Stains A-E
(routine stains, immunostains and molecular markers)
Last revised 26 October 2008
Copyright © 2002-2008 PathologyOutlines.com, Inc.
See also CD Marker chapters
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Note: stains/proteins are in alphabetical order, with numbers before letters, and ignoring dashes and spaces
Primary references, immunohistochemistry basics, common panels, biopsy interpretation, enzyme cytochemistry
Note: the chapter has many more topics than are included in the table of contents, which is being updated
A: acid fast bacilli; actins: general, muscle specific; alpha-1-antitrypsin
B:
C: caldesmon, calponin, calretinin
Cytokeratins: general, CK1, CK2, CK3, CK4, CK5, CK6, CK7, CK8, CK9, CK10, CK11, CK12, CK13, CK14, CK15, CK16, CK17, CK18, CK19, CK20, CK21, CK22, CK23, CK24, 34betaE12, 35betaH11, AE1, AE3, AE1-AE3, CAM5.2, KL-1, MNF116
D:
E:
Go to Stains F-Z and cell cycle
American Journal of Surgical Pathology (AJSP)
Archives of Pathology and Lab Medicine (Archives)
Human Pathology (Hum Path)
Modern Pathology (Mod Path)
Rosai, J: Ackerman’s Surgical Pathology (9th Ed); Mosby, 2004
University of Pittsburgh Medical Center Case Reports
Please refer to these primary references for more detailed discussions and photographs
Antibodies are often useful beyond their recommended expiration dates
Focus on what type of cells are staining (tumor cells, endothelial cells, stromal cells)
Note the number (percent) of cells staining, the intensity of staining and the pattern of staining (cytoplasmic, membranous, nuclear, dot like)
Pattern of immunoreactivity must follow the anatomic distribution of the antigen before it is called positive / immunoreactive
Repeating or performing additional tests may be important when reviewing slides of patients with prior diagnosis of cancer (AJSP 2002;26:1222)
Sources of error in interpretation are ectopic antigen expression, cross reactions, less specificity than thought
Recommended to obtain contemporaneous H&E section (AJSP 2007;31:1627)
Note: diagnosis should be based on H&E morphology, with confirmation by immunohistochemistry or molecular testing; it is dangerous to use immunohistochemistry alone to make the diagnosis
Note: combining results from different studies may be hazardous, as studies may use different antibodies and different standards of interpretation
Steps in immunohistochemistry:
1. pretreatment, often with microwaving of tissue in citrate buffer to unmask antigens hidden by formalin cross-links or other fixative
2. other agents for pretreatment (antigen retrieval) are pepsin, proteases, trypsin
3. apply primary antibody (monoclonal antibodies usually are more specific); antibody binds to antigens of interest
4. wash off excess primary antibody
5. add biotinylated anti-IgG antibody (secondary antibody), which binds to the primary antibody present
6. add avidin-biotin-peroxidase complex, which binds to secondary antibody
7. add 3, 3’ diaminobenzidine (DAB) as a chromagen (color changing reagent), with hematoxylin counterstaining
Other enzyme complexes besides avidin-biotin are horseradish peroxidase, alkaline phosphatase with naphthol phosphate and glucose oxidase with nitroblue tetrazolium
Other chromagens besides DAB are AEC (water soluble, sensitive to light)
Most important steps are selection of appropriate antibodies, correct interpretation, technical quality and integration of results into final diagnosis (AJSP 2002;26:873)
Common panels of immunohistochemistry stains
Epithelial markers: low molecular weight keratin (CAM 5.2), AE1-AE3 cytokeratin cocktail, CK7, CK20, CEA, EMA
Melanocytic markers: S100 (also a mesenchymal marker), HMB45, MelanA/Mart1
Mesenchymal markers: vimentin, Factor XIIIa, Factor VIII, CD31, CD34, HHF35, smooth muscle actin, desmin
Lymphoid markers: CD3, CD20, CD15, CD30, various others
Histiocytic markers: CD68, lysozyme, CD1a (Langerhans cells)
Neuroendocrine markers: neuron specific enolase, chromogranin, synaptophysin
Cell proliferation/apoptosis markers: Ki-67, bcl2
Recommended to interpret immunohistochemical stains in small needle core biopsy specimens based on the area with the greatest immunoreactivity (AJCP 2007;127:273)
Detects enzymatic activity in cytoplasm
Enzyme product unites with coupler, which produces localized color at site of enzyme activity
Fresh smears are preferred, especially for myeloperoxidase; if not possible, store unstained slides away from light
3 beta hydroxysteroid dehydrogenase
Critical enzyme in biosynthesis of all steroid hormones
Positive staining (normal): testicular Leydig cells
Negative staining: normal seminiferous tubules
Micro images: 3 beta-hydroxysteroid dehydrogenase immunohistology in adrenal gland
7-amino-actinomycin D (7 AAD)
DNA-binding, fluorescent dye is excited by 488 nm laser line commonly used in flow cytometry
Intact cells exclude 7 AAD; dead cells allow 7 AAD entry, which binds to DNA
Used in flow cytometry to reduce non-specific staining by eliminating 7 AAD positive cells (dead cells) from further analysis
14-3-3 sigma protein
Member of highly conserved family of acidic proteins
Phosphoserine binding protein that mediates G2/M arrest; also other cellular signaling pathways
May be a tumor suppressor, induced by DNA damage and p53
Cytoplasmic staining
Micro images: expression in normal and malignant tissue
Positive staining (normal): urothelium, prostate and breast periductal and periglandular cells, uterus (strong in squamous epithelium, weak in endometrial and endocervical glands)
Positive staining-tumors: bladder urothelial carcinoma (98%), cervical squamous cell carcinoma (67%), endometrial adenocarcinoma (57%), prostatic adenocarcinoma (55%), ovarian carcinoma (33%), testicular tumors (27%), breast carcinoma (23%), renal carcinoma (12%)
Negative staining: germinal cells of testis and ovary, kidney (sporadic expression in tubules)
References: Mod Path 2005;18:340
45M1
Recognizes peptide core of M1 gastric mucin antigen
Positive staining (normal): normal gastric epithelium
Positive staining (disease): intestinal metaplasia in Barrett’s esophagus, AJSP 2001;25:87
Negative staining: mature small intestinal goblet cells
A beta 42
42 amino acid protein; variant of APP
May be prone to forming plaques in Alzheimer’s
Deposited early in plaques; may be a seed for other plaques
abl
also called c-abl; gene at 9q34.1, named after abelson murine leukemia virus
Functions as a tyrosine kinase / signal transducer and a negative regulator of apoptosis
Overexpression causes resistance to apoptosis induction by Fas, ceramide or chemotherapy
Overexpressed in chronic myelogenous leukemia
Acid fast bacilli (AFB) - Stains chapter
last updated October 2008
Acid fast refers to microorganisms whose cell wall has a high lipid content of mycolic acids and long chain fatty acids (diagram), which causes them to bind and retain the complex basic dye carbol-fuchsin even after strong decolorization with acid-alcohol (thus “acid-fast”)
Acid fast organisms: Mycobacteria, Cryptosporidium parvum, Isospora and Cyclospora cysts, hooklets of cysticerci
Partially acid fast organisms: exhibit both acid fast and non acid fast bacilli and filaments in a single strain - nocardiae (Univ Texas Med Branch), Dietzia (Int J Syst Evol Microbiol 2006;56:1667), Rhodococcus (South Med J 1991;84:1217), Gordonia (Emerg Infect Dis 2000;6:382), Tsukamurella (J Med Case Reports 2008;2:207), rarely Mycobacterium peregrinum (J Clin Microbiol 2005;43:2015)
Recommended that laboratories standardize and optimize their particular processes (J Clin Pathol 2003;56:613)
Note: nucleic acid based tests can rapidly detect and speciate mycobacteria (Archives 2008;132:1333, Thorax 2008;63:317)
Acid-fast methods:
Ziehl-Neelsen (classic) - commonly used; bacteria stain bright red due to retention of carbol-fuchsin dye, against blue background due to methylene blue counterstain; procedure involves heat (procedure #1, #2)
Ziehl-Neelsen (modified bleach) - more sensitive than classic (Acta Cytol 2008;52:325)
Kinyoun - commonly used; uses more concentrated fuchsin dye and lipid solvent, but no heat; bacteria stain bright red against green background (procedure #1, #2)
Fite - used for detecting M. leprae (leprosy) and Rhodococcus (Diagn Cytopathol 2001;24:244); combines peanut/vegetable oil with xylene to minimize exposure of bacteria cell wall to organic solvents and protect precarious acid-fastness of organism (procedure #1, #2)
Ellis and Zabrowarny - excludes phenol; procedure (J Clin Pathol 1993;46:559)
Auramine-rhodamine - mixture of Auramine O and Rhodamine B dyes, auramine binds to mycolic acid in cell wall; detection requires a fluorescence microscope (mercury vapor lamp or LED), but is the most sensitive stain for mycobacteria (Hum Path 1984;15:1085) [nucleic acid methods are more sensitive but are not stains]; saves time in searching for microorganisms (Clin Infect Dis 2008;47:203); procedure
Water filters are recommended to reduce false positives due to nonTB mycobacteria (Appl Environ Microbiol 2007;73:6296)
In Mexico, nontuberculous mycobacteria cause superinfection of lipoid pneumonia (caused by aspiration), associated with malnourished infants (Fetal Pediatr Pathol 2006;25:107)
Acid fast bacilli (AFB) - Stains chapter (continued)
Micro images:
Cryptosporidium - oocysts-modified acid-fast #1; #2; stool specimen (Ziehl-Neelsen); oocysts-auramine-rhodamine stain
Mycobacterium tuberculosis - site
unknown (Ziehl-Neelsen); cervix; lung
(Ziehl-Neelsen); lung
(auramine), small
intestine #1 (method unknown); #2
Mycobacterium avium-complex - Ziehl-Neelsen;
bone marrow #1;
#2-M. kansasii;
breast (Ziehl-Neelsen); colon
(Ziehl-Neelsen); liver; lymph
node post bCG vaccination (Ziehl-Neelsen); spleen; stomach-modified
Fite stain
Mycobacterium leprae - liver-Fite stain; skin-Fite stain
Nocardia - lung-Fite-Faraco Modified Acid Fast stain #1; #2; #3; #4
Rhodococcus - Kinyoun modified acid-fast stain of non-lysed sputum, AFB+ cocci can be seen in a macrophage
Other - Kayexalate particles at site of aspiration pneumonia (Ziehl-Neelsen with light counterstain)
Acid phosphatase
Enzyme histochemistry technique
Positive staining: osteoclasts
Enzyme cytochemistry: T-ALL (focal paranuclear), AML (variable)
Acridine Orange
Used for staining low numbers of bacteria; examine under ultraviolet light
Actin is globular protein found in all eukaryotic cells except nematode sperm
Highly conserved, differs by at most 20% between algae and humans
Monomeric subunit of microfilaments, one of 3 major components of cytoskeleton (also microtubules and intermediate filaments); also a component of thin filaments (part of contractile apparatus of muscle cells)
Mammals have at least 6 actin isoforms - two smooth muscle (alpha smooth muscle and gamma smooth muscle), two sarcomeric (alpha cardiac and alpha skeletal) and two nonmuscle (beta cytoplasmic and gamma cytoplasmic)
Muscle cells contain alpha and gamma smooth muscle actin, alpha cardiac and alpha skeletal actin
Nonmuscle cells contain beta and gamma cytoplasmic actin
Functions: muscle cells - contraction; all cells - forms part of cytoskeleton, associated with motility
Actin and myosin in muscle: drawing #1; #2; animation
References: Wikipedia
Actin, alpha cardiac type
There are two types of alpha sarcomeric/striated actin: cardiac type and skeletal muscle type; both are expressed in cardiac and skeletal muscle, but the proportions vary at different developmental periods (J Biol Chem 1994;269:12212) or with disease (Rapid Commun Mass Spectrom 2003;17:1467)
Mutations in cardiac type may cause dilated or hypertrophic cardiomyopathy (J Mol Cell Cardiol 2000;32:1687), atrial septal defects (Hum Mol Genet 2007 Oct 18 [Epub ahead of print])
Positive staining (normal): myocardium (adult and fetal), skeletal muscle (fetal), skeletal muscle (adult-muscle spindle myocytes), vascular smooth muscle (occasional)
Positive staining (disease): skeletal muscle (regenerating skeletal muscle cells [Differentiation 1996;60:245], Duchenne muscular dystrophy, degenerative atrophy), rhabdomyosacoma, Wilm’s tumor-rhabdomyomatous cells, occasional smooth muscle tumors
Negative staining (normal): skeletal muscle (adult, but muscle spindle myocytes are positive)
References: Virchows Arch 2006;449:175
Actin, alpha skeletal type
There are two types of alpha sarcomeric/striated actin: cardiac type and skeletal muscle type; both are expressed in cardiac and skeletal muscle, but the proportions vary at different developmental periods (J Biol Chem 1994;269:12212) or with disease (Rapid Commun Mass Spectrom 2003;17:1467)
Absence causes nemaline myopathy (Ann Neurol 2007;61:175)
Positive staining: rhabdomyosarcoma (but not commonly used, AJSP 1985;9:467)
Actin, alpha smooth muscle type
Also called smooth muscle actin, SMA; clone 1A4 or sm-1
Discovered in 1986 (J Cell Biol 1986;103:2787)
Antibodies to alpha smooth muscle actin do not detect the other actin isoforms
Reduced expression in brain blood vessels in Alzheimer patients (J Neuropathol Exp Neurol 2004;63:735)
No apparent deficiency in intestinal pseudoobstruction (J Clin Pathol 2004;57:1168)
Uses:
(a) identify smooth muscle cells and myofibroblasts in normal, reactive (Am J Respir Cell Mol Biol 1999;20:582) or neoplastic tissue (Am J Dermatopathol 2006;28:105)
(b) identify myoepithelial cells in normal, neoplastic or diseased breast, salivary glands or sweat glands; may be helpful to rule out invasion; may be particularly important in cytology specimens (Anticancer Res 2003;23:4175)
(c) identify pericytes, which are associated with mature microvessels and better prognosis in colorectal carcinoma (Oncology 2005;69:159)
(d) help distinguish pleuropulmonary desmoid tumors (SMA+) from solitary fibrous tumor (SMA-, Archives 2006;130:1503)
Note: in breast papillary lesions, p63 is a more sensitive and specific marker because smooth muscle actin also stains stromal cells (J Clin Pathol 2007;60:315)
Interpretation: membranous or cytoplasmic staining
Positive staining (normal): myoepithelial cells of breast (most but not all, Breast Cancer Res 2003;5:R151), salivary glands, sweat glands and tracheobronchial glands (J Histochem Cytochem 1988;36:659); myofibroblasts (except alveolar-J Histochem Cytochem 1992;40:1955 and some granulation tissue/scars-Lab Invest 1989;60:275, Int J Legal Med 1992;105:99), pericytes (J Histochem Cytochem 1989;37:315), smooth muscle, vascular smooth muscle; also chondrocytes (Folia Biol (Praha) 2006;52:167), choroidal non-vascular smooth muscle cells (J Anat 2005;207:381), decidual stromal cells (Hum Reprod 1999;14:1599), fibroblastic reticulum cells (J Cancer Res Clin Oncol 1981;101:149), glomus coccygeum (Archives 1999;123:905), hepatic stellate cells (Virchows Arch 1997;430:195), osteoblasts (J Orthop Res 2002;20:622)
Actin, alpha smooth muscle (continued)
Positive staining (disease): adenoid cystic carcinoma (Archives 1999;123:801), angiomyofibroblastoma (occasionally focal, Hum Path 1997;28:1046), angiomyolipoma, atypical teratoid/rhabdoid tumor (J Neurosurg 1996;85:56), collagenous spherulosis (Mod Path 2006;19:1351), endometrial stromal sarcoma (65%, Gynecol Oncol 2004;92:71), endometriosis-stroma (Pathol Int 2003;53:371), epithelial-myoepithelial carcinoma (AJSP 2007;31:44), epithelioid sarcoma-proximal type (33%, AJSP 1997;21:130), fibromatosis (56%, AJSP 2002;26:1296), fibroblastic reticulum cell tumor (AJSP 1998;22:1048), gastric carcinoma stromal cells (J Clin Pathol 2002;55:741), GIST (45%, AJSP 2002;26:1296), glomus tumor (Hum Path 1999;30:1259), granulosa cell tumors of ovary-adult (variable, Mod Path 1995;8:25), hemangiopericytoma (AJSP 2003;27:737), kidney-focal segmental glomerulosclerosis (Braz J Med Biol Res 2001;34:985), inflammatory myofibroblastic tumor (Ann Diagn Pathol 2001;5:335, AJSP 1992;16:896), leiomyoma, leiomyosarcoma, liposarcoma (focal in some cases, AJSP 2004;28:1257), melanoma-desmoplastic (Am J Dermatopathol 1999;21:537), mesothelioma-sarcomatoid (60%, Histopathology 2003;42:270), MFH (30%, J Clin Pathol 2003;56:666), myoepithelioma (57%, Hum Path 2004;35:14), myofibroblastoma (occasionally focal, Pathology 2005;37:144, AJSP 2001;25:1022), myofibroblastic sarcoma (Chin Med J (Engl) 2007;120:363), nodular fasciitis (Ann Diagn Pathol 2002;6:94), ossifying fibromyxoid tumor (some, J Laryngol Otol 1993;107:75), pancreatic stellate cells post-obstruction (J Surg Res 2003;114:6), plexiform fibrohistiocytic tumor (Histopathology 1991;19:503), pulmonary lymphangioleiomyomatosis (J Clin Pathol 1993;46:479), renal mixed epithelial and stromal tumor (Archives 2006;130:80), rhabdomyoma (focal/rare, Hum Path 1993;24:754, Hum Path 1993;24:608), rhabdomyosarcoma (botryoid type, Pediatr Dev Pathol 2005;8:427), spindle cell carcinoma (AJSP 2001;25:1009), synovial sarcoma (25%, Mod Path 2007;20:760)
Negative staining (normal): cardiac muscle (positive during development-J Cell Sci 2007;120:229), skeletal muscle (J Cell Biol 1985;100:807)
Negative staining (disease): carcinomas (usually), schwannoma, solitary fibrous tumor (Archives 2006;130:1503)
Micro images:
Normal: arterial wall; chondrocytes-ear; decidual stromal cells; eye-choroid and sclera
Breast: myoepithelial cells #1; #2 (fig C/G); adenoid cystic carcinoma (fig B) vs collagenous spherulosis (fig G); basal-like carcinoma (fig a); cellular fibroadenoma (fig a-black cytoplasmic staining) and phyllodes tumor (fig b); fibromatosis; hamartomas-myoid; leiomyoma; metastases (various) due to myofibroblasts; myoepithelial cell disruptions in normal and hyperplastic epithelium
Actin, alpha smooth muscle (continued)
Micro images (continued):
GI: colon-carcinosarcoma; colon-leiomyoma #1 (top); #2; colon-Peutz Jeghers polyp (fig 4); gastric carcinoma-diffuse type (fig A); gastric carcinoma-intestinal type (fig A); liver epithelioid angiomyolipoma; liver glomangioma (fig 5); small intestine, colon and cases of intestinal pseudoobstruction; various sites-inflammatory fibroid polyp
Lung: normal and early pulmonary hypertension; late pulmonary hypertension; lymphangioleiomyomatosis;
inflammatory myofibroblastic tumor: bone; gallbladder; pleura; salivary gland (fig 3b)
leiomyosarcoma: bladder leiomyosarcoma (fig C); cervix; esophagus #1 (fig b); #2; face; mandible (fig D); skin; thyroid
Other: bladder-PEComa; cardiac scar tissue; Crohn’s disease-obliterative muscularization; desmoid tumor of lung (fig D); eye-MFH of conjunctiva (top left); glomus coccygeum (fig d); glomus tumor #1-bone; #2-nasal cavity; heart from fetus with heart block; hemangiopericytoma-sinonasal; kidney-atypical epithelioid angiomyolipoma #1; #2 (fig 3B); kidney-focal segmental glomerulosclerosis; larynx-spindle cell carcinoma (fig 1e); lip-angiomyolipoma (fig 2); melanoma-desmoplastic (fig 7); myofibroblastic sarcoma (left: smooth muscle actin, right: muscle specific actin); myofibroblastoma of lymph node-interstitial cells are SMA+; placenta accreta (fig 3); plexiform fibrohistiocytic tumor (staining of tumor cells around nests); pleomorphic sarcoma/MFH #1; #2 (fig B); salivary gland duct carcinoma-myoepithelial layer rules out invasion; salivary gland sialometaplasia in parotid node; submandibular gland-adenoid cystic carcinoma
Actin, muscle specific - Stains chapter
last updated October 2008
Also called HHF35, MSA
Recognizes all alpha actins (skeletal, smooth, cardiac) and gamma smooth muscle actin; not beta cytoplasmic or gamma cytoplasmic
Recognizes actin expressed in all cells with muscle differentiation (cardiac, smooth and skeletal muscle), myoepithelial cells, myofibroblasts, pericytes and myogenic tumors
Discovered in 1987 (Am J Pathol 1987;126:51)
Uses: