（共享）免疫组化常用标记抗体

上皮及间皮类常用标记抗体

EPITHELIAL AND MESOTHELIAL MARKERS

CK (MNF 116)
- MNF-116 is a pan-cytokeratin antibody.
- Source: Dakopatts.
- Dilution 1:50.
- Antigen retrieval method: HIER (pressure cooking) using EDTA.
- Staining pattern: Cytoplasmic, typically fibrillary, with or without accentuation beneath the cell membrane or in paranuclear region. A diffuse flocculent pattern in the cytoplasm is often produced by endogenous biotin rather than real staining.
- This is an excellent antibody which reacts with a broad spectrum of cytokeratins of different molecular weights (including cytokeratins 5, 6, 8, 17, and possibly 19), i.e. it is considered a "pan-cytokeratin antibody" (although this antibody may not cover the cytokeratins of very low M.W. or very high M.W. well). This antibody will stain up practically all histological types of epithelial tumor, including glandular tumors, undifferentiated carcinomas, keratinizing squamous cell carcinoma, neuroendocrine carcinomas, small cell carcinomas and hepatocellular carcinomas.
- For general purposes, it is adequate to order "CK" alone to detect epithelial differentiation. Order the following cytokeratin antibodies with more restricted reactivities only in selected circumstances, e.g. hepatocellular carcinoma versus other carcinoma types; small cell carcinoma. May consider replacing CK with CAM5.2 for suspected small cell carcinoma.
- Main applications of pan-CK:
a) Diagnosis of "undifferentiated" malignant neoplasm

b) For mucosal sites, diagnosis of subtle carcinoma through demonstration of positive cells that are not part of the normal epithelial architecture

c) Detection of occult carcinoma in lymph node, bone or bone marrow

d) Diagnosis of certain types of mesenchymal tumors, e.g. synovial sarcoma, chordoma

AE1/AE3 (pan-cytokeratin)
- A mixture of two anti-cytokeratin monoclonal antibodies AE1 and AE3.
- Source: Biogenex.
- Dilution 1: 400.
- Antigen retrieval method: HIER (pressure cooking) using EDTA.
- Staining pattern: Cytoplasmic, typically fibrillary, with or without accentuation beneath the cell membrane or in paranuclear region.
- This antibody combination reacts with both low and intermediate M.W. cytokeratins, although it does not stain high M.W. cytokeratins well in paraffin sections.
- In the liver, the hepatocytes are typically negative, while the bile ducts are positive.



CAM 5.2
- Monoclonal antibody against low M.W. cytokeratin (including CK8 and CK18).
- Source: Becton-Dickinson.
- Dilution 1:8.
- Antigen retrieval method: HIER (pressure cooking) using EDTA.
- Staining pattern: Cytoplasmic, typically fibrillary, with or without accentuation beneath the cell membrane or in paranuclear region.
A monoclonal antibody that stains low M.W. cytokeratin best, particularly neuroendocrine cells and glandular epithelium. It does not stain the normal stratified squamous epithelium. In the liver, both the hepatocytes and bile ducts are positive.
Stains most epithelial tumors, and particularly useful for neuroendocrine tumors (e.g. small cell carcinoma), hepatocellular carcinoma and renal cell carcinoma.
- In fact, a combination of CAM 5.2 and AE1/AE3 is helpful for suggesting a diagnosis of hepatocellular carcinoma if CAM 5.2 is positive (cell membrane staining) and AE1/AE3 is negative.
- Major applications:
a) May use CAM5.2 instead of CK for small cell carcinoma and neuroendocrine carcinomas

b) May consider using CAM5.2 in addition to CK for thymomas and spindle cell
carcinomas

c) Aid in diagnosis of hepatocellular carcinoma

d) Diagnosis of mammary and extramammary Paget"s disease

34bE12
- Monoclonal antibody against high M.W. cytokeratin, including cytokeratins 1, 5, 10 and 14.
- Source: Dakopatts
- Dilution 1:10
- Antigen retrieval method: HIER (pressure cooking) using EDTA.
- Staining pattern: Cytoplasmic, typically fibrillary, with or without accentuation beneath the cell membrane or in paranuclear region.
- This antibody stains stratified squamous epithelium and squamous cell carcinomas particularly well.
- Major applications:
a) 34 E12 stains the basal cells of the prostate, i.e. normal/reactive acini or PIN versus invasive adenocarcinoma

b) Staining the myoepithelial cells of salivary glands (hence of some value in study of salivary gland tumors, although neoplastic myoepithelial cells may show inconsistent CK14 reactivity).

c) Staining Mallory hyaline.

Epithelial membrane antigen (EMA)
- Monoclonal antibody E29.
- Source: Dakopatts.
- Dilution 1:10
- Antigen retrieval method: HIER (pressure cooking) using EDTA.
- Staining pattern: Cell membrane and/or cytoplasm
- A marker for epithelial cells. However, since it does not stain carcinomas as consistently as the cytokeratin antibodies, it is generally not as useful for detection of epithelial differentiation. Nonetheless, this may be used as an additional "epithelial marker" in very poorly differentiated carcinoma.
- Plasma cells are also EMA positive -- they should provide an excellent internal positive control to ascertain that the stain is working properly.
- Major applications:
a) Synovial sarcoma: EMA is more sensitive than cytokeratin antibodies for detecting the "epithelial differentiation".

b) Detection of perineurial cell differentiation (EMA+, S100-)

c) Anaplastic large cell CD30+ lymphoma (commonly EMA+)

d) L&H cells in nodular lymphocyte predominant Hodgkin lymphoma

e) Meningeal cells and meningiomas are often EMA+, CK-.

f) Plasmacytomas are not uncommonly EMA+.

Cytokeratin 7 (CK7) and Cytokeratin 20 (CK20)
- Monoclonal antibodies: CK7 (OV-TL12/30) and CK20 (Ks20.8).
- Source: Dakopatts.
- Dilution 1:150 for CK7, and 1:100 for CK20.
- Antigen retrieval method: HIER (pressure cooking) using EDTA.
- Staining pattern: Cytoplasmic, typically fibrillary, with accentuation beneath cell membrane and around nucleus.
- CK7 labels a wide range of epithelial cells and their tumors, except those of the gastrointestinal tract.
- CK20 has very limited and selective distribution, and is normally only found the gastrointestinal epithelium, urothelium and Merkel cells. Their corresponding tumors are also positive. Thus CK20 is of particular value in diagnosis of Merkel cell carcinoma versus small cell neuroendocrine carcinomas of other sites.
- Use of CK7 and CK20 together is of great help in predicting the likely origin of a carcinoma, especially adenocarcinoma (Wang et al, Appl Immunohistochem 1995;3:99-107; Chu et al, Mod Pathol 2000;13:962-972). Focal staining or staining of only a small percentage of cells should be considered NEGATIVE.

Tumor type CK7/CK20 profile Percentage of cases positive
Colorectal adenocarcinoma +/+
-/+
-/- 5-10%
75-95%
0-15%
Colonic mucinous carcinoma +/+
-/+ 9%
91%
Gastric adenocarcinoma
+/+
+/-
-/+
-/- 13-38%
17-25%
35-37%
10-25%
Gastrointestinal carcinoid +/-
-/+
-/- 13%
7%
80%
Pancreatic adenocarcinoma
+/+
+/-
-/+
-/- 62-65%
26-30%
0-9%
0-8%
Lung adenocarcinoma +/+
+/-
-/- 10-11%
72-90%
0-17%
Cholangiocarcinoma
+/+
+/-
-/- 43%
50%
7%
Hepatocellular carcinoma +/+
+/-
-/+
-/- 0-7%
9-17%
0-9%
77-82%
Ovary serous & endometrioid adenocarcinoma
+/+
+/- 0-4%
96-100%
Endometrial carcinoma +/+
+/-
-/- 0-12%
80-100%
0-8%
Ovarian mucinous tumors +/+
+/- 93%
7%
Breast carcinoma +/+
+/-
-/+ 0-14%
84-96%
2%
Mucinous (colloid) carcinoma of breast +/-
-/- 93%
7%
Prostatic adenocarcinoma +/+
+/-
-/+
-/- 0-8%
0-8%
0-23%
100-62%
Renal cell carcinoma +/-
-/+
-/- 11-24%
0-6%
71-89%
Malignant mesothelioma +/-
-/- 65-69%
31-35%
Adrenal cortical tumor -/- 100%
Bladder transitional cell carcinoma +/+
+/-
-/+
-/- 25-89%
11-63%
0-4%
0-8%
Esophagus squamous cell carcinoma +/-
-/- 21%
79%
Salivary gland tumor +/- 100%
Epithelioid sarcoma -/- 100%
Thyroid follicular, papillary and medullary carcinoma +/-
-/- 98%
2%
Thymus thymoma -/- 100%

CK5/6 (cytokeratins 5 and 6)
- Monoclonal antibody clone D6/16-B4.
- Source: Roche Diagnostics
- Dilution 1:25.
- Antigen retrieval method: HIER (pressure cooking) using EDTA.
- Staining pattern: Cytoplasmic, with a fibrillary quality, often with subplasmalemmal or perinuclear accentuation.
- The antibody reacts with human cytokeratins 5 and 6, and weakly with cytokeratin 4, but not other cytokeratin types.
- CK5/6 is normally expressed in the basal cells and some stratum spinosum cells of the stratified squamous epithelium, as well as subpopulations of mesothelial cells, but not glandular cells.
- The main application of CK5/6 antibody is to aid in the diagnosis of mesothelioma. >90% of epithelial mesotheliomas are CK5/6 positive. Desmoplastic mesotheliomas are usually negative. Adenocarcinomas are almost always negative (if positive, the staining is weak and focal). Thus if the differential diagnosis is between mesothelioma and adenocarcinoma, strong staining for CK5/6 supports a diagnosis of mesothelioma.
- Squamous cell carcinoma may be positive for CK5/6, and thus this antibody is not helpful if the differential diagnosis is between mesothelioma and squamous cell carcinoma.
- The potential use of CK5/6 for distinction between poorly differentiated squamous cell carcinoma and poorly differentiated adenocarcinoma remains to be explored.

BerEP4
- Monoclonal antibody
- Source: Dakopatts.
- Dilution 1:5
- Antigen retrieval method: Protease I digestion for 8 minutes.
- Staining pattern: Cell membrane +/- weak cytoplasmic.
- BerEP4 reacts with an epithelial antigen distinct from cytokeratin, EMA and CEA. It shows a broad pattern of reactivity with the majority of human epithelial tissues, except superficial layers of stratified squamous epithelium, hepatocytes and parietal cells. Mesothelial cells are generally negative (with rare exceptions).
- However, BerEP4 is not commonly used for demonstration of epithelial differentiation, but is rather used for specific purposes.
- Main applications:
a) Distinction between epithelial cells (carcinoma cells) and mesothelial cells (reactive or neoplastic) in effusion. The former are commonly positive, while the latter are commonly negative.

b) Distinction between papillary adenocarcinoma (commonly positive) and choroid plexus carcinoma (commonly negative).

MOC-31, epithelial-related antigen
- Monoclonal antibody
- Source: Dakopatts
- Dilution 1:20
- Antigen retrieval method: Protease I treatment for 12 minutes
- Staining pattern: Cell membrane
- MOC-31 reacts with a transmembrane glycoprotein present on most normal and malignant epithelia. In the spring-roll positive control block used at QEH, the colonic mucosa, thyroid and breast cancer cells show positive reaction. Approximately 80-90% of adenocarcinomas are positive for MOC-31, and squamous cell carcinomas are also frequently positive. Thus this antibody is more sensitive than Ber-EP4 in staining carcinomas. Only about 5% of mesotheliomas are positive for this marker. Normal or reactive mesothelial cells are negative.
- Main applications:
a) Distinction between carcinoma (commonly positive) and reactive mesothelial cells or mesothelioma in an effusion or pleural biopsy (negative).

b) Distinction between hepatocellular carcinoma (almost always negative) and other carcinomas (commonly positive).

E-cadherin
- Monoclonal antibody 4A2-C7
- Source: Zymed
- Dilution 1:25
- Antigen retrieval method: HIER (pressure cooking) in EDTA
- Staining pattern: Cell membrane staining (apical cell membrane is negative)
- E-cadherin is a form of calcium-dependent cell adhesion molecule expressed in most epithelial cells, but not mesothelial cells. It is a transmembrane glycoprotein. Thus most epithelial cells, including myoepithelial cells, are positive. Rarely, E-cadherin expression can also be found in osteoblasts, peripheral nerve Schwann cells and Langerhans cells.
- The monoclonal antibody reacts strongly with human E-cadherin, and weakly with human P-cadherin.
- Main applications:
a) Distinction between infiltrating lobular carcinoma of breast (almost always negative) and infiltrating ductal carcinoma (almost always positive, although expression may be reduced)

b) The value for distinction between carcinoma (positive) and mesothelial cells/mesothelioma (usually negative) in an effusion or pleural biopsy is currently controversial.

c) In some carcinoma types (such as the breast, lung, G.I. tract), reduced expression of E cadherin is associated with a less favorable prognosis.


N-cadherin
- Monoclonal antibody 3B9
- Source: Zymed
- Dilution 1:25
- Antigen retrieval method: HIER (pressure cooking) using EDTA
- Staining pattern: Cell membrane
- N-cadherin is a form of cell adhesion molecule normally expressed in mesothelial cells, nerve cells, developing skeletal muscle, and embryonic and mature cardiac muscle cells.
- This antibody does not show cross reaction with E-cadherin or P-cadherin.
- The value for distinction between carcinoma (usually negative) and mesothelial cells/mesothelioma (usually positive) in an effusion or pleural biopsy is currently controversial. Some carcinomas can aberrantly express N-cadherin.



Calretinin
- Polyclonal antiserum (catalogue 7696)
- Source: Swant, Bellinzona, Switzerland.
- Dilution 1:1300
- Antigen retrieval method: HIER (pressure cooking) using EDTA.
- Staining pattern: Nuclear + cytoplasmic (Nuclear staining must be present to be considered true positive)
- Calretinin is a 29 kD protein belonging to the EF-hand family of calcium binding proteins.
- It is normally expressed in mesothelial cells strongly. Also reacts with steroid-producing cells of the testis/ovary and mast cells.
- Main applications:
a) Distinction between mesothelioma (calretinin positive) and adenocarcinoma (usually calretinin negative)

b) Distinction between sarcomatoid mesothelioma (calretinin positive, although the staining is often patchy) versus sarcoma of the serosal membranes (calretinin negative)

c) Distinction between carcinoma cells (calretinin negative) and reactive mesothelial cells (calretinin positive).

d) Confirmation of mesothelial nature of cells in tissue specimens (such as entrapped cells and adenomatoid tumor)

e) Marker of neuronal differentiation in CNS tumors, especially central neurocytoma.

f) Aid in the diagnosis of cardiac myxoma - practically all cases show positive staining