Immunohistochemical Antibody Selection Scheme for Spleen Diseases (Part 1)

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6 2 月, 2026

On 6 2 月, 2026

Introduction:

Currently, immunohistochemistry has become a routine tool in pathological differential diagnosis, but the interpretation of immunohistochemistry in splenic diseases remains confusing. This article introduces the latest knowledge on the immunophenotypes of normal spleen and benign and malignant splenic diseases. Due to space limitations, it will be published in five parts, hoping to be helpful to colleagues in their daily work.

Histology of the Spleen

To correctly interpret immunohistochemical results of the spleen, we first need to understand the histology and function of the normal spleen. This also helps us determine which part has changed when assessing splenic lesions.

The spleen is a hematopoietic and lymphoid organ that develops from the mesoderm of the embryo in the fifth week of gestation. Firstly, it is a site for the maturation of hematopoietic precursor cells; secondly, it plays a role in the development of the immune system. The spleen consists of red pulp and white pulp. The red pulp primarily functions to filter blood, removing foreign substances, damaged and aged red blood cells; simultaneously, it serves as a reservoir for iron, red blood cells, and platelets. The white pulp acts as the initiator of immune responses to various antigens in the blood, making the spleen the largest secondary lymphoid organ in the human body. The red pulp and white pulp contain different cell types, hence they have different immunophenotypes. The spleen also has significant stromal and vascular components, which play roles in the physiology and anatomy of the spleen and may also exhibit abnormal hyperplasia.

The red pulp of the spleen consists of splenic cords and venous sinuses. The splenic cords are composed of reticular myofibroblasts and macrophages. The splenic sinuses are lined by prominent endothelial cells with partial histiocytic functions, known as littoral cells. Within the red pulp, myofibroblasts are immunoreactive to smooth muscle actin (SMA), and macrophages can express macrophage markers (such as CD68 and CD163). Littoral cells are unique to the spleen and can be identified by their distinctive immunophenotype to recognize ectopic spleen. Splenic littoral cells express vascular endothelial markers such as CD31, WT1, Factor VIII, ERG, CD68, and variably express CD21. The most unique marker for littoral cells is CD8, typically associated with cytotoxic T cells. The expression of CD8 in littoral cells shows a dendritic growth pattern, thereby revealing the structure of the red pulp. CD8 positivity indicates that the splenic structure is intact; conversely, loss of CD8 suggests that the original splenic structure has been replaced by tumors or other space-occupying lesions.

The white pulp of the spleen consists of small arteries surrounded by lymphocytes, known as the periarteriolar lymphoid sheath (PALS). PALS is mainly composed of T cells and flattened reticular cells. T cells express common T lymphocyte antigens (CD2, CD3, CD5, and CD7), most are CD4+, with little expression of CD8. In pediatric spleens, especially in PALS, scattered TDT+ (terminal deoxynucleotidyl transferase positive) cells are also found.

Splenic lymphoid follicles are mainly composed of B cells, with scattered small numbers of CD4+ T lymphocytes. Lymphoid follicles are divided into primary and secondary follicles. Primary follicles consist of mature B cells expressing standard B lymphocyte markers (CD19, CD20, CD79a, PAX5, and BCL2). Secondary follicles consist of germinal centers containing B cells and occasional T lymphocytes, surrounded by a mantle zone with follicular dendritic cells. Germinal center B lymphocytes express CD19, CD20, CD79a, PAX5, CD10, and BCL6, and express immunoglobulins IgM and IgD. However, they are negative for BCL2 expression. Mantle zone B lymphocytes express CD19, CD20, CD5, DBA.44, BCL2, and IgD, but are negative for CD10 and CD23. Follicular dendritic cells forming the follicular network express follicular dendritic cell markers such as CD21 and CD35.

The marginal zone is located between the red pulp and white pulp and is rich in macrophages. Marginal zone B lymphocytes have a characteristic IgM+/IgD- immunophenotype; they are positive for B cell markers (CD19, CD20, CD22) and express BCL2. Marginal zone B cells are negative for CD5, CD10, CD23, and DBA.44. Additionally, the marginal zone contains a large number of SMA-positive myofibroblasts, with SMA staining pattern resembling a marginal band that separates the white pulp from the red pulp. Thrombomodulin staining can also highlight this phenomenon.

The basic immunohistochemistry panel for the spleen can include CD8, CD20, CD3, and appropriate vascular markers. This panel allows for some basic phenotypic analysis of the white pulp by comparing the numbers of CD20+ B cells and CD3+ T cells. CD8 is crucial for determining whether the red pulp structure of the spleen is intact (Table 1).

Table 1. Spleen Immunohistochemistry Panel

Basic Spleen Immunohistochemistry Panel:

CD3, CD8, CD20,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 ,CD3

TCell orNKCell Lymphoma/Leukemia:

CD2, CD3, CD5, CD7, EBER, TIA1, granzyme B, TCL1,CD56, CD4, CD8, CD30

Cysts:

CD8, cytokeratin AE1/AE3, calretinin, GMS ERG

There are clear advantages and disadvantages among vascular markers. ERG is a newer marker with multiple advantages, including nuclear localization (easier interpretation); extremely high staining intensity and sensitivity (essentially all vascular endothelial and vascular tumors are positive). WT1 is also an excellent vascular marker with high sensitivity, clean background staining, and cytoplasmic staining, staining most vascular endothelial and vascular tumors. CD34 is a very good vascular marker, but when used for the spleen, it cannot distinguish splenic littoral cells (e.g., red pulp sinuses), vascular hyperplasia (e.g., hamartoma), or tumors derived from littoral cells (littoral cell angioma). Although CD34 can be expressed in poorly differentiated vascular tumors, most cases are negative. CD31 is sensitive to vascular endothelium but not specific, as it also stains macrophages (Table 2). Factor VIII has moderate sensitivity and specificity (Figure 1, A to O).

Table 2. Comparison of Vascular Markers in the Spleen

Antibody	Specificity	Sensitivity	Characteristics
ERG	↑↑↑	↑↑↑↑	Nuclear positive, marks vascular endothelial and vascular tumors
WT1	↑↑↑	↑↑↑	Cytoplasmic positive, background staining isCD34light
CD34	↑↑↑	↑↑	Cannot mark splenic littoral cells, splenic hamartoma, or littoral cell angioma
CD31	↑↑	↑↑↑	Non-specific, also marks histiocytes and macrophages
VIII Factor	↑↑	↑↑	Second-line antibody, less used, moderate sensitivity and specificity

↑↑Indicates lower specificity and/or sensitivity;↑↑↑Indicates moderate specificity and/or sensitivity;↑↑↑↑Indicates highest specificity and/or sensitivity.

Figure1 Immunophenotype of normal spleen.

Figure1A, white pulp, showing germinal center and mantle zone/Marginal zone;

Figure1B，CD20showing white pulpBcell positive；

Figure 1C, CD3 is expressed in scattered T cells of the white pulp.

Figure1D，Bcl-2Shows negative in germinal centers of normal white pulp, but usually marginal zone/mantle zone positive；

Figure1E，CD43Expressed inTcells of normal white pulp；

Figure1F，CD21Highlights the follicular dendritic reticular structure in the white pulp；

Figure1GtoM, red pulp;

Figure1G, red pulp showing splenic sinuses；

Figure1H，VIIIFactor stains endothelial cells；

Figure1I，CD8Highlights the splenic sinus structure；

Figure1J，CD68Shows positive in histiocytes；

Figure1K，ERGMarks endothelial cells；

Figure1L，CD34Also marks endothelial cells；

Figure1M，WT1is a very sensitive endothelial marker；

Figure1NandO, showingTzone.N, periarteriolar lymphoid sheath, surrounded by lymphocytes；

Figure1O，CD4Shows helperTcells.

Flow Cytometry

When performing flow cytometry analysis on normal splenic tissue, some abnormal lymphocyte subsets may be encountered. Approximately20% to30% of non-neoplastic splenicBlymphocytes expressCD5. In human immunodeficiency virus (HIV) and autoimmune diseases, the number of theseCD5-positive non-neoplasticBlymphocytes can increase. These non-neoplasticBcells should not be confused with mantle cell lymphoma or chronic lymphocytic leukemia//small lymphocytic lymphoma, both of which expressCD5. A small number ofCD4 ＋/ CD8＋Tcells are usually present in normal spleen, with an average number accounting forT% of3.3cells, up to6%; usuallyCD4fluorescencestaining is bright, whileCD8fluorescencestaining is dim, and should not be misdiagnosed asTcell lymphoblastic leukemia//lymphoma. SomeTcell antigens may be lost in splenicTcells, so they should not be considered neoplastic. A portion of normalTlymphocytes in the spleen do not expressCD7. The proportion ofγδTcells in the spleen (12.5％±8.1%) is higher than in peripheral blood (4.0％±3.1%); theseγδTcells usually expressCD3, but notCD4，CD8andCD5. These findings are related to neoplasia in theαβTcell population, notγδTcells.Studies have shownthat approximately5%of splenicTcells areCD5 -; most of these cells areCD8+ or areγδTcells.

The number of immature phenotype (NK-) natural killer (CD56＋ / CD16 ) cells in the spleen is higher than in peripheral blood. Additionally,CD2–NKcells constitute an important normal cell subset in splenic tissue. AlthoughCD7expression is considered a characteristic ofNKcells, it is common in the spleen.CD7expression is widespread.

Although flow cytometry is an excellent method for immunophenotyping, such as in lymphomas, for splenic lesions (such as vascular hyperplasia or stromal lesions), histological section examination is essential to assess splenic structure and immunohistochemical expression.

To be continued.…..

Maxin Related Antibodies

Antibody Name	Product Number	Clone Number	Positive Location
^CD3*	MAB-0740	MX036	Cell Membrane
CD3	Kit-0003	SP7	Cell Membrane
CD8	RMA-0514	SP16	Cell Membrane
CD8	MAB-0021	C8/144B	Cell Membrane
CD20	Kit-0001	L26	Cell Membrane
CD34	Kit-0004	QBEnd/10	Cell Membrane/Cytoplasm
ERG*	RMA-0748	MXR004	Nucleus
WT1*	MAB-0678	MX012	Nucleus

*Marked as Maxin clone products

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