A Primer on Immunostains

Department of Pathology, Sinai Hospital of Baltimore Pathology, Baltimore, MD, USA


CytokeratinMelanomaCarcinomaSarcomaLymphomaMesotheliomaGerm cell tumorVascularNeuralMyoepithelial

The use of immunostains is a highly complex field in and of itself and one that is changing so rapidly that no printed text can really keep up. This chapter is meant as an introduction to the most commonly used stains so that you can at least follow the thread of conversation when the acronyms begin to fly. Stains are organized by the organ system in which they are most often used. A much more comprehensive reference is Rekhtman and Bishop’s Quick Reference Handbook for Surgical Pathologists.

Table 31.1.
Blood vessels (endothelium).


Normal tissues stained

When it is used


Endothelial cells and megakaryocytes (cytoplasmic and membranous), also macrophages

To identify endothelial differentiation or angiosarcoma; most specific endothelial marker


Endothelial cells, fibroblasts, and hematopoietic blasts (cytoplasmic and membranous)

To identify vascular sarcomas, Kaposi sarcoma, solitary fibrous tumor, DFSP, epithelioid sarcoma, plus some other soft tissue tumors. Synovial sarcoma is negative


Lymphatic endothelium (cytoplasmic)

To identify vascular differentiation or lymphatics; also marks mesothelium


Endothelial cells, megakaryocytes, platelets (cytoplasmic)

To identify endothelial differentiation, specific but not very sensitive

DFSP dermatofibrosarcoma protuberans

Table 31.2.
Brain and meninges.


Normal tissues stained

When it is used


Epithelial, perineural, meningothelial cells (cytoplasmic or membranous)

To identify meningioma, perineuroma, chordoma. Germ cell tumors (excluding some teratomas) are negative


Glial cells (cytoplasmic)

To identify astrocytoma and ependymoma; also myoepithelial tumors of salivary gland


None: antibody stains mutant IDH1 (cytoplasmic)

To subclassify glial tumors (IDH1 mutated)


All normal cells (nuclear)

To identify atypical teratoid/rhabdoid tumor (loss of staining)

NSE (neuronal-specific enolase)

Neuroectodermal and neuroendocrine cells (cytoplasmic)

To identify neural differentiation but not very specific (not the same as nonspecific esterase, an enzyme assay for heme path). Sensitive for neuroblastoma


Glial cells, Schwann cells, dendritic and Langerhans cells, melanocytes, and other mesenchymal cells (nuclear and cytoplasmic)

To identify cellular schwannoma, astrocytomas, granular cell tumor, chordoma, ependymoma, MPNST, and melanocytic lesions (all types)


Neuroendocrine cells (cytoplasmic)

To identify neuroendocrine tumors, paraganglioma, pheochromocytomas, small cell carcinoma, neuroblastoma, and others. Differentiates neural differentiation (positive) from glial (negative)

MPNST malignant peripheral nerve sheath tumor

Table 31.3.


Normal tissues stained

When it is used

CK903 (CK5/6 is similar)

Myoepithelial cells (cytoplasmic and membranous) and usual duct hyperplasia

To differentiate usual ductal hyperplasia (positive) from ductal carcinoma in situ (negative). Also stains metaplastic carcinoma


Normal ductal and lobular cells (membranous)

Loss of staining identifies lobular carcinoma (in situ and invasive); ductal lesions are positive

ER and PR

Estrogen receptor (nuclear) and progesterone receptor (nuclear)

For breast cancer prognosis (predicts response to tamoxifen) and to identify metastatic breast cancer, some gynecologic tumors, and others


Breast epithelium (nuclear)

To identify breast differentiation in carcinoma; sensitive but not specific


Apocrine metaplasia of the breast and apocrine sweat glands (cytoplasmic)

To identify breast differentiation in carcinoma, also sweat and salivary gland carcinoma


Growth factor receptor that is only weakly expressed in normal epithelial cells (membranous)

To evaluate breast carcinomas (overexpression is a poor prognostic sign but can be treated with Herceptin)


Normal breast tissue (cytoplasmic)

To identify breast differentiation in carcinoma, also sweat and salivary gland carcinoma

Stains that identify myoepithelial cells to rule out invasive carcinoma


Myoepithelial cells (cytoplasmic)

To delineate myoepithelial layer


Tumor suppressor gene (nuclear)

Stains myoepithelial cells but not endothelium and fibroblasts. Also stains metaplastic carcinoma

Smooth muscle myosin heavy chain (SMMHC)

Myoepithelial cells, blood vessels, myofibroblasts (cytoplasmic)

To delineate myoepithelial layer

Smooth muscle actin (SMA)

Smooth muscle: myoepithelial cells, blood vessels, myofibroblasts (cytoplasmic)

To delineate myoepithelial layer; also stains myofibroblasts

Table 31.4.


Normal tissues stained

When it is used

AE1–AE3 (pankeratin)

Wide panel of keratins stains most epithelial cells (cytoplasmic), except cytokeratins 8 and 18

To identify carcinomas in general; used in conjunction with cam 5.2 to screen for carcinoma


Epithelial, perineural, meningothelial cells (cytoplasmic or membranous)

To identify meningioma, many carcinomas, plus some sarcomas (synovial sarcoma, epithelioid sarcoma), and plasma cell neoplasms. Germ cell tumors (excluding some teratomas) are negative

Entities that are EMA-positive, keratin-negative: meningioma, perineuroma, plasma cell myeloma

High-molecular-weight keratins


High-molecular-weight keratins, mainly in squamous and urothelial epithelia (cytoplasmic)

To differentiate squamous cell carcinoma (positive) or mesothelioma (positive) from adenocarcinoma (negative)

CK903 (34ßE12)

High-molecular-weight keratins, mainly in squamous and urothelial epithelia (cytoplasmic and membranous)

To identify prostatic basal cells (loss of staining indicates carcinoma) and urothelial carcinoma (positive); also metaplastic breast carcinoma

Low-molecular-weight keratins


A specific low-molecular-weight cytokeratin (cytoplasmic, membranous)

CK7 and CK20 are used in combination to narrow the differential of carcinoma of unknown origin. CK7 is generally positive in above-the-diaphragm carcinomas (see below on CK7 and CK20)


A specific low-molecular-weight cytokeratin (cytoplasmic, membranous)

Generally positive in below-the-diaphragm carcinomas and in Merkel cell carcinoma (see below on CK7 and CK20)

cam 5.2

Low- and intermediate-molecular-weight keratins 8, 18, and 19, in nonsquamous epithelia (cytoplasmic)

Used in conjunction with AE1/AE3 to screen for carcinoma. Also to identify hepatocellular carcinoma, some adrenal cortical tumors, and some carcinomas that are negative for other keratins (undifferentiated carcinoma)

7/20 matrix

CK20 +


CK7 +

Urothelial carcinoma

Breast carcinoma

Pancreatic carcinoma

Lung carcinoma, non-small cell

Ovarian mucinous carcinoma

Ovarian serous carcinoma

Endometrial carcinoma

Epithelial mesothelioma



Colorectal carcinoma

Hepatocellular carcinoma

Merkel cell carcinoma

Renal cell carcinoma, clear cell type

Prostate carcinoma

Neuroendocrine small cell carcinoma

Squamous cell carcinoma

Table 31.5.
Germ cell and testis.


Normal tissues stained

When it is used


Human chorionic gonadotropin β-chain (cytoplasmic) in syncytiotrophoblasts

To identify choriocarcinoma and germ cell tumors, some adenocarcinoma


Activated lymphocytes (cytoplasmic)

To identify embryonal carcinoma, Hodgkin lymphoma, and ALCL

c-kit (CD117)

Germ cells, mast cells, interstitial cells of Cajal (cytoplasmic or membranous)

To identify seminoma (membranous) and mature teratoma, plus GIST in the stomach


Developing brain and stem cells (nuclear)

To identify seminoma, embryonal carcinoma, and GCNIS


Placenta (cytoplasmic)

To identify germ cell tumors, GCNIS


Fetal tissues, germ cells (nuclear)

To identify germ cell tumors (sensitive but not specific)


Stem cells (nuclear)

To identify embryonal carcinoma; also stains squamous carcinomas

ALCL anaplastic large cell lymphoma, GCNIS germ cell neoplasia in situ, GIST gastrointestinal stromal tumor

Table 31.6.


Normal tissues stained

When it is used

Actin, desmin

Smooth muscle cells, myometrium (cytoplasmic)

To identify leiomyomas or leiomyosarcomas


Syncytiotrophoblasts (cytoplasmic)

To identify choriocarcinoma and germ cell tumors


Endometrial stroma (cytoplasmic)

To identify endometrial stroma and stromal sarcoma


Ovarian and endometrial tissues (nuclear)

To differentiate between some carcinomas and to identify treatment responsiveness


Granulosa cells, Sertoli cells, and others (cytoplasmic)

To identify sex cord stromal tumors (granulosa, Sertoli, and Leydig cells) and hydatidiform moles, choriocarcinomas, fibrothecomas, and adrenal cortical tumors

Napsin A

Pneumocytes, lung (cytoplasmic)

To identify clear cell carcinoma of the ovary, as well as lung carcinomas


Renal and Mullerian tissues (nuclear)

To identify nonmucinous tumors of GYN origin, also stains renal and thyroid tumors


Cells infected by HPV (nuclear), also benign tubal metaplasia

To identify HSIL and HPV lesions of the cervix and to differentiate between endocervical (positive) and endometrial (negative) adenocarcinoma; serous carcinoma also positive


Tumor suppressor gene variant that should be absent in normal cells (nuclear)

To identify serous carcinoma of endometrium or the ovary, to identify STIC


Expressed in trophoblastic cells (nuclear)

To confirm complete hydatidiform mole (negative); only expressed in maternally derived chromosomes

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Jan 30, 2018 | Posted by in PATHOLOGY & LABORATORY MEDICINE | Comments Off on A Primer on Immunostains
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