International Journal of Surgical Pathology

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Subcellular Localization of Immunohistochemical Signals : Knowledge of the Ultrastructural or Biologic Features

of the Antigens Helps Predict the Signal Localization and Proper Interpretation of Immunostains
W. Cheuk and John K. C. Chan
INT J SURG PATHOL 2004 12: 185
DOI: 10.1177/106689690401200301

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 International Journal of Surgical Pathology 12(3):185–206, 2004

Subcellular Localization of
Immunohistochemical Signals
Knowledge of the Ultrastructural or Biologic Features
of the Antigens Helps Predict the Signal Localization
and Proper Interpretation of Immunostains

W. Cheuk, MBBS, FRCPA, and John K. C. Chan, MBBS, FRCPA

In the literature, sufficient attention has not been paid to the precise subcellular lo-
calization of immunohistochemical signals, the knowledge of which is essential for
proper interpretation of immunostains and distinction of genuine staining from bi-
otin-associated or other nonspecific stainings. The subcellular localization of the sig-
nals can in fact be easily deduced from the known biologic or ultrastructural charac-
teristics of the antigens. Extracellular antigens obviously are located in the
extracellular compartment. Cellular antigens fall into 3 major groups: membranous,
nuclear, and cytoplasmic. Membranous antigens include cell adhesion molecules
(such as E-cadherin, N-CAM), cell surface/transmembrane receptors and proteins
(such as tyrosine kinase receptors, most leukocyte antigens, CD10, CEA), and mole-
cules linking surface molecules to cytoskeleton (such as β-catenin, dystrophin). Nuclear
antigens include cell cycle-associated proteins (such as cyclins, p16, Ki-67), nuclear en-
zymes (such as TdT), transcription factors (such as TTF-1, CDX-2, myogenin, PAX-5),
tumor suppressor gene products (such as p53, p63, WT1, Rb), steroid hormone recep-
tors (such as ER, PR), calcium-binding proteins (such as S-100 protein, calretinin), and
some viral proteins (such as CMV, herpes). Cytoplasmic antigens can take up a granu-
lar pattern due to localization in organelles, granules, or secretory vesicles (such as
chromogranin, hormones, lysozyme, HMB-45), fibrillary pattern attributable to the fil-
amentous nature of the molecules (intermediate filaments and microfilaments), or dif-
fuse or patchy pattern due to localization in the cytosol or large vesicles (such as myo-
globin, albumin, thyroglobulin). Aberrant localization of the molecules, when present,
can provide important insight into disease processes and aid in their diagnosis, such as
loss of membranous E-cadherin expression in lobular breast carcinoma, aberrant nu-
clear localization of β-catenin in colorectal adenocarcinoma, pattern of ALK staining in
anaplastic large cell lymphoma correlating with the different types of chromosomal
translocations, presence of additional cytoplasmic CD10 staining in the enterocytes in-
dicative of microvillous inclusion disease, and “reversed” staining for EMA in mi-
cropapillary mammary carcinoma. Int J Surg Pathol 12(3):185–206, 2004
Key words: immunohistochemistry, subcellular localization, false-positive im-
munostaining, endogenous biotin, membrane staining, cell adhesion molecule, cell
surface receptor, nuclear staining, cell cycle protein, hormone receptor, cytoplasmic
staining, aberrant localization.

Department of Pathology, Queen Elizabeth Hospital, Hong Kong.

Address for correspondence: Dr. John K. C. Chan, Department of Pathology, Queen Elizabeth Hospital, Wylie Road, Kowloon,
Hong Kong.

185

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186 International Journal of Surgical Pathology Vol. 12 No. 3 July 2004
The practice of surgical pathology has been revo-
lutionized by the widespread use of immunohisto-
chemistry because of the ability to stain a wide va-
riety of molecules in a consistent fashion on routine
diagnostic materials. This is attributable to the avail-
ability of effective antigen retrieval techniques, sen-
sitive detection systems, and an ever-growing list of
antibodies [1]. Despite these advances, wrong inter-
pretations and discrepancies of immunohistochem-
ical assays are still frequently encountered, such as
misinterpretation of biotin nuclear inclusions in
gestational endometrium as herpesvirus infection
[2], misreporting expression of inhibin in hepato-
cellular carcinoma [3,4], and controversies of c-kit
expression in desmoid tumor and other soft tissue
tumors [5–7]. Many discrepancies in the reported
literature have been attributable to methodologic
divergence and technical reasons, particularly the
choice of fixatives, duration of fixation, and tissue
processing [8,9]. Consequently, some investigators
have called for the use of “only validated, standard-
ized methods for diagnostic immunohistology, to
the absolute exclusion of others” and supplemented
by a vigorous quality assurance program, indepen-
dent validation (such as electron microscopy or
molecular techniques), and ultimately, accredita-
tion of laboratories specialized in performing im-
munostaining [10]. These proposals are indeed
worth exploring, but realization of these goals may
not be possible for most laboratories in the near fu-
ture. A more fundamental aspect in improving the
results of immunohistochemistry, which is also
much more easily tackled at this time, lies in the
proper interpretation of immunostains. In this arti-
cle, a critical evaluation of interpretation of im-
munostains with emphasis on subcellular localiza-
tion is discussed.
Using Immunohistochemistry
to Visualize Antigens
There are 3 principal variables in interpretation of
immunostains: the presence or absence of labeling,
microanatomic distribution of the signals, and mor-
phologic characteristics of the staining products.
Unfortunately many pathologists pay attention only
to the first of these variables. This practice would
significantly decrease the specificity and, to a lesser
extent, sensitivity of immunostaining as a diagnos-
tic tool. In fact, without proper subcellular localiza-
tion and appropriate appearance, mere presence of
“positive staining” is almost meaningless in im-
munohistochemistry.
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While it may seem daunting having to memorize
the subcellular localizations of the ever-increasing
number of antibodies available in the market, this
is actually quite simple because the immunolocal-
ization can be predicted by reasoning from the bi-
ological or ultrastructural features of the individ-
ual antigens.
Categorization of Antigens Based
on Intracellular Localization
Cellular antigens can be categorized by their pre-
dominant intracellular distribution into membra-
nous, nuclear, and cytoplasmic types. The extracel-
lular antigens such as extracellular substances (e.g.,
amyloid P) or components of extracellular matrix
(e.g., laminin, type IV collagen, osteocalcin) will not
be further discussed in this article.
Membranous Antigens
The cell membrane forms a barrier between the
cell and its external environment. It consists of a
lipid bilayer within which are embedded proteins
that act as receptors, transporters, or ligands. The
major groups of membranous antigens include the
following (Table 1):
• Cell adhesion molecules, e.g., cadherins,
platelet endothelial cell adhesion molecule
(PECAM), neural cell adhesion molecules (N-
CAM), epithelial cell adhesion molecule (Ep-CAM)
• Cell surface or transmembrane receptors and
proteins, e.g., c-erbB-2 (homologue of epidermal
growth factor receptor), c-kit (tyrosine kinase re-
ceptor), most leukocyte antigens, EGFR (epidermal
growth factor receptor), PDGF (platelet-derived
growth factor), EBV-LMP1
• Molecules that cross-link membrane proteins to
the underlying cytoskeleton network, e.g., β-catenin
(in epithelial cells), dystrophin (in muscle cells),
spectrin (in erythrocytes). These proteins apparently
show cell membrane immunoreactivity at the light
microscopic level owing to their linear localization
along the membrane-cytoplasmic interface.
Antibodies against the membrane antigens usu-
ally produce circumferential staining, which may be
accompanied by paranuclear Golgi staining because
the Golgi is the site where posttranslation modifica-
tion of proteins occurs before they are transported
onto the cell surface (Figs. 1–4). Exceptionally, the
staining is purely Golgi when the density of mole-
cules on the cell surface is too low for immunohis-
tochemical detection.
 Subcellular Localization of Immunohistochemical Signals • Cheuk and Chan 187

Table 1. Molecules with Membranous Pattern of Immunostaining*

Category of Molecules Examples Explanation or Special Features

Cell adhesion molecules
• Cadherins, e.g., E-cadherin, N- Since there is no cell interaction at the
cadherin, P-cadherin luminal surface, E-cadherin is
• CD31 (PECAM) expressed only on the basolateral sur-
• CD56 (N-CAM) faces of the normal glandular
• Intercellular cell adhesion epithelium. Ep-CAM also commonly
molecule (I-CAM) shows similar basolateral distribution
• Epithelial cell adhesion molecule in glandular epithelium.
(Ep-CAM, including Ber-EP4,
MOC-31)
• P-selectin
• Integrin
• CD44

Cell membrane receptors • c-erbB-2 EMA, CEA, villin, and CA125

and proteins • Epidermal growth factor receptor commonly show selective expression
• CD117/c-kit in the luminal side of glandular
• Platelet derived growth factor epithelium. CD10 staining is
receptor circumferential on neutrophils and
• Most leukocyte antigens, e.g., lymphoid cells, but limited to the
CD2, surface CD3, CD4, CD5, luminal-free surface of enterocytes
CD7, CD8, CD10, CD20, CD21, and bile canaliculi. Staining for EBV-
CD23, CD25, CD30, CD45, CD138 LMP1 is on the cell membrane, but
• Epithelial membrane antigen often with a granular quality, owing
(EMA) probably to uneven distribution of the
• Carcinoembryonic antigen (CEA) molecules on the cell surface.
• Villin
• CA125
• Glycophorin A
• EBV-LMP1
• Placental alkaline phosphatase

Molecules that cross-link with • β-catenin Since these molecules bind with cell
cell surface proteins • Dystrophin membrane proteins, they show an ap-
• Spectrin parent membranous distribution at
the light microscopic level.

*Some molecules with fibrillary cytoplasmic staining may mimic membranous staining owing to condensation of the filaments beneath
the cell membrane.

The epithelial cells are normally polarized. Ac- Leukocyte Antigens

cordingly, some membranous antigens show re-
stricted or predominant expression on the apical
side (e.g., epithelial membrane antigen, carcinoem-
bryonic antigen, villin) or basolateral surfaces (e.g.,
E-cadherin, β-catenin, Ep-CAM) of the glandular
epithelium (Figs. 5–7). Nevertheless, the polarized
staining pattern of these antigens may be replaced
by circumferential staining in malignant neoplasms
attributable to disturbances in cell polarity.
Immunohistochemical demonstration of mem-
branous antigens in tumors is assuming increasing
importance in clinical practice because of the avail-
ability of novel biologic response modifiers or ther-
apeutic antibodies against specific targets located in
the cell membrane. Obviously, tumors that strongly
express the corresponding targets are expected to
show better response to such therapies.
The great majority of leukocyte antigens are cell
surface receptors or ligands involved in cell recogni-
tion, cell interaction, cell adhesion, signal transduc-
tion, or interaction with soluble proteins/glycopro-
teins and extracellular matrix. Examples are CD1,
CD2, surface CD3, CD4, CD5, CD7, CD8, CD19,
CD20, CD43, and CD45. Immunostaining for these
antigens produces circumferential membrane stain-
ing due to uniform distribution of these antigens on
the cell surface (Fig. 8). The expression level can be
weaker in neoplastic cells compared with normal
lymphoid cells.
The cell membrane leukocyte antigens can be tar-
geted by therapeutic antibodies, which cause de-
struction of the lymphoma or leukemia cells that
bear them. The most widely used is anti-CD20 anti-
body (such as Rituximab), which has been success-

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188 International Journal of Surgical Pathology Vol. 12 No. 3 July 2004

Fig. 1. Schematic drawing to illustrate the different pat-

terns of membranous staining. Fig. 2. Membranous pattern. CD138 high-
lights the cell membranes and Golgi regions
of the neoplastic cells in a plasmacytoma.

Fig. 4. Membranous pattern. EBV LMP1 im-

Fig. 3. Membranous pattern. CD21 outlines munostaining characteristically produces a
the cell membranes of the complex dendritic discontinuous, dot-like staining along the cell
cell processes of a follicular dendritic cell membrane (arrows). This is an example of in-
sarcoma. fectious mononucleosis involving tonsil.

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 Subcellular Localization of Immunohistochemical Signals • Cheuk and Chan 189

Fig. 6. Polarized membranous pattern. Ber-

Fig. 5. Polarized membranous pattern. Ep- EP4 stains the basolateral surfaces of the thy-
ithelial membrane antigen is preferentially roid follicular epithelial cells.
expressed along the luminal surface of the
glandular spaces in pleomorphic adenoma.

Fig. 7. Polarized membranous pattern. E- Fig. 8. Membranous pattern. CD45RO

cadherin immunostaining highlights the ba- highlights the cell membranes of the neo-
solateral surfaces of the ductal epithelial cells plastic cells in this example of peripheral T-
in the breast, as well as the outer layer of my- cell lymphoma.
oepithelial cells.

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190 International Journal of Surgical Pathology Vol. 12 No. 3 July 2004

fully used to treat B-cell lymphoma and nodular

lymphocyte predominant Hodgkin lymphoma [11].
Of interest, the neoplastic cells can lose expression
of the targeted molecules after treatment, probably
as an “escape” mechanism. Another newly available
therapeutic antibody is anti-CD52 (such as Cam-
path-1H), which has been successfully used for
treating chronic lymphocytic leukemia [12].

c-kit (CD117)
c-kit (CD117) is a transmembrane tyrosine kinase
receptor normally expressed in hematopoietic stem
cells, mast cells, germ cells, melanocytes, and inter-
stitial cells of Cajal [13]. Mutation resulting in con-
stitutive activation of c-kit is a critical event in the
pathogenesis of most gastrointestinal stromal tu-
mors (GISTs) and constitutes one of the major diag-
nostic criteria of the tumor [13,14]. Immunohisto-
chemical demonstration of c-kit currently is
important not only in the diagnosis of GISTs but also
Fig. 9. Membranous pattern. c-kit im-
in the selection of patients who may benefit from munostaining highlights the cell membranes
treatment with the tyrosine kinase receptor inhibitor of the tumor cells of a gastrointestinal stromal
STI571 (such as Gleevec, Imatinib) [15]. There are tumor, and there is also simultaneous weak
conflicting reports on the expression of c-kit in clear cytoplasmic staining.
cell sarcoma [16], intraabdominal desmoid tumors
[6], angiomyolipoma [17], and other soft tissue tu-
mors [7]. One cause of these discrepancies appears
to be the different criteria adopted for interpretation
of c-kit immunostaining. For instance, some investi-
gators consider an “unequivocal cytoplasmic stain-
ing” as positive, whereas some do not even mention
the criteria for positive staining at all [6,17,18]. By
nature, c-kit is a transmembrane receptor, and thus
genuine staining should be primarily along the cell
membrane, or at least cytoplasmic with cell mem-
brane accentuation (Fig. 9). Apparently many of the
reports on immunoreactivity for c-kit (such as in an-
giomyolipoma) do not fulfill such stringent criteria
of interpretation because there is diffuse cytoplasmic
staining only.

c-erbB2
In some earlier studies, there is confusion on
what constitutes positive staining for c-erbB-2; for
example, cytoplasmic staining has been accepted as
positive [19–22]. As understood from c-erbB-2 be-
ing a form of epidermal growth factor receptor, only
Fig. 10. Membranous pattern. Strong mem-
membranous staining should be considered gen-
branous staining for c-erbB-2 in an invasive
uine or significant, especially when this molecule is
ductal carcinoma of the breast.
to be targeted by monoclonal antibody therapy
(e.g., Herceptin, Trastuzuamb) (Fig. 10) [23]. The
most widely used scoring system for c-erbB2 over- considered positive) does correctly emphasize the
expression in breast cancer (HercepTest, with scores need to identify circumferential cell membrane
0-1 being considered negative, and scores 2-3 being staining as the basic requirement of a positive result.

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 Subcellular Localization of Immunohistochemical Signals • Cheuk and Chan 191

Epidermal Growth Factor Receptor (EGFG) Nuclear Antigens

Epidermal growth factor receptor (EGFR) is nor-
mally expressed in the basal and suprabasal layers of
the stratified squamous epithelium [24]. It is over-
expressed in many carcinomas, usually attributable
to an increase in gene copy number. Anti-EGFR
(such as Gefitinib) is increasingly used for the treat-
ment of lung carcinoma, head and neck carcinoma,
and some other carcinomas, and thus the surgical
pathologist is often requested to assess tumors for
EGFR overexpression [25]. There is as yet no uni-
versally accepted scoring system for assessment of
EGFR overexpression, but that proposed by Putti et
al [24] may be used—the final score is obtained by
multiplying the extent of staining score (0,1,2,3)
and intensity of staining score (0,1,2,3).
The nucleus is the repository of DNA where gene
expression and cell replication take place. The ma-
jor groups of nuclear antigens include the following:
cell-cycle-associated proteins, mismatch repair gene
proteins, nuclear enzymes, transcription factors, tu-
mor suppressor gene products, steroid hormone re-
ceptors, calcium-binding proteins, and some viral
nuclear proteins (Table 2).
The staining in the nucleus is typically homoge-
neous, with the notable exceptions of human her-
pes 8 virus latent nuclear antigen where there is su-
perimposed speckled pattern, and Ki-67 protein as
well as nucleophosmin with accentuation in the
nucleoli (Figs. 11–13). Nuclear staining can be ac-
companied by variable cytoplasmic staining, proba-

Table 2. Molecules with Nuclear Pattern of Immunostaining*

Category of Molecules Examples

Cell cycle-associated proteins • Cyclins, e.g., cyclin A, cyclin B, cyclin D1, cyclin D2, cyclin D3,
cyclin E
• Cyclin-dependent kinases
• Cyclin-dependent kinase inhibitors, e.g., p16, p27
• Proliferation-associated molecules, e.g., Ki-67, PCNA

Nuclear enzymes and proteins • TdT

• Nucleophosmin
• Mismatch repair gene products, e.g., HMLH1, HMSH2

Transcription factors • Muscle, e.g., MyoD1, myogenin

• Thyroid or lung, e.g., thyroid transcription factor-1 (TTF-1)
• Intestine, e.g., CDX-2
• Leukocyte, e.g., PAX-5, Oct-2, Bob.1

Tumor suppressor gene products • Retinoblastoma (Rb)

• Wilms tumor (WT1)
• p53
• p63

Steroid hormone receptors • Estrogen receptor

• Progesterone receptor
• Androgen receptor

Calcium-binding proteins • S-100 protein

• Calretinin

Some viruses • Cytomegalovirus

• Adenovirus
• Herpes simplex (HSV)
• Human papillomavirus (HPV)
• Human herpesvirus 8 (HHV-8)
• HBcAg
• Parvovirus

*May be accompanied by concurrent cytoplasmic staining.

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192 International Journal of Surgical Pathology Vol. 12 No. 3 July 2004

Fig. 11. Schematic drawing to illustrate the different patterns of nuclear staining.

Fig. 12. Nuclear pattern. Progesterone re- Fig. 13. Nuclear pattern. Ki-67 typically pro-
ceptor immunostaining typically produces duces granular nuclear staining with accen-
homogeneous staining of the nuclei (an ex- tuation of the chromatin clumps and nucleoli
ample of ductal carcinoma of breast). (an example of large B-cell lymphoma).

bly related to diffusion of molecules or partial cyto-
plasmic localization of the molecules in the cytosol.
The calcium-binding proteins, S-100 protein and
calretinin, for instance, typically produce simulta-
neous nuclear and cytoplasmic staining (Fig. 14).
On the other hand, some nuclear antigens are prac-
tically never accompanied by cytoplasmic staining,
such as Ki67 and TdT. As illustrated in Figure 15,
paying attention to the pattern of nuclear staining
can avert instances of wrong interpretation.
Cytoplasmic Antigens
The cytoplasm contains variable numbers of
membrane-bound organelles, granules, and vesicles
submerged in the cytosol. Ultrastructurally, there is
a structural lattice within the cytosol forming an in-

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 Subcellular Localization of Immunohistochemical Signals • Cheuk and Chan 193

ternal scaffolding called the cytoskeleton (interme-

diate filaments and microfilaments), which is re-
sponsible for the rigidity, intracellular transport, and
mobility of the cell. Cytoplasmic antigens exhibit 3
patterns of staining: granular, fibrillary, and homo-
geneous (Table 3) (Fig 16).

Granular Cytoplasmic Staining

Fig. 14. Nuclear pattern. In additional to nu-
clear staining, S-100 protein staining is typi-
cally also observed in the cytoplasm (an ex-
ample of malignant melanoma).
A granular pattern of cytoplasmic staining is ex-
pected for antigens contained in organelles (e.g., an-
timitochondrial antibody, lysosomal enzymes and
proteins, cytotoxic granules), secretory granules (e.g.,
BRST-2, surfactant, chromogranin, synaptophysin,
hormones), and intracytoplasmic microorganisms
(Figs. 17, 18). The staining can appear homogeneous
when the cells are densely packed with the organelles
or granules, but careful examination will reveal gran-
ular staining in at least a proportion of cells.
The cytoplasmic granules are not necessarily uni-
formly distributed throughout the cells but can be
polarized depending on the cell type. For example,
the neuroendocrine granules of the endocrine cells
in the gastrointestinal tract are concentrated along
the base of the cells, which is the pole facing the
blood vessels in the stroma. On the other hand, ex-
ocrine or apocrine secretory granules are accentu-
ated beneath the apical portion of the cells.

Fig. 15. Technical artifact revealed by careful analysis of staining pattern. A. A duc-
tal carcinoma of the breast immunostained for Ki67 showed a very high percentage
of positive cells. Two features suggested that the staining was spurious: The nuclear
staining pattern was too homogeneous and there were too many positive cells for
this low-grade tumor. Further investigation revealed that the antibody dispenser for
progesterone receptor antibody was leaky, resulting in some progesterone receptor
antibody having been dropped onto the slide within the automated immunostainer.
B. Realizing the problem, the test was repeated. This time, the Ki67 index was ob-
viously low. Note the typical coarse granular staining of Ki67 (arrows).

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194 International Journal of Surgical Pathology Vol. 12 No. 3 July 2004

Table 3. Molecules with Cytoplasmic Pattern of Immunostaining

Category of Molecules Examples

Cytoplasmic granular pattern*

Organelles or organelle-associated • Anti-mitochondrial antibody

molecules • HEP-PAR1 (in mitochondria of hepatocytes)
• Factor VIII-related antigen (in Weibel-Palade bodies)
• Melanosomes and premelanosomes (e.g., HMB-45, Melan-A)
• Lysosome-associated (e.g., CD68, lysozyme, α1-antitrypsin, α1-
antichymotrypsin)
• Cytotoxic granules (e.g., granzyme B, TIA-1, perforin)
• Neutrophil granules (e.g., myeloperoxidase, neutrophil elastase)

Secretory granules • BRST-2

• Prostatic specific antigen
• Surfactant
• Synaptophysin (in presynaptic vesicles)
• Chromogranin (in neurosecretory granules)
• Various hormones, e.g., parathyroid hormone, insulin, glucagon,
calcitonin, serotonin, ACTH, prolactin, growth hormone, HCG

Cytoplasmic microorganisms • Toxoplasma

Cytoplasmic fibrillary pattern*

Intermediate filaments† • Cytokeratin

• Vimentin
• Desmin
• Neurofilament
• Glial fibrillary acidic protein

Microfilaments • Actin

Proteins associated with myofilaments • Myosin

• Calponin
• H-caldesmon

Diffuse or patchy cytoplasmic pattern

Proteins or enzymes in cytosol or large • Myoglobin

vesicles • Hemoglobin
• Albumin
• Neuron-specific enolase
• Thyroglobulin
• Alpha-fetoprotein
• Immunoglobulin
• CD79a
• Cytoplasmic CD3
• Bcl-2
• VS38 (rough endoplasmic reticulum-associated protein)

Viral antigens • HBsAg

*Dense staining due to presence of large numbers of organelles, granules, or filaments may produce apparently diffuse cytoplasmic
staining.
†Staining can occasionally take up a paranuclear globular pattern.

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 Subcellular Localization of Immunohistochemical Signals • Cheuk and Chan 195

Fig. 16. Schematic drawing to illustrate the different patterns of cytoplasmic staining.

Fig. 17. Granular cytoplasmic pattern. Im- Fig. 18. Granular cytoplasmic pattern. Chro-
munostaining for cytotoxic molecule TIA-1 mogranin immunostaining highlights the
shows the granules in the lymphoma cells in neurosecretory granules in the islet cells of
an NK/T cell lymphoma. the pancreas.

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196 International Journal of Surgical Pathology Vol. 12 No. 3 July 2004

Fibrillary Cytoplasmic Staining

The cytoskeleton comprises filamentous assem-
blies of intermediate filaments (cytokeratin, vi-
mentin, desmin, glial fibrillary acidic protein, neu-
rofilament), actins, and microtubules. The
cytoskeletal fibers form a meshwork stretching from
the nuclear envelope to the cell membrane, and
thus immunostaining for these proteins characteris-
tically exhibits a fibrillary quality, often with sub-
membrane accentuation attributable to the lami-
nating layers of fibers beneath the cell membrane
(Figs. 19, 20). Although the fibrillary quality of the
intermediate filaments can be difficult to discern in
cells with very dense cytoplasm, it can always be ap-
preciated in at least some cells.
Cytokeratin immunostaining typically takes the
form of dense fibrillary staining. It also clearly high-
lights the quantity of cytoplasm as well as the shape
of the positive cells, e.g., dendritic cell processes in
type B1/B2 thymoma (Fig. 21) and cytokeratin-
positive dendritic cell tumor of lymph node. The
staining commonly shows accentuation beneath
the cell membrane, and sometimes the almost ex- Fig. 20. Fibrillary cytoplasmic pattern. Im-
clusive submembranous localization may mimic munostaining for actin in the intestinal mu-
cosa highlights pericytes and smooth muscle
membranous staining pattern (Fig. 22). Accentua-
cells in a fibrillary pattern with accentuation
tion of staining in a perinuclear location is also com- beneath the cell membranes.
mon. Some tumor types, particularly small cell car-

Fig. 19. Fibrillary cytoplasmic pattern. Al-

though the immunostaining for cytokeratin Fig. 21. Cytoplasmic pattern. Cytokeratin
in this carcinoma is rather dense, a fibrillary immunostaining can also delineate the retic-
quality is evident. There is also accentuation ular-dendritic quality of the epithelial cells in
of staining beneath the cell membranes. thymoma.

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 Subcellular Localization of Immunohistochemical Signals • Cheuk and Chan 197

Fig. 22. Fibrillary cytoplasmic pattern. In
this piece of endometrium, the staining for
cytokeratin is predominantly localized to the
submembrane region, mimicking membra-
nous staining.
Fig. 23. Cytoplasmic pattern. Immunostain-
ing for cytokeratin in this example of olfac-
tory neuroblastoma shows paranuclear dots
in some tumor cells.

cinoma, Merkel cell carcinoma, and malignant

rhabdoid tumor, frequently exhibit paranuclear
punctate/globular staining due to presence of
whorls or aggregates of cytokeratin intermediate fil-
aments in the paranuclear region as revealed by
electron microscopy (Fig. 23).

Diffuse or Patchy Cytoplasmic Staining

Antigens that produce patchy or diffuse cytoplas-
mic staining include proteins in the cytosol or large
vesicles (e.g., hemoglobin, albumin, myoglobin,
thyroglobulin, neuron-specific enolase, im-
munoglobulin, cytoplasmic CD3, CD79a, bcl-2) and
some viral proteins (e.g., HBsAg, which produces
homogeneous staining usually in a paranuclear or
perisinusoidal region) (Fig. 24). The staining for
these molecules can sometimes exhibit a granular
quality. Staining for immunoglobulin, cytoplasmic
CD3, CD79a, and bcl-2 often shows accentuation
around the nuclear membrane.

Problem of False-Positive Staining:

Endogenous Biotin or Biotin-Like
Substance and Other Nonspecific Stainings Fig. 24. Homogeneous cytoplasmic pattern.
Immunostaining for immunoglobulin pro-
Heat-induced epitope retrieval usually greatly en- duces homogeneous cytoplasmic staining in
hances endogenous biotin and biotin-like activity the plasma cells.

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198 International Journal of Surgical Pathology Vol. 12 No. 3 July 2004

(avidin-binding activity), producing false-positive

staining if an avidin-biotin immunohistochemical de-
tection system is used [26,27]. Some investigators,
unaware of this form of false-positive staining, have
incorrectly reported in the literature on the im-
munoreactivities of certain antibodies, such as inhibin
immunoreactivity in hepatocellular carcinomas and
herpes simplex in gestational endometrium [2–4].
Biotin is a low-molecular-weight protein that acts
as a cofactor in carboxylation reaction and is part of
the vitamin B complex. It occurs in abundance in mi-
tochondria, and hence, in mitochondria-rich cells,
such as proximal renal tubular cells, hepatocytes, on-
cocytes, apocrine cells of the breast, and Hurthle cells
of the thyroid (Figs. 25, 26). The biotin staining is
most frequently located in the cytoplasm, often ap-
pearing dull-brown granular or fluffy. Rarely, the bi-
otin is located in the nuclei, as seen in gestational en-
dometrium, morules in endometrioid carcinoma,
pulmonary blastoma, and cribriform-morule variant
of papillary thyroid carcinoma, pancreatoblastoma,
and colonic adenoma [2,28–30]. On the other hand,
it practically never produces a cell membrane or cy- Fig. 26. Endogenous biotin staining. Im-
toplasmic fibrillary pattern of staining. munostaining for desmin reveals many tu-
To avoid misinterpreting endogenous biotin or bi- mor cells with granular to fluffy cytoplasmic
otin-like activity as positive staining, the pathologist staining, which is contradictory to the ex-
should be vigilant about this possibility and pay at- pected fibrillary staining for desmin. Thus this
represents nonspecific staining consistent
tention to the expected subcellular localization and
with endogenous biotin.

pattern of the antigen to be demonstrated. This pos-

Fig. 25. Endogenous biotin in the mitochon-
dria-rich striated duct of salivary gland. If an
avidin-biotin immunohistochemical detec-
tion system is used, the striated ducts are
stained in a granular pattern attributable to
the presence of abundant endogenous biotin,
irrespective of what antibody is applied.
sibility should be strongly suspected whenever an
identical pattern of staining is seen with several an-
tibodies against different antigens, particularly if the
staining is dull-colored. Most problems involve an-
tibodies that produce diffuse, patchy, or granular cy-
toplasmic staining. If there are uncertainties, the
immunostain should be repeated with a prior
avidin-biotin block or an alternative detection sys-
tem that does not involve avidin-biotin, such as
polymer detection system or peroxidase-antiperox-
idase (Fig. 27). Nonetheless, even nonavidin-biotin
detection systems are not totally free of nonspecific
stainings; for example, it is common to observe
weak diffuse cytoplasmic staining of smooth muscle
cells by using the polymer immunohistochemical
detection system (e.g., EnVision), and antibodies
themselves can also produce nonspecific staining.
Aberrant Patterns of
Immunolocalization in Disease
The normal subcellular localization of the mole-
cules is altered in some pathologic conditions.

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 Subcellular Localization of Immunohistochemical Signals • Cheuk and Chan 199

A B
Fig. 27. Endogenous biotin staining eliminated by avidin-biotin block. A. Immunostain-
ing for prostatic specific antigen in a poorly differentiated carcinoma of the bladder. It is
unclear whether the cytoplasmic staining is genuine or merely due to endogenous biotin.
B. The staining is completely abolished with prior avidin-biotin block, indicating false-pos-
itive staining due to endogenous biotin.

When present, this can provide important insight
into disease processes and can furthermore be uti-
lized to aid in diagnosis.
Pattern of Anaplastic Lymphoma Kinase
Immunostaining Reflects the Underlying
Molecular Event
Anaplastic lymphoma kinase (ALK) gene, encod-
ing a transmembrane tyrosine kinase receptor, is lo-
cated at chromosome 2p23. In anaplastic large cell
lymphoma (ALCL) with the classical t(2;5) translo-
cation, the ALK gene is fused with the nucleophos-
min gene (NPM) located at chromosome 5 encoding
for a ubiquitously expressed housekeeping nuclear
protein [31,32]. The gene fusion results in the pro-
duction of a chimeric protein with the amino-ter-
minal of NPM linked to the cytoplasmic domain
ALK. This leads to constitutive activation of the ty-
rosine kinase activity, which is believed to play a
key role in the genesis of ALCL [33,34]. Since ALK
is absent in normal tissues except occasional cells in
the nervous system [32], the presence of ALK
translocation can be inferred whenever there is pos-
itive immunostaining of this protein. Thus, ALK im-
munohistochemistry represents a valuable aid in
the diagnosis of ALCL. Furthermore, ALK expres-
sion identifies a subset of ALCL with predominant
occurrence in young patients, good response to
chemotherapy, and a favorable prognosis [35–37].
Of interest, the subcellular distribution of ALK is in-
formative on the translocation partner genes be-
cause it is dictated by the normal localization of the
partner gene product (Table 4). In ALCL with t(2;5),
ALK expression is observed in the nucleus and cy-
toplasm (Fig. 28A, B), because dimerization of the
NPM segment of the NPM-ALK protein with the
wild-type NPM shuttling protein results in transfer
of the chimeric protein into the nucleus [38]. Gran-
ular staining for ALK indicates that the partner gene
is clathrin gene, which encodes a granule-associated
protein [39]. Cytoplasmic fibrillary staining with ac-
centuation beneath the cell membrane, on the
other hand, suggests that the partner gene is
tropomyosin [40].
In the rare type of ALK+ large B-cell lymphoma,
the pattern of ALK immunostaining also correlates
well with the partner gene, being cytoplasmic gran-
ular when the partner gene is CLTCL (clathrin), and
nuclear-cytoplasmic when the partner gene is NPM
[41–43].
Translocation of ALK gene has also been demon-
strated in a subset of inflammatory myofibroblastic
tumors [44–47]. Like ALCL, the partner genes are

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200 International Journal of Surgical Pathology Vol. 12 No. 3 July 2004

Table 4. Correlation of Translocation Partners of ALK Gene and Subcellular Distribution

of Chimeric Proteins in ALK+ Anaplastic Large Cell Lymphoma [76]
Fusion Gene Chromosomal Translocation Staining Pattern of ALK Protein

NPM-ALK t(2:5)(p23;q35) Nuclear and cytoplasmic

TPM3-ALK t(1;2)(q21;p23) Cytoplasmic, often with membranous accentuation
TPM4-ALK t(2;19)(p23;p13.3) Cytoplasmic, often with membranous accentuation
TFG-ALK t(2;3)(p23;q21) Cytoplasmic
ATIC-ALK Inv(2)(p23;q35) Cytoplasmic
CLTCL-ALK t(2;22)(p23;q11.2) Cytoplasmic granular
MSN-ALK t(X;2)(q11-12;p23) Membranous

Fig. 28. Different patterns of ALK immunostaining in anaplastic large cell lymphoma. A.
In this example, the signal is localized in the nucleus and cytoplasm, suggestive of a t(2;5)
translocation. B. In this example, the staining is cytoplasmic with membrane accentuation,
suggestive of translocation of ALK with tropomyosin gene.

variable but include, among others, TPM3 and
CLTCL, which are also known to be involved in
ALCL [40,45]. Again the subcellular localization of
the ALK immunostain reflects the normal localiza-
tion of the partner gene product.
Loss of E-Cadherin Expression is the
Hallmark of Lobular Breast Cancer
E-cadherin is a transmembrane cell adhesion
molecule of epithelial cells, mediating cell-to-cell
adhesion via calcium-dependent homophilic bind-
ing. It plays a major role in maintaining the organi-
zation and integrity of epithelial tissues [48].
On immunostaining, practically all lobular breast
carcinomas and about half of the cases of diffuse-
type gastric adenocarcinomas show complete loss of
staining for E-cadherin [49,50]. The loss of cell ad-
hesion molecule therefore provides a plausible ex-
planation for the observed highly permeative and
noncohesive growth patterns of these tumor types
[51]. The loss of E-cadherin expression results from
silencing of the E-cadherin (CDH1) gene by inacti-
vating mutations or hypermethylation [51,52]. Of
interest, for gastric adenocarcinoma showing a
mixed diffuse-intestinal morphology, the E-cad-
herin gene mutation is confined to the diffuse com-
ponent [53]. Lack of immunoreactivity for E-cad-

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 Subcellular Localization of Immunohistochemical Signals • Cheuk and Chan 201

A B

Fig. 29. Carcinoma of breast. A. The tumor grows in the form of single files comprising
apparently noncohesive cells, raising the possibility of invasive lobular carcinoma. B. Pos-
itive membranous staining for E-cadherin suggests that this is an invasive ductal rather
than lobular carcinoma.

herin can be utilized to distinguish invasive lobular

from invasive ductal carcinoma of the breast, as well
as lobular carcinoma in situ or atypical lobular hy-
perplasia from nonspecific lobular hyperplasia (Figs.
29, 30) [54,55]. Furthermore, this feature has been
utilized to define the pleomorphic variant of inva-
sive or in situ lobular carcinoma of the breast [56].

Aberrant Nuclear Staining for Beta-Catenin

in Selected Tumor Types
β-catenin belongs to a group of proteins that is as-
sociated with the cytoplasmic domain of E-cadherin
to mediate intercellular adhesion [57]. It is also an
important component of the Wnt signaling path-
way [58]. In normal epithelial cells, β-catenin ex-
hibits membranous staining due to its strategic loca-
tion in close association with E-cadherin. Free
β-catenin in the cytoplasm interacts with a complex
of proteins including adenosis polyposis coli, axin,
and glycogen synthase kinase 3β, and is subse-
quently degraded [59,60]. In transformed cells with
mutation or downregulation of these components Fig. 30. Loss of E-cadherin immunostaining
of the Wnt signaling pathway, excess β-catenin ac- is noted in this case of lobular carcinoma in
cumulates in the cytoplasm owing to impairment of situ. The myoepithelial cells at the periphery
its degradation [61]. This leads to translocation of β- of the lobular units serve as internal positive
catenin into the nucleus, where it can form tran- controls.

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202 International Journal of Surgical Pathology Vol. 12 No. 3 July 2004

Fig. 31. Aberrant localization of β-catenin. A. Adenocarcinoma of colon shows

abnormal localization of the signals to the nuclei. B. Cribriform-morule variant
of papillary thyroid carcinoma. In contrast to the membranous staining of the
normal thyroid follicles (upper field), the tumor cells show aberrant β-catenin
immunostaining in the nuclei.

Fig. 32. Large B-cell lymphoma with fibrillary matrix and pseudorosettes.
A. The pseudorosettes are formed by fibrillary cores surrounded by tumor cells.
B. Since the fibrillary materials are composed mostly of cell membranes (inter-
digitating cell processes), the rosettes can be highlighted by CD20, which stains
a membrane-associated molecule.

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 Subcellular Localization of Immunohistochemical Signals • Cheuk and Chan 203

A B

Fig. 33. Signet ring B-cell lymphoma. A. This tumor is composed of cells with clear cyto-
plasmic vacuoles that indent the nuclei. B. The vacuoles are highlighted by CD20 im-
munostain since they are delimited by cell membrane.

scriptionally active complexes with T-cell

factor/lymphoid enhancer factor, resulting in activa-
tion of several genes such as C-MYC and CCND1
[62,63]. Thus, aberrant nuclear expression of β-
catenin is indicative of a disturbance of the Wnt sig-
nal transduction pathway. Most colorectal adenocar-
cinomas show this phenomenon attributable to
mutations in APC (Fig. 31A), and this feature can be
utilized to distinguish metastatic colorectal adeno-
carcinoma from primary adenocarcinoma in the uri-
nary bladder or primary ovarian mucinous carci-
noma [64–66]. Desmoid fibromatosis, which can
occur sporadically or as a component of Gardner
syndrome (familial adenomatous polyposis with fi-
bromatosis) also commonly exhibits aberrant nu-
clear translocation of β-catenin [67]. The cribriform-
morular variant of papillary thyroid carcinoma (Fig.
31B), adamantinomatous craniopharyngioma, and
calcifying odontogenic cyst also commonly show
aberrant nuclear expression of β-catenin, but attrib-
utable to mutations in β-catenin gene [68–72].
Fig. 34. Invasive micropapillary carcinoma
Aberrant CD10 Staining Pattern in of breast. Epithelial membrane antigen is ex-
Microvillous Inclusion Disease pressed on the peripheral surface of the glan-
dular structures instead of the central lu-
Microvillous inclusion disease is a rare autosomal mens, suggesting that these tumor islands
recessive disease of infancy presenting as intractable represent micropapillae instead of tubules.

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204 International Journal of Surgical Pathology Vol. 12 No. 3 July 2004
secretory diarrhea. CD10 is a membrane-associated
neutral peptidase that is normally expressed at the
brush borders of the enterocytes, producing pure
linear luminal membrane staining. The presence of
additional apical cytoplasmic CD10 immunoreactiv-
ity in the enterocytes, which reflects the disorga-
nized microvilli and cytoplasmic microvillous inclu-
sions on electron microscopy, can aid in the
diagnosis of microvillous inclusion disease [73].
Pattern of CD20 Staining in Rare Variants
of B-cell Lymphoma Provides Clue to the
Nature of the Aberrations
In the rare examples of B-cell lymphomas with
fibrillary matrix, positive staining of the matrix for
CD20 in a striated pattern indicates that the mater-
ial is formed by cell membrane material, in specific,
interdigitating cell processes (Fig. 32) [74]. In signet
ring B-cell lymphoma of the clear vacuole-type,
positive staining of the cytoplasmic vacuole for
CD20 indicates that the vacuole is lined by cell
membrane material, that is, representing “private”
luminal space (Fig. 33) [74].
Pattern of EMA Staining Helps Distinguish
Micropapillary Variant of Breast Cancer
from Conventional Breast Cancer with
Retraction Artifact
The micropapillary variant is an aggressive form
of breast carcinoma, but it is not always easy to di-
agnose. It can be difficult to distinguish between mi-
cropapillae formed by cellular aggregates or gland-
like structures in a fibrous stroma and tumor islands
with retraction artifact in conventional breast carci-
noma. Since EMA often selectively or predomi-
nantly decorates the free surface of the cells, posi-
tive immunostaining of the periphery instead of the
central lumens of the cell clusters indicates that they
are micropapillary structures (Fig. 34) [75].
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