IHC is a valuable tool in the evaluation of splenic disorders. It provides unique challenges, however, because of the functional complexity of the spleen and the variety of its histological components. The purpose of this section is to provide general guidelines on evaluating the complex splenic microanatomy, stressing in particular the morphologic and the immunohistological characteristics of the white pulp and red pulp compartments (44). The lymphoid components will not be specifically discussed.

As a functional compartment, the stroma of the spleen is often overlooked. It does, however, give rise to both neoplastic and nonneoplastic proliferations, which may be complex and difficult to evaluate. In addition, the stroma of the spleen may in itself, provide insight into the development of splenic disorders (e.g., myeloid metaplasia).

In terms of architecture, the splenic stroma can be divided into three main components: the vascular component, the reticuloendothelial or monocyte/ macrophage component, and the remainder (45). This remaining portion consists of various mesenchymal cells and extracellular matrix (ECM), which forms the structural framework of the spleen.

The large arteries, arterioles, and veins are similar to other sites in the body, and can be evaluated with the battery of immunostains used to stain endothelial cells as well as vascular tumors at other sites, i.e., CD34, CD31, factor VIII (von Willebrand factor), and, more recently, FLI-1 (Figs. 22 and 23). A specialized endothelial cell unique to the spleen is the littoral cell, which lines the vascular sinuses of the spleen. The littoral cells share features of both endothelial cells and monocyte/macrophages. They are usually flat, inconspicuous cells along the edges of sinuses. When activated, they become plump and appear larger. Some of the interesting features of the cells are that they are strongly and uniformly positive for CD8, a marker that is more commonly associated with cytotoxic or suppressor T-cells (Fig. 24). Therefore, CD8 is a useful stain in delineating the sinusoidal architecture in the spleen; however, the strong vascular reactivity with CD8 can make evaluation of CD8-positive T-cells somewhat

Fig. 22. CD34 expression in small vessels of the spleen. Note that the sinus-lining cells (e.g., littoral cells) are CD34 negative.
Fig. 23. Factor VIII antigen staining in a normal spleen. All vascular elements stain with factor VIII antigen.
Splenic Blood Vessel Cd34 Cd8
Fig. 24. CD8 staining of littoral cells in spleen. In addition, numerous circulating T-cells of the suppressor type (e.g., CD8 positive) are seen.

more difficult. CD8 is only inconsistently positive in the neoplastic counterpart of the sinusoidal endothelium, the littoral cell angioma.

The monocyte/macrophage system (MMS) is one of the more important anatomic and functional components of the spleen; it forms the bulk of the red pulp (cordal macrophages), but often receives little specific attention. Only when there is a significant derangement of the MMS, is it even noticed. Normally, the MMS serves as a functional filter for effete red blood cells and other less than desirable components of the circulating blood. In states in which there is an increase in destruction of circulating blood components, as in an autoimmune hemolytic anemia or immune thrombocytopenic purpura, the cordal macrophages will become engorged and hyperplastic within the spleen producing a picture of red pulp congestion. The cordal macrophages can also become engorged with metabolic products in storage diseases, infectious agents, or even abnormally produced immunoglobulins.

Evaluation of the MMS using IHC can be accomplished by a number of antibodies, which include CD68, lysozyme, antichymotrypsin, and others. When there is accumulation of intracellular material in macrophages, histological stains may be of considerable value, depending on the accumulated product. Most abnormal storage proteins are PAS positive. Infectious organisms can be evaluated by the judicious use of AFB, GMS, tissue Gram, and PAS stains. In

Fig. 25. Nerve growth factor receptor (NGFR) staining of red pulp of spleen. NGFR stains stromal elements of the spleen.

the case of immunoglobulin accumulation, IHC for K and X light chains may be of benefit.

A considerable portion of the spleen (white pulp) is devoted to its function as a lymphoid organ. Components that are notable parts of this are dendritic cells (FDC and IDC). Distinction of dendritic cells is usually accomplished by IHC. Dendritic cells are varyingly positive for monocyte/macrophage-associ-ated markers such as CD68, S100, CD1a, lysozyme and, a-1-antitrypsin (as previously described).

Evaluation of structural mesenchymal cells and ECM by IHC can be performed using various antibodies. Low-affinity nerve growth factor receptor (ME20-4) identifies reticulum cells in the splenic cords, periarteriolar and peri-capillary adventitial cells as well as FDCs (Figs. 25 and 26). The low-affinity nerve growth factor receptor positive cell subsets all share fibroblastic or spindle cell morphology and perform mainly structural functions. Smooth muscle actin (SMA; 1A4) can be used to identify pericytes and cells with myofibro-blastic differentiation (myoid cells). SMA staining highlights a concentric reticular meshwork of SMA-positive cells present at the interface between red pulp and marginal zones (Fig. 27). SMA staining of perivascular cells within the red pulp can also be observed. The ECM can be evaluated by IHC for collagen IV (CIV22; basement membrane type collagen) and by reticulin staining. Collagen IV highlights the "ring fibers," which incompletely surround the littoral cells of

Fig. 26. Nerve growth factor receptor (NGFR) staining of white pulp of spleen. NGFR stains dendritic cells of white pulp.
Fig. 27. Smooth muscle actin (SMA) staining in normal spleen. SMA highlights cells with myoid differentiation that define the boundaries between red and white pulp.
Fig. 28. Collagen type IV staining in normal spleen. This stain highlights the ring fibers of the splenic sinusoids.

the splenic sinusoids (Fig. 28). Other types of collagen are collectively stained by Gomori silver impregnation technique also known as reticulin stain. Various patterns of stromal cell hyperplasia or alterations in the architectural distribution of ECM proteins can be observed in a variety of splenic disorders.


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