CAP Laboratory Improvement Programs
Practical Applications in Immunohistochemistry
An Immunophenotypic Approach to the Spleen
William R. Borch, MD; Nadine S. Aguilera, MD; Mark D. Brissette, MD; Dennis P. O’Malley, MD; Aaron Auerbach, MD, MPH
 Context.—Even though immunohistochemistry is rou-
tinely used by pathologists, evaluation of immunohisto-
chemistry in splenic lesions remains difficult for many.
Classification of benign and splenic lesions often requires a
combination of hematoxylin-eosin evaluation, immuno-
phenotyping, and sometimes molecular testing. Immuno-
histochemical staining is essential in evaluating many
splenic lesions, and requires an understanding of the
normal compartments of the spleen.
Objective.—To address different immunohistochemical
features used for identification and subclassification of
different lesions of the spleen, as well as in the normal
compartments of the spleen.
D espite the routine use of immunohistochemistry as a
tool to assist in the diagnosis of surgical pathology and
hematopathology specimens, immunohistochemical evalua-
tion of the spleen remains confusing for many. Unusual
combinations of markers and reactivities may be present that
are not seen in other, comparable locations such as lymph
node. As such, pathologists may find immunohistochemical
evaluation of the spleen challenging. There is a paucity of
literature on this subject, and the purpose of this article is to
review current knowledge about the immunophenotype of
the various compartments of the normal spleen, as well as
benign and malignant diseases of the spleen.
COMPARTMENTS OF THE SPLEEN
To be able to interpret immunohistochemistry or flow
cytometric immunophenotyping of the spleen, one needs to
Accepted for publication November 21, 2018.
Published online March 27, 2019.
From the Department of Pathology, Walter Reed National Military
Medical Center, Bethesda Maryland (Dr Borch); the Department of
Hematopathology, University of Virginia, Charlottesville (Dr Agui-
lera); the Department of Hematopathology, Joint Pathology Center,
Silver Spring, Maryland (Drs Brissette and Auerbach); and the
Department of Hematopathology, NeoGenomics, Aliso Viejo,
California (Dr O’Malley).
The authors have no relevant financial interest in the products or
companies described in this article.
Supported by the Joint Pathology Center (JPC). The views
expressed in this article are those of the authors and do not
necessarily reflect the official policy or position of the Department of
Defense or the US Government.
Corresponding author: Aaron Auerbach, MD, MPH, Department of
Hematopathology, Joint Pathology Center, 606 Stephen Sitter Avenue,
Silver Spring, MD 20814 (email: aaron.auerbach.civ@mail.mil).
Arch Pathol Lab Med—Vol 143, September 2019
Data Sources.—The information outlined in this review
article is based on our experiences with a variety of
spleen cases, on the current World Health Organization
classification of hematopoietic and lymphoid tumors, and
on a review of English-language articles published during
2018.
Conclusions.—Features for phenotyping normal spleen
as well as a variety of splenic lesions, including littoral cell
angioma and splenic marginal zone lymphoma, are
discussed. Suggested immunopanels are provided to assist
in the diagnosis of different lesions of the spleen.
(Arch Pathol Lab Med. 2019;143:1093–1105; doi:
10.5858/arpa.2018-0211-CP)
understand the different compartments and functions of the
normal spleen. This also helps to determine which
compartment of the spleen is altered in a given disease.
The spleen is a hematopoietic and lymphoid organ that
arises in the fifth week of development from the embryonic
mesoderm. Initially, it functions as a site of maturation for
hematopoietic precursors, and later, it functions in immune
system development.1,2 The spleen is an encapsulated organ
composed of 2 major compartments: the red pulp and the
white pulp. The red pulp functions primarily as a blood filter
for foreign materials and damaged and senescent erythro-
cytes, though it also is a storage site for iron, erythrocytes,
and platelets. The white pulp functions as an immune-
response initiator to blood-borne antigens, making it the
body’s largest secondary lymphoid organ. The red pulp and
white pulp contain different cells, and thus have a different
immunophenotype. The spleen also has prominent stromal
and vascular compartments that play a role in spleen
physiology and anatomy, and may present as abnormal
cellular proliferations.
The red pulp of the spleen is composed of cords and
venous sinuses; the cords are composed of reticular
myofibroblasts and macrophages, and the sinuses are lined
by a distinctive endothelial cell with partial histiocytic
functions called the splenic littoral cell.3 Within the red pulp,
the myofibroblasts are immunoreactive for smooth muscle
actin (SMA), and the macrophages stain with macrophage
markers such as CD68 and CD163.4,5 The sinus-lining
littoral cells are unique to the spleen with a characteristic
immunophenotype. In fact, their immunophenotype is so
distinctive, that if present, it can identify splenic tissue in
unusual sites (ie, ectopic/heterotopic spleen). Splenic littoral
cells express endothelial markers such as CD31, Wilms
An Immunophenotypic Approach to the Spleen—Borch et al 1093
 Table 1. Immunohistochemistry Panels
Basic spleen immunohistochemistry panel
CD8, CD20, CD3, CD34, ERG, WT1
Red pulp lymphoid lesions
CD8, CD68, CD34, DBA.44, annexin A1, BRAF V600E,
CD25, CD103
White pulp lymphoid lesions
CD20, CD3, CD5, CD10, BCL6, BCL1 (cyclin D1), BCL2,
Ki-67, MUM1, EBER, CD30, CD8
Vascular lesions
ERG, CD31, CD34, factor VIII, D2-40, CD68, CD163, CD8,
WT1
Stromal/histiocytic lesions
CD21, CD23, CD35, LMP1, EBER, SMA, S100, ERG, CD31,
CD34, WT1, CD68, CD163, CD1a and CD3
T-cell or NK-cell lymphoma/leukemia
CD2, CD3, CD5, CD7, EBER, TIA1, granzyme B, TCL1,
CD56, CD4, CD8, CD30
Cysts
CD8, cytokeratin AE1/AE3, calretinin, GMS ERG
Abbreviations: EBER, Epstein-Barr virus–encoded RNA; GMS, Grocott-
Gomori methenamine silver stain; SMA, smooth muscle actin; WT1,
Wilms tumor protein 1.
tumor protein 1 (WT1), factor VIII, and ERG, as well as
CD68 and variably, CD21.5,6 Their most distinctive marker,
though, is CD8. Typically associated with cytotoxic T cells,
CD8 expression by littoral cells highlights the architectural
framework of the red pulp in a dendritic pattern. CD8
positivity shows that the framework of the spleen is intact.
Conversely, loss of CD8 can be used to demonstrate that the
architecture is being replaced by a neoplasm or other space-
occupying lesion.
The splenic white pulp consists of arterioles that are
surrounded by lymphoid cells, the so-called periarteriolar
lymphoid sheath (PALS) and adjacent outpouchings of
nodular lymphoid tissue.6 The PALS is composed of
predominantly T cells and flattened reticular cells.3 The T
cells express usual T-lymphocyte antigens (CD2, CD3, CD5,
and CD7) and most are CD4þ, with few staining for CD8.6
Scattered TDTþ (terminal deoxynucleotidyl transferase-
positive) cells have also been seen in pediatric spleens
particularly in the PALS.7
The lymphoid follicles are composed mostly of B cells
along with fewer scattered CD4þ T lymphocytes. Lymphoid
follicles are divided into primary and secondary follicles.3
Primary follicles are composed of small mature B cells that
express standard B-lymphocyte markers (CD19, CD20,
CD79a, PAX5, and BCL2). Secondary follicles are composed
of germinal centers, containing B and occasional T
lymphocytes and follicular dendritic cells, surrounded by
mantle zones. The B lymphocytes of the germinal centers
express CD19, CD20, CD79a, PAX5, CD10, and BCL6 with
coexpression of immunoglobulin (Ig) M and IgD; however,
they are negative for BCL2. Mantle zone B lymphocytes
express CD19, CD20, CD5, DBA.44, BCL2, and IgD, but are
negative for CD10 and CD23. The follicular dendritic cells
that compose the follicular meshwork express follicular
dendritic cell markers such as CD21 and CD35.6
The marginal zone is a macrophage-rich zone located at
the interface between the red and white pulp. Marginal zone
1094 Arch Pathol Lab Med—Vol 143, September 2019
Table 2. Characteristics of Vascular Markers in the
Spleen
Antibody Specificity Sensitivity Characteristics
ERG   Nuclear stain, stains
vascular cells and
vascular tumors
WT1   Cytoplasmic reactivity, less
background staining than
CD34
CD34   Does not highlight splenic
littoral cells, splenic
hamartoma, or littoral
cell angioma
CD31   Nonspecific, will also stain
histiocytes and
macrophages
Factor VIII   Older antibody, less used,
less sensitive and
specific
Abbreviation: WT1, Wilms tumor protein 1.
 indicates some specificity and/or sensitivity;  indicates moderate
specificity and/or sensitivity;  indicates maximum specificity and/
or sensitivity.
B lymphocytes have a characteristic IgMþ/IgD phenotype;
they are positive for B-cell markers (CD19, CD20, CD22)
and coexpress BCL2. Marginal zone B cells are negative for
CD5, CD10, CD23, and DBA.44.3,6,8 Additionally, the
marginal zone has a high concentration of SMA-positive
myofibroblasts, and staining may show a partial ‘‘barrier’’ of
cells that separates red from white pulp. This may also be
highlighted by staining for thrombomodulin.4
A basic panel of immunohistochemical stains for initial
study of the spleen could include CD8, CD20, CD3, and
appropriate vascular markers. This panel allows for some
basic phenotyping of the white pulp by comparing the
amounts of CD20þ B cells and CD3þ T cells. CD8 is essential
to determine if the red pulp architecture of the spleen is
intact (Table 1).
The choice of vascular markers should be based on those
available, but there are significant strengths and weaknesses
among them. ERG is a newer marker that has several
advantages including nuclear localization, making positive
identification of positive cells easy; robust staining; and
extremely high sensitivity (essentially all vascular cells and
vascular tumors are positive).9 WT1 is also an excellent
vascular marker, staining most vascular cells and vascular
neoplasms, with high sensitivity, low background staining,
and cytoplasmic reactivity.10 CD34 is a very good vascular
marker, but has an important caveat in spleen. It does not
highlight splenic littoral cells (eg, red pulp sinuses),
proliferations (eg, hamartoma), or neoplasms derived from
littoral cells (littoral cell angioma).11 In cases of poorly
differentiated vascular neoplasms, it is present in most, but a
significant proportion are negative. CD31 is sensitive for
vascular structures, but not specific, as it will stain
macrophages as well (Table 2). Factor VIII antigen is an
adequate stain, but has only moderate sensitivity and
specificity (Figure 1, A through O).
FLOW CYTOMETRY
When normal splenic tissue is sampled for flow cytometry,
several unusual lymphoid subsets can be encountered.
An Immunophenotypic Approach to the Spleen—Borch et al
Figure 1. Immunophenotype of normal spleen. A though F, White pulp. A, White pulp showing a germinal center and marginal/mantle zone. B,
CD20 shows positivity in B cells of white pulp. C, CD3 is expressed in scattered T cells of white pulp. D, Bcl-2 shows negativity in small germinal
center but typically stains the marginal/mantle zone. E, CD43 is expressed in T cells in normal white pulp. F, CD21 highlights follicular dendritic cell
meshwork in white pulp. G through M, Red pulp. G, Red pulp showing splenic sinuses. H, Factor VIII stains endothelial cells. I, CD8 highlights the
splenic sinuses. J, CD68 shows positivity in histiocytes. K, ERG marks endothelial cells. L, CD34 also marks endothelial cells. M, WT1 is a very
sensitive endothelial marker. N and O, The T zone. N, Periarteriolar lymphoid sheath with surrounding lymphocytes. O, CD4 highlights helper T cells
(hematoxylin-eosin, original magnifications 3200 [A through F, and O], 3400 [G through M], and 3100 [N]).

Approximately 20% to 30% of nonneoplastic splenic B
lymphocytes express CD5 and this number can be increased
in human immunodeficiency virus (HIV) and autoimmune
diseases.12–14 These nonneoplastic B cells should not be
confused for mantle cell lymphoma or chronic lymphocytic
leukemia/small lymphocytic lymphoma, both of which also
express CD5.15 A small population of CD4þ/CD8þ T cells is
typically present within the normal spleen, which comprises
on average 3.3% of the T cells and at most 6% of the T cells;
they are typically CD4 bright and CD8 dim and should not
be misinterpreted as a T-cell lymphoblastic leukemia/
lymphoma.12 Some T-cell antigens may be lost in splenic
T cells and should not necessarily be considered neoplastic.
A subset of normal T lymphocytes in the spleen does not
express CD7. The proportion of cd T cells is higher in the
spleen (12.5% 6 8.1%) than in peripheral blood (4.0% 6
3.1%); these typically show bright CD3 expression without
expression of CD4, CD8, and CD5. These findings would be
concerning for neoplasia in a population of ab T cells, but
are not concerning in cd T cells.16 In one study,17
approximately 5% of T cells in the spleen were CD5; most
of these are either CD8þ or are cd T cells as described above.
Natural killer (NK) cells with an immature phenotype
(CD56 brightþ/CD16) are markedly increased in splenic
tissue when compared to peripheral blood. Additionally,
CD2 NK cells compose an important normal subpopula-
tion within splenic tissue, and although CD7 expression is
Arch Pathol Lab Med—Vol 143, September 2019
considered a defining feature of NK cells, variably intense
CD7 expression is common in the spleen.17
Although flow cytometry is an excellent way of immuno-
phenotyping some entities such as lymphoma, splenic
lesions such as vascular proliferations or stromal lesions
should be examined as tissue sections to evaluate architec-
ture and immunohistochemical staining pattern.
PHENOTYPING SPLENIC DISEASES
Splenic diseases are often located in 1 specific compart-
ment of the spleen, either the red pulp or the white pulp.
Splenic hamartomas, littoral cell angiomas, hemangiomas,
and hairy cell leukemia (HCL) are diseases typically found in
the red pulp, while other B- and T-cell lymphomas, such as
splenic marginal zone lymphoma, are commonly detected in
the white pulp18,19 (Table 3). After determining by hema-
toxylin-eosin light microscopy whether it is the red pulp
and/or white pulp that appears abnormal, one can target a
more specific immunohistochemical panel aimed at the
differential diagnosis that would be expected in that
compartment of the spleen. If there is a red pulp
abnormality, then CD8, CD68, CD163, CD31, CD34, ERG,
WT1, DBA.44, annexin A1, T-bet, CD25, and BRAF would
be helpful. If there is a white pulp abnormality, then a B-cell
panel including CD20, CD3, CD5, CD10, BCL6, BCL1
(cyclin D1), BCL2, and Ki-67 (also MUM1, EBER [Epstein-
Barr virus–encoded RNA], and CD30 in large cell infiltrate)
An Immunophenotypic Approach to the Spleen—Borch et al 1095
 Table 3. Distribution of Diseases Within Red Pulp
and White Pulp
Common Diseases Common Diseases
in Red Pulp in White Pulp
Splenic hamartoma Splenic marginal zone
lymphoma
Littoral cell angioma Diffuse large B-cell lymphoma
Hemangiomas Chronic lymphocytic leukemia/
small lymphocytic lymphoma
Hairy cell leukemia Measles
Splenic diffuse red pulp Typhoid
small B-cell lymphoma
Chronic myeloid leukemia Castleman disease
Infectious mononucleosis Idiopathic thrombocytopenic
purpura
Angiosarcoma Felty syndrome
would be more helpful for the distinction of small and large
B-cell lymphomas (Table 1).
MESENCHYMAL DISEASES
Although splenic mesenchymal lesions have a low
incidence in the population, they are some of the most
common space-occupying lesions in the spleen, and
frequently are difficult to classify. The most common splenic
mesenchymal lesion, splenic hemangioma, has an estimated
incidence of 0.02% to 0.16%.20 Hemangiomas are benign
vascular tumors of interconnected blood vessels with
variable lumen sizes, lined by variably shaped endothelial
cells. By definition, hemangioma lacks features of malig-
nancy, such as solid areas, mitotic figures, cellular atypia,
and necrosis, which are instead features seen in splenic
angiosarcoma. There are 2 vascular neoplasms of interme-
diate malignant potential: hemangioendothelioma and
Kaposi sarcoma. Kaposi sarcoma appears similar to that
seen in other sites and is typically identified in immuno-
compromised patients, most often in HIV/AIDS (acquired
immunodeficiency syndrome). Kaposi sarcoma will be
positive for general vascular endothelial markers (ie, ERG,
WT1, CD31, CD34, and factor VIII), but it is also uniformly
positive for the lymphatic endothelial marker D2-40. In
addition, most cases are positive for human herpesvirus 8.
Hemangioendothelioma appears in many patterns, and the
neoplastic cells can be epithelioid to spindled and myoid,
such that they may be mistaken for epithelial or myoid
tumors. Splenic hemangiomas, hemangioendotheliomas,
and angiosarcomas are all vascular neoplasms, so they will
stain positively for vascular markers, such as ERG, WT1,
CD31, CD34, and factor VIII.21 Higher-grade neoplasms,
especially angiosarcoma, may lack some or most of the
vascular markers; ERG is the most commonly retained
marker in these neoplasms. Additionally, hemangioendo-
theliomas may be positive for cytokeratins if epithelioid or
positive for SMA if myoid.6,22
Lymphangioma is frequently in the differential diagnosis
of hemangioma because of a similar histologic appearance.
The lumens of lymphangiomas tend to be filled with pink
proteinaceous fluid, although some erythrocytes may be
present, whereas the lumens of hemangiomas are usually
filled with blood. Lymphangiomas stain positively for
general vascular endothelial markers, and are also positive
for D2-40, while hemangiomas do not express the latter.23
1096 Arch Pathol Lab Med—Vol 143, September 2019
Littoral cell angioma is a relatively rare vascular neoplasm
that is found only in the spleen and is thought to arise from
the red pulp sinus lining (littoral) cells. This neoplasm is
based within the red pulp and composed of multiple
irregular cystic channels filled with blood. It may be
multifocal and is most often incidentally discovered on
imaging of the abdomen. The lumens are lined by tall and
flat cells of red pulp derivation that protrude into the lumen
of the vascular spaces. The tall lining cells express histiocytic
markers (CD68 and CD163), whereas the flat cells express
vascular markers (CD31 and factor VIII).21 These tumors
also express ERG, but typically lack staining for WT1 and
CD34. In contrast to the normal splenic littoral cell, littoral
cell angioma does not express CD8. Coexpression of
markers of both histiocytic and vascular differentiation is
highly distinctive and useful in the identification of these
tumors.
Splenic hamartoma is also unique to the spleen; this
lesion may represent a developmental anomaly but its
true etiology is unknown. The lesion is composed
exclusively of red pulp and can be recognized microscop-
ically by a mass of red pulp with notable absence of white
pulp. On cut section, it often bulges from the surface,
suggesting that the nodular mass is compressed against
adjacent normal spleen. It is unencapsulated, but often
has a well-defined border from adjacent normal spleen.
Architecturally, it is composed of variably organized cords
and sinuses. The cells lining the sinuses are CD8
immunopositive and also reactive for general vascular
endothelial markers (ERG, WT1, CD31, and factor VIII).
Additionally, they can be focally positive for histiocyte
markers24 (Figure 2, A through P).
Hemangioma, lymphangioma, littoral cell angioma, and
splenic hemangioma are in the differential diagnosis of a
splenic vascular lesion. An immunohistochemistry panel of
ERG, WT1, CD31, CD34, factor VIII, D2-40, CD68, CD163,
and CD8 can be used for this differential diagnosis (Table 1).
In this differential diagnosis, D2-40 serves to distinguish
lymphangioma, whereas the tall cells of littoral cell angioma
express CD68 and CD163.
SPINDLE CELL DISEASES PRIMARY TO THE SPLEEN
Splenic lesions with spindled mesenchymal cells and
admixed lymphoplasmacytic infiltrates may occur in the
spleen and have a broad differential diagnosis. Inflamma-
tory pseudotumor of the spleen is benign but frequently
mimics a malignant process. These lesions are generally well
circumscribed and can be composed of mixed chronic
inflammatory cells, with fibrosis and vascular tissue.
Immunohistochemically, there is a mixed pattern of
staining, corresponding to the mixture of cells involved:
myofibroblasts stain for SMA and/or S100, vascular tissue is
positive for typical vascular markers (ERG, WT1, CD31,
CD34, and factor VIII), histiocytes stain with CD68 and
CD163, and lymphocytes are T-cell predominant with most
highlighted by CD3.25 Any entrapped normal red pulp will
stain for CD8. A variant of splenic pseudotumor—post-
chemotherapy histiocyte-rich pseudotumor of spleen—
arises after treatment of a chemotherapy-sensitive malig-
nant mass in the spleen, usually an aggressive lymphoma,
metastatic carcinoma, or ‘‘small blue cell’’ pediatric malig-
nancy. This entity is composed of mixed inflammatory cells,
CD68þ/CD163þ histiocytes and necrotic malignant cells.
Immunohistochemical stains specific to the primary malig-
An Immunophenotypic Approach to the Spleen—Borch et al
Figure 2. Vascular lesions involving the spleen. A through D, Hemangioma. A, Well-demarcated hemangioma. B, Abnormal dilated vessel with
prominent endothelial cells. C, CD34 shows positivity in the endothelial cells. D, CD31 shows positivity in the endothelial cells. E through H,
Hamartoma. E, Low power shows the absence of white pulp. F, CD8 highlights the sinuses. G, CD34 highlights normal splenic vessels. H, Lysozyme
stains histiocytic cells in the sinuses. I through L, Littoral cell angioma. I, lining cells sloughing into the vascular lumen. J, CD31 staining the flat
endothelial lining cells. K, Factor VIII stains the flat lining cells. L, CD8 shows negativity in the lining cells but stains the sinuses. M through O, Peliosis.
M, Sinus dilatation near a white pulp nodule. N, Dilation of the sinus by red blood cells. O, CD34 shows negativity in the lining. P, Factor VIII stains
the lining of the sinuses (hematoxylin-eosin, original magnifications 320 [A and E], 3400 [B, I, and N]; 3400 [C, D, F through H, K, L, O, and P], and
3200 [J and M]).

nancy may help to highlight any residual viable malignant
cells within the mass.26
Inflammatory pseudotumors must be differentiated from
other spindle cell mesenchymal lesions such as follicular
dendritic cell sarcoma, inflammatory pseudotumor-like
follicular/fibroblastic dendritic cell tumor (IPFDC/FRC),
and inflammatory myofibroblastic tumor. Follicular dendrit-
ic cell sarcoma (FDCS) is a low-grade malignancy thought
to arise from the follicular dendritic cell network of the
splenic white pulp. It is composed of spindled to ovoid cells
Arch Pathol Lab Med—Vol 143, September 2019
in sheets, fascicles, and whorls that stain for CD21, CD23,
CD35, D2-40, and Ki-M4P.27 IPFDC/FRC is a variant with
admixed inflammatory cells (sometimes with striking
eosinophil-rich and granulomatous areas that can mimic
infectious lesions). The lesion is well demarcated and
usually at least partially encapsulated, and the neoplastic
cells are positive for the same follicular dendritic cell
markers as the usual FDCS. Unlike classic FDCS, however,
the pathophysiology of this variant is linked to Epstein-Barr
virus, and the neoplastic cells are always positive for EBER
An Immunophenotypic Approach to the Spleen—Borch et al 1097
Figure 3. Stromal/mesenchymal and histiocytic lesions. A through D, Inflammatory pseudotumor. A, Spindle-appearing proliferation. B, Smooth
muscle actin shows positivity. C, Muscle-specific actin stains the spindle cells. D, CD8 shows negativity, indicating there is no normal splenic tissue. E
through H, Langerhans cell histiocytosis. E, Granulomatous appearance at low power. F, Histiocytic cells with longitudinal nuclear grooves. G, CD1a
shows positivity in the neoplastic cells. H, S100 shows positivity in the neoplastic cells (hematoxylin-eosin, original magnifications 3200 [A through
C], 3400 [D, and F through H]), and 320 [E]).
and sometimes for EBV-LMP1.28,29 Cases similar to IPFDC/
FRC have been reported to have increased IgG4þ plasma
cells, which has brought into question whether this entity is
an IgG4-related sclerosing disease.30,31
Inflammatory myofibroblastic tumor (IMT) is a myofibro-
blastic spindle cell neoplasm of predominantly children and
young adults that classically features a mixed chronic
inflammatory infiltrate and rarely involves the spleen. By
immunohistochemistry, the spindle cells are positive for SMA
and desmin (variable), with a subset showing focal keratin
expression. ALK immunohistochemistry shows cytoplasmic
positivity in 50% to 60% of cases outside of the spleen, but is
reported to be negative in splenic IMTs. Fluorescence in situ
hybridization testing will also show rearrangement of 2p23
ALK in ~50% of cases. ALK partners with a variety of genes
including EML4, ATIC, TPM3, TPM4, CLTC, and RANBP2.
Some of the ALK-negative IMTs harbor fusions involving
ROS1, RET, PDGFRB, or ETV6 and NTRK3.
Spindle cell lesions primary to the spleen include
inflammatory pseudotumors (if there are conspicuous
spindled myofibroblasts), IMT, FDCS, and IPFDC/FRC. An
immunohistochemical panel to differentiate these lesions
includes CD21 (and other FDC markers, such as CD23,
CD35, Ki-M4P), EBER, SMA, cytokeratin AE1/3, ALK, S100,
ERG, CD34, CD68, CD163, and CD3 (Figure 3, A through
H). Inflammatory pseudotumor expresses varying amounts
of SMA, S100, ERG, CD31, CD34, CD68, CD163, and CD3
depending on the density of each type of inflammatory cell.
FDCS expresses CD21, CD23, CD35, and Ki-M4P. IPFDC/
FRC is positive for EBER and variably positive for CD21 and
other FDC markers. Inflammatory myofibroblastic tumor
expresses SMA, sometimes desmin, and occasionally
keratins. Rarely, other spindle cell tumors, such as
1098 Arch Pathol Lab Med—Vol 143, September 2019
interdigitating dendritic cell tumor (S100þ) or mastocytosis
(CD117þ, tryptase positive), are encountered.
Histiocytic neoplasms may also occur in the spleen.
Langerhans cell histiocytosis is a clonal proliferation of
Langerhans cells, which typically form aggregates that
expand the red pulp. The lesional cells have abundant
eosinophilic cytoplasm with grooved to lobulated nuclei;
they stain for CD1a, langerin (CD207), and S100 (both
cytoplasmic and nuclear staining).32,33 Histiocytic sarcoma,
or malignant histiocytosis, is a high-grade neoplasm with a
widely variable appearance. The neoplastic cells are large
and discohesive, and can appear bland to overtly malignant.
The role of immunohistochemistry is often to exclude other
high-grade malignancies; the cells are positive for histiocytic
markers (CD68, CD163), and negative for melanoma,
carcinoma, myeloid, and Langerhans cell markers.6,34 In
any histiocytic neoplasm, testing for mutant BRAF is wise, as
this may be a therapeutic target and can support a diagnosis.
This can be accomplished by immunohistochemistry for the
mutant BRAF V600E antibody.
CYSTS AND PELIOSIS
Immunohistochemistry can assist in differentiating the
different cysts in the spleen and peliosis. Cysts in the spleen
can be divided into epithelial cysts (true cysts), mesothelial
cysts, pseudocysts, and parasitic cysts. Epithelial cysts of the
spleen are typically lined by a stratified squamous epithe-
lium (epidermoid), a squamous epithelium with adnexal
structures (dermoid), or a cuboidal to columnar epithelium.
Often, if long-standing, the cystic lining may be attenuated
or absent, and careful sampling and a diligent search for
flattened epithelial cells are necessary. Mesothelial cysts may
exhibit squamous metaplasia. The squamous epithelial
lining stains positively for cytokeratins, carcinoembryonic
An Immunophenotypic Approach to the Spleen—Borch et al
Figure 4. Splenic cysts. A through D, Epithelial cyst. A, Normal spleen tissue below a cyst with an epithelial lining. B, Cytokeratin AE1/AE3þ lining
cells. C, CD34 lining cells. D, CD68 lining cells. E through H, Mesothelial cyst. E, Cyst showing lining cells. F, Cytokeratin AE1/AE3 shows positivity.
G, WT1 shows positivity. H, Calretinin shows positivity in the lining cells (hematoxylin-eosin, original magnifications 3100 [A and B], 3400 [C and
D]), and 3200 [E through H]).
antigen, and CA 19-9; the mesothelial lining stains
positively for mesothelial markers, such as calretinin. The
epithelium of these cysts is negative for vascular mark-
ers.6,35,36 Pseudocysts have a wall composed of dense fibrous
tissue without an epithelial lining and are thought to be
sequelae of traumatic hematomas. Negative staining for
cytokeratins helps confirm the lack of an epithelial lining,
which differentiates a pseudocyst from an epithelial cyst37
(Figure 4, A through H).
Parasitic cysts (hydatid cysts) in the spleen are most often
due to infection with Echinococcus tapeworm larvae. The cyst
is often multiloculated with a laminated (alternating
between light and dark areas) membrane. Within the
laminated membrane is a germinal membrane, upon which
protoscoleces develop. The scolex of the worm is the
anterior end, composed of a sucker and 2 rows of hooklets.
The role of immunohistochemistry is limited in parasitic
cysts, but special stains, such as Grocott-Gomori methena-
mine silver (GMS) stain, can highlight hooklets in suspi-
cious cases6 and differentiate an echinococcal cyst from an
epithelial cyst or pseudocyst.
Peliosis is a rare condition in which there are multiple
blood-filled cavities within parenchymal organs—usually the
spleen and liver. The origin may be a congenital vascular
malformation, worsened by a particular exogenous insult. It
has been seen in association with tuberculosis, liver cirrhosis,
hematologic malignancies, alcoholism, and anabolic steroid
treatment. Histologically, the blood-filled cavities have no
definitive endothelial lining; stains for vascular markers
(ERG, CD31, CD34, and factor VIII) should show negativity
around the cystic spaces; positive staining would favor
another vascular entity, such as a hemangioma. Of note,
vascular spaces or ‘‘blood lakes’’ in the spleen may become
lined by neoplastic B cells in HCL, and stains for B-cell
Arch Pathol Lab Med—Vol 143, September 2019
markers should not show positivity in the lining of the
peliotic lesions.6,38,39 An immunohistochemistry and special
stain panel including cytokeratin AE1/3, calretinin, GMS, and
ERG could be used in this differential of cystic splenic
diseases and peliosis. Mesothelial cysts are distinguished
from epithelial cysts because mesothelial cysts are positive for
both calretinin and cytokeratin AE1/3, but epithelial cysts are
calretinin negative and cytokeratin AE1/3 positive. GMS
staining can be positive in an echinococcal cyst. Peliosis
would express one of the vascular markers such as ERG, and
a pseudocyst would not express any of these markers.
LYMPHOPROLIFERATIVE DISORDERS
Small B-cell lymphomas, large B-cell lymphomas, and T-
cell lymphomas may all involve the spleen. Many of these
primarily involve the white pulp, some are seen in both red
and white pulp (eg, chronic lymphocytic leukemia/small
lymphocytic lymphoma), and others occur in the red pulp
(eg, HCL).
Splenic marginal zone lymphoma (SMZL) is a primary
splenic low-grade B-cell lymphoma composed of small to
medium-sized B lymphocytes present in micronodules
distributed throughout the white pulp in a miliary pattern.
Circulating neoplastic cells often have characteristic short
cytoplasmic villi that are usually unevenly distributed. In
most cases, the cells express IgM or IgD and are CD20þ,
CD22þ, FMC7þ, and CD79aþ. A subset of cases may express
DBA.44, CD11c, CD23, CD103, CD25, and CD5 by flow
cytometry and/or immunohistochemistry6,40 (Tables 4 and
5). Follicular dendritic cell meshworks, which can be
highlighted by CD21, CD23, and CD35, are often disrupted
and expanded in SMZL (Figure 5, A through L).
An Immunophenotypic Approach to the Spleen—Borch et al 1099
 Table 4. Immunohistochemistry in Splenic Marginal Zone Lymphoma
Antibody Staining Pattern Results Additional Information
Annexin A1 Not applicable Negative If positive, consider hairy cell leukemia
BCL2 Membranous and Positive Detected in tumor cells, but not in residual reactive GC cells
cytoplasmic
BCL6 Nuclear Negative Positive in residual GC cells; may be positive in transformed SMZL
CD5 Membranous Positive Positive in ~20% cases; usually faint reactivity
CD10 Membranous Negative Positive only in residual GC cells, may be diffusely positive in transformed SMZL
CD20 Membranous Positive Strong expression by neoplastic cells
CD23 Membranous Negative Positive in expanded follicular dendritic cell meshworks; negative in lymphocytes;
if positive in lymphocytes, consider CLL/SLL
CD43 Membranous þ/ May be coexpressed in the malignant B cells
Cyclin D1 (BCL1) Nuclear Negative If positive, consider mantle cell lymphoma
DBA.44 Cytoplasmic Positive ~50% of cases are positive
IgD Membranous Positive ~50% of cases are positive
IgM Membranous Positive .90% of cases are positive
Ki-67 Nuclear Positive Low proliferative rate; cells usually found at marginal zone
PAX5 Nuclear Positive
Sox 11 Nuclear Positive
Abbreviations: CLL/SLL, chronic lymphocytic leukemia/small lymphocytic lymphoma; GC, germinal center; Ig, immunoglobulin; SMZL, splenic
marginal zone lymphoma.
þ/ indicates that CD43 is expressed in some, but not all of the cases.
This table was modified from 6Auerbach A. Diagnostic Pathology: Spleen. 1st ed. Altona: Amirsys, Lippincott Williams & Wilkins; 2014.

Some B-cell lymphoproliferative disorders primarily in- this entity, however, the cells are typically positive for B-cell
volve the red pulp. Hairy cell leukemia is composed of small markers and DBA.44 with a similar phenotype to SMZL, but
to medium-sized B lymphocytes with oval nuclei; it is named are negative for TRAP, annexin A1, CD25, CD103, CD11c,
for its ‘‘hair-like’’ projections, distributed evenly around the CD10, and CD123, distinguishing it from HCL and HCL-v.44
cytoplasm seen on touch preparations and in peripheral Other small B-cell lymphomas can involve the splenic
blood smears. Unlike SMZL, the neoplastic lymphocytes of white pulp with or without the red pulp and can be
HCL are present throughout and diffusely efface the splenic characterized by their phenotype. When the spleen is
red pulp. The cells are characteristically positive for B-cell involved by chronic lymphocytic leukemia/small lympho-
markers, annexin A1, tartrate-resistant acid phosphatase cytic lymphoma, it is characterized by diffuse white pulp
(TRAP), BRAF V600E, cyclin D1, DBA.44, CD11c, CD25, expansion by aggregates of the neoplastic lymphocytes,
CD103, and CD123. Annexin A1 is the most specific, but is which typically express B-cell markers, CD5, CD43, CD23,
often the hardest to interpret, especially in the bone marrow LEF1, and CD200.33 Mantle cell lymphoma and follicular
where it also shows positivity in erythroid cells. In the lymphoma can also involve the spleen, and their pheno-
uncommon hairy cell leukemia variant (HCL-v), cells types are similar to these lymphomas in nonsplenic
typically express B-cell markers, CD11c, DBA.44, and locations. A basic small B-cell lymphoma panel can typically
CD103, but may lack CD25, CD123, annexin A1, and distinguish these neoplasms (Figure 6, A through I).
TRAP.6,41–43 CD10 may show positivity in up to 20% of Large B-cell lymphomas may also involve the spleen.
HCLs, but it typically shows negativity in HCL-v. Splenic Diffuse large B-cell lymphoma (DLBCL) of the spleen
diffuse red pulp small B-cell lymphoma also shows diffuse typically has the gross appearance of multiple, large, nodular
involvement by cells with villous cytoplasmic projections; in masses. The neoplastic B cells are large and express mature
B-cell markers; expression of BCL6, MUM1, and CD10 is
variable but carries prognostic significance and is frequently
Table 5. Flow Cytometry in Splenic Marginal Zone used to classify a lymphoma into germinal center and
Lymphoma nongerminal center subtypes. The cells can have variable
Antibody Results Additional information expression of CD5, CD23, BCL2, and CD43, but the tumor
nodules should lack follicular dendritic cell meshworks. This
CD5 Positive Positive in 20% of cases
helps to separate DLBCL from higher-grade follicular
CD10 Negative
lymphomas.6,33
CD11c Positive Positive in 50% of cases
High-grade B-cell lymphoma is a new entity in the 2017
CD20 Positive Bright World Health Organization classification. This was previ-
CD22 Positive Bright ously classified as B-cell lymphoma, unclassifiable, with
CD23 Positive Positive in ~30% of cases features intermediate between DLBCL and Burkitt lympho-
CD25 Positive Positive in 25% of cases ma. These lymphomas morphologically resemble DLBCL or
CD103 Usually negative Rare positive in ,10% of cases have features between DLBCL and Burkitt lymphoma. The
FMC7 Positive Positive in most cases neoplastic cells are positive for B-cell markers and usually
IgD Positive Positive in 50% of cases BCL6. CD10 expression is variable, while TDT shows
IgM Positive Positive in most cases negativity. High-grade B-cell lymphoma is split into 2
This table was modified from 6Auerbach A. Diagnostic Pathology: subgroups: high-grade B-cell lymphoma with MYC and
Spleen. 1st ed. Altona: Amirsys, Lippincott Williams & Wilkins; 2014. BCL2 and/or BCL6 rearrangements (‘‘double-hit’’ or ‘‘triple-
1100 Arch Pathol Lab Med—Vol 143, September 2019 An Immunophenotypic Approach to the Spleen—Borch et al
Figure 5. B-cell lymphomas involving the white pulp. A, Follicular lymphoma, low grade. B, Predominantly cleaved centrocytes with rare
centroblasts. C, CD10þ malignant lymphoid cells. D, BCL6 stains the malignant cells in a nuclear pattern. E through H, Splenic marginal zone
lymphoma. E, There is expansion of the marginal zone of the white pulp. F, CD20 stains the malignant lymphocytes. G, BCL2 stains the malignant
lymphocytes. H, CD43 shows negativity in the malignant lymphocytes. I through L, Small lymphocytic lymphoma/chronic lymphocytic leukemia. I,
There is a focal area of splenic involvement. J, The cells are monotonous, small, and round with clumped chromatin. K, CD5 expression in the
neoplastic lymphocytes. L, CD23 expression in the neoplastic lymphocytes (hematoxylin-eosin, original magnifications 320 [A, E, and I], 3400 [B
through D, and J through L], and 3100 [F through H]).

hit lymphoma’’ and high-grade B-cell lymphoma, not
otherwise specified).
T-cell lymphomas and leukemias that frequently involve
the spleen include hepatosplenic T-cell lymphoma (HSTCL),
T-cell prolymphocytic leukemia, T-cell large granular lym-
phocytic leukemia, and peripheral T-cell lymphoma, not
otherwise specified. HSTCL in the spleen shows infiltration
of the splenic sinusoids by malignant T cells and also involves
the sinuses of the liver and bone marrow. The neoplastic T
cells are typically TCR-cdþ, CD3þ, TIA1þ, CD56þ/, and
granzyme Mþ, while typically negative for CD5, CD4, CD7,
granzyme B, perforin, and B-cell markers.
In the aggressive T-cell prolymphocytic leukemia (T-PLL),
marked lymphocytosis accompanies diffuse splenic enlarge-
ment. The neoplastic T cells are positive for pan T-cell
markers, TCL1, and are usually CD4þ/CD8, though T-PLL
can coexpress both CD4 and CD8 or be CD4/CD8þ. The
more indolent T-cell large granular lymphocytic leukemia is
typically positive for TCR-ab, CD3, CD2, CD8, CD57, TIA1,
granzyme B, and granzyme M. Peripheral T-cell lymphoma
Arch Pathol Lab Med—Vol 143, September 2019
(PTCL), not otherwise specified, also presents with diffuse
involvement of splenic parenchyma. The neoplastic T cells
are positive for T-cell markers (CD2, CD3, and usually CD4)
with loss or downregulation of CD5 and/or CD7, and
frequent loss of BCL2 expression. Rarely, aberrant expres-
sion of some B-cell markers (CD79a and CD20) can be
seen.6,33 PTCL is diagnosed when a T-cell lymphoma does
not fit another more specific T-cell lymphoma subtype.
Aggressive NK-cell leukemia in the spleen is typically
manifested by diffuse and variable splenic enlargement. The
cells express NK-cell markers with an activated cytotoxic
phenotype: CD2, CD56, TIA1, granzyme B, and perforin.
NK- and T-cell lymphomas and leukemias of the spleen are
rare; an immunohistochemistry panel for these neoplasms
would include CD2, CD3, CD5, CD7, EBER, TIA1, granzyme
B, TCL1, CD56, CD4, and CD8 (Figure 7, A through H).
Classic Hodgkin lymphoma can involve the spleen. It
was previously more frequently encountered when
staging laparotomy including splenectomy was per-
formed. Presently, however, it is rarely encountered, as
An Immunophenotypic Approach to the Spleen—Borch et al 1101
Figure 6. Lesions involving red pulp. A through C, Hairy cell leukemia. A, Red pulp involvement without prominent white pulp. B, CD20 positivity.
C, DBA.44 positivity. Other markers that show positivity include CD25, CD123, annexin A1 (not shown). D through F, Extramedullary hematopoiesis.
D, Megakaryocytes, nucleated red blood cells, and myeloid cells within splenic parenchyma. E, Hemoglobin peroxidase in erythroid precursors. F,
CD61 highlights a megakaryocyte. G through I, Hemophagocytic lymphohistiocytosis. G, Extensive erythrophagocytosis in the histiocytes. H, CD68þ
histiocyte showing erythrophagocytosis. I, CD20 shows white pulp deletion due to steroids (hematoxylin-eosin, original magnifications 340 [A], 3200
[B, C, and G], 3400 [D through F, and H], and 340 [I]).

imaging studies will typically identify splenic Hodgkin
lymphoma, and it is rarely biopsied. Hodgkin lymphoma
in the spleen typically presents as a solitary or a few
discrete, well-circumscribed nodules. The Reed-Sternberg
cells of classic Hodgkin lymphoma are positive for CD30,
MUM1, usually CD15 and PAX5 (weak staining), EBER
(~50%), and sometimes CD20 (variable), but are negative
for CD45. Admixed nonneoplastic T cells are typically
CD3þ and CD4þ.
1102 Arch Pathol Lab Med—Vol 143, September 2019
BONE MARROW ELEMENTS
Proliferations of marrow-based cells are not infrequently
encountered in the spleen, especially since splenomegaly
associated with myeloid metaplasia is a common sequela of
myeloproliferative neoplasms. These bone marrow elements
may not be a clinically significant finding, but it is of
important clinical consequence to determine if there is an
associated neoplastic myeloid proliferation.45 Extensive
An Immunophenotypic Approach to the Spleen—Borch et al
Figure 7. T-cell lymphomas. A through D, Hepatosplenic T-cell lymphoma. A, Diffuse effacement of the splenic architecture. B, Absence of white
pulp. C, CD3 shows positivity. D, CD56 shows positivity with a sinus pattern. E through H, Peripheral T-cell lymphoma. E, Diffuse splenic infiltrate. F,
Lymphocytes showing abundant cytoplasm. G, CD3 shows positivity in the neoplastic cells. H, CD20 shows negativity (hematoxylin-eosin, original
magnifications 320 [A and E], 3200 [B through D, and F], and 3100 [G and H]).
extramedullary hematopoiesis in the spleen may raise
concern for a myeloid neoplasm.46
The presence of all or some of the 3 usual hematopoietic
cell lines depends on the underlying condition. When
proliferations of neoplastic myeloid cells occur in the context
of myeloid neoplasms, the splenic red pulp can be markedly
expanded by hematopoietic precursors, including immature
and dysplastic forms. Immunohistochemistry can be of
particular use to identify these bone marrow elements.
Megakaryocytes are highlighted by CD42b and CD61.
Erythroid precursors are highlighted by CD71 or E-cadherin.
Myeloid precursors are highlighted by myeloperoxidase,
lysozyme, CD15, and CD33, although markers of myeloid
immaturity, such as CD34, TDT, and CD117, help identify
more immature forms and can be diagnostic of acute
myeloid leukemia or blast transformation of a chronic
myeloid disorder involving the spleen.6,45 Of the myeloid
markers, CD33 is the newest marker and is thought to be
very specific. Lysozyme is more sensitive than myeloperox-
idase and will often stain more cells that myeloperoxidase.
Of the markers of immaturity, CD34 is thought to be more
specific, but may not be immunoreactive with certain types
of acute leukemia such as acute myelomonocytic leukemia,
acute promyelocytic leukemia, and acute megakaryocytic
leukemia. Regarding acute leukemia, TDT most commonly
highlights lymphoid leukemia such as B-lymphoblastic
leukemia/lymphoma or T-lymphoblastic leukemia/lympho-
ma. Additionally, if there is clinical concern for involvement
by Hodgkin lymphoma (the Reed-Sternberg cells of classic
Hodgkin lymphoma may look similar to megakaryocytes),
Reed-Sternberg cells are CD30þ and should be negative for
CD42b and CD61.6
Arch Pathol Lab Med—Vol 143, September 2019
CHRONIC MYELOPROLIFERATIVE NEOPLASMS
Myeloproliferative neoplasms typically manifest with
splenic enlargement. Chronic myeloid leukemia, BCR-
ABL1 positive (CML) shows massive splenic enlargement.
The role of immunohistochemistry is limited, but it can be
used to help identify myeloid cells (myeloperoxidase, CD33)
as well as the small hypolobated megakaryocytes (CD42b or
CD61). Determining the blast count in the bone marrow of
CML is necessary to help determine if the CML is in chronic
phase, accelerated phase, or blast phase. Blastic transfor-
mation often occurs in the spleen. CD34 and CD117 can be
used to highlight blasts. In chronic myelomonocytic
leukemia, in addition to background trilineage hematopoi-
esis, the splenic cords are often distended with immature
monocytic cells, positive for lysozyme, CD68, and CD163.6,33
Primary myelofibrosis and polycythemia have prominent
splenic involvement, but essential thrombocythemia rarely
demonstrates splenomegaly. When the spleen is involved in
essential thrombocythemia, there may be abnormal triline-
age hematopoiesis. The megakaryocytes present may be
large and complex with hyperlobated nuclei. If necessary,
they can be highlighted by CD61 or CD42b. Primary
myelofibrosis may present with spleen fibrosis later in the
disease.6,33
Splenic involvement is frequently seen in systemic
mastocytosis. The mast cells form aggregates in splenic
cords and around white pulp and express mast cell markers,
such as tryptase (highly sensitive and specific) and CD117
(highly sensitive), as well as other less specific myeloid
markers (eg, CD33 and CD43). Neoplastic mast cells will
typically express CD2 and/or CD25, which are not seen in
normal mast cells.6,33
An Immunophenotypic Approach to the Spleen—Borch et al 1103
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