Histopathology of the Spleen

Abstract

The spleen contains hematopoietic and lymphoid elements, is a primary site of extramedullary hematopoiesis, and removes degenerate and aged red blood cells as well as particulate materials and circulating bacteria from the blood supply. Lesions of this important component of the immune system may center on the red pulp, the white pulp or involve both compartments The spleen is the site of direct and indirect toxicity, a target for some carcinogens, and also a site for metastatic neoplasia. Many systemic or generalized diseases have splenic involvement. This paper documents spontaneous background and treatment-induced lesions seen in rodent toxicity and carcinogenicity studies.

Introduction

The spleen contains vascular and lymphoid elements and is a site of hematopoiesis, and in some species, removal of effete, degenerate and aged red blood cells as well as particulate materials and circulating bacteria from the blood supply. The spleen is the site of direct and indirect toxicity and a target for some carcinogens and also a site for metastasis of malignant neoplasms arising in other sites.

In many laboratories routine histopathological evaluation of the spleen involves examination of a single cross-section. While a cross-section may be adequate to diagnose pathological changes in the red pulp, a sufficient amount of white pulp on which to base a diagnosis may not be present in a cross-section. A longitudinal section will increase the amount of lymphoid tissue (white pulp) available for assessment (Figure 1) but it should be kept in mind that due to branching of the periarteriolar lymphoid sheaths (PALS), the size of the PALS will differentially vary in the hilar versus the parietal regions of the spleen. It is recommended that care be taken to sample longitudinal sections consistently so that meaningful comparisons may be made between animals.

Non-Proliferative Lesions

Degenerative lesions can occur spontaneously, often as an age-related change. However, degenerative lesions, such as atrophy and fibrosis, may occur as direct or indirect treatment-related changes.

Atrophy

Atrophy can affect the red pulp, the white pulp or both (Figure 2). As a spontaneous change, it is more common in older rats and mice. However, it has been seen as a direct treatment-related effect and can result as an indirect effect secondary to reduced body weight gain or loss of body weight (Figures 3–6).

Pigment

The appearance of pigment in the red pulp (Figures 7–12) is a common background lesion in rodents. Pigment, can be ceroid/lipofuscin or hemosiderin, is usually harbored in macrophages and may be present in the marginal zone in addition to being in the red pulp. In both rats and mice, hemosiderin is more prominent in females. Iron stains, e.g., Perl’s Prussian blue, can demonstrate the iron in hemosiderin-containing macrophages. Hemosiderin pigment can be increased in treatment-induced hemolytic anemia or methemoglobinemia. Focal deposits of hemosiderin may be associated with areas of hemorrhage secondary to malignant neoplasms. Ceroid/liposuscin pigment occurs scattered throughout the red pulp in macrophages, is less common than hemosiderin deposition, and is acid-fast positive. In some pigmented mice, melanin pigment may be seen in the spleen (Figure 13).

Parenchymal and Capsular Fibrosis

Both parenchymal (Figures 14 and 15) and capsular fibrosis (Figures 16–19) can occur as a reparative process following injury or inflammation. Fibrosis can be induced by a variety of chemicals and may lead to sarcoma formation (Goodman et al., 1984; Ward et al., 1980). Parenchymal fibrosis is primarily localized to the red pulp, although extension into the white pulp may occur when there is extensive fibrosis. The fibrosis may be focal or diffuse. Capsular fibrosis is more common and is typically focal. It is seen in untreated rats, and may be a consequence of trauma to the surface of the spleen. Capsular fibrosis may also occur in association with inflammatory, toxic, or neoplastic lesions of the spleen. Fibrotic regions may contain areas of hemorrhage and/or hemosiderin pigment deposition.

Lipidosis/Fatty Infiltration/Lipid Metaplasia

Lipidosis/ metaplasia (Figures 20–23) of the red pulp is seen in rats after exposure to aniline-related compounds and in rats with mesenchymal neoplasms in the spleen. It is also seen as an occasional finding in chronic toxicity/carcinogenicity studies and may not necessarily be related to treatment. The lesion consists of well differentiated adipocytes in a fibrous stroma although the fibrosis is not always obvious. The lesion is believed to represent metaplastic splenic stromal cells.

Vacuolization of Splenic Histiocytes

In contrast to the lipidosis described in rats (above), a similar appearing but less extensive lesion was observed in B6C3F1 mice treated with Elmiron (Figure 24). Based upon detailed study of this change, which was also present in lymph nodes and liver, it was determined that the affected cells were vacuolated histiocytes. Histochemically, neutral and acidic mucins were present in the vacuoles, which were identified as lysosomal structures by transmission electron microscopy.

Amyloidosis

The incidence of amyloidosis in mice varies varies between strains. It is rare in B6C3F1 mice, intermediate in frequency in C57BL mice, and common in CD-1 mice. It is a systemic disease and is usually present in other organs besides the spleen. Amyloid is not seen in rats. Amyloid is deposited in the red pulp and adjacent to the white pulp (Figures 25 and 26) and when severe can result in atrophy of the white pulp.

Splenic Necrosis/Infarct

Splenic infarcts are not common but can occur secondary to vascular obstruction (Figure 27), trauma (Figure 28), or neoplasms. Hemorrhage may occur in and around infracted areas with a compensatory increased extramedullary hematopoiesis (Figure 27).

Lymphoid Necrosis/Apoptosis

Compounds inducing lymphocyte toxicity may cause necrosis of the white pulp. With the exception of vascular obstruction (see infarct above) and marked inflammation, necrosis in the splenic white pulp is typically characterized by apoptosis of lymphocytes (Figures 29 and 30). The preferred diagnostic term is apoptosis. This topic is also discussed by Elmore (2006). A diagnosis of lymphoid necrosis is preferred when the predominant cytomorphology is consistent with a classical form of necrosis, such as coagulation necrosis.

Hyperplastic Lesions

Extramedullary Hematopoiesis (EMH)

EMH may be seen as a diffuse (non-neoplastic) hyperplasia of the red pulp. EMH (Figures 31–34) is present in the normal spleen, more commonly in young compared to aged rodents and, more common in females compared to males. EMH consists of erythroid precursors, myeloid precursors, megakaryocytes or all three. The erythroid component may predominate (erythroid hyperplasia) secondary to hemorrhage or erythrocyte destruction (hemolytic or autoimmune anemia), the myeloid component may predominate (myeloid or granulocyte hyperplasia) secondary to inflammatory conditions. Myeloid hyperplasia shares some histological similarities to granulocytic leukemia (Long et al., 1986). While some degree of extramedullary hematopoiesis is present in normal rodents, especially in mice, increased extramedullary hematopoiesis can result from hematotoxic insult, systemic anemia, and infections elsewhere in the body.

Focal Red Pulp Hyperplasia

Focal hyperplasia of the red pulp has been reported in aging Fischer rats (Stefanski et al., 1990). The lesion is typically a solitary, well demarcated nodule protruding from the spleen (Figure 35). It consists of a monotonous sheet of normal-appearing red pulp containing blood-filled sinusoids, foci of hematpoietic cells, and hemosiderin. There is an absence of white pulp elements within the expansile lesion. Focal hyperplasia of the red pulp may also be categorized as a hamartoma or malformation.

Focal White Pulp Hyperplasia

Focal White Pulp Hyperplasia (syn. lymphohistiocytic hyperplasia; nodular hyperplasia of the white pulp) is seen as a well demarcated, focal expansile lesion which occasionally occurs in the F344 rat as a spontaneous lesion (Stefanski et al., 1990) (Figures 36 and 37) but may occur as a treatment-related change as in rats treated with Elmiron (Figure 38). It can become large with compression of adjacent splenic tissue and distortion of the capsule. The lesion consists of an admixture of lymphocytes and aggregates of macrophages. This form of focal white pulp hyperplasia is distinct from the more diffuse lymphocyte hyperplasia affecting the PALS and lymphoid follicles (see Lymphocyte Hyperplasia next).

Neoplastic Lesions

Lymphocyte Hyperplasia and Lymphosarcoma

Diffuse hyperplasia of the white pulp, referred to here as lymphocyte hyperplasia (syn. lymphoid hyperplasia) and lymphosarcoma (syn. lymphoma, malignant lymphoma) are less common in the rat, than in the mouse. Hyperplasia is reported to occur more frequently in female mice than in male mice.

Because lymphocyte hyperplasia and lymphosarcoma can be part a continuum, they are presented together here. Lymphocyte hyperplasia is a polyclonal increase in the number of lymphocytes whereas lymphosarcoma is made up of a monoclonal lymphocyte population. This distinction, however, is not obvious by light microscopic evaluation of a hemotoxylin and eosin stained section of spleen. Table 1 summarizes the major features that should be considered when evaluating a spleen with an increased lymphocyte population.

In lymphocyte hyperplasia (Figures 39 and 40) there is an increase in the area and number of white pulp elements but with normal red pulp compared to lymphosarcoma where, in advanced cases, both white pulp and red pulp structure is lost. Low magnification viewing of the spleen can often provide a perspective on the relative structure and proportions of white pulp. However, there will undoubtedly be cases where the distinction between lymphocyte hyperplasia and lymphosarcoma is not clear (Figure 41). Higher magnification will often help to determine whether the increased white pulp is comprised of a normal mixture of cells or is comprised of either a monotonous cell population or a pleomorphic population of abnormal lymphoid cells (Figures 42–46). While the presence of lymphosarcoma in other lymphoid tissues may provide some comfort in evaluating the splenic changes, difficult cases may best be resolved by assessing antigen expression using immunohistochemistry (Ward et al., 1999, 2006). While many pathologists lump all lymphosarcomas into one category, they may also be sub-classified based upon published criteria (Taddesse-Heath and Morse, 2000; Morse et al., 2002; Valli et al., 2002). Some B-cell tumors have abundant cytoplasm which can be demonstrated to contain immunoglobulins. Histiocytic lymphoma has been reported in mice, consisting of an admixture of histiocytes and lymphoma cells, however some of these lesions may represent two distinct tumors—histiocytic lymphoma and lymphoma.

Granulocytic Leukemia

Neoplasms of granulocytes (Figure 47) and their precursors are not as common as lymphoid neoplasms. Granulocytic leukemia may be relatively common in some strains of old rats (Harleman and Jahn, 1990). It has a spontaneous rate of occurrence in C3H/He mice of up to 1% (Seki and Inoue, 1990) while it is extremely rare in other strains. As a systemic disease, granulocytic leukemia is typically accompanied by a high circulating leukocyte count and marked splenomegaly (Gal et al., 1990; Seki and Inoue, 1990). Based upon the cytomorphology of the neoplastic cells, granulocytic leukemia can be subclassified as mature or immature.

Erythroleukemia

Erythroleukemia is a rare neoplasm that arises spontaneously in mice of different strains and stocks (Figure 48). The TgAC mouse spontaneously develops erythroleukemia characterized by expansion of the splenic red pulp with small basophilic erythroid progenitor cells. Treatment of Tg.AC mice may also induce erythroleukemia (Figure 49). Erythroleukemia appears to arise in the splenic red pulp. It may also be induced by retroviruses in the mouse.

Mononuclear Cell Leukemia (Large granular lymphocyte leukemia, LGL)

Mononuclear cell leukemia (MCL), also known as large granular lymphocyte (LGL) leukemia (Reynolds et al., 1984; Rosol et al., 1990; Stromberg et al., 1990, 1983, 1988; Ward et al., 1980; Ward and Reynolds, 1983) occurs commonly in the F344 rat (Stefanski et al., 1990) but is also reported in other strains of rats. Affected rats show clinical pathologic signs indicative of hemolytic anemia and disseminated intravascular coagulation (thrombocytopenia and hypofibrinogenemia). Splenomegally is the most characteristic gross pathologic finding (Figure 50). MCL arises in and expands the red pulp and effaces the white pulp with the malignant LGL cells (Figure 51A). LGLs are pleomorphic with round to irregular and often eccentric nuclei and abundant pale staining cytoplasm (Figure 51B). LGLs are considered to be natural killer (NK) cells that exhibit characteristics of both T-lymphocytes and macrophages. They contain distinctive azurophilic cytoplasmic granules; lysosomes containing perforins and granzymes (Slauson and Cooper, 2002). These LGL cytoplasmic granules can be seen in blood smears (Figure 52). In advanced stages of MCL, hepatic sinusoids may be expanded with neoplastic large granular lymphocytes (Figure 53) and hepatocyte necrosis may be present.

Staging Mononuclear Cell Leukemia

In National Toxicology Program lifetime studies using the F344 rat, chemical treatment has been shown to either increase or decrease the relatively high spontaneous incidence of mononuclear cell leukemia. Hence, staging of the disease may be useful in assessing whether or not treatment has enhanced or reduced the spontaneous occurrence of this neoplasm. In very early stages before leukemia develops, diagnosis can be performed using a tissue smear from the spleen to identify the neoplastic LGLs in the red pulp. The major histological features of the early- and late-stage disease are summarized in Table 2 and shown in Figure 54.

In an unusual case of advanced mononuclear cell leukemia in a F344 rat, areas of inflammation peripheral to the thrombotic region had morphological similarities to hemangiosarcomas. This was ultimately considered a rim of early fibrosis surrounding the thrombotic area (Figure 55).

Histiocytic Sarcoma

Histiocytic sarcomas (Figures 56 and 57) are common in mice, especially in the B6C3F1 hybrid. They are rare in the F344 rat. These tumors usually arise in other tissues, particularly the liver and uterus, and frequently metastasize to the spleen. Histologically, there are uniform sheets of histiocytes, sometimes exhibiting pleomorphism and multinucleated giant cells (Figure 56 B and C). A useful diagnostic feature for histiocytic sarcoma is the presence of hyaline droplets in the renal tubules which consist of lysozyme derived from the histiocytes.

Mast Cell Tumor

Mast cell tumors (Figures 58–60) are rare in the mouse and are highly malignant (Ward etal., 1999). They usually arise in tissues where mast cells are found, including the skin and lymph nodes (Rehm et al., 1990). They may be systemic but can arise in the spleen as solitary masses or with uniform infiltration. This neoplasm consists of a homogeneous population of cells with centrally located nuclei and abundant faintly amphophilic cytoplasm. Growth is typically in closely packed clusters or in rows. Metachromatic cytoplasmic granules can be demonstrated with Giemas or toluidine blue stains.

Hemangioma and Hemangiosarcoma

Both hemangomas (Figure 61) and hemangiosarcomas (Figures 62 and 63) are more common in mice than rats. These neoplasms may arise in the red pulp from endothelial cells lining the venous sinusoids. Splenic hemangiomas are less common in rodents compared to their malignant counterpart. They consist of irregular blood-filled spaces lined by normal or enlarged, crowded endothelial cells. Stroma is generally scant but there can be proliferation of fibroblasts with collagen deposition within hemangiomas. Hemangiosarcomas consist of prominent, variable sized, blood-filled vascular spaces lined by pleomorphic endothelial cells, often with hyperchromatic nuclei. Mitoses, stromal fibrosis, and thrombosis are more common in hemangiosarcomas than in hemangiomas.

Mesothelioma

Mesothelioma is occasionally seen in aged F344 rats, especially in males and is reported to arise in the tunica vaginalis of the testis. It can be seen on the splenic capsule in some of these cases (Figure 64).

Other Tumors

A variety of other tumors are reported occasionally in the spleen of rodents. These include fibrosarcomas, giant cell sarcomas, and osteosarcomas (Figures 65–67), schwannomas (Figure 68), and a variety of metastatic tumors including pheochromocytomas and prostatic carcinomas (Figures 69 and 70).