marker, demonstrable by immunoperoxidase staining, for neural-derived tumors as well as selected endocrine neoplasms with neural differentiation. Hanganitziu-Deicher antigen is an altered ganglioside present in certain human neoplasms (CD3, GM1, and terminal 4NAcNeu).


Immunoperoxidase staining of pituitary adenomas with antibodies to the pituitary hormones ACTH, GH, prolactin, FSH, and LH facilitates definition of their clinical phenotype. Adrenocorticotrophic hormone (ACTH) antibody is a polyclonal antibody preparation useful in immunoperoxidase procedures to stain corticotroph cells of the pituitary gland and benign and malignant tumors arising from these cells, in formalin-fixed, paraffin-embedded tissue biopsies. ALZ-50 is a monoclonal antibody that serves as an early indicator of Alzheimer’s disease by reacting with Alzheimer’s brain tissue, specifically protein A-68. Antihuman follicle-stimulating hormone (FSH) antibody (Figure 28.12) is a rabbit antibody that labels gonadotropic cells in the pituitary. Positive staining for adenohypophyseal hormones assists in the classification of pituitary tumors. FSH is an adenohypophyseal glycoprotein hormone found in gonadotropic cells of the anterior pituitary gland of most mammals. Gonadotropic cells average about 10% of anterior pituitary cells. This antibody can be used for immunohistochemical staining. Chromogranin monoclonal antibody (Figure 28.13) is used to recognize chromogranin A (68 kDa) and other related chromogranin polypeptides from human, monkey, and pig. It is designed for the specific and quantitative localization of human chromogranin in paraffin-embedded and frozen tissue sections. It aids the localization of secretory storage granules in endocrine cells. Chromogranin A is a large, acidic protein present in catecholamine-containing granules of bovine adrenal medulla. It may be widely distributed in endocrine cells and tissues, which share some common characteristics and are known as APUD cells. Dispersed throughout the body, they are also referred to as

FIGURE 28.13 Chromogranin — pancreas.

FIGURE 28.14 HMB-45. Melanoma in lymph node.

the diffuse neuroendocrine system (DNES). Chromogranin has been demonstrated in several elements of the DNES, including anterior pituitary, thyroid parafollicular C cells, parathyroid chief cells, pancreatic islet cells, intestinal enteroendocrine cells, and tumors derived from these cells. Chromogranin immunoreactivity has also been observed in the thymus, spleen, lymph nodes, fetal liver, neurons, the inner segment of rods and cones, the submandibular gland, and the central nervous system. Chromogranin is a widespread histological marker for polypeptide producing cells (APUD) and the tumors derived from them. Antigrowth hormone (GH) antibody is a rabbit polyclonal antibody against human growth hormone that positively stains the growth hormone-producing cells and somatotrophs of the pituitary gland and malignant and benign neoplasms arising from these cells.

FIGURE 28.12 Follicle-stimulating hormone (FSH) — pituitary.

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Antiprolactin antibody is a rabbit antibody that gives positive staining of the prolactin cells of the anterior pituitary


and benign and malignant neoplasms derived from these cells. Antimelanoma primary antibody is a mouse monoclonal antibody (clone HMB-45) raised against an extract of pigmented melanoma metastases from lymph nodes directed against a glycoconjugate present in immature melanosomes. This antibody may be used to aid in the identification of cells of melanocytic lineage. The antibody is for qualitative staining in sections of formalinfixed paraffin-embedded tissue. This antibody binds specifically to antigens located on immature melanosomes. Unexpected antigen expression or loss of expression may occur, especially in neoplasms. Clinical interpretation must be complemented by morphological studies and evaluation of proper controls. Anti-BRST-2 (GCDFP-15) monoclonal antibody is specific for BRST-2 antigen expressed by apocrine sweat glands, eccrine glands (variable), minor salivary glands, bronchial glands, metaplastic epithelium of the breast, benign sweat gland tumors of the skin, and the serous cells of the submandibular gland. Breast carcinomas (primary and metastatic lesions) with apocrine features express the BRST-2 antigen. BRST-2 is positive in extramammary Paget’s disease. Other tumors are negative. Anti-BRST-3 (B72.3) monoclonal antibody recognizes TAG-72, (Figure 28.15) a tumor-associated oncofetal antigen expressed by a wide variety of human adenocarcinomas. This antigen is expressed by 84% of invasive ductal breast carcinoma and 85 to 90% of colon, pancreatic, gastric, esophageal, lung (non-small cell), ovarian, and endometrial adenocarcinomas. It is not expressed by leukemias, lymphomas, sarcomas, mesotheliomas, melanomas, or benign tumors. TAG-72 is also expressed on normal secretory endometrium but not on other normal tissues.

FIGURE 28.16 Estrogen receptor — carcinoma of the breast.

Estrogen/progesterone receptor protein: Monoclonal antibodies against estrogen receptor protein and against progesterone receptor protein permit identification of tumor cells by their preferential immunoperoxidase staining for these markers, whereas stromal cells remain unstained. This method is claimed by some to be superior to cytosol assays in evaluating the clinical response to hormones. (Figure 28.16) Antiestrogen receptor antibodies are mouse monoclonal specific for estrogen receptors. The estrogen receptor (ER) content of breast cancer tissue is an important parameter in the prediction of prognosis and response to endocrine therapy. Monoclonal antibodies to ER permit the determination of receptor status of breast tumors to be carried out in routine histopathology laboratories. Although monoclonal antibodies that recognize ER were only effective on frozen sections initially, currently available monoclonal antibodies are effective on formalin-fixed, paraffinembedded tissues to allow the determination of ER in routinely processed and archival material. CA-15-3 is an antibody specific for an antigen frequently present in the blood serum of metastatic breast carcinoma patients. CA-125 (Figure 28.17) is a mucinous ovarian carcinoma cell surface glycoprotein detectable in blood serum. Increasing serum concentrations portend a grave prognosis. It may also be found in the blood sera of patients with other adenocarcinomas, such as breast, gastrointestinal tract, uterine cervix, and endometrium.

FIGURE 28.15 Tag 72 — carcinoma of the breast.

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CA-125 antibody is a mouse monoclonal antibody that reacts with malignant ovarian epithelial cells. The antigen is formalin resistant, permitting the detection of ovarian cancer by immunohistochemistry, although serum assays for this protein are widely used to monitor ovarian cancer.


FIGURE 28.17 CA125 — papillary carcinoma of the ovary.

CA-125 also reacts with antigens in seminal vesicle carcinoma and anaplastic lymphoma. c-erb-B2 murine monoclonal antibody is specific for cerb-B2 oncoprotein which is expressed by tumor cell membranes at a level detectable by immunohistochemistry in up to 20% of adenocarcinomas from various sites including ovary, gastrointestinal tract, and breast. Immunohistochemical staining correlates with gene amplification. In the case of breast cancer, c-erb-B2 expression has been shown to be associated with poor prognosis. Between 15 and 30% of invasive ductal cancers are positive for c-erbB2. Almost all cases of Paget’s disease and approximately 70% of cases of in situ ductal carcinoma are positive. Cu-18 is a glycoprotein of breast epithelium. Immunoperoxidase staining identifies this marker in most breast tumors and a few tumors of the ovary and lung. Stomach, pancreas, and colon tumors do not express this antigen. Lactalbumin is a breast epithelial cell protein demonstrable by immunoperoxidase staining that is found in approximately one half to two thirds of breast carcinomas for which it is relatively specific. More than 50% of metastatic breast tumors and some salivary gland and skin appendage tumors stain positively for lactalbumin. Gross cystic disease fluid protein 15 (GCDFP-15) antigen is a 15-kDa glycoprotein that is demonstrable with immunoperoxidase staining and expressed by primary and metastatic breast carcinomas with apocrine features and extramammary Paget’s disease. Normal apocrine sweat glands, eccrine glands (variable), minor salivary glands, bronchial glands, metaplastic breast epithelium, benign sweat gland tumors of skin, and submandibular serous cells express GCDFP-15 antigen. GCDFP-15 (23A3), mouse: Gross cystic disease fluid protein-15 is a 15,000-Da glycoprotein that was localized

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FIGURE 28.18 Placental alkaline phosphatase (PLAP) — placenta.

in the apocrine metaplastic epithelium lining breast cysts and in apocrine glands in the axilla, vulva, eyelid, and ear canal. Approximately 70% of breast carcinomas stain positive with antibody to GCDFP-15. Colorectal carcinomas, as well as mesotheliomas, do not stain with this antibody. Lung adenocarcinoma rarely stains with this antibody. Human milk-fat globulin (HMFG) is a human milk glycoprotein on secretory breast cell surfaces. Many breast and ovarian carcinomas are positive for HMFG. In diagnostic immunology, estradiol is a marker identifiable in breast carcinoma tissue by monoclonal antibody and the immunoperoxidase technique that correlates, to a limited degree, with estrogen receptor activity in cytosols from the same preparation. Antiplacental alkaline phosphatase (PLAP) antibody (Figure 28.18) is normally produced by syncytiotrophoblasts after the twelfth week of pregnancy. Human placental alkaline phosphatase is a member of a family of membrane-bound alkaline phosphatase enzymes and isoenzymes. It is expressed by both malignant somatic and germ-cell tumors. PLAP immunoreactivity can be used in conjunction with epithelial membrane antigen (EMA) and keratin to differentiate between germ cell and somatic tumor metastases. Germ cell tumors appear to be universally reactive for PLAP, whereas somatic tumors show only 15 to 20% reactivity. Antiprogesterone receptor antibody is a mouse monoclonal antibody against human progesterone receptor. A mouse monoclonal antihuman progesterone receptor antibody that specifically recognizes the A and B forms of the receptor in Western blot purified recombinant receptor, normal endometrium, and cell lysates of the progesterone receptor-rich T47D human breast carcinoma cell line.


FIGURE 28.19 Prostatic acid phosphatase (PSAP) — prostate.

FIGURE 28.20 Prostate-specific antigen (PSA) — prostate.

No reactivity was observed with lysate of the progesterone receptor-negative MDA-MB-231 breast carcinoma cells. No crossreactivity was found with androgen receptor, estrogen receptor, or glucocorticoid receptor. The antibody binds an epitope found between amino acids 165 and 534, in the N-terminal transactivation domain of the progesterone receptor molecule. Various tumors of the female reproductive tract have been shown to express progesterone receptor. Immunoreactivity has been demonstrated in breast carcinoma, uterine papillary serous carcinoma, endometrial carcinoma, ovarian serous borderline tumor, endometrial stromal sarcoma, uterine adenomatoid tumor, and ovarian thecoma. Other tumors that have been shown to stain positively include medullary carcinoma of the thyroid and meningioma.

with PSA levels greater than 4.0 µg/l and 60% of the individuals with PSA levels greater than 10 µg/l.

Prostatic acid phosphatase (PAP)/prostatic epithelial antigen are prostate antigens, identifiable by immunoperoxidase staining, that are prostate-specific and -sensitive. Used together, they detect approximately 99% of prostatic adenocarcinomas. Antihuman prostatic acid phosphatase (PSAP) (Figure 28.19) is a rabbit antibody that reacts with prostatic ductal epithelial cells — normal, benign hypertrophic, and neoplastic. This antibody labels the cytoplasm of prostatic epithelium, secretions, and concretions. PSA (prostate-specific antigen) (Figure 28.20) is a substance secreted only by the prostate epithelium and is a 34-kDa glycoprotein serine protease that lyses seminal coagulum. Individuals with benign prostatic hypertrophy have a 30 to 50% elevation in PSA levels, whereas those with prostatic carcinoma have a 25 to 92% elevation. It is a more reliable indicator of prostatic carcinoma than is serum prostatic acid phosphatase (PAP). PSA levels are also valuable in signifying recurrence of prostatic adenocarcinoma. Prostate cancer may occur in 22% of the individuals

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Prostate-specific antigen (PSA) is a marker in serum or tissue sections for adenocarcinoma of the prostate. PSA is a 34-kDa glycoprotein found exclusively in benign and malignant epithelium of the prostate. Men with PSA levels of 0 to 4.0 ng/ml and a nonsuspicious digital rectal examination are generally not biopsied for prostate cancer. Men with PSA levels of 10.0 ng/ml and above typically undergo prostate biopsy. About one half of these men will be found to have prostate cancer. Certain kinds of PSA, known as bound PSA, link themselves to other proteins in the blood. Other kinds of PSA, known as free PSA, float by themselves. Prostate cancer is more likely to be present in men who have a low percentage of free PSA relative to the total amount of PSA. This finding is especially valuable in helping to differentiate between cancer and other, benign, conditions, thus eliminating unnecessary biopsies among men in that diagnostic gray zone, who have total PSA levels between 4.0 and 10.0 ng/ml. The PSA molecule is smaller than prostatic acid phosphatase (PAP). In patients with prostate cancer, preoperative PSS serum levels are positively correlated with the disease. PSA is more stable and shows less diurnal variation than does PAP. PSA is increased in 95% of new cases of prostatic carcinoma compared with 60% for PAP. It is increased in 97% of recurrent cases compared with 66% of PAP. PAP may also be increased in selected cases of benign prostatic hypertrophy and prostatitis, but these elevations are less than those associated with adenocarcinoma of the prostate. It is inappropriate to use either PSA or PAP alone as a screen for asymptomatic males. Transurethral resection (TUR), urethral instrumentation, prostatic needle biopsy, prostatic infarct, or urinary retention may also result in increased PSA values. PSA is critical for the prediction of recurrent adenocarcinoma in postsurgical patients. PSA


identification of cells of epithelial lineage. The antibody is intended for qualitative staining in sections of formalinfixed, paraffin-embedded tissue. Anti CEA antibodies specifically bind to antigens located in the plasma membrane and cytoplasmic regions of normal epithelial cells. Unexpected antigen expression or loss of expression may occur, especially in neoplasms. Occasionally, stromal elements surround heavily stained tissue and/or cells which show immunoreactivity. Clinical interpretation of any staining or its absence must be complemented by morphological studies and evaluation of proper controls.

FIGURE 28.21 CEA — carcinoma of the colon.

is also a useful immunocytochemical marker for primary and metastatic adenocarcinoma of the prostate. Antiprostate specific antigen (PSA) antibody is a rabbit antibody that reacts with prostatic ductal epithelial cells — normal, benign hypertrophic, and neoplastic. This antibody labels the cytoplasm of prostatic epithelium, secretions, and concretions. Carcinoembryonic antigen (CEA) (Figure 28.21) is a 200-kDa membrane glycoprotein epitope that is present in the fetal gastrointestinal tract in normal conditions. However, tumor cells, such as those in colon carcinoma, may reexpress it. CEA was first described as a screen for identifying carcinoma by detecting nanogram quantities of the antigen in serum. It was later shown to be present in certain other conditions as well. CEA levels are elevated in almost one third of patients with colorectal, liver, pancreatic, lung, breast, head and neck, cervical, bladder, medullarythyroid, and prostatic carcinoma. However, the level may be elevated also in malignant melanoma, lymphoproliferative disease, and smokers. Regrettably, CEA levels also increase in a variety of nonneoplastic disorders, including inflammatory bowel disease, pancreatitis, and cirrhosis of the liver. Nevertheless, determination of CEA levels in the serum is valuable for monitoring the recurrence of tumors in patients whose primary neoplasm has been removed. If the patient’s CEA level reveals a 35% elevation compared to the level immediately following surgery, this may signify metastases. This oncofetal antigen is comprised of one polypeptide chain with one variable region at the amino terminus and six constant region domains. CEA belongs to the immunoglobulin superfamily. It lacks specificity for cancer, thereby limiting its diagnostic usefulness. It is detected with a mouse monoclonal antibody directed against a complex glycoprotein antigen present on many human epithelial derived tumors. This reagent may be used to aid in the

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Serotonin (5-hydroxytryptamine [5-HT]) is a 176-molwt catecholamine found in mouse and rat mast cells and in human platelets that participates in anaphylaxis in several species such as the rabbit but not in humans. It induces contraction of smooth muscle, enhances vascular permeability of small blood vessels, and induces large blood vessel vasoconstriction. 5-HT is derived from tryptophan by hydroxylation to 5-hydroxytryptophan and decarboxylation to 5-hydroxytryptamine. In man, gut enterochromaffin cells contain 90% of 5-HT, with the remainder accruing in blood platelets and the brain. 5-HT is a potent biogenic amine with wide species distribution. 5-HT may stimulate phagocytosis by leukocytes and interfere with the clearance of particles by the mononuclear phagocyte system. Immunoperoxidase staining for 5-HT, which is synthesized by various neoplasms, especially carcinoid tumors, is a valuable aid in surgical pathologic diagnosis of tumors producing it. CA-19-9 is a tumor-associated antigen found on the Lewis A blood group antigen that is sialylated or in mucincontaining tissues. In individuals whose serum levels exceed 37 U/ml, 72% have carcinoma of the pancreas. In individuals whose levels exceed 1000 U/ml, 95% have pancreatic cancer. Anti-CA-19-9 monoclonal antibody is useful to detect the recurrence of pancreatic cancer following surgery and to distinguish between neoplastic and benign conditions of the pancreas. However, it is not useful for pancreatic cancer screening. Antihuman glucagon antibody is a rabbit antibody that labels A cells of the endocrine mammalian pancreas. Antihuman chorionic gonadotropin (HCG) antibody is an antibody that reacts with the beta chain of human chorionic gonadotropin (HCG). HCG is a polypeptide hormone synthesized in the syncytiotrophoblastic cells of the placenta and in certain trophoblastic tumors. HCG is a marker for the biochemical differentiation of trophoblastic cells, which often precedes their morphological differentiation. The antibody aids detection of HCG in trophoblastic elements of germ cell tumors of the ovaries, testes, and extragonadal sites. It crossreacts with luteinizing hormone.


O125 (ovarian celomic) is a nonmucinous ovarian tumor antigen demonstrable with homologous antibody by immunoperoxidase staining. Selected mesotheliomas express this antigen as well. Colon–ovary tumor antigen (COTA) is a type of mucin demonstrable by immunoperoxidase staining in all colon neoplasms and in some ovarian tumors. COTA occurs infrequently in other neoplasms. Normal tissues express limited quantities of COTA. Antisomatostatin antibody is a rabbit antibody that can be used for the immunohistochemical staining of somatostatin in tumors and hyperplasias of pancreatic islets. Antipancreatic polypeptide (PP) antibody is a polyclonal antibody that detects pancreatic polypeptide in routinely fixed paraffin embedded or frozen tissue sections. Hyperplasia of pancreatic polypeptide-containing cells (PP cells) is often seen in patients with juvenile diabetes, chronic pancreatitis, and islet cell tumors. Hyperplasia of PP cells (greater than 10% of the islet cell population) in the nontumoral pancreas has been observed in nearly 50% of islet cell tumors. Demonstration of increased numbers of cells secreting pancreatic polypeptide found both within the islets and between the islets is characteristic of type II hyperplasia of pancreatic islets. Monoclonal antiinsulin antibody is an antibody used for the immunohistochemical localization of the polypeptide hormone insulin that is the most reliable means to accurately characterize the functional repertoire of islet cell tumors. Islet cell neoplasms of the pancreas appear as solitary or multiple circumscribed lesions that contrast sharply with the neighboring pancreatic parenchyma. These tumors are grouped on the basis of their predominant secretory hormone. This monoclonal antibody is used for the specific and qualitative localization of insulin in routinely fixed paraffin-embedded or frozen tissue sections.

FIGURE 28.22 Calcitonin — medullary carcinoma of the thyroid.

Calcitonin (Figure 28.22) is a hormone that influences calcium ion transport. Immunoperoxidase staining demonstrates calcitonin in thyroid parafollicular or C cells. It serves as a marker characteristic of medullary thyroid carcinoma and APUD neoplasms. Lung and gastrointestinal tumors may also form calcitonin. Antihuman gastrin is a rabbit antibody that labels G-cells of antropyloric mucosa of the stomach. It permits immunohistochemical detection of gastrin-secreting tumors and G-cell hyperplasia. Antihuman thyroglobulin is a rabbit antibody that reacts with human thyroglobulin. It labels the cytoplasm of normal and neoplastic thyroid follicle cells. Some staining of colloid may also be observed.

Antihuman thyroid-stimulating hormone (TSH) is a rabbit antibody used for the immunochemical detection of thyroid stimulating hormone (TSH) in thyrotrophic cells and in certain pituitary tumors.

Common leukocyte antigen (LCA) (CD45) is an antigen shared in common by both T and B lymphocytes and expressed, to a lesser degree, by histiocytes and plasma cells. By immunoperoxidase staining, it can be demonstrated in sections of paraffin-embedded tissues containing these cell types. Thus, it is a valuable marker to distinguish lymphoreticular neoplasms from carcinomas and sarcomas (Figure 28.23).

Antiparathyroid hormone (PTH) antibody is a polyclonal antibody against parathyroid hormone (PTH). PTH controls the concentration of calcium and phosphate ions in the blood. A decrease in blood calcium stimulates the parathyroid gland to secrete PTH, which acts on cells of bone, increasing the number of osteoclasts and leading to absorption of the calcified bone matrix and the release of calcium into the blood. Hyperparathyroidism may be caused by adenomas, rarely by carcinomas and by ectopic PTH production. PTH is released by renal adenocarcinomas as well as by squamous cell cancers of the bronchus.

Anti-CD45R (Leukocyte common antigen) is a mouse monoclonal antibody specific for an epitope present on the majority of human leukocytes. This reagent may be used to aid in the identification of cells of lymphocytic lineage. The antibody is intended for qualitative staining in sections of formalin-fixed, paraffin-embedded tissue. It specifically binds to antigens located predominantly in the plasma membrane and to a lesser degree in the cytoplasm of lymphocytes, with variable reactivity to monocytes/ histiocytes, and polymorphonuclear leukocytes. Unexpected antigen expression or loss of expression may occur,

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FIGURE 28.23 CD45 — tonsil.

especially in neoplasms. Occasional stromal elements surrounding heavily stained tissues and or cells would show immunoreactivity. The clinical interpretation of any staining or its absence must be complemented by morphological features and evaluation of proper controls. Anti-T cell (CD45RO) antibody reacts with CD45RO determinant of leukocyte common antigen. It reacts with most T lymphocytes, macrophages, and Langerhans cells of normal tissues. It also reacts with peripheral T cell lymphomas, T cell leukemia, histiocytosis, and monocytic leukemia with mature phenotype. It reacts very rarely with B cell lymphoma and leukemia. UCHL1 antihuman T cell, CD45RO is a mouse monoclonal antibody that recognizes specifically the 180-kDa isoform of CD45 (leukocyte common antigen). The 180kDa glycoprotein occurs on most thymocytes and activated T cells, but only a proportion of resting T cells. This antibody and antibodies to the high-molecular-weight form of CD45 (CD45R) seem to define complementary, largely nonoverlapping populations in resting peripheral T cells demonstrating heterogeneity within the CD4 and CD8 subsets. The antibody labels most thymocytes, a subpopulation of resting T cells within both the CD4 and CD8 subsets, and mature activated T cells. Cells of the myelomonocytic series, e.g., granulocytes and monocytes, are also labeled, whereas most normal B cells and NK cells are consistently negative. Weak cytoplasmic staining is however seen in cases of centroblastic and immunoblastic lymphoma. CD3 antibody has been considered the best all around Tcell marker. This antibody reacts with an antigen present in early thymocytes. The positive staining of this marker may represent a sign of early commitment to the T cell lineage. Anti-CD1a is a murine monoclonal antibody that reacts with CD1a, a nonpolymorphic MHC class-I related cell

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FIGURE 28.24 CD20 — tonsil.

surface glycoprotein, expressed in association with β2 microglobulin. In normal tissues the antibody reacts with cortical thymocytes, Langerhans cells, and interdigitating reticulum cells. It also reacts with thymomas, Langerhans histiocytosis cells (histiocytosis X), and some T cell lymphomas and leukemias. The staining is localized on the membrane. Anti-CD5 monoclonal antibody detects CD5 antigen, which is expressed in 95% of thymocytes and 72% of peripheral blood lymphocytes. In lymph nodes, the main reactivity is observed in T cells. CD5 antigen is expressed by many T cell leukemias, lymphomas, and activated T cells. CD5 antigen is also expressed on a subset of B cells. CD5 is recommended for the identification of mantle cell lymphomas. Antibodies to CD5 may prove to be of particular use in the detection of T cell acute lymphocytic leukemias (T-ALL), some B cell chronic lymphocytic leukemias (B-CLL), as well as B and T cell lymphomas. CD5 does not react with granulocytes or monocytes. Ki-67 or -780 are nuclear antigens expressed by both normal and neoplastic-proliferating cells. They are demonstrable by immunoperoxidase staining. A relatively high percentage of positive cells in a neoplasm implies an unfavorable prognosis. Anti-Ki-67 (MIB) is a mouse monoclonal antibody directed against the Ki-67 nuclear antigen. This reagent may be used to aid in the identification of proliferating cells in normal and neoplastic cell populations. It is intended for qualitative staining in sections of formalin-fixed, paraffinembedded tissue (some form of antigen enhancement is required for paraffin-embedded samples), frozen tissue, and cytologic preparations. Ki-67 antibody specifically binds to nuclear antigen(s) associated with cell proliferation which is present throughout the active cell cycle (G1, S, G2, and M phases) but absent in resting (G0) cells. Unexpected


FIGURE 28.26 Hodgkin’s disease.

FIGURE 28.25a Ki-67 — Carcinoma of the breast.

antigen expression or loss of expression may occur, especially in neoplasms. Occasionally, stromal elements surrounding heavily stained tissues and or cells will show immunoreactivity. The clinical interpretation of any staining or its absence must be complemented by morphological studies and evaluation of proper controls. Ki-1 (CD30 antigen) is a marker of Reed-Sternberg cells found in Hodgkin’s disease of the mixed-cellularity, nodular-sclerosing, and lymphocyte-depleted types and in selected cases of large-cell non-Hodgkin’s lymphomas (Figure 28.27). Antihuman Ki-1 antigen, CD30 is a mouse monoclonal antibody that reacts with a 595-amino acid transmembrane, 121-kDa glycoprotein. It contains six cysteine-rich motifs in the extracellular domain and is homologous to members of the nerve growth factor receptor superfamily. The CD30 gene was assigned to the short arm of chromosome 1 at position 36. The CD30 antigen was initially designated Ki-1. The antibody detects a formalin-resistant epitope on the 90-kDa precursor molecule. This molecule

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FIGURE 28.27 CD30 — Hodgkin’s disease.

is processed in the Golgi system into the membrane-bound phosphorylated mature 120-kDa glycoprotein and into the soluble 85-kDa form of CD30, which is released from the supernatant and appears in serum at detectable levels in conditions such as infectious mononucleosis or neoplastically amplified CD30-positive blasts. The CD30 antigen is expressed by Hodgkin’s and Reed-Sternberg cells in Hodgkin’s disease, by the tumor cells of a majority of anaplastic large-cell lymphomas, and by a varying proportion of activated T and B cells. It is also expressed on embryonal carcinomas. CD15 (Leu M1) is a monoclonal antibody that recognizes the human myelomonocytic antigen lacto-N fucopentose III. It is present on greater than 95% of mature peripheral blood eosinophils and neutrophils and is present at low density on circulating monocytes. In lymphoid tissue, CD15 reacts with Reed-Sternberg cells of Hodgkin’s disease and with granulocytes. CD15 reacts with few tissue macrophages and does not react with dendritic cells.


Leu-M1 (CD15) (Figure 28.26) is a granulocyte-associated antigen. Immunoperoxidase staining detects this marker on myeloid cells but not on B or T cells, monocytes, erythrocytes, or platelets. It can be detected in Hodgkin’s cells and Reed-Sternberg cells. CD20 primary antibody (Figure 28.24) is a mouse monoclonal antibody (Clone L26) directed against an intracellular epitope of the CD20 antigen present on human B lymphocytes. This reagent may be used to aid in the identification of cells of B lymphocytic lineage. The antibody is intended for qualitative staining in sections of formalinfixed paraffin-embedded tissue. Anti-CD20 antibodies specifically bind to antigens located in the plasma membrane and cytoplasmic regions of normal B lymphocytes which may also be expressed in Reed-Sternberg cells. Unexpected antigen expression or loss of expression may occur, especially in neoplasms. Occasionally, stromal elements surrounding heavily stained tissue and/or cells may show immunoreactivity. The clinical interpretation of any staining, or its absence, must be complemented by morphological studies and evaluation of proper controls. The CD21 antigen is a restricted B cell antigen expressed on mature B cells. The antigen is present at high denisty on follicular dendritic cells (FDC), the accessory cells of the B zones. It shows moderate labeling of B cells and a strong labeling of FDC in cryostat sections, whereas the staining of B cells is reduced or abolished in paraffin sections. However, the labeling of FDC in paraffin sections is as strong as on cryostat sections. The antibody reacts with FDC meshwork in normal and hyperplastic lymph nodes and tonsils. Sharply defined, dense meshwork of FDC in germinal centers is revealed. Follicular mantles of secondary and primary follicles show a loosely textured and ill-defined meshwork of FDC. Immunohistological analysis of FDC in paraffin sections of non-Hodgkin’s lymphomas demonstrates a nodular and usually a dense and sharply defined FDC meshwork in follicular lymphomas (e.g., centroblastic/centrocytic lymphoma) and a loose, ill-defined FDC meshwork of varying size in some diffuse lymphoma types (e.g., centrocytic lymphoma). Precursor B cell lymphomas (lymphoblastic lymphomas), Burkitt’s lymphomas, plasmacytomas, and hairy cell leukemias constantly lack FDC. FDC in nonHodgkin’s lymphomas is mainly restricted to peripheral T cell lymphomas of angioimmunoblastic lymphadenopathy (AILD) type and some cases of pleomorphic T cell lymphomas. The FDC meshwork in AILD contains constant hyperplastic venules in contrast to pleomorphic T cell lymphomas. In contrast to B cell lymphomas, the FDC meshworks in T cell lymphomas and AILD contain only a relatively small number of B cells.

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CD10 is a mouse monoclonal antibody that reacts with common acute lymphoblastic leukemia antigen (CALLA/CD10) as a useful marker for the characterization of childhood leukemia and B cell lymphomas. This antibody reacts with antigen of lymphoblastic, Burkitt’s, and follicular lymphomas; and chronic myelocytic leukemia. Also, CD10 detects the antigen of glomerular epithelial cells and the brush border of the proximal tubules. This characteristic may be helpful in interpreting renal ontogenesis in conjunction with other markers. Other nonlymphoid cells that are reactive with CD10 are breast myoepithelial cells, bile canaliculi, neutrophils, and small population of bone marrow cells, fetal small intestine epithelium, and normal fibroblasts. Common acute lymphoblastic leukemia antigen (CALLA/CD10) is a useful marker for the characterization of childhood leukemia and B cell lymphomas. This antibody reacts with antigen of lymphoblastic, Burkitt’s, and follicular lymphomas; and chronic myelocytic leukemia. Also, CD10 detects the antigen of glomerular epithelial cells and the brush border of the proximal tubules; this characteristic may be helpful in interpreting renal ontogenesis in conjunction with other markers. Other nonlymphoid cells that are reactive with CD10 are breast myoepithelial cells, bile canaliculi, neutrophils, and small population of bone marrow wells, fetal small intestine epithelium, and normal fibroblasts. Cyclin D1 (polyclonal), rabbit: Anti-Cyclin D1 is a rabbit polyclonal antibody that detects Cyclin D1, one of the key cell-cycle regulators that is a putative protooncogene overexpressed in a wide variety of human neoplasms. Cyclins are proteins that govern transitions through distinct phases of the cell cycle by regulating the activity of the cyclin-dependent kinases. In mid to late G1, Cyclin D1 shows a maximum expression following growth factor stimulation. Cyclin D1 has been successfully employed and is a promising tool for further studies in both cellcycle biology and cancer-associated abnormalities. This antibody is useful for separating mantle cell lymphomas (Cyclin D1-positive) from SLLs and small cleaved-cell lymphomas (Cyclin D1-negative). Antihuman kappa light chain (Figure 28.28) is a rabbit antibody that reacts with free kappa light chains as well as kappa chains in intact immunoglobulin molecules. This antibody may be used for typing of free and bound monoclonal light chains by immunoelectrophoresis and immunofixation. It may also be used for immunohistochemistry. Anti-LN1is a mouse monoclonal antibody against a sialoglycan antigen (CDw75) on cell membranes. In lymphoid tissues, the antibody reacts strongly with the B lymphocytes in the germinal centers, but only faintly with B lymphocytes of the mantle zone. No reaction is observed


nodular lymphocyte predominant Hodgkin’s disease. In contrast, anti-BCL-6 rarely stains mantle cell lymphoma, and MALT lymphoma bcl-6 expression is seen in approximately 45% of CD30+ anaplastic large cell lymphomas but is consistently absent in other peripheral T cell lymphomas. CD23(1B12): Anti-CD23 mouse monoclonal antibody is a B cell antibody that is useful in differentiating between B-CLL and B-SLLs that are CD23 positive from mantle cell lymphomas and small cleaved lymphomas that are CD23 negative. This antibody reacts with the antigen that is found on a subpopulation of peripheral blood cells, B lymphocytes, and on EBV transformed B-lymphoblastoid cell lines. FIGURE 28.28 Kappa light chain — tonsil.

with T lymphocytes. LN1 also reacts with certain epithelial cells, including cells of the distal renal tubules, breast, bronchus, and prostate. Immunoglobulin is demonstrable by immunoperoxidase staining of plasma cell and B lymphocyte cytoplasm in frozen or paraffin-embedded sections. B-5 fixative is preferable to formalin for demonstration of intracellular IgG or light chains in paraffin sections. Monoclonal cytoplasmic staining for either κ or λ light chains aids the diagnosis of B cell lymphomas. Antihuman lambda light chain is a rabbit antibody that reacts with free lambda light chains as well as the lambda light chains in intact immunoglobulin molecules. Anti-bcl-2 primary antibody contains a mouse monoclonal antibody. The bcl-2 oncoprotein expression is inhibited in germinal centers where apoptosis forms a part of the B cell production pathway. In 90% of follicular lymphomas, a translocation occurs which justaposes the bcl-2 gene at 19q21 to an immunoglobulin gene, with subsequent deregulation of protein synthesis and cell proliferation. The bcl-2 product is considered to act as an inhibitor of apoptosis. This observation has turned out to have clinical implications. Distinction of follicular hyperplasia from follicular lymphoma is a common problem in histopathology. Reactive follicles show no staining for bcl2, whereas the cells in neoplastic follicles exhibit membrane staining. Anti-BCL-6 (PG-B6p) mouse monoclonal antibody: This is a transcriptional regulator gene which codes for a 706-amino-acid nuclear zinc finger protein. Antibodies to this protein stain the germinal center cells in lymphoid follicles, the follicular cells and interfollicular cells in follicular lymphoma, diffuse large B cell lymphomas, and Burkitt’s lymphoma, and the majority of the Reed-Sternberg cells in

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CD31 (JC/70A): Anti-CD31 mouse monoclonal antibody detects CD31 expressed by stem cells of the hematopoietic system and is primarily used to identify and concentrate these cells for experimental studies as well as for bone marrow transplantation. Endothelial cells also express this marker; therefore, antibodies to CD31 have been used as a tool to identify the vascular origin of neoplasms. CD31 has shown to be highly specific and sensitive for vascular endothelial cells. Staining of nonvascular tumors (excluding hematopoietic neoplasms) has not been observed. Anti-CD34 (Figure 28.29) is a murine monoclonal antibody, raised by immunization with human placental endothelial cells, that has a specificity for the CD34 glycoprotein, which is considered the earliest known CD marker and is expressed on virtually all human hematopoietic progenitor cells. Anti-CD43 is a murine monoclonal antibody directed against an epitope present on human monocytes, granulocytes, and lymphocytes. This reagent may be used to aid in the identification of cells of lymphoid lineage. It is

FIGURE 28.29 CD34 — highly vascular tumor.


FIGURE 28.31 Myeloperoxidase — bone marrow. FIGURE 28.30 CD68 — tonsil.

intended for qualitative staining in sections of formalinfixed, paraffin-embedded tissue. Anti-CD43 antibody specifically binds to antigen located in the plasma membrane and cytoplasmic regions of normal granulocytes or T lymphocytes. Anti-CD68 (human macrophage marker) (Figure 28.30) is a murine monoclonal antibody that stains macrophages and a wide variety of human tissues, including Kupffer cells and macrophages in the red pulp of the spleen, in the lamina propria of the gut, in lung alveoli, and in bone marrow. Antigen-presenting cells, such as Langerhans cells, are either negative or show weak and/or restricted areas of reactivity, e.g., interdigitating reticulum cells. Resting microglia in the normal white matter of the cerebrum and microglia in areas of infarction react with the antibody. Peripheral blood monocytes are also positive, with a granular staining pattern. The antibody reacts with myeloid precursors and peripheral blood granulocytes. The antibody also reacts with the cell population known as “plasmacytoid T cells” which are present in many reactive lymph nodes and which are believed to be of monocyte/macrophage origin. The antibody stains cases of chronic and acute myeloid leukemia, giving strong granular staining of the cytoplasm of many cells, and also reacts with rare cases of true histiocytic neoplasia. The positive staining of normal and neoplastic mast cells is seen with the antibody as well as staining of a variable number of cells in malignant melanomas. Neoplasms of lymphoid origin are usually negative, although some B cell neoplasms, most frequently small lymphocytic lymphoma and hairy cell leukemia, show weak staining of the cytoplasm, usually in the form of a few scattered granules. CD99 (HO36-1.1): Anti-CD99 mouse monoclonal antibody reacts with MIC-2 antigen present on the cell membrane of Ewing’s sarcoma and primitive peripheral neuroectodermal tumors (PNET). It is also present on some

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bone marrow, lymph nodes, spleen, cortical thymocytes, granulosa cells of the ovary, most beta cells, CNS ependymal cells, Sertoli’s cells of the testis, and a few endothelial cells. MIC-2 has also been identified in lymphoblastic lymphoma, rhabdomyosarcoma, mesenchymal chondrosarcoma, and thymoma. CD117 (c-kit) (polyclonal), rabbit: Anti-CD117 is a purified immunoglobulin fraction of rabbit antiserum that detects CD117, a tyrosine kinase receptor found on interstitial cells of Cajal, germ cells, bone marrow stem cells, melanocytes, breast epithelium, and mast cells. This receptor is found on a wide variety of tumor cells (follicular and papillary carcinoma of thyroid; adenocarcinomas from endometrium, lung, ovary, pancreas, and breast; and malignant melanoma, endodemal sinus tumor, and small cell carcinoma) but has been particularly useful in differentiating gastrointestinal stromal tumors from Kaposi’s sarcoma and tumors of smooth muscle origin. Antihuman myeloperoxidase antibody (Figure 28.31) is an antibody that is used to discriminate between lymphoid leukemias and myeloid leukemias in formalin-fixed paraffin-embedded tissues. Antihuman luteinizing hormone (LH) is a rabbit antibody that labels gonadotropic cells of the pituitary. Positive staining for adenohypophyseal hormones assists in classification of pituitary tumors. Lutenizing hormone (LH) is an adenohypophyseal glycoprotein hormone found in gonadotropic cells of the anterior pituitary gland of most mammals. Gonadotropic cells average about 10% of anterior pituitary cells. α-1 antichymotrypsin is a histiocytic marker. By immunoperoxidase staining, it is demonstrable in tumors derived from histiocytes. It may also be seen in various carcinomas. Factor VIII (Figure 28.32) is a coagulation protein produced by endothelial cells, which makes it a useful marker


FIGURE 28.32 Factor VIII — placenta.

FIGURE 28.33 Cytomegalovirus (CMV) — placenta.

for vascular tumors. It is demonstrable by immunoperoxidase staining. Megakaryocytes and platelets also stain for factor VIII.

although perinuclear cytoplasmic staining of koilocytotic cells may also be seen.

Antifactor VIII is a mouse monoclonal antibody that gives positive staining in the cytoplasm of normal vascular endothelial cells of arteries, veins, capillaries, and endocardial cells. Factor VIII related antigen is also present in megakaryocytes and platelets. Antihuman hemoglobin is a rabbit antibody against hemoglobin A, isolated from erythrocytes of normal adults, that reacts with hemoglobin A and, due to a common alpha chain, also with hemoglobin A2 and hemoglobin F. 200-4 nuclear matrix protein is a marker expressed preferentially by malignant cells rather than by normal cells. It is demonstrated by immunoperoxidase staining. Anticytomegalovirus antibody (Figure 28.33) is a mouse monoclonal antibody that reacts with CMV infected cells giving a nuclear staining pattern with early antigen and a nuclear and cytoplasmic reaction with the late viral antigen. The antibody shows no crossreactivity with other herpesviruses or adenoviruses. Antihepatitis B virus core antigen (HBcAg) antibody labels the nuclei and occasionally cytoplasm of virus infected cells. HBcAg is expressed predominantly in the nuclei of infected liver cells, although variable staining may also be seen in the perinuclear cytoplasm. Antipapillomavirus is a rabbit antibody against papillomavirus. The structural antigens on this virus can be detected in a variety of proliferative squamous lesions. Only 50 to 60% of lesions caused by papillomavirus will express the structural antigens. This antibody staining is predominantly intranuclear in a focal or diffuse pattern,

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Collagen Type IV (CIV22): Anti-Collagen Type IV is a mouse monoclonal antibody that detects Collagen Type IV, the major component of the basal lamina. Antibodies to this molecule confirm its presence and reveal the morphological appearance of the structure. Normal tissue stains with this antibody in a fashion consistent with the sites of mesenchymal elements and epithelial basal laminae. Collagen IV can also be useful in the classification of soft tissue tumors, Schwanomas, and leiomyomas, and their well-differentiated malignant counterparts usually immunoreact to this antibody. The vascular nature of neoplasma, hemangiopericytoma, angiosarcoma, and epitheliod hemangioendothelioma can be revealed by this antibody with greater reliability than nonspecific stains (e.g., silver reticulum). Polyclonal rabbit anti-calretinin is intended to qualitatively detect normal and malignant mesothelial cells in formalin-fixed, paraffin-embedded tissue sections using light microscopy. Calretinin, a calcium-binding protein with a mol wt of 29 kDa, is a member of the large family of EF-hand proteins that also include S-100 protein. EFhand proteins are characterized by a helix–loop–helix fold that acts as the calcium-binding site. Calretinin contains six such EF-hand stretches. It is abundantly expressed in central and peripheral neural tissues, especially in the retina and neurons of the sensory pathways. Calretinin is also consistently expressed in normal and reactive mesothelial cell lining of all serosal membranes, eccrine glands of skin, convoluted tubules of kidney, Leydig and Sertoli cells of the testis, endometrium and ovarian stromal cells, and adrenal cortical cells. Calretinin is also a sensitive and specific indicator of normal and reactive mesothelial cells in effusion cytology. This antibody is useful as part of an immunohistochemical marker panel to distinguish


mesothelioma from adenocarcinoma. The combination of calretinin and E-Cadherin was shown to have high sensitivity and specificity in differentiating malignant mesothelioma from metastatic adenocarcinoma to the pleura in one study. P63 (ap53 Homolog at 3q27–29) Ab-4 (cocktail) mouse monoclonal antibody recognizes a 63-kDa protein, identified as p63. Ths p63 gene, a homolog of the tumorsuppressor p53, is highly expressed in the basal or progenitor layers of many epithelial tissues. Protein p63 shows remarkable structural similarity to p53 and to the related p73 gene. Unlike p53, the p63 gene encodes multiple isotypes with remarkable divergent abilities to transactivate p53 reporter genes and induce apoptosis. NeoMarkers’ Ab-4 recognizes all known isotypes of p63. TTF-1 (8G7G3/a), mouse: Thyroid transcription factor1 is useful in differentiating primary adenocarcinoma of the lung from metastatic carcinomas from the breast and malignant mesothelioma. It can also be used to differentiate small-cell lung carcinoma from lymphoid infiltrates. Myogenin (F5D), mouse: Anti-Myogenin monoclonal antibody labels the nuclei of myoblasts in developing muscle tissue and is expressed in tumor cell nuclei of rhabdomyosarcoma. Positive nuclear staining may occur in Wilms’ tumor, as well as in some myopathies. Neurofilament (2F11), mouse: Neurofilament antibody stains an antigen localized in a number of neural, neuroendocrine, and endocrine tumors. Neuromas, ganglioneuromas, gangliogliomas, ganglioneuroblastomas, and neuroblastomas stain positively for neurofilament. Neurofilament is also present in paragangliomas and adrenal and extra-adrenal pheochromocytomas. Carcinoids, neuroendocrine carcinomas of the skin, and oat cell carcinomas of the lung also express neurofilament. Anti-p53 primary antibody (clone Bp53-11) is a mouse monoclonal antibody directed against both the mutant and wild-type of the p53 nuclear phosphoprotein. Very rare normal cells express p53, but alterations in the p53 suppressor gene result in an overproduction of this protein in malignancies. This reagent may be used to aid in the identification of abnormally proliferating cells in neoplastic cell populations. The antibody is intended for qualitative staining in sections of formalin-fixed, paraffin-embedded tissue on a Ventana automated slide staining device. Some form of antigen enhancement is required for paraffin-embedded samples. The p53 antibody specifically binds to nuclear

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antigen(s) associated with the normal downregulation of cell division. Increased expression of p53 in actively dividing cells is an indication of loss of function due to mutation of the p53 gene. Inhibin, alpha (R1), mouse: Anti-Inhibin alpha is an antibody against a peptide hormone which has demonstrated utility in differentiation between adrenal cortical tumors and renal cell carcinoma. Sex cord stromal tumors of the ovary, as well as trophoblastic tumors, also demonstrate cytoplasmic positivity with this antibody. MART-1 (M2-7C10), mouse: MART-1 (also known as Melan A) is a melanocyte differentiation antigen. It is present in melanocytes of normal skin and retina, nevi, and in more than 85% of melanomas. This antibody is very useful in establishing the diagnosis of metastatic melanomas. BRST-2 (GCDFP-15), monoclonal antibody (murine) detects BRST-2 antigen expressed by apocrine sweat glands, eccrine glands (variable), minor salivary glands, bronchial glands, metaplastic epithelium of the breast, benign sweat gland tumors of the skin, and the serous cells of the submandibular gland. Breast carcinomas (primary and metastatic lesions) with apocrine features express the BRST-2antigen. BRST-2 is positive in extramammary Paget’s Disease. Other tumors tested are negative. E-Cadherin (ECH-6), mouse: Anti-E-Cadherin mouse monoclonal antibody detects E-Cadherin, an adhesion protein expressed in cells of epithelial lineage. It stains positively in glandular epithelium as well as adenocarcinomas of the lung and GI tract and ovary. It has been useful in distinguishing adenocarcinoma from mesothelioma. It has also been shown to be positive in some thyroid carcinomas. Fascin (55k-2), mouse: Fascin is a very sensitive marker for Reed-Sternberg cells and variants in nodular sclerosis, mixed cellularity, and lymphocyte depletion Hodgkin’s disease. It is uniformly negative in lymphoid cells, plasma cells, and myeloid cells. Fascin is positive in dendritic cells. This marker might be helpful in distinguishing between Hodgkin’s disease and non-Hodgkin’s lymphoma in difficult cases. Also, the lack of expression of fascin in the neoplastic follicles in follicular lymphoma can be helpful in distinguishing these lymphomas from reactive follicular hyperplasia in which the number of follicular dendritic cells is normal or increased.

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