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Histopathology of the Spleen
Andrew W. Suttie
Covance Laboratories Inc., Vienna, Virginia 22182, USA
Correspondence: Address correspondence to: Andrew W. Suttie, Covance Laboratories Inc., 9200 Leesburg Pike, Vienna, VA 22182, USA; e-mail:andrewsuttie{at}covance.com
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Abstract
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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.
Key Words: Red pulp • white pulp • lymphoma • lymphoid hyperplasia • mononuclear cell leukemia • LGL leukemia
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Introduction
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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.
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Figure 1 Cross and longitudinal sections of spleen from a F344 rat. Some pathologists prefer the cross-section as a means of assessing splenic enlargement, which would show up as rounded edges of the cross-sectional profile.
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Non-Proliferative Lesions
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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).
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Figure 2 This low (A) and higher (B) magnification of the spleen shows depletion of white and red pulp. There is an absence of follicles and decreased size of the PALS in this female B6C3F1 mouse from a subchronic AZT/methadone study. The cellularity and size of the red pulp is also reduced.
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Figures 3. & 4 Spleen from a F344 rat on 50% caloric restriction for two weeks. Figures 5. & 6. Spleen from an ad libitum control on the same study as Figures 3 and 4.
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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).
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Figure 7 Low magnification of the spleen from a F344 rat. When large amounts of pigment are present as in this example, it can usually be detected at low magnification in the red pulp. 8.— Higher magnification of Figure 7 showing brown granular hemosiderin pigment predominantly within macrophages in the red pulp. 9. — High magnification of a portion of Figure 8. 10. —This iron stain on the spleen from a F344 rat shows blue staining hemosiderin pigment throughout the red pulp. 11. —In this higher magnification of Figure 10 most of the blue staining hemosiderin pigment is localized within macrophages. 12. —This is a higher magnification of a portion of Figure 11.
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Figure 13 Dark brown finely granular pigment consistent with melanin is present in the spleen of this mouse. Photomicrograph courtesy of Dr. Michael Leach.
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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.
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Figure 14 A–C. A large focal area of the spleen from a male F344 rat treated with 2-butoxyethanol probably represents a healed infarct. The fibrotic area is contracted causing distortion of the splenic contour. The parenchymal fibrosis is sufficiently extensive to involve the marginal zone and follicular areas of the white pulp and the PALS has a reduced number of lymphocytes. Focal areas representing hemorrhage and pigment-laden macrophages are present in the fibrotic area.
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Figure 15 A–D. There is extensive generalized parenchymal fibrosis in this male F344 rat treated with glycidol. Both red and white pulp are being replaced by fibroblasts and early collagen deposition. Remnants of the PALS and follicular area are evident. In some areas of fibrosis there is deposition of pigment consistent with hemosiderin.
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Figure 16 Focal capsular fibrosis can occur as an incidental finding in long-term rodent safety assessment studies and is more commonly seen in rats. 17. —Hemorrhage and fibrin deposition are present in this focal capsular fibrotic area from a F344 rat with advanced mononuclear cell leukemia (large granular lymphocytic leukemia). 18. —Focal capsular fibrosis from a F344 rat with advanced mononuclear cell leukemia. Leukemic cells have infiltrated the capsular fibrosis.
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Figure 19 A–C. This example of a more diffuse capsular fibrosis was present at the 13-week interim time point in a male F344 rat treated with o-nitroanisole. The affected capsule is of relatively uniform thickness and is lined by prominent mesothelial cells. Atrophy of the white pulp is also evident in Figure 19A.
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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.
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Figure 20 This spleen is from a F344 rat from a 2-year carcinogenicity study and consists of a localized area of lipidosis surrounded by a band of fibrotic red pulp. 21. —In this example much of the splenic red pulp has been replaced by adipocytes without associated fibrosis. There is also depetion of the white pulp. 22. —Another example of lipidosis in the spleen of a male F344 rat. A large region of the spleen has been replaced by adipocytes with absence of red pulp and atrophy of the white pulp in the affected as well as in the unaffected regions.
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Figure 23 (A) In this spleen with a smaller and localized region of lipidosis, there is capsular fibrosis and depletion of the white pulp. (B) Higher magnification of Figure 23A.
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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.
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Figure 24 A–C. Low and higher magnifications of splenic histiocytic vacuolization in the spleen of a B6C3F1 mouse treated with Elmiron for 14 weeks. Histochemically, neutral and acidic mucins were present in the vacuoles, which were identified as lysosomal structures by transmission electron microscopy.
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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.
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Figure 25 Low (A) and higher (B) magnifications of amyloid deposits in the red pulp in this mouse spleen. The amyloid appears homogeneously eosinophilic with hematoxylin and eosin stains. Photomicrographs courtesy of Dr. Michael Leach.
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Figure 26 In these low (A), medium (B), and high (C) magnifications of hematoxylin & eosin stained sections from a 17-month old CD-1 mouse, amyloid appears as a pink to light pink amorphous material in the red pulp. Its presence can be confirmed with special stains.
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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).
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Figure 27 A large infarct is present in this rat spleen. The finely basophilic stippling in the pink necrotic area of the infarct represents some remaining lymphocytes. There is a narrow zone of hemorrhage present along the edge of the infarct. There is extramedullary hematopoiesis in the viable portions of the splenic parenchyma. Photomicrograph courtesy of Dr. Michael Leach. 28. —This iatrogenic lesion (pseudo-infarct) in the spleen of a male F344 rat is attributed to needle damage from an intraperitoneal injection of phenobarbital.
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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.
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Figure 29 (A) In this spleen from a female B6C3F1 mouse treated with bis(2-chloroethoxy) methane for 13 weeks, there is apoptosis characterized by chromatin condensation and karyorrhexis of lymphocytes and an increase in tingible body macrophages in the PALS (B) and a small follicle (C).
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Figure 30 (A) Apoptosis in the PALS of the spleen from a F344 rat treated with cyclophosphamide 48 hours earlier (B). Higher magnification of A. (C) Concurrent control F344 rat with a normal PALS for comparison to B.
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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.
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Figure 31 (A) Spleen from a F344 rat treated with methylene blue trihydrate for 2 years. There is marked erythropoiesis secondary to treatment-related hemolytic anemia characterized by poorly demarcated aggregates of intensely basophilic cells throughout the red pulp. (B) Higher magnification of A. In addition to the erythropoiesis, there is a tag of capsular fibrosis present. (C) High magnification of B. The EMH is essentially entirely erythryoid with erythryoblasts and rubricytes present. Hemosiderin pigment is also present.
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Figure 32 (A) Low magnification cross-section of the spleen from a normal B6C3F1 mouse. The distinction between the red and white pulp is clear. Mature erythrocytes are evident in the red pulp. (B) Low magnification cross-section of the spleen from a normal B6C3F1 mouse. Islands of darkly staining cells in the red pulp represent aggregates of erythyroid hematopoiesis. 33. — (A) The subcapsular area of the spleen from a B6C3F1 mouse with a mild to moderate amount of extramedullary hematopoiesis. (B) Both darkly stained erythyroid precursors and megakaryocytes are present in this higher magnification of A. 34. —This high magnification of the spleen is from a B6C3F1 mouse with increased extramedullary hematopoiesis. In addition to erythyroid and myeloid precursors, a large number of megakaryocytes are present.
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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.
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Figure 35 (A) A well demarcated nodule protruding trom the surface of the spleen represents focal hyperplasia of the red pulp. (B) Higher magnification of A showing normal appearing red pulp in the protruding nodule. There is an absence of white pulp within the nodule. (C) Higher magnification of B showing the demarcation between the normal spelenic architecture on the right and the hyperplastic red pulp on the left. (D) High magnification of C showing detail of hyperplastic red pulp.
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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).
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Figure 36 (A) A small focal area of lymphohistiocytic hyperplasia is present in the spleen of a female F344 rat. (B) At slightly higher magnification, small pale staining foci are evident within the hyperplastic lesion. (C) A higher magnification of B reveals the pale staining areas to be aggregates of histiocytes. (D) High magnification of C showing cellular detail.
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Figure 37 (A) This example of focal white pulp hyperplasia was a spontaneous lesion in a male F344 rat. It was originally diagnosed as nodular hyperplasia of the white pulp and consists of large areas of histiocytes and smaller aggregates of lymphocytes. (B)–Higher magnification of A.
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Figure 38 Focal lymphohistiocytic hyperplasia was a treatment-related lesion in the spleens of rats dosed with Elmiron. Since this lesion was seen in the prechronic as well as the chronic study and the histiocytes contained neutral and acidic mucins and lipid material within lysosomes, it could be considered granulomatous inflammation. (A) Low magnification of the spleen from a F344 male treated with Elmiron for 2-years. There is a focal area of lymphohistiocytic hyperplasia characterized as an enlarged area of white pulp. (B) An area of lymphohistiocytic hyperplasia from a male F344 rat treated with elmiron. The hyperplastic lesion in this case is more extensive than that in A and is comprised of a mixture of lymphocytes and focal aggregates of histiocytes. (C) Higher magnification of B. The focal histiocytic aggregates are more apparent at this magnification.
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Neoplastic Lesions
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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.
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Figure 39 (A) The lymphocyte hyperplasia is characterized by expansion and partial bridging of some areas of white pulp in this vehicle female B6C3F1 mouse from a 2-year carcinogenicity study. (B) In this enlarged view of A, the paler staining central areas represent PALS while the darker staining more peripheral portions of the white pulp are follicular areas. (C) A higher magnification of B shows the mixed population of lymphocytes in the areas of lymphocyte hyperplasia.
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Figure 40 (A) In this mid-dose female B6C3F1 mouse from a citral carcinogenicity bioassay, the lymphocyte hyperplasia consists of enlargement of most of the areas of white pulp. (B) The mixed cell population in the hyperplastic white pulp contains expansion of both PALS and follicular compartments. (C) In this higher magnification of B the edge of a follicular area is seen to be contained within the marginal sinus.
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Figure 41 This composite figure of splenic cross sections from multiple mice spans a range of morphology from normal to lymphosarcoma and is meant to illustrate the continuum of changes between lymphocyte hyperplasia and lymphosarcoma. While clear diagnoses may be evident at low magnifications for normal, lymphocyte hyperplasia, and lymphosarcoma, the borderline lesions present diagnostic challenges and require evaluation of cytological detail at high magnification. The consensus diagnoses on these various samples are as follows: (A) normal, (B) normal with evidence of extramedullary hematopoiesis, (C) lymphocyte hyperplasia, (D) lymphocyte hyperplasia, (E) a borderline lesion showing expansion and early bridging of the white pulp, (F) this borderline lesion was diagnosed as lymphosarcoma based upon cytological detail of the proliferating lymphocytes (see Figure 42), (G) another borderline lesion that was diagnosed as lymphosarcoma based upon cytological features of the proliferating lymphocytes, (H) lymphosarcoma, (I) lymphosarcoma, and (J) lymphosarcoma.
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Figure 42 (A) This borderline lesion (see Figure 41 F) from a low dose female B6C3F1 mouse in the citral cancer bioassay was diagnosed as lymphosarcoma. (B) A higher magnification shows that the expanding PALS is marginating and invading into the follicular compartment and there is loss of the marginal sinus. (C) Based upon the irregular nuclear profiles and immature features of the lymphoid cells in the expanding PALS, this lesion was considered an early lymphosarcoma.
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Figure 46 (A) In this enlarged spleen from a vehicle control female B6C3F1 mouse in a 2-year carcinogenicity bioassay, the white pulp is markedly expanded by pale staining cells. This lesion was diagnosed as a histiocytic lymphosarcoma. (B) An expanding edge of the neoplastic white pulp is encroaching upon the red pulp present in this photomicrograph. (C) Cytological variability in nuclear size and shape plus the presence of abundant pale staining cytoplasm is evident at this magnification. (D) A higher magnification of C showing cellular pleomorphism and presence of mitotic figures. (E) Higher magnification of a portion of D to show cellular detail.
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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.
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Figure 47 (A) A low magnification photomicrograph of granulocytic leukemia in the spleen from a vehicle control male in a subchronic study shows total effacement of the normal splenic architecture with only occasional trabecular structures evident among the proliferating neoplastic cells. (B) The proliferating cells are predominantly granulocytes with only occasional small clusters of erythyroid cells and some megakaryocytes present. (C) Higher magnification of B to show cellular detail.
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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.
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Figure 48 (A) The low magnification photomicrograph of erythroleukemia in this vehicle male CD-1 mouse resembles the low magnification view of granulocytic leukemia shown in Figure 47A. (B) At higher magnification there is a relatively uniform appearing population of cells that have replaced normal splenic compartments with only traces of trabecular structures evident. (C) Cytologic detail reveals numerous erythrocyte precursor cells and small clusters of darkly staining rubricytes.
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Figure 49 (A) In this case of erythroleukemia in a Tg.AC mouse on a FVB/N background, the white pulp compartments as well as the red pulp has been replaced by neoplastic cells but with trabecular structures still evident. (B) Cytological detail shows erythroid progenitor cells at various stages of development.
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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.
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Figure 50 Grossly enlarged spleen (bottom) from a F344 rat with mononuclear cell leukemia (large granular lymphocyte leukemia) compared to an age-matched normal rat (top).
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Figure 51 (A) In this case of mononuclear cell leukemia, the red pulp from this F344 rat has been expanded by the proliferating neoplastic large granular lymphocytes and has resulted in effacement of the white pulp. (B) In this higher magnification of A, the morphological features of neoplastic large granular lymphocytes include cellular pleomorphism, round to irregular and often eccentric nuclei, and relatively abundant eosinophilic cytoplasm. 52. —In perpheral blood smears, large granular lymphocytes contain asurophilic cytoplasmic granules which represent perforins and granzymes. 53. —Liver from a F344 rat with mononuclear cell leukemia. The sinuoids contain circulating neoplastic large granular lymphocytes. Liver involvement is characteristic of advanced stages of mononuclear cell leukemia.
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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.
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Figure 54 (A) In an early stage of mononuclear cell leukemia, the red pulp is diffusely infiltrated and expanded by neoplastic large granular lymphocytes and white pulp elements are still discernable as are foci of extramedullary hematopoiesis. (B) This section of liver from the same rat as in A contains very few large granular lymphocytes in hepatic sinuosoids. (C) In this advanced case of mononuclear cell leukemia, the white pulp is not sharply demarcated from the red pulp. (D) This liver from the same rat as in C has dilated sinusoids containing large granular lymphocytes with early atrophy of some hepatic cords.
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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).
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Figure 55 (A) The splenic parenchyma has been effaced by proliferating large granular lymphocytes throughout the red pulp with atrophy of the white pulp in this advanced case of mononuclear cell leukemia. The poorly stained area in the lower left of the photomicrograph represents an area of necrosis secondary to thrombosis. (B) In this higher magnification of A, there is parenchymal fibrosis surrounding the region of necrosis which was originally misdiagnosed as hemangiosarcoma.
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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.
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Figure 56 (A) In this histiocytic sarcoma, irregular patches of pale staining neoplastic cells have infiltrated the splenic parenchyma and have begun to replace the normal structures of the spleen. The basophilic cells in the intervening red pulp represents extramedullary hematopoiesis. (B) In this higher magnification of A the proliferating histiocytic cells have proliferated within an area of white pulp with margination of the remaining lymphocytes. (C) A higher magnification of B shows the multinucleated giant cells present in this neoplasm. The presence of a sufficient number of giant cells, especially in the absence of histiocytic sarcoma elsewhere in the body, might justify a diagnosis of giant cell sarcoma.
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Figure 57 (A) This case represents another histiocytic sarcoma in a mouse and is characterized by irregular patches of pale staining neoplastic histiocytes with margination of existing splenic lymphocytes. (B) Higher magnification of A showing a uniform population of malignant histiocytic cells.
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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.
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Figure 58 (A) In this spleen pale staining neoplastic mast cells are proliferating in the red pulp where they are admixed with hematopoietic cells. (B) In this higher magnification of A sheets of uniform mast cells containing abundant, lightly eosinophilic cytoplasm are evident along with a few other hematopoietic cells. (C) Higher magnification of B. The mast cell cytoplasm contains granules which are not usually visible in hematoxylin and eosin stained sections. (D) In this toluidine blue stained section the metachromatic granules in the mast cell cytoplasm are dark purple in contrast to the light blue staining of splenic lymphocytes in the upper portion of the photomicrograph.
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Figure 59 (A) In this example of mast cell neoplasia, there is more extensive involvement of the splenic red pulp. (B) In this higher magnification of A, a few hematopoietic erythrocytes and megakaryocytes are evident within the red pulp where the mast cells have proliferated. 60. — (A) Virtually all of the red pulp has been replaced by malignant mast cells in this neoplasm. (B) The higher magnification shows the uniform cytological features of the mast cells. A few megakaryocytes remain in the areas of mast cell proliferation.
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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.
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Figure 61 (A) This hemangioma of the spleen is fairly discrete with expansile growth containing small and large blood filled spaces. (B) The higher magnification of A shows the uniform endothelial cell lining of the blood-filled spaces and copious intervening connective tissue. Diagnosis of hemangiomas in the spleen is typically difficult as the stromal reaction favors a diagnosis of hemangiosarcoma. 62. — (A) This hemangiosarcoma has effaced and expanded the spleen and has invaded the capsular surfaces. (B) In this higher magnification of A, irregular vascular spaces are lined by hyperchromatic plump endothelial cells and proliferating epithelial cells are infiltrating the connective tissue that is also part of this neoplasm. 63. — (A) Another example of an expansile hemangiosarcoma in a treated male B6C3F1 mouse from the cumene cancer bioassay. The spleen is enlarged with the normal parenchyma largely replaced by the malignant neoplasm. (B) In this higher magnification of A the small vascular spaces lined by endothelial cells and solid areas of proliferating neoplastic endothelial cells form a large part of the neoplastic growth.
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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).
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Figure 64 (A) Low magnification of a mesothelioma located on the splenic capsule and comprised of both solid and papillary cellular proliferations. (B) Higher magnification of A showing cellular detail of the mesothelioma.
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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).
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Figure 65 (A) This fibrosarcoma is localized in the red pulp with extension into white pulp compartments. (B) Cellular features of A include a highly cellular proliferation of fibroblasts with a moderate amount of collagen deposition. 66. —Cellular features of this giant cell sarcoma include variable poor differentiation, variable cell size, anaplasia, and presence of occasional giant cells. 67. —This osteosarcoma is comprised of large areas of undifferentiated mesenchymal cells with occasional focal areas of osteoid formation. In this photomicrograph there is mineralization of the osteoid.
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Figure 68 (A) The low magnification photomicrograph of this schwannoma features a area of tumor growth that has completed replaced the splenic parenchyma. A small areas of red pulp and atrophic white pulp is present at the right edge of the photomicrograph. (B) A higher magnification of A shows the characteristic growth pattern of a schwannoma. 69. —A metastatic pheochromocytoma has localized in the spleen and replaced an area of normal splenic parenchyma. The malignant adrenal medullary cells are extending into the adjacent unaffected splenic tissue at the bottom of the photomicrograph. 70. —An anaplastic prostatic carcinoma is present on the left half of this photomicrograph. Without knowledge of the location of the primary neoplasm, it would be difficult to make a definitive diagnosis based solely on the cytologic features of the neoplastic cells.
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Figure 43 (A) In this lymphosarcoma there is expansion and merging of adjacent white pulp areas in the spleen from this high-dose female B6C3F1 mouse in the citral cancer bioassay. This example is similar to Figure 42 above but with greater expansion of the white pulp areas. (B) A higher magnification of the central portion of A shows an expanding PALS and loss of the marginal sinus at points where adjacent areas of white pulp have merged. (C) Higher magnification of B to show cellular features of the expanding white pulp. Normal dark staining lymphocytes are present on the right edge of the photomicrographs. (D) High magnification of the central portion of C shows a relatively uniform population of immature lymphoid cells.
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Figure 44 (A) In this case of lymphosarcoma in an untreated male B6C3F1 mouse, the enlarged areas of white pulp are comprised of pale staining cells. (B) A higher magnification of the proliferating white pulp cells is suggestive of cytological variability and possible anaplastic lymphoid cells. (C) Cytological detail shows the variation in nuclear size and shape, varying degree of hyperchromasia, and greater than normal amounts of pale pink staining cytoplasm in the hematoxylin and eosin stained proliferating lymphoid cells.
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Figure 45 (A) This spleen from a mid-dose female B6C3F1 mouse in the citral cancer bioassay was diagnosed as a lymphosarcoma (see Figure 41I). The red pulp is partially obliterated by the expanding white pulp areas. (B) This higher magnification of A shows an absence of mature lymphocytes in the expanded white pulp. (C) The cytomorphological features of the malignant lymphocytes includes irregular nuclear profiles with prominent nucleoli in some of the lymphocytes.
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Footnotes
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This work was supported by NIEHS contracts N01ES35513 and N01ES95435. The author wishes to acknowledge the assistance of the staffs of Integrated Laboratory Systems Inc., Experimental Pathology Laboratories Inc., and the National Institute of Environmental Health Sciences with preparation of photographs included in this paper and the helpful comments from Dr. Jerrold Ward.
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References
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Toxicologic Pathology, Vol. 34, No. 5,
466-503 (2006)
DOI: 10.1080/01926230600867750
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Abstract
Introduction
