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Hematology/Clinical Pathology Mary Jo Burkhard DVM, PhD, DACVP The Ohio State University Section I. Just Scraping the Surface: Cytology of Cutaneous Samples (LN and Skin) LYMPH NODES Assessment of neoplastic lymphocytes has traditionally relied on morphologic characterization either by cytology or histopathology. However, using the morphology revealed by light microscopic examination to make a cell type diagnoses and prognosis is not always straightforward. Lymphocyte morphology is typically divided by cell size, nuclear maturation, and cytoplasmic features into the categories below. Lymphocyte morphology Small lymphocytes: These cells are smaller in size than a neutrophil and have a round nuclei that takes up the majority of the cell. The nuclei contain densely aggregated chromatin forming large chromocenters (condensed chromatin). Nucleoli are not seen. The cytoplasm is scant (sometimes only a very thin rim is visible) and lightly basophilic in color. These are typically called 'mature lymphocytes'. However, early lymphoid progenitor cells, hematopoietic stem cells, certain stages and types of dendritic cells, and other immature precursor cells may have a very similar morphology to 'mature, well-differentiated, small, resting lymphocytes'. Intermediate to large lymphocytes: These cells are range in size from slightly larger than small lymphocytes to the size of neutrophils. The nuclei still takes up the majority of the cell, however more abundant cytoplasm is visible in these cells. Often, the nuclei is placed eccentrically within the cytoplasm. The nuclear chromatin is finely clumped to granular. Typically, nucleoli are not seen although strands of loosely clumped nuclear chromatin may be mistaken for nucleoli. The cytoplasm is lightly basophilic in color. Occasionally these cells contain azurophilic granules suggestive of a natural killer (NK) phenotype. Lymphoblasts: Lymphoblasts are as large as a neutrophil or larger. Size alone does not indicate neoplasia. Very large lymphoblasts (2-4x the size of neutrophils) may be seen in reactive and hyperplastic processes. Lymphoblasts contain round to oval nuclei with fine or stippled chromatin (loosely aggregated chromatin). One or more nucleoli may be visible. The cytoplasm is moderately to deeply basophilic. Occasionally (seen more in cats than dogs) the cytoplasm may contain punctate vacuoles. Reactive lymphocytes: These cells are similar in morphology to small lymphocytes but are slightly larger and have more abundant, more basophilic cytoplasm. Plasma cells: Plasma cells are intermediate sized cells that contain small, round, eccentrically placed nuclei with condensed chromatin. Cytoplasm is abundant, deeply basophilic, and often contains a prominent, eccentric, perinuclear, clear zone that corresponds to the Golgi. Most maturation charts show lymphocyte development as starting at the lymphoblast stage. Cells then become progressively smaller with a more condensed chromatin pattern as they mature. Thus lymphoblasts become intermediate cells which transition into small 'mature' lymphocytes. However more detailed immunologic analysis into the phenotype and structure of activated, resting, memory, effector, regulatory, and precursor lymphocytes suggests substantial overlap in morphologic features between these categories. Morphologic features and a knowledge of 'typical findings' has been used by both clinical and anatomic pathologists to help characterize underlying cell type and the disease process. Some of these are described below. Morphologic features and typical findings used to characterize lymphocytes Lymphoglandular bodies: Lymphoglandular bodies are round, homogeneous, basophilic structures comprised of cytoplasmic fragments. The presence of lymphoglandular bodies is seen in cytologic preparations of lymphoid tissue that contains increased numbers of lymphoblasts. This can be due to neoplasia (lymphoma) or hyperplasia. Golgi clearing zone: The presence of an eccentric, perinuclear clearing zone is often suggested as a feature of B-cells and plasma cells. The clearing zone is the Golgi and it is a prominent feature in plasma cells. However, the Golgi apparatus is an organelle found in most cells, including T-cells and myeloid cells. Sezary cells: In humans, Sezary cells are described as medium to large lymphocytes with ceribriform nuclei. These neoplastic T-cells are characteristic features of Sezary syndrome which encompasses mycosis fungoides, an epitheliotropic variant of cutaneous lymphoma. A similar syndrome occurs in dogs but has been rarely reported in cats. In dogs, epitheliotropic T-cell lymphoma is also seen in the gastrointestinal tract [Coyle, 2004 #1594]. T-cells predominate in both the cutaneous and GI variants. Interestingly, expression of protein gene product 9.5 (PGP 9.5), a marker previously considered specific for neural and neuroendocrine tissues, was recently detected in over 8/14 cases of canine cutaneous mycosis fungoides [Ramos-Vara, 2007 #1593] suggesting that there may be other biologic differences between the human and canine variants. Flower cells: In humans, the presence of flower cells or cloverleaf cells is most often associated with T-cell disease and is particularly a feature of infection with human T-lymphotrophic virus-1 (HTLV-1). In dogs and cats, similar morphology has been seen in both B-cell and T-cell lymphoproliferative disease as well as myeloproliferative disease. Chronic lymphocytic leukemia: Unlike that seen in humans where CLL is considered a disease of B-cells, CLL of dogs and cats is primarily a T-cell disease. In dogs, CD8+ (cytotoxic) CLL predominate while in cats, CD4+ (T-helper) CLL is more common. However, there is variation in the disease in both dogs and cats and B-cell, CD4+ T-cell, and CD8+ T-cell CLL have all been diagnosed in small animals. Chronic lymphocytosis: In dogs, a chronic lymphocytosis comprised of intermediate sized lymphocytes with small azurophilic granules has been reported in association with Ehrlichiosis. Interpretation of lymph node cytology Reactive/hyperplastic lymph node: Small lymphocytes predominate although the lymphoid population is heterogeneous with increased numbers of plasma cells, medium lymphocytes, and lymphoblasts. Macrophages, neutrophils, eosinophils, and mast cells may also be increased. Mandibular and mesenteric lymph nodes are often "normally reactive" due to drainage of the oral cavity and intestine. Lymphadenitis: Lymphadenitis is characterized by increased numbers of inflammatory cells. The cell type varies with the type of inflammation.
Not compatible with lymph node: Samples without lymphoid tissue can be obtained in two scenarios. The first is when the lymph node has been effaced by metastatic neoplasia. The second is when the lymph node is not aspirated (e.g. fat, salivary gland, muscle). The latter is relatively common and should be excluded before considering effacement of the lymph node. CUTANEOUS LESIONS Inflammatory cells Inflammatory lesions can occur due to infectious or non-infectious causes. Compared to tissue cells, inflammatory cells are readily collected by aspirates and scrapings. Inflammation is characterized by the predominant cell type which helps to predict the underlying etiology. Neutrophils indicate suppurative or purulent inflammation. Non-infectious causes include immune-mediated disorders, chemical irritation, neoplasia, and acute trauma. Infectious causes include most bacterial infections and some fungal organisms. Degenerative neutrophils (swollen, karyolitic nuclei) are commonly associated with the toxic environment induced by bacterial infections however degenerative changes also occur as cells become exposed on mucosal or external surfaces. Macrophages may be seen in mixed, granulomatous, and pyogranulomatous inflammatory processes. Causes include yeast and fungal agents, foreign body reactions, certain bacteria (Mycobacteria spp.), hemorrhage, and chronic inflammatory conditions. Epithelioid macrophages have a wispy to epithelial appearance. These, and multinucleated cells, and are typical of granulomas making granulomatous inflammation difficult to distinguish from histiocytic neoplasia. Lymphocytes and plasma cells are associated with allergic or immune reactions, some viral diseases, feline cholangitis, feline stomatitis and gingivitis, gastroenteritis, and vaccine reactions. Their presence can usually be differentiated from neoplasia by the heterogeneity of the lymphocyte population. Eosinophils are associated with tissue parasites, neoplasia (lymphoma, mast cell tumor, melanoma, others), fungal organisms, protozoal organisms, eosinophilic granulomas, collagen necrosis, and hypereosinophilic syndrome. Mast cells in low numbers are often seen in inflammatory lesions. Infectious inflammatory conditions Bacteria: Bacterial agents may be found in abscesses, contaminated surgical sites, and secondary to numerous underlying metabolic, allergic, and neoplastic disorders. Specific agents that may be recognized include Actinomyces/Nocardia (filamentous), Mycobacterium (negative staining), and Yersinia (bipolar 'safety pin'). Scrutiny of diplococci has increased recently, with additional concerns over MRSAs. Bacterial infection primarily induce a suppurative response, however the filamentous bacteria and Mycobacterium sp may induce a mixed to pyogranulomatous response. Fungi: Fungal diseases can be present as either cutaneous or subcutaneous lesions. The cutaneous diseases include those induced by dermatophytes (Microsporium, Tricophyton, or Epidermophyton), Candida, and Malassezia. Dermatophytosis typically results in hairless lesions on the head, paws, ant occasionally the tail. The organism are best detected through examination of skin scrapings taken from the periphery of the lesion as they may are often seen in association with hair shafts. Dermatophytes induce a mixed inflammatory response. Cutaneous or mucocutaneous candidiasis is uncommon, but when seen is detected as septate branching hyphae that induce a pyogranulomatous to granulomatous response. Malassezia can be detected by scraping, swab imprints, or impression touch prints of the lesion. As Malassezia can be found on normal dogs and cats and dermatitis is often seen as a secondary disease (e.g. to atopy, pyoderma, hypothyroidism). Subcutaneous mycoses include sporotrichosis, histoplasmosis, cryptococcus, blastomycosis, and coccidioides. Most of these are detected through the identification of yeast forms (see Infectious Disease section for detailed descriptions) however fungal infections resulting in hyphal forms can be seen. Algae: Although Prototheca is often mentioned with the fungi and the organisms have several morphologic features, it is actually an algae (see Infectious Disease section for detailed description). Protothecosis typically produces a strong macrophagic to granulomatous inflammation. Neutrophils may also be seen. Protozoal: Leishmania is the most commonly seen protozoal organism in skin and subcutaneous lesions (see Infectious Disease section for detailed description). Non-infectious inflammatory conditions Hematoma: The appearance depends on the age of the lesion. Acutely, a hematoma appears as blood without platelets however within a short time period, erythrophagocytic macrophages are seen and blood breakdown products (hemosiderin, hematoidin) begin to form. Reactive mesenchymal cells (fibrosis) may be present as the hematoma organizes. Hygroma: A hygroma is a subcutaneous swelling usually over bony prominences. It forms as a consequence of repeated trauma and is most common in older large breed dogs. The fluid has low cellularity fluid, primarily comprised of macrophages and few reactive mesenchymal cells, and possible evidence of hemorrhage. Sialocele: A sialocele is typically a fluctuant, cool, painless swelling associated with the rupture of a salivary duct and accumulation of saliva within subcutaneous tissue. Siaoloceles may become firm or painful with inflammation or infection. The fluid is clear to bloody and often has a thick and stringy texture. Cytologically, there will be a pink to basophilic, mucoproteinaceous background with moderate numbers of individualized, large, vacuolated mononuclear cells that are difficult to differentiate between macrophages or salivary glandular epithelial cells. Non-degenerate neutrophils and evidence of hemorrhage are also common. Hematoidin crystals are frequently found in sialoceles. A seroma is a fluid pocket that forms at the site of an injury or incision site. Fluid seeps from proliferating (and thus leaky) capillaries resulting in a low cellularity specimen, comprised primarily of activated, phagocytic macrophages with a mix of other inflammatory cell types. Calcinosis cutis is associated with hypercortisolemia and represents dystrophic mineralization of collagen. Calcinosis circumscripta results from dermal deposits of mineral surrounded by fibrosis and inflammation at pressure points, sites of trauma and the tongue and is most common in large dogs and young German Shepherds. The cytologic appearance of both is similar. Slides often appear chalky when examined grossly before staining. After staining, a fine, granular, basophilic background is seen with variable amounts of irregular, refractile, non- or pale yellow-green staining mineral. The inflammatory response is mixed, chronic or pyogranulomatous. Vaccine reactions induce a mixed inflammatory infiltrate typically comprised of macrophages, lymphocytes, and plasma cells. Occasionally multinucleate giant cells may be seen. Eosinophils and mast cells are often present. Occasionally, magenta or basophilic material thought to represent the adjuvant material may be seen extracellarly in the background. The vaccine reaction may induce moderate to marked reactive fibroplasias that can be difficult to differentiate from early fibrosarcoma. Steatitis or nodular panniculitis can be induced by a number of causes ranging from immune mediated and idiopathic to foreign body reactions. The inflammatory reaction will be seen against a background of adipocytes and lipid. In chronic cases, a marked fibrotic reaction may also be seen. Eosinophilic plaque/granuloma are characterized by large numbers of eosinophils. However mast cells and lymphocytes may also be present necessitating differentiation of these diseases. Acral lick dermatitis (lick granuloma) induces a chronic inflammatory infiltrate typically comprised of macrophages, lymphocytes, and plasma cells. The presence of squamous epithelial cells is common as the chronic inflammation induces squamous hyperplasia (acanthosis). Parasitic bite reactions induce a mixed inflammatory infiltrate that often includes increased numbers of eosinophils as part of the hypersensitivity response. Foreign bodies also induce a mixed inflammatory infiltrate. The specific type varies dependent on the type of foreign material, however macrophages including the presence of multinucleated giant cells are common. Secondary bacterial infection may complicate the diagnosis. Pemphigus foliaceus can be diagnosed through the cytologic examination of pustule contents. Acantholytic cells will be seen in a background of non-degenerate neutrophils and occasional eosinophils. Acantholytic cells are basilar squamous epithelial cells that appear as individualized, deeply basophilic, round keratinocytes. Non-inflammatory/non-neoplastic conditions Epidermal and follicular cysts are common, non-neoplastic cysts located in the dermis of dogs and cats. The cyst is lined by mature squamous epithelial cells and keratinized epithelial cells while cellular debris accumulates in the center. Cytologically, they may appear similar to epithelial tumors with follicular differentiation. The cysts are comprised of mature squamous epithelial cells and cell fragments that are angular, flat, basophilic cells without a nucleus, cytoplasm is glassy in appearance. Keratinaceous debris and cholesterol crystals may be seen. If the cyst ruptures, it will induce an intense pyogranulomatous inflammation. Sebaceous hyperplasia is more common in older dogs and is often appears as a raised, hairless lesion with a cauliflower appearance. The sample may appear oily or greasy when unstained. Uniform clusters of epithelial cells will be seen with moderate amounts of foamy cytoplasm and small central nuclei. There may also be small numbers of more basal-appearing cells (reserve cells). Cytology alone can not differentiate sebaceous hyperplasia from sebaceous adenoma - this is likely of little significance as adenomas have similar clinical features and usually a benign behavior. A meibomian gland adenoma of the eye may look similar cytologically. Epithelial neoplasia Hair follicle tumors and keratoacanthoma. There are several types of hair follicle tumors, typically benign, with follicular differentiation. These include tricoepithelioma, trichoblastoma, pilomatrixoma, etc and cannot be differentiated by cytology. Keratoacanthoma (intracutaneous cornifiying epithelioma) arises from the epithelium between hair follicles and often has a pore that opens to the surface. Hair follicle tumors contain abundant keratin and debris and often resemble the material seen in follicular cysts. Depending on the tumor type, the material will range from primarily mature non-nucleated squamous epithelial cells, to a mixture of cells and debris, to a predominance of basophilic debris. "Ghost cells" which are keratinized epithelial cells with an empty hole in the center where the nucleus used to be may be seen in certain processes. Small numbers of uniform basilar epithelial cells may be seen if the wall of the tumor is aspirated. Rupture induces intense suppurative to pyogranulomatous inflammation Basal cell tumor is the most common skin tumor of cats, also common in dogs. It is usually benign and typically presents as a solitary intradermal mass. The cells are arranged in tight clusters, sheets or rows and have a uniform cuboidal shape with scant amounts of deeply basophilic cytoplasm that may be pigmented. If the tumor is cystic, the cystic material will resemble that seen in epidermal or follicular cysts. Sebaceous or squamous differentiation may be seen as individualized or small clusters of sebaceous cells or foci of keratinized epithelial cells. The latter needs to be differentiated from a well differentiated squamous cell carcinoma and may require histopathology to examine the architecture. Meibomian gland adenoma of the eye may look like a basal cell tumor with sebaceous differentiation. Sebaceous gland tumors are a diverse set of sebaceous proliferation that ranges from hyperplasia to epithelioma to adenoma to carcinoma. Hyperplasia and adenoma cannot be differentiated cytologically as both consist primarily of clusters of mature, uniform, sebaceous epithelial cells. An epithelioma is essentially a basal cell carcinoma with areas of sebaceous differentiation and contains a mixture of basal cells (sometimes pigmented) and sebaceous epithelial cells. Sebaceous carcinomas are rare and may lack cytologic features of sebaceous differentiation. Sweat gland tumors are uncommon in both dogs and cats. When seen, these masses are often solitary, firm, and well circumscribed and tend to be malignant (adenocarcinomas). Cells have round to oval nuclei, fine to granular chromatin, and lightly basophilic cytoplasm. The appear in clusters, sometimes forming acinar structures and have variable features of malignancy. Papillomas "warts" are rarely seen in cats but relatively common in dogs. These are thought to be associated with different types of papilloma viruses and clinically appear as multiple cauliflower like growths. Cytologically, these samples contain large numbers of large polygonal cells with abundant cytoplasm which is basophilic to granular and/or foamy depending on the degree of cornification. The N:C ratio is low. Nuclei are round and often eccentric with smoothly clumped chromatin. As would be expected of hyperplastic squamous epithelial cells, these cells appear individually rather than in cohesive clusters. Perianal gland adenoma is most common in intact, male dogs and rare in cats. Malignant tumors are uncommon and usually are associated with greater cellular pleomorphism although the two can be very difficult to differentiate and often require a biopsy to examine the invasive nature of the tumor. A perianal gland adenoma usually appear as raised smooth to lobulated masses near the anus but may also be found in other locations (tail, perineum, prepuce, thigh). Cytologically, it is identified by the presence of clusters of relatively uniform, large, oval to polygonal cells (hepatoid cells) with round, somewhat eccentric nuclei and abundant finely granular, pink-blue cytoplasm. Small numbers of reserve cells also may be seen Apocrine gland adenocarcinoma is more commonly seen in dogs than cats. It is a highly malignant tumor that arises from the anal sac apocrine glands and often metastasize to regional lymph nodes, even when the tumor is small. Hypercalcemia is seen in approximately half the cases. The tumor has a neuroendocrine to epithelial appearance comprised of loosely cohesive clusters of cells with round nuclei and indistinct cell margins. In some tumors, cells are easily ruptured - "naked nuclei" similar to that seen in neuroendocrine tumors. Cells range from relatively uniform to moderately pleomorphic. Acinar-like arrangements may be seen. Squamous cell carcinoma is commonly seen in both cats and dogs. This malignant tumor of squamous epithelium is often ulcerated and accompanied by suppurative inflammation that can make diagnosis difficult. Preps from squamous cell carcinomas are often very cellular. Cells are usually individualized but may occur in clusters if they are more basilar. Cells may be tadpole shaped, angular, to round in shape. They often have evidence of keratinization as indicated by the presence of hyalinized, pale blue cytoplasm and fine, perinuclear vacuolization. Keratinized material often induces strong inflammatory responses, such that neutrophils may markedly outnumber epithelial cells. Emperipolesis (the migration of neutrophils through the cell cytoplasm) is common. Cells may be highly individualized and can be mistaken for mesenchymal or round cell tumors requiring a search for small sheets with definitive cell-cell junctions, other features of epithelial cells, or evidence of keratinization. Well differentiated tumors with inflammation can be difficult to distinguish from dysplastic changes induced in non-neoplastic cells secondary to chronic inflammation or irritation. Other types of carcinomas can also keratinize (thyroid, mammary, transitional cell, respiratory, etc.) Mesenchymal neoplasia A diagnosis of mesenchymal neoplasia warrants histologic evaluation as sarcomas can be easily confused with granulation tissue, fibrosis, endothelial cell proliferation, or granulomatous inflammation secondary to inflammatory processes. Some carcinomas induce a marked scirrhous response which can strongly resemble mesenchymal neoplasia. Sarcomas can sometimes be difficult to differentiate from poorly differentiated carcinomas and some discrete cell neoplasia. Many sarcomas have similar biologic and cytologic features requiring histopathology for diagnosis, however those with more unique features are described below. Fibroma and other benign lesions such as collagen nevus and fibroadnexal dysplasia require histopathology for diagnosis. Typically, very few cells are obtained by aspiration of these lesions and the cells that are present are uniform spindled cells with lightly basophilic cytoplasm, round to oval nuclei, lacy chromatin, and indistinct nucleoli. Fibrosarcoma are more commonly seen in cats, especially at vaccine sites, but may also be diagnosed in dogs. Cells from fibrosarcomas tend to be very pleomorphic ranging from straplike to fusiform to stellate to oval. Marked anisocytosis and anisokaryosis may be seen. Multinucleate cells are common, particularly in vaccine induced sarcomas. The chromatin pattern is coarse with single to multiple, prominent, variably sized and shaped nucleoli. Cytoplasm is moderately to deeply basophilic. Hemangioma/hemangiosarcoma often contain only blood or evidence of chronic hemorrhage as the cells do not always exfoliate well. When spindled cells are seen cytologically, it may be difficult to differentiate them from other malignant mesenchymal neoplasia or fibrosis. Soft tissue sarcomas (hemangiopericytomas, neurofibromas, etc) are more common in dogs than in cats. They frequently occur on limbs, especially near the joints. Aspiration of these samples typically results in a moderately cellular sample. When in clumps, cells often have a swirling or fronding pattern. Individual cells range from round to stellate to spindled with wispy grey-blue cytoplasm that may contain small vacuoles. The nucleus round to oval with fine chromatin and indistinct nucleoli. The cells are usually relatively homogeneous. Giant cell tumors such as malignant fibrous histiocytoma and giant cell tumors of soft parts can be seen in dogs and cats. There may be a predilection for the scapular and dorsal thoracic regions, however they can be found anywhere. Cytologically, these are characterized by the presence of large multinucleated cells containing as many as 40-50 nuclei although those with 10-20 nuclei are more common. Cells with individual nuclei are also seen and range in appearance from histiocytic to spindled. The cells have moderate to marked anisocytosis and anisokaryosis. Anisokaryosis may also be seen within a single multinucleated cell. Nuclei are round to oval with coarse chromatin and prominent, often multiple nucleoli. Lipoma/liposarcoma. Lipoma is a commonly seen benign tumor while liposarcomas more closely resemble sarcomas, rather than lipomas. Lipomas are soft, well-circumscribed, freely movable subcutaneous masses. When aspirated, the material will appear greasy and does not dry. Cytologically, lipomas appear as appear as clusters of signet ring shaped cells (adipocytes) with abundant clear cytoplasm. However, normal subcutaneous or perinodal adipose tissue appear the same. Alcohol fixatives used in Romanowsky-type stains will dissolve away free lipid and may clear away cells as well, however usually a few cells remain on the slide for examination. A wet mount stain such as new methylene blue or lipid stains such as Sudan can be used as an alternative if needed. "Round cell tumors" or discrete cell neoplasia Round cell tumors are commonly detected as cutaneous or subcutaneous masses. More differentiated tumors have very unique features that help to identify the specific type of neoplasia. However, poorly differentiated round cell tumors can be very difficult to identify even by histopathology and diagnosis often requires phenotypic marking by immunocytochemistry or flow cytometry. Mast cell tumor are common tumors in both dogs and cats. Cytologic features include the presence of many individualized mast cells. Well differentiated cells have abundant small blue to purple granules that often obscure the nucleus. Poorly differentiated cells can be highly pleomorphic with multiple criteria of malignancy and few to no granules or large granules. Eosinophilic inflammation often present, but is not always seen. Mesenchymal cells (sometimes with granules) may also be seen. Diff Quick may degranulate the granules of some mast cell and any diagnosis of round cell neoplasia using Diff-Quik that doesn't clearly fall into another category should be further examined by staining new slides (counterstaining previously stained slides will not work) with Wright's or Giemsa based stains. Lymphoma may be a primary cutaneous disease or part of disseminated disease. Cells may be small to large but are uniform in appearance (vs. heterogeneous suggesting lymphoid hyperplasia or inflammation). Nuclei may be round, indented or highly convoluted, the latter being a feature of epitheliotropic lymphomas and Sezary syndrome. Chromatin is often fine, even in smaller to intermediate cells. Nucleoli are often seen, but may be indistinct in appearance. The amount and basophilia of the cytoplasm varies with the stage of the neoplastic lymphocytes. Plasmacytomas are more common in dogs than cats and the cutaneous forms have a predilection for the oral cavity, ears, digits, and forelimbs. The cells exfoliate readily and may be well differentiated and easily recognized as plasma cells or can exhibit prominent atypia. The often contain deeply basophilic cytoplasm with discrete margins that is darker at the margins and paler in the perinuclear zone creating a perinuclear clear zone. Nuclei are eccentrically placed, are round to oval, and contain coarsely clumped chromatin that may give a 'cartwheel' appearance to the nuclei. Binucleated cells are commonly seen and multinucleated cells may be present. Histiocytoma is a common tumor of young dogs but may also be seen in older dogs. The tumor originates from the Langerhans cell of the epidermis. Most will spontaneously regress following infiltration by CD8+ T-cells, thus the presence of small lymphocytes is often seen. The neoplastic cells exfoliate as individualized round cells with a moderate amount of lightly basophilic cytoplasm. Nuclei are oval or indented with fine chromatin and indistinct nucleoli. The outer edges of the cytoplasm often stains lighter than the perinuclear area. Cutaneous and systemic histocytosis (histiocytic sarcoma, malignant histiocytosis) are proliferative histiocytic disorders of dogs and cats. Cutaneous involvement is variably reported in both species. The cells are pleomorphic individualized cells that often have many features of malignancy. Nuclei may be round, indented, or pleomorphic in appearance with fine to lacy chromatin and prominent single to multiple nucleoli. Micronuclei and multinucleation is common. The cytoplasm is often vacuolated and erythrophagocytosis is common. Transmissible venereal tumor is a transmissible neoplasia of dogs. The cells have only 59 chromosomes compared with normal canine karyotype of 78. The tumors typically occur as soft, pedunculated, friable masses on the mucus membranes of the external genitalia, although other sites and metastasis have been uncommonly reported. Cytologically, these tumors appear as a uniform population of round cells with round nuclei, coarse chromatin and one to two prominent nucleoli and variable amounts of basophilic cytoplasm. A characteristic feature of TVT is the presence of numerous punctate vacuoles in the cytoplasm. Melanoma can be seen in both dogs and cats. These arise due to a proliferation of melanocytes and therefore are of neural crest origin. However, cells range in morphology from discrete round cells to epithelioid to spindled cells and these cells can be highly variable in both benign and malignant forms. If poorly granulated, these cells are easily mistaken for other tumor types. Pigmentation varies both from tumor to tumor and within tumors. Well differentiated cells have abundant melanin granules which are fine and stain green-black to brown-black and the granules may obscure nuclear and cytoplasmic morphology. Poorly differentiated tumors may have no granules or only a very fine dusting. In these cases, the cytology may appear 'dirty' or that there is some background staining. Even in well differentiated tumors, cells from the deeper margins tend to be less well granulated and thus the cytologic impression may depend on the sampling site. Features of malignancy will vary, however cells from poorly differentiated melanomas are often markedly pleomorphic with variable nuclear to cytoplasmic ratios, anisocytosis and anisokaryosis, finely granular to coarse to lacy chromatin, with prominent nucleoli. Amelanotic melanomas are difficult to differentiate from other round cell neoplasia or mesenchymal neoplasia and sometimes may even have features of epithelial origin (clusters). Melanophages can be mistaken for melanocytes, especially in lymph nodes. Melanophages tend to have coarse aggregates of melanin pigment rather than fine granules. Section II. Going deeper: Fine Needle Aspiration Cytology of Commonly Sampled Internal Organs with Side Commentary on the Perils of Ultrasound Gel Cytology of internal organs as a diagnostic tool Fine needle aspiration (FNA) cytology is an excellent adjunctive tool for evaluating internal organs. It is often diagnostic, requires little specialized equipment, and can rapidly provide useful information. While aspiration of internal body cavities, organs, and masses is considered an invasive procedure and has greater risk of complications than does sampling of cutaneous masses and lymph nodes, it often provides diagnostic information that would otherwise require surgery. In addition, current ultrasound techniques allow guided placement into the lesion of question and visualization of the site both prior to and after aspiration. This had substantially improved the information that can be gained from cytologic evaluation of FNA samples. However, understanding the limitations of FNA cytology of abdominal organs is equally as important. The limitations of FNA cytology of internal organs are amplified because of the relatively 'blind' technique necessitated by their anatomic location, even with ultrasound guidance. The sample may be inadequate for evaluation because of low cellularity; a limitation often associated with more fibrous tissues or tissues of relatively low parenchymal cell density, e.g., kidney and lung, respectively. Even if cellularity is adequate, the sample may not represent the primary lesion. In contrast to a biopsy sample, a cytologic specimen does not reflect the tissue architecture which may be critical for differentiation of neoplastic versus hyperplastic or reactive tissue. A well differentiated malignant neoplasm may not demonstrate morphologic features of malignancy necessary for cytologic diagnosis. However, by combining interpretive expertise with appropriate site selection, ultrasound guided aspirates correlate well with histologic diagnosis, The primary use of cytology of internal organs should be to differentiate inflammatory from neoplastic lesions. Cytology is not as useful as histology for determining the type or origin of epithelial or mesenchymal cells, however discrete cell tumors can often be accurately diagnosed. If inflammation is present, the type of inflammation can be identified. Cytology is often useful for the detection and identification of infectious agents. General approach to cytology of internal organs Cytologic examination begins by evaluating the sample cellularity. If the sample cellularity is inadequate either re-aspiration or an alternative diagnostic procedure should be considered. Some types of disease processes, such as those with mesenchymal proliferation fibrosis do not exfoliate well and repeated aspiration is not useful. Next, cells should be evaluated to determine if they are consistent with the expected sample. When observations are inconsistent with expected findings, the challenge is to determine if the specimen accurately represents the site of interest or is an unexpected finding. Although an organ may be effaced by a neoplastic or inflammatory lesion, another possibility is that non-representative tissue was sampled. This is more likely to occur with 'blind' aspirates but occasionally happens with ultrasound guided samples. Inflammatory cells should be characterized. Neutrophils indicate a suppurative process and should prompt examination for micro-organisms that may be located within the cell (bacteria) or extracellularly (mycotic agents, filamentous bacteria). Foamy macrophages may be seen in mixed inflammatory processes and more epithelioid-like macrophages in granulomatous disease. Increased numbers of eosinophils are typically associated with allergic or hypersensitivity reactions, parasites, or neoplasia. Significant numbers of eosinophils are seen in mast cell tumors, however the presence of eosinophils is not unique to mast cell neoplasia. Many fungal, protozoal, or parasitic organisms are more easily identified at lower magnifications and time should be spent at both low and high magnifications. Small lymphocytes and plasma cells are often present as part of an inflammatory response. The presence of immature lymphocytes in a non-lymphoid organ raises the suspicion for neoplasia however lymphoblasts may be seen if long standing inflammation allows the formation of lymphoid follicles. Thus if the lymphoid population is heterogeneous, the presence of lymphoblasts should be interpreted with caution. Small cell lymphoma is less common than the lymphoblastic form and needs to be considered as a differential when large numbers of small lymphocytes are seen. However, diagnosis requires histologic evidence of architectural effacement as cytologically the cells appear morphologically normal. Caution should be taken when interpreting changes to either mesenchymal and epithelial tissue when there is evidence of inflammation, necrosis, or hemorrhage. Both mesenchymal and epithelial cells can become reactive. Reactive cells will be mild to moderately pleomorphic; they may be larger, more basophilic, have coarser chromatin, and prominent nucleoli and can be very difficult to differentiate from neoplastic cells. Neoplasia can develop from any of the normal cell populations that comprise an organ. Neoplastic changes include both nuclear and cytoplasmic changes such as anisokaryosis, variable nuclear to cytoplasm ratio, multiple nuclei, coarse chromatin, large variable sized or angular nucleoli, abnormal mitotic, increased cytoplasmic basophilia and the presence of punctate vacuoles. A diagnosis of neoplasia is strengthened through the detection of multiple changes as well as the variability of these findings. Pleomorphism is a 'hallmark' of neoplasia however some tumors, such as neuroendocrine tumors, are notable for their bland appearance. In most cases, an attempt should be made to categorize neoplastic cells as epithelial, mesenchymal, or discrete (round) although poorly differentiated and anaplastic tumors defy classification. Sampling techniques Ultrasonography allows a rapid, relatively inexpensive procedure to identify and sample lesions. The combination can be an effective alternative strategy to thoracic or abdominal surgery. Solid masses can be differentiated from cystic or necrotic masses based on tissue density, lesions can be viewed in multiple planes, and needle placement can be viewed to ensure correct sampling. However, because of the generalized nature of many diseases, 'blind' aspirates will often provide diagnostic material. If ultrasound is used to guide the sampling procedure, care needs to be taken to thoroughly clean the ultrasound gel off the sampling site prior to sampling. Even small amounts of ultrasound gel in the sample will induce lysis of cells that contact the gel and too much gel artifact will render the entire slide unreadable. In general, a 22 gauge, 1-1/2 inch needle or a 22 gauge 2-1/2 inch spinal needle with stylet attached to a 3-5 cc syringe is adequate to aspirate most internal lesions. If a spinal needle is used, the stylet is removed once the needle is inserted into the tissue and the syringe attached. Approximately 3 cc's of vacuum is applied and the needle is passed through the organ or mass several times at slightly different angles. The cellular material will usually be confined to the needle. Release the negative pressure before removing the needle from the animal, then remove the needle from the syringe, draw air into the syringe, replace the needle, and then expel the material onto a glass slide. Gentle squash and pull preps are ideal techniques to separate the cells and provide a readable slide. Slides should be air dried prior to staining. Exposure to formalin fumes and excessive heat will change the cell pH and alter the staining characteristics imparting a green hue to cells making it very difficult to make a diagnosis. Contraindications and complications While no procedure is without the potential for complications, fine needle aspiration is regarded as a safe and effective diagnostic tool. The risk of hemorrhage should always be considered and a platelet count and coagulation profile should be performed to identify those patients with increased risk. Accidental aspiration of the biliary system and gut may result in leakage of bile and gut contents, respectively. If this occurs, abdominocentesis should be performed at several time points after the initial sampling to monitor for peritonitis. Pneumothorax is always a risk when obtaining thoracic samples. Clinical observation and post-sampling radiographs are recommended to help identify early pneumothorax. Seeding of the needle tract by neoplastic cells is a potential, but uncommonly reported, complication. Effusions Fluid analysis should include gross evaluation of color and turbidity, nucleated cell count (NCC), determination of total solids, and cytologic evaluation. Additionally, biochemical analysis may also be useful, e.g. triglycerides in chylous effusions, creatinine in ruptured bladders. Effusions can be classified into transudates, modified transudates, or exudates based on cell count and protein concentration. Transudates are characterized by minimal cell counts comprised primarily of mesothelial cells and macrophages and a protein concentration less than 2.5 g/dl resulting from hypoalbuminemia or leakage of low protein intestinal lymph due to portal hypertension. Differentials include severe hepatic disease, protein losing enteropathy or protein losing nephropathy. Modified transudates have similar cell numbers and distribution to transudates but have protein concentration greater than 2.5 g/dl resulting from leakage of high protein hepatic lymph or occasionally inflammatory proteins. Modified transudates have a variety of causes, one of the most common non-inflammatory disease is right sided heart failure however neoplastic effusions may also fall in this category. Exudates are characterized by an increased protein concentration and marked cellularity - usually comprised of neutrophils. Intracellular bacteria are found in septic exudates. Bacterial toxins are responsible for presence of degenerate neutrophils characterized by swollen nuclei and indistinct, smudged chromatin patterns. Streams of lightly eosinophilic nuclear debris may be encountered if these cells rupture during the slide-making process. The exudate associated with FIP is somewhat different than the traditional exudates and is distinguished by a remarkably high protein concentration (often greater than 5 g/dL) with a relatively low number of neutrophils and macrophages. Neoplastic effusions often fall into the exudates category. Hemorrhage is particularly common in pericardial effusions but may also be seen in thoracic and abdominal samples. However, hemorrhage needs to be differentiated from blood contamination. Rapid clotting of the specimen is more common with blood contamination. The presence of platelets in a unclotted specimen is more indicative of fresh blood, either as a consequence of the sampling procedure or ongoing hemorrhage. Erythrophagocytosis and hemosiderin laden macrophages indicate true hemorrhage. Chylous effusions are milky in color and contain increased concentrations triglycerides. Small lymphocytes predominate early in the pathogenesis, however over days to weeks, neutrophils and macrophages will increase. Fluid triglycerides are usually much greater than serum triglycerides in true chylous effusions. Bile peritonitis is characterized by large clouds of green material and macrophages containing yellow-green pigment. Uroabdomen initially results in a fluid with low protein and increased creatinine. With chronicity, the protein will increase with the ensuing suppurative inflammatory process. Urea nitrogen is a less reliable indicator of uroabdomen as it equilibrates more rapidly. Neoplastic effusions will generally fall into the exudate category but occasionally cell counts are within the modified transudate category. Neoplastic cells seen in effusions are typically those that exfoliate well: lymphomas, carcinomas, mast cell tumors, and mesothelial cell tumors. Mesothelial cells, which occur both singly and in clumps, resembling epithelial cells and may demonstrate reactive changes that mimic neoplastic changes. These should be interpreted with caution. Lung Fine needle aspiration of the lung parenchyma is most rewarding in cases of diffuse interstitial disease or when large focal lesions can be localized and sampled. The right caudal lung lobe is typically sampled with diffuse disease. Normal lung is characterized by the presence of columnar to cuboidal respiratory epithelial cells that are often ciliated. These cells have basilar oval nuclei with granular chromatin and lightly basophilic. Goblet cells may contain pink to purple granules. Mucus is often present in respiratory samples as ribbons of eosinophilic material. Occasionally this material is found in tightly wound formations called Curschman's spirals. Increased mucus is a non-specific finding and may be associated with many pathologic processes. "Not lung". The two most common non-respiratory cells seen with lung aspirates are mesothelial cells and hepatocytes. Sheets of monomorphic mesothelial cells with angular, cohesive borders resembling fish scales are seen if the lung surface is scraped during the aspiration process. Aspiration of the liver occurs when the chest is entered too far caudally. Various inflammatory changes can be seen in respiratory samples. Suppurative inflammation is commonly seen with infectious organisms. Even if bacterial or fungal organisms are not seen, a culture or other diagnostics are often still indicated. Large vacuolated alveolar macrophages are present in both acute and chronic inflammation. In chronic disease, binucleate and multinucleate forms may be seen. Increased numbers of eosinophils are typically associated with allergic or hypersensitivity reactions although occasionally parasites such as lungworms may be seen. Other inflammatory cells include small numbers of mast cells, lymphocytes, and plasma cells. Hemorrhage is characterized cytologically by the presence of erythrophagocytosis, hemosiderin-laden macrophages, and hematoidin crystals. Hemorrhage is a complication of fine needle aspirate, so the presence of erythrophagia, preferably with hemosiderin, is important to distinguish pathologic from iatrogenic hemorrhage or blood contamination. Prussian blue stain can be used to distinguish hemosiderophages from other pigment laden macrophages. Pulmonary atelectasis or collapse is suggested by finding large numbers of relatively normal respiratory epithelial cells. Necrosis is characterized cytologically by abundant amounts of basophilic granular to amorphous background material usually admixed with inflammatory or neoplastic cells. However, acellular aspirates or those with only remnant cell membranes or "ghost cells" may occasionally be obtained from necrotic centers. Reactive hyperplasia is common in the lung. Respiratory epithelial cells become more cuboidal in shape and develop several morphologic features than can be seen in neoplastic cells including increased cytoplasmic basophilia, clumped nuclear chromatin, and prominent nucleoli. Caution should be taken if evidence of inflammation, necrosis, or hemorrhage is present. Metaplasia is the replacement of normal cells with a secondary, but non-neoplastic, population. Squamous metaplasia is the most common type seen in the lung and occurs in response to chronic disease. Aspirates from areas of squamous metaplasia are moderately cellular, yielding large, round to polygonal squamous epithelial cells that may be seen in sheets or individually. Nuclei are relatively small in comparison to the cell size (low nuclear-to-cytoplasmic ratio) and may be pyknotic. Lightly basophilic cytoplasm is abundant and may become folded or angular as the cells become keratinized. Anuclear superficial cells and keratin flakes may also be seen. Squamous metaplasia is difficult to differentiate from squamous neoplasia and is complicated by the fact that pulmonary SCC often originates from areas of squamous metaplasia. Neoplasia of the lung can be primary or metastatic. In dogs and cats, especially young animals, the lung is more often affected by metastatic neoplasia than by primary lung tumors although both are reported. Metastatic tumors are more likely to present as multiple nodules scattered throughout all lung lobes, particularly the periphery, whereas a solitary lesion is more typical of primary pulmonary neoplasia. Carcinomas are the most common neoplasia diagnosed in the lung although hemic lymphatic neoplasia and sarcomas are also seen. Carcinomas can arise from any level of the respiratory tract and multiple types of lung carcinomas have been identified in dogs and cats. While adenocarcinomas of bronchogenic or bronchio-alveolar origin predominate as primary lung carcinomas, numerous carcinomas also metastasize to the pulmonary parenchyma and cytologic differentiation of most carcinomas is not possible. Aspirates contain moderate numbers of epithelial cells in sheets, aggregates, and clusters with lesser numbers of individualized cells. Acinar formation indicates glandular origin suggesting an adenocarcinoma. Moderate to marked pleomorphism, anisocytosis, and anisokaryosis is commonly seen between clumps of cells as well as within cells of the same cluster. Nuclei tend to be round, are frequently eccentrically placed, and contain coarsely clumped chromatin and prominent, single to multiple nucleoli. The cytoplasm of carcinomas is deeply basophilic and often contains punctate vacuoles in the perinuclear region. Other criteria of malignancy that may be seen include nuclear molding, signet ring cell formation, cell or nuclear gigantism, and the presence of binucleate and multinucleate cells. Squamous cell carcinoma has distinguishing cytologic features. Cells often occur individually and less commonly in sheets or clusters. There will be moderate to marked variation in cell and nuclear size, nuclear-to-cytoplasmic ratios, amount of cytoplasm, and degree of keratinization. Individual cell morphology ranges from basal squamous cells with little or no keratinization to fully keratinized squamous cells. The basal cells are cuboidal to round with deeply basophilic cytoplasm, large central nuclei, coarse chromatin, and prominent nucleoli. Mature squamous cells are large with abundant homogenous cytoplasm and pyknotic or karyorrhectic nuclei. Dysynchrony of cytoplasmic and nuclear maturation is common in SCC. Hemolymphatic neoplasia can appear as diffuse infiltrative disease or as discrete nodules. Multiple diseases have been reported in both dogs and cats, some of the more common include lymphoma, malignant histiocytosis, and lymphomatoid granulomatosis. For lymphoma, airway samples may be more sensitive for detection than lung aspirates, however this may depend on the pattern of infiltration. Because bronchial associated lymphoid tissue is present within the normal lung, the presence of a monomorphic population of lymphoblasts cells is helpful to distinguish a reactive from a neoplastic population of lymphocytes. Histopathology with immunophenotyping may be helpful in establishing the malignant nature of the lymphoid population in cases where small cells predominate or a heterogeneous population is seen. Malignant histiocytic neoplasia commonly affects the lung of both dogs and cats. The lung is one of the primary sites of infiltration in malignant histiocytosis of both dogs and cats although less so in cats than in dogs. In dogs, histiocytic neoplasia in the dog has been defined as a spectrum of diseases characterized by proliferation of Langerhans dendritic cells, interstitial dendritic cells, or macrophages. The cell of origin in cat is unclear, however a recent report suggests that at least in some cases, malignant histiocytic neoplasia in the cat may originate in the skin. On cytology, malignant histiocytes are large, pleomorphic discrete cells that contain abundant, often vacuolated, deeply basophilic cytoplasm. Nuclei are oval to reniform and contain lacy chromatin and prominent nucleoli. A continuum between discrete histiocytic cells and spindled mesenchymal cells may be seen; the appearance frequently varies in masses from the same animal and may even vary from different sites of the same mass. Variable numbers of multinucleated cells are often seen. Cells may also exhibit phagocytosis of erythrocytes and leukocytes, which helps to suggest a histiocytic origin, however, phagocytosis is not a consistent feature and phagocytosis can be seen in other malignant cell types. Malignant histiocytosis can be cytologically (and sometimes histologically!) difficult to differentiate from granulomatous inflammation, anaplastic carcinomas, large cell T-cell lymphoma, pulmonary lymphomatoid granulomatosis, and plasmacytoma or extramedullary myeloma. Positive immunoreactivity to lysozyme can aid in this differentiation. Pulmonary lymphomatoid granulomatosis is an uncommon, but aggressively infiltrative, pleocellular T-cell lymphoid neoplasia of dogs. It is characterized by variable numbers of large pleomorphic mononuclear cells, which range from lymphoid to plasmacytoid to histiocytic in appearance; binucleate cells and mitoses are common and they are easily confused with malignant or reactive histiocytes. Neoplastic cells may actually compose the minority of the cell population present and are admixed with numerous small lymphocytes, eosinophils, and plasma cells. Peripheral basophilia and canine dirofilariasis have been inconsistently associated with lymphomatoid granulomatous.. Mesenchymal neoplasia in the lung is often metastatic. Tumors arising from the pulmonary connective tissue are relatively rare in dogs and cats. Tumors resembles those seen in the more common sites. Thymus Thymoma, lymphoma, and other primary and metastatic neoplasia may appear as anterior mediastinal masses. Thymoma is characterized by numerous small lymphocytes, a variable proportion of thymic epithelial cells (the neoplastic population), and occasional mast cells. By flow phenotyping, a significant proportion of the lymphocytes will be CD4+CD8+ double positive thymic precursors that helps to differentiate thymoma from lymphoid neoplasia. Mediastinal lymphoma appears similar to lymphoma at other sites. Liver When diffuse or generalized liver disease is suspected, aspiration of the left liver lobe can be performed by non-guided or "blind" techniques with the animal in right lateral recumbency or standing position. Ultrasound guided aspiration improves the sensitivity of aspiration when focal or multifocal lesions are identified. Normal hepatocytes are large polygonal cells primarily found in cohesive clumps. Round nuclei are surrounded by abundant lightly basophilic to acidophilic cytoplasm. Hepatocytes from older animals often contain lipofuscin pigment. Small numbers of binucleate cells are present in normal liver samples. Sheets of biliary epithelium may also be seen in liver aspirates. These tightly clumped cuboidal-shaped cells are smaller than hepatocytes with central round nuclei and scant basophilic cytoplasm. Hyperplasia is characterized by the presence of hepatocytes with mild to moderate cytologic atypia. Hyperplastic changes include anisokaryosis, binucleate cells, multiple nucleoli, and increased cytoplasmic basophilia. These morphologic findings can be associated with nodular hyperplasia, common in older animals, or as a response to hepatitis. Histologic examination of the hepatic architecture is required to differentiate neoplasia from hyperplasia when cytologic findings are equivocal. Cholestasis is defined by the presence of thick plugs of bile pigment in the canalicular space. Fine crystalline to clumps of dark bile pigment may also be retained in the hepatocyte cytoplasm during hepatobiliary disease but the pigment needs to be differentiated from lipofuscin. Hepatocellular vacuolation can be seen with lipidosis or glycogen deposition. Lipid appears as distinctly round clear vacuoles that push the nucleus to the periphery of the cell. An individual cell may have multiple small vacuoles or a single large vacuole. Corticosteroids, exogenous or endogenous, cause moderate to marked glycogen accumulation in the canine liver. Glycogen deposition results in a wispy appearance to the cytoplasm. Hepatitis. The presence of leukocytes suggests inflammation but should be differentiated from extramedullary hematopoiesis or hemic lymphoma. The presence of neutrophils, epithelioid macrophages, and/or eosinophils should prompt a search for organisms. The numbers of neutrophils needs to be considered in light of the number of circulating neutrophils as blood contamination is inevitable when obtaining a hepatic aspirate. Lymphocytes and plasma cells associated with inflammation will be well-differentiated and spread throughout the sample. Large clusters or atypical lymphocytes raise concern for lymphoma or plasmacytoma. Extramedullary hematopoiesis may be present in the liver although it is typically present in low numbers. Hematopoietic progenitors can be differentiated from lymphoblasts by the presence of erythroid or myeloid progression. Myeloid precursors have more abundant cytoplasm, indented nuclei, and cytoplasmic granules while erythroid progenitors often demonstrate hemoglobinization. Occasionally megakaryocytes may be seen and greatly assist in characterization of the cytologic findings as extramedullary hematopoiesis. Hepatocellular neoplasia can be difficult to diagnose cytologically and sometimes even histologically. The morphologic differences between nodular hyperplasia, adenoma, and well-differentiated hepatocellular carcinoma can be minimal. Well-differentiated hepatocytes are seen in all cases. Occasionally cells will have increased basophilia and increased numbers of binucleates may be seen. Poorly differentiated neoplasia is readily identified by the presence of multiple criteria of malignancy, however identification of the cells as hepatocellular origin can be difficult. Biliary neoplasia is often seen as very dense clusters of small epithelial cells. Cell characteristics are often difficult to identify due to the thickness of the clusters, but cells are smaller than hepatocytes with high nuclear to cytoplasm ratios and prominent nucleoli. Organization of cells into tubular or acinar structures may be seen especially if the edge of the clump is examined. Non-hepatic neoplasia and metastasis to the liver are common. Carcinomas, sarcomas, and discrete cell tumors may all be identified in cytologic aspirates. Metastatic carcinomas and sarcomas are more typically focal to multifocal and are best sampled by ultrasound aided techniques. In contrast, discrete cell proliferation often results in diffuse disease, hepatomegaly, and ready exfoliation. Spleen Similar to the liver, splenic aspirates may be obtained by blind aspiration or ultrasound guided techniques. Fine needle aspiration of a diffusely enlarged spleen can be obtained by pressing the spleen against the abdominal wall with the animal in lateral or dorsal recumbency. The potential for hemorrhage must always be considered, however aspirates are often obtained from animals, even those with thrombocytopenia, with minimal complications. Splenic aspirates often contain peripheral blood elements due to hemodilution of the sample. Normal spleen contains primarily blood and lymphoid cells. Occasionally, a few monomorphic spindled cells representing fibrocytes or endothelial cells are seen. Lymphocytes are evaluated similarly to those seen in lymph nodes. Most of the lymphoid cells are small lymphocytes with a lesser population of prolymphocytes and lymphoblasts. Occasional plasma cells, macrophages, and mast cells may also be present. Reactive hyperplasia is identified through the detection of increased numbers of prolymphocytes, lymphoblasts, and plasma cells. A marked plasmacytosis, resembling multiple myeloma, may be seen in canine Ehrlichiosis, leishmaniasis, and feline immunodeficiency infections. Extramedullary hematopoiesis (EMH) is commonly seen cytologically in splenic aspirates. Erythroid precursors and megakaryocytes are seen more frequently, with granulocytic EMH less common. Immature cells are easily confused with lymphoblasts and can lead to the erroneous diagnosis of lymphoma. Visualization of erythroid progression (including hemoglobinization) is helpful for differentiating erythroid and lymphoid precursors. Lymphoma is characterized by a monomorphic population of lymphoid cells. Typically, these are lymphoblasts, however occasionally prolymphocyte or small cell variants are seen which require histologic evaluation of splenic architecture. Extraskeletal multiple myeloma and plasmacytoma may be seen in both the spleen and the liver. These are characterized by a population of plasma cells. Well-differentiated plasma cells have eccentric nuclei, clumped "cartwheel" chromatin, deeply basophilic cytoplasm, and a paranuclear clear area. Binucleate cells and nuclei with less clumped chromatin may be seen. Poorly differentiated plasma cells need to be differentiated from amelanotic melanoma, osteosarcoma, and malignant histiocytosis. Myeloproliferative disease involving any of the cell lines or the stem cells may involve the spleen and must be differentiated from EMH. Generally, large numbers of a relatively monomorphic population of cells are seen in cases of myeloproliferative disease. Concurrent evaluation of peripheral blood and bone marrow including the use of special stains is often necessary to fully characterize the neoplastic process. Splenic mast cell tumors are common, particularly in cats. Primary splenic neoplasia such as hemangiosarcoma is difficult to diagnosis by aspiration cytology, but some of the more solid hemangiosarcomas exfoliate well resulting in large numbers of atypical mesenchymal cells. However most aspirates of hemangiosarcomas result in blood and only rare spindled cells. Gastrointestinal tract Scrapings of gastrointestinal tract lesions taken via endoscopy may be evaluated cytologically. Due to the nature of the sample taken, cells are more likely to represent surface lesions and not indicate deeper processes. Examination of ultrasound guided aspirates of mass lesions is often more rewarding. The expected cell type should be a monomorphic cuboidal to columnar epithelial cell with some variability in phenotype depending on the location within the GI tract. Inflammation is characterized by the type of inflammatory cells. Predominantly lymphocytic-plasmacytic or eosinophilic infiltrates can be identified from deep scrapings. Neutrophils indicate a suppurative response and may be seen with ulcerative lesions and pathogenic organisms. Bacterial and fungal organisms are easily identified cytologically; however bacteria need to be considered in light of the expected normal flora. For example, Helicobacter species have been associated with gastritis in dogs and cats and can be identified on cytologic examination of gastric samples, however presence alone does equal disease. Primary GI neoplasia is not common, however both epithelial and mesenchymal neoplasia of the GI tract can be seen in both dogs and cats and most are malignant. However diagnosis can be challenging because of difficulty in obtaining a quality sample, the fact that the normal epithelial and mesenchymal cells often have marked reactive responses to GI neoplasia, ulceration and associated inflammation is common, and the poor exfoliation of many of the tumor types. Evaluation of the draining lymph node for atypical cells may be more definitive than evaluation of the gastrointestinal mass itself. Epithelial neoplasia can be seen at all levels of the GI tract. Cells may appear in clusters and sheets, but may also present more individualized. Features of malignancy are common and cells often have granules or vacuoles suggesting a secretory nature. Neuroendocrine tumors (APUD tumors, carcinoids) are uncommonly seen in the GI tract. These tumors tend to exfoliate well, but the cells themselves are often fragile, therefore care must be taken when preparing slides from suspected neuroendocrine tumors. The cells tend to have a more 'bland' monomorphic appearance similar to other neuroendocrine tumors. The most common mesenchymal tumor of the GI tract is the GIST (GI stromal tumor). Previously, these were primarily diagnosed as leiomyosarcomas based on H&E, however more recent analysis using c-kit has resulted in a reclassification of these tumors. These arise from the interstitial cells of Cajal but are morphologically similar to leiomyoma/sarcoma having an elongated cigar shaped nucleus, thin spindled morphology, and scant amounts of cytoplasm. However, differentiation is important as GISTs are susceptible to treatment with c-kit inhibitors. Infiltrative neoplasia such as lymphoma or mast cell tumor are more easily identified than epithelial or mesenchymal proliferations. However, it should be noted that aggregates of lymphoid nodules are located in the small intestine, especially the ileum. Cytologically, they often appear as reactive lymphoid tissue and may confound the interpretation. Lymph node Thoracic and abdominal lymph nodes are evaluated similarly to peripheral lymph nodes. However, the mesenteric lymph nodes are antigenically stimulated by material from the lymphatics draining the gastrointestinal tract and, like the mandibular lymph node, are "normally" reactive. Examination of lymph nodes aspirates is also useful for diagnosing thoracic and abdominal lesions. Atypical cells representing metastatic neoplasia are often more easily identified and characterized within the lymph node environment than in the mass lesion itself where inflammation, necrosis, and reactive tissue hyperplasia can confuse the diagnosis. Lymph nodes draining inflammatory lesions also serve as a site for accumulation of organisms. Pancreas Ultrasound guided FNA of the pancreas is not commonly performed, however touch preps of biopsy material or FNA at surgery can readily provide diagnostic samples. Insulinoma is the most commonly diagnosed tumor of the pancreas in dogs and less commonly in cats. Cytologically, this endocrine beta islet cell tumor is characterized by the presence of cellular samples with many fragile cells and naked nuclei. While most tumors are malignant, the cells often only exhibit mild to moderate anisocytosis, anisokaryosis, and features of malignancy consistent with other neuroendocrine tumors. Nuclei are round with clumped chromatin and occasionally a single prominent nucleoli. The cytoplasm is pale to moderately basophilic and often contains large numbers of punctate vacuoles. Exocrine pancreatic neoplasia is often malignant. Cytologic examination of samples reveal clusters of cells that are moderately to markedly pleomorphic. These cells often have marked anisocytosis, anisokaryosis, nuclear molding, multiple nuclei, coarse chromatin, multiple prominent nucleoli, and deeply basophilic cytoplasm that often contains punctate perinuclear vacuolation. Pancreatic adenocarcinomas often metastasize to the local lymph nodes, liver, and abdominal effusions. Particularly in fluids, these cells often become individualized and take on a round appearance. Adrenal gland Adrenal neoplasia can arise from the cortical or medullary regions. In normal tissue, each region has different morphologic features that readily differentiate the cell types, however in neoplasia these differences may be blurred and the non-neoplastic population may become reactive. Aspirates typically obtain at least small numbers of both cell types which can further confound the diagnosis. Pheochromocytomas arise from the adrenal medullar. These are more common in dogs than in cats. Cells have a classic neuroendocrine appearance and naked nuclei are common. The cells often appear to be in sheets, although because the cytoplasm is frequently disrupted, the cells may have a more 'discrete' appearance. Nuclei have mild to moderate anisokaryosis, are round to oval in shape, with coarse chromatin and occasional nucleoli. The cytoplasm is grey blue to amphophilic in color and may have a granular appearance. Adrenocortical tumors can be either benign (adenoma) or malignant (adenocarcinoma). Both are more frequently seen in dogs than cats. The cytologic appearance depends to some degree on the degree of malignancy. Adenomas resemble normal adrenocortical cells which have abundant pale cytoplasm that often contains punctate vacuoles. Nuclei will be uniform in size, small, round to oval, granular to finely clumped chromatin, and prominent nucleoli. Adenocarcinomas frequently display numerous criteria of malignancy included moderate to marked anisocytosis and anisokaryosis, multiple nuclei, coarse chromatin, prominent multiple nucleoli, and more deeply basophilic cytoplasm. Cells with more malignant features may be less fragile. However, some adenocarcinomas are well-differentiated and the cytologic appearance may not represent the tumor's behavior. Kidney Aspiration of the renal parenchyma is indicated when diffuse disease is suspected. The kidney is a fibrous organ and only a few cells and moderate amounts of blood are aspirated from a non-diseased kidney. Therefore, a cellular specimen is meaningful. Normal renal epithelial cells are large, round to polyhedral in shape, with eccentric nuclei, and abundant light blue-gray cytoplasm. Feline renal epithelial cells often contain multiple clear lipid vacuoles. Inflammation is diagnosed by increased numbers of inflammatory cells. However, the presence of neutrophils in a renal aspirate must be evaluated concurrently with the number seen in the peripheral blood. Increased numbers indicates suppurative inflammation and should prompt a cytologic search for intracellular bacteria. Small lymphocytes may be markedly increased with chronic, non-suppurative inflammation. Occasionally plasma cells may also be present. Neoplasia may originate from the renal parenchyma or be infiltrative. Lymphoma is the most common infiltrative neoplasia and is characterized by a monomorphic population of lymphocytes, typically large lymphoblasts. Carcinomas and sarcomas can be diagnosed cytologically if a significant population of cells demonstrating morphologic atypia are present. Aspirates from renal neoplasia may contain characteristic tubular like structures. Bladder Samples from the bladder can be obtained by examining exfoliated cells in the urine sediment or through traumatic catheterization or ultrasound guided aspirates of mass lesions. Because urine has a low protein concentration, cells tend to spread out more than in other fluid preparations. Cystitis is characterized by increased numbers of neutrophils. Neutrophils in urine become very swollen with round karyolytic nuclei and can be confused with epithelial or mononuclear cells. The presence of pale cytoplasm and bacterial organisms are more consistent with neutrophils. Transitional epithelial cells which line the bladder and urethra surface are the most common origin of lower urinary tract tumors of the dog and cat. However, transitional cells readily undergo reactive changes and should be interpreted with caution with concurrent inflammation. Neoplastic cells exfoliate individually and in large clusters. Cells may appear round, oval, tadpole, or stellate in appearance. Marked pleomorphism is common with variation in cell and nuclear size, coarse nuclear chromatin, prominent large nucleoli, deeply basophilic cytoplasm, and eosinophilic cytoplasmic vacuoles. Occasionally these cells will undergo squamous differentiation and demonstrate keratinizing, lightly basophilic, foamy cytoplasm. Prostate The prostrate can be sampled by lavage, ejaculate, perirectal aspiration, or transabdominal aspiration. Normal prostatic cells are often found in clumps, have round nuclei, and acidophilic cytoplasm. Prostatic hyperplasia results in cells are that are mildly pleomorphic with basophilic cytoplasm and an increased nuclear to cytoplasm ratio. Prostatitis is characterized by the presence of neutrophils and occasionally macrophages. Bacterial organisms are often present but may be a contaminant from elsewhere in the urogenital tract. A search for intracellular organisms should be made to differentiate septic inflammation versus contamination. Epithelial hyperplasia is commonly induced secondary to inflammation. Squamous metaplasia occurs in the presence of endogenous or exogenous estrogen or in cases of chronic inflammation. The cells demonstrate typical keratinization changes ranging from basophilic basilar cells with large nuclei to large lightly basophilic angular keratinized cells with pyknotic nuclei. Prostatic neoplasia can originate from either prostatic or transitional epithelium. Transitional cell carcinoma appears similar to that seen in the bladder and often originates from the prostatic urethra. Cells from prostatic carcinoma are pleomorphic, deeply basophilic, with an increased nuclear to cytoplasm ratio, and may form acinar or tubular structures. Section III. Identification and Differentiation of Common Infectious Agents Bacteria Mycobacteria do not stain with routine cytologic stains and can be difficult to visualize. However, careful examination of the cells and background material reveals the presence of distinctive negatively stained thin rod present both intra- and extracellularly. The organisms can be confirmed by acid fast staining. Siamese cats appear to have increased susceptibility to mycobacteriosis. Mycobacteriosis often induces a granulomatous or pyogranulomatous inflammatory response rather than the suppurative response seen against most bacteria. Langhans' multinucleate giant cells and large epithelioid macrophages are often seen. Actinomycosis/Nocardiosis infection is often seen as subcutaneous masses or intrathoracic lesions due to penetrating wounds. The thin organisms have a characteristic branching beaded filamentous appearance that often appear in large mats that resemble amorphous debris. Clostridia can be identified as large gram positive rods (1 x 4um) that often contain a clear, oval spore at one pole of the bacterial. Clostridium is associated with cellulites, penetrating wounds, and GI overgrowth. Yersinia pestis is an uncommonly detected organism, the zoonotic potential of this organism makes identification of the organism crucial. Yersinia pestis is a gram-negative bacillus cytologically recognizable as bipolar coccobacilli present both intra- and extracellularly with large numbers of degenerate neutrophils. In cats, pneumonic plague occurs in about 10% of cases and can be seen with or without the classic bubonic presentation. Fungal Coccidioides immitis is a soil-borne dimorphic fungus, found mainly in the arid, acid-soil regions of the United States (Arizona and California) and in some regions of South America. In endemic areas, infected appears to be relatively common while development of clinical signs is relatively uncommon. It is primarily a respiratory pathogen and infection is by inhalation of spores. The incubation period for development of respiratory infection is 1-3 weeks after exposure. Disseminated disease occurs after primary lung infection, especially in dogs. Boxers and Doberman pinschers may be predisposed to disseminated disease. Until recently, cats were thought to be resistant to infection with Coccidioides, but both susceptibility to infection and development of clinical signs has been reported in endemic areas. In both dogs and cats with disseminated disease, lesions in long bones (especially the metaphyses area) and skin lesions are common. Coccidioidomycosis is induces a pyogranulomatous or granulomatous inflammation. Coccidioides immitis spherules (sporangium) are large organisms seen extracellularly. Spherules range in size from 10 to 100 µm in Romanowsky-stained preparations and contain a thick double-contoured wall with finely granular, blue-green protoplasm. Occasionally internal endospores of 2 to 5 µm may be seen. Organisms are scarce in cytologic preparations and multiple slides may need to be examined to find the organism. Due to the organism's large size, scanning is best done at low magnification (e.g., 10X). Mycelia may rarely be seen in tissue. Blastomyces dermatitidis is a soil-borne, dimorphic fungus. The mycelial phase occurs in nature and the yeast form in vivo, although hyphal stages may be seen (albeit rarely). It probably has worldwide distribution, although the number of cases reported outside of North America is relatively small. A second strain has been isolated from cases in Africa and probably represents a distinct serotype with geographic diversity. The endemic area in the United States includes the middle western, southeastern and Appalachian states. Blastomyces can infect numerous tissues, but the lung is the most frequently involved organ in primary infection. Infection tends to be via inhalation of spores. Although direct puncture and skin wounds can lead to cutaneous lesions. most cutaneous lesions are derived from pulmonary infection. In dogs, infection more often occurs in young, large-breeds. Blastomyces is uncommon in cats, however when seen in cats the disease tends to be systemic rather than localized and Siamese appear to be over-represented in the literature. The incubation period for Blastomycosis is relatively long and variable (weeks to months). The extracellular yeast forms are dark blue, round, and 5 to 20 µm in diameter, with a thick biconcave wall having a granular internal structure. Broad-based budding may be seen. The organisms are likely found in aggregates of mucus and necrotic debris and induce granulomatous or pyogranulomatous inflammation. Cryptococcus neoformans is the primary pathogenic species among the genus Cryptococcus however species have been identified to induce disease. C. neoformans a soil-borne, yeast-like fungus found worldwide. It multiplies to large numbers in the feces of pigeons and other birds where it can remain viable for months (note, it does not live in the intestinal tract of birds). Infection is via inhalation of spores. However both Cryptococcus neoformans and Cryptococcus gattii have been reported in the nasal passages of dogs and cats in the absence of local or systemic infection, and sub-clinical infection and/or asymptomatic carriers have been reported. Cryptococcosis is seen more frequently in cats than in dogs. The disease is manifested most commonly as nasal and frontal sinus infections, particularly in cats, that often extends to the meninges, brain and lungs. The characteristic tumor-like granulomatous lesions may also involve the skin and subcutis. Organisms are readily identified in swabs of nasal exudates or imprints/aspirates from nasal masses. New methylene blue and India ink can be used to demonstrate the negative staining capsule, however, care must be taken not to mistake air bubbles and fat droplets for organisms. Cryptococcus sp. are round to oval yeast that range in diameter from 8-40u (including the capsule). The organism has a granular internal structure that stains eosinophilic to purple and is surrounded by a thick, non-staining, mucoid capsule. The capsule material can give the sample a mucinous texture. Occasionally, narrow based budding may be seen. Unencapsulated or rough forms are 4-8µm and are difficult to distinguish from H. capsulatum. Fungal culture and serology are useful in this case. The presence and type of inflammation ranges from the observation of minimal to no inflammatory cells amid a field full of organisms to pyogranulomatous inflammation. The degree and type of inflammation may be related to characteristics of the capsule. The polysaccharide capsule inhibits phagocytosis and the capsular polysaccharide have been associated with the induction of suppressor T-cells. . Histoplasma capsulatum is a oil-borne, dimorphic fungus with world-wide distribution that infects both cats and dogs. It is seen more frequent in certain geographic regions, e.g., in the United States, primarily in the northeast, central and south-central states. The primary route of infection is through the respiratory tract, but systemic spread occurs quickly in most cases. The incubation period for histoplasmosis is relatively short (12-16 days). Infection of the reticuloendothelial system results in the formation of tubercle-like granulomas in infected tissues. Pulmonary disease is common in affected cats. In addition to systemic histoplasmosis seen in both species, a self-limiting syndrome of pulmonary histoplasmosis is also seen in dogs. The small yeast-like organisms are round to oval and 1 to 4 µm in diameter with a purple nucleus and lightly basophilic protoplasm surrounded by a thin, clear halo. Organisms are seen within macrophages and neutrophils as well as extracellularly. A narrow-necked budding may be seen. Histoplasma induces a mixed to pyogranulomatous reaction. Sporothrix schenckii is a dimorphic fungus found worldwide. It typically enters via cutaneous wounds of the limbs and induces suppurate nodules in the skin, subcutis, superficial lymphatics and lymph nodes. Dissemination to internal organs occurs occasionally, particularly in the dog. Localized lesions are sometimes seen on the face of cats. Sporothrix has also been isolated in the nasal cavity of cats with sporotrichosis and is more commonly detected in those with cutaneous lesions. The organisms are similar in size and morphology to Histoplasmosis. Round to oval to cigar-shaped 2 X 10-µm organisms with a thin, clear halo, slightly eccentric purple nucleus, lightly basophilic cytoplasm, and thin clear halo are observed both within macrophages and extracellularly. The presence of cigar-shaped organisms differentiates Sporotrichosis from Histoplasmosis. In cats, organisms are often abundant. However in dogs, organisms are rarely seen and identification may require searching over several slides. Aspergillosis and penicilliosis can occur as focal or disseminated infections in dogs and cats. While the respiratory tract is a primary target of infection, both fungi are frequent contaminants of the respiratory tract, therefore diagnosis should be supported by a combination of culture, cytological or histological identification of the organism, and the presence of an inflammatory reaction. Both fungi are morphologically similar, necessitating culture for differentiation. German Shepherd dogs are frequently afflicted with systemic aspergillosis. While aspergillosis often appears as an opportunistic infection, there are reported cases without obvious predisposing factors. Infection with Aspergillus sp. can be associated with purulent, granulomatous, or pyogranulomatous inflammation. Cytologically, fungal hyphae are branching, septate, 5-7µ in width, with straight parallel walls and globose terminal ends. Hyphae can stain either intensely basophilic with a thin clear outer cell wall or appear as negatively staining images against a cellular background. Hyphae may be difficult to identify when found in low numbers or in dense mats admixed with mucus, inflammatory cells, and cellular debris. Occasionally, round to ovoid blue-green fungal spores may also be observed. Rhinosporidium seeberi can infect the nasal cavity of dogs resulting in single to multiple polyps in which numerous small, miliary sporangia can be observed on the surface. Infection appears to be related to contact with water and/or trauma to the nasal mucous membranes. Cytologically, preparations contain variable numbers of magenta staining spores that range in diameter from 5 to 15µm with slightly refractile capsules and contain numerous, round, eosinophilic structures (spherules). In some cases, the spores stain deeply eosinophilic, preventing visualization of the internal structures. Sporangia are variably sized (30-300µ), well defined, globoid structures that undergo sporulation to contain numerous small, round endospores. Sporangia are not commonly observed in stained smears because they are only present in low numbers and the wall of the sporangia are slightly refractile and do not stain well. Three different maturation stages of endospores can be seen cytologically. Mature endospores predominate in cytologic preparations. These structures are 8-15 µm in diameter, with a thick, hyalinized cell wall and a pale, magenta to non-staining halo. Internal structure can be difficult to visualize in thick areas of the prep, but when endospores are spread out, numerous small spherical eosinophilic globular internal structures can be seen. Immature endospores are approximately 2-4 µm in diameter with lightly basophilic cytoplasm, a pink-purple nucleus encompassing 1/3 to 1/2 the endospore, and 1-2 smaller round magenta structures. Intermediate endospores are rarely seen spherical, granular, basophilic structures approximately 5-8 µm in diameter with eosinophilic to globular internal structures and a variably sized, clear halo. Rhinosporidiosis incites a mixed inflammatory response consisting of neutrophils, plasma cells, and lymphocytes as well as lesser numbers of . macrophages, mast cells, and eosinophils . Rosetting of inflammatory cells, particularly neutrophils, around the spores may be seen. Phaeohyphomycosis is an opportunistic infection of pigmented (dematiaceous) fungi such as Alternia, Curvularia, and Bipolaris spp that commonly reside in soil or water. The morphology of the fungi varies with the specific organism and typically requires culture for final diagnosis. Hyphal structures are typically septate and may produce globose structures that can be confused with yeast forms. Algae/Mold Protothecosis is an unpigmented algae most often found in sewage contaminated water. Infection is seen more often in animals that are immunocompromised due to therapy or other diseases. In dogs, both a cutaneous and systemic forms of the disease have been recognized, while in cats the disease tends to remain cutaneous. In dogs, the organism is often detected in scrapings from the rectum or colon. The organism can be found extracellularly or occasionally within macrophages and ranges from 5-10 um in diameter and have a round to oval shape. Internal septation results in the production of variable numbers (2-20) of basophilic endospores that contain granular internal material, a dark nuclei, and a clear halo. Prototheca induces a chronic pyogranulomatous inflammatory response. Pythiosis is a water mold. Within the United States, the organism is found primarily within the southeastern states. Animals become infected by drinking or cutaneous exposure to contaminated water. GI exposure frequently leads to systemic dissemination while cutaneous exposure may result in localized dermal nodules and draining tracts. Cytologically, the organism is recognized by the presence of broad, poorly septate and branching hyphae. Pythiosis induces pyogranulomatous inflammation that often includes eosinophils. Protozoa Toxoplasma gondii infects virtually all mammals and is of importance to both human and veterinary health. T. gondii is a member of the protozoan phylum Apicomplexa, which includes the Plasmodium spp., Cryptosporidium and Eimeria spp. The life cycle of toxoplasmosis includes two phases. The intestinal phase occurs only in cats and produces oocysts. The extraintestinal phase occurs in all infected animals (including cats) and produces tachyzoites and then bradyzoites. Toxoplasmosis can be transmitted by ingestion of oocysts (in cat feces) or bradyzoites (in raw or undercooked meat). Tachyzoites of Toxoplasma gondii look similar to the tachyzoites of Neospora. Tachyzoites are 1 to 4 µm, crescent-shaped bodies with lightly basophilic cytoplasm and a central metachromatic nucleus. Organisms may be found intracellularly (primarily within macrophages) and extracellularly. Additional diagnostics are needed to differentiate Toxoplasma and Neospora. Neospora caninum infection is most commonly detected in animals less than 1 year of age or immunocompromised animals and results in progressive, frequently fatal, ascending paralysis. The disease in older animals is diverse but characterized by systemic involvement, including marked pulmonary infiltration with associated pneumonia. On cytology, tachyzoites are indistinguishable from those of Toxoplasma gondii and are 1 to 5 µm by 5 to 7 µm, oval to crescent-shaped structures with a central metachromatic nucleus and lightly basophilic cytoplasm. They may be found intracellularly and extracellularly and induce a mixed inflammatory response. Leishmania is a protozoa that is more commonly detected in dogs than cats. At least two species of Leishmania have been detected in animals in the United States. The amistigotes are most commonly seen on cytologic preparations and have a characteristic appearance. These are primarily intracellular organisms but may be seen extracellularly if the cell ruptures. Amistigotes are 1-5 um in size, contain a magenta nucleus and a bar shaped kinetoplast placed perpendicularly to the nucleus. Skin, bone marrow, and lymph nodes are common sites of involvement. Pneumocystis carinii is a protozoal organism that is most commonly reported in young dogs, primarily miniature dachshunds and Cavalier King Charles spaniels. Several immune defects have been identified that may predispose some dogs to with Pneumocystis carinii including hypogammaglobulinemia, decreased lymphocyte proliferation, and reduced numbers of B lymphocytes. Infection results in a diffuse interstitial pneumonia. The abundant foamy fluid present in the alveoli often contains trophozoite and cyst forms. Trophozoites are pleomorphic in morphology, ranging from 2-7 um in length with pale blue cytoplasm and dark punctate nuclei. Trophozoites can be seen within macrophages or extracellularly. Cysts are less commonly seen and are extracellular, 5-10µ in diameter and contain 4-8 round, 1- to 2-µm basophilic bodies (sporozoites) arrange din a circumferential cluster. Section IV. Correlation (or lack thereof) Between Cytologic Morphology, Flow Phenotype, and Receptor Rearrangement in Lymphoid Neoplasia Diagnosis by lymphocyte morphology Assessment of neoplastic lymphocytes has traditionally relied on morphologic characterization either by cytology or histopathology. However, using the morphology revealed by light microscopic examination to make a cell type diagnoses is not always straightforward. Lymphocyte morphology is typically divided by cell size, nuclear maturation, and cytoplasmic features into the categories below. Small lymphocytes are smaller in size than a neutrophil and have a round nuclei that takes up the majority of the cell. The nuclei contain densely aggregated chromatin forming large chromocenters (condensed chromatin). Nucleoli are not seen. The cytoplasm is scant (sometimes only a very thin rim is visible) and lightly basophilic in color. These are typically called 'mature lymphocytes'. However, early lymphoid progenitor cells, hematopoietic stem cells, certain stages and types of dendritic cells, and other immature precursor cells may have a very similar morphology to 'mature, well-differentiated, small, resting lymphocytes'. Intermediate to large lymphocytes range in size from slightly larger than small lymphocytes to the size of neutrophils. The nuclei still takes up the majority of the cell, however more abundant cytoplasm is visible in these cells. Often, the nuclei is placed eccentrically within the cytoplasm. The nuclear chromatin is finely clumped to granular. Typically, nucleoli are not seen although strands of loosely clumped nuclear chromatin may be mistaken for nucleoli. The cytoplasm is lightly basophilic in color. Occasionally these cells contain azurophilic granules suggestive of a natural killer (NK) phenotype. Lymphoblasts are as large as a neutrophil or larger. Size alone does not indicate neoplasia. Very large lymphoblasts (2-4x the size of neutrophils) may be seen in reactive and hyperplastic processes. Lymphoblasts contain round to oval nuclei with fine or stippled chromatin (loosely aggregated chromatin). One or more nucleoli may be visible. The cytoplasm is moderately to deeply basophilic. Occasionally (seen more in cats than dogs) the cytoplasm may contain punctate vacuoles. Reactive lymphocytes are similar in morphology to small lymphocytes but are slightly larger and have more abundant, more basophilic cytoplasm. Plasma cells are intermediate sized cells that contain small, round, eccentrically placed nuclei with condensed chromatin. Cytoplasm is abundant, deeply basophilic, and often contains a prominent, eccentric, perinuclear, clear zone that corresponds to the Golgi. "Diagnosis by typical findings" Most maturation charts show lymphocyte development as starting at the lymphoblast stage. Cells then become progressively smaller with a more condensed chromatin pattern as they mature. Thus lymphoblasts become intermediate cells which transition into small 'mature' lymphocytes. However more detailed immunologic analysis into the phenotype and structure of activated, resting, memory, effector, regulatory, and precursor lymphocytes suggests substantial overlap in morphologic features between these categories. Morphologic features and a knowledge of 'typical findings' has been used by both clinical and anatomic pathologists to help characterize underlying cell type and the disease process. Some of these are described below. Lymphoglandular bodies are round, homogeneous, basophilic structures comprised of cytoplasmic fragments. The presence of lymphoglandular bodies is seen in cytologic preparations of lymphoid tissue that contains increased numbers of lymphoblasts. This can be due to neoplasia (lymphoma) or hyperplasia. Golgi clearing zone. The presence of an eccentric, perinuclear clearing zone is often suggested as a feature of B-cells and plasma cells. The clearing zone is the Golgi and it is a prominent feature in plasma cells. However, the Golgi apparatus is an organelle found in most cells, including T-cells and myeloid cells. Sezary cells are described as medium to large lymphocytes with ceribriform nuclei. In humans, these neoplastic T-cells are characteristic features of Sezary syndrome which encompasses mycosis fungoides, an epitheliotropic variant of cutaneous lymphoma. A similar syndrome occurs in dogs but has been rarely reported in cats. In dogs, epitheliotropic T-cell lymphoma is also seen in the gastrointestinal tract. T-cells predominate in both the cutaneous and GI variants. Interestingly, expression of protein gene product 9.5 (PGP 9.5), a marker previously considered specific for neural and neuroendocrine tissues, was recently detected in over 8/14 cases of canine cutaneous mycosis fungoides suggesting that there may be other biologic differences between the human and canine variants. In humans, the presence of flower cells or cloverleaf cells is most often associated with T-cell disease and is particularly a feature of infection with human T-lymphotrophic virus-1 (HTLV-1). In dogs and cats, similar morphology has been seen in both B-cell and T-cell lymphoproliferative disease as well as myeloproliferative disease. Chronic lymphocytic leukemia (CLL). Unlike that seen in humans where CLL is considered a disease of B-cells, CLL of dogs and cats is primarily a T-cell disease. In dogs, CD8+ (cytotoxic) CLL predominate while in cats, CD4+ (T-helper) CLL is more common. However, there is variation in the disease in both dogs and cats and B-cell, CD4+ T-cell, and CD8+ T-cell CLL have all been diagnosed in small animals. In dogs, a chronic lymphocytosis comprised of intermediate sized lymphocytes with small azurophilic granules has been reported in association with Ehrlichiosis. Diagnosis by flow cytometry One of the goals of flow cytometric analysis is to define cells through a panel of phenotypic markers and receptors (usually surface) and thereby provide a more objective characterization of these abnormal lymphocyte populations. Lymphocyte phenotyping by flow cytometric analysis has become an established diagnostic assay for assessment of abnormal hematopoietic populations in small animal patients. In both dogs and cats, a number of well-characterized antibodies are available for evaluation of lymphoid populations with fewer antibodies available for examination of histiocytic, myeloid, erythroid, and megakaryocytic cells. Antibodies Commonly Used for Flow Cytometric Analysis of Canine and Feline Hematpoietic Neoplasia Stem cell markers CD34. CD34 is a glycosylated surface protein expressed on non-committed (stem) hematopoietic progenitor cells. CD34 expression does not differentiate myeloid versus lymphoid origin but can be used to help differentiate acute lymphoid leukemia (ALL) and acute myeloid leukemia (AML) from more differentiated neoplasia such as chronic lymphocytic leukemia (CLL), the leukemic phase of lymphoma, or chronic myelogenous leukemia (CML). CD117. CD117 is a transmembrane tyrosine kinase growth factor receptor expressed on some stem cells and mast cells. CD172. CD172 or SIRP1-alpha is found on bone marrow progenitor cells. It recently was found to be expressed on early B-cells and expression appears to be loss with the rearrangement of Ig heavy chain genes. Hematopoietic markers CD45. CD45 is a pan-hematopoietic marker with several isoforms and is found on all hematopoietic cells (including TVT) except erythrocytes. It is expressed on lymphoid as well as myeloid precursors and mature cells. CD45 intensity coupled with side scatter can be used to subgate cells. Mature lymphocytes have the brightest expression of CD45 while normal lymphoblasts have much dimmer expression and neoplastic lymphocytes can lose CD45 expression entirely. B cell markers B-cells express a variety of markers as they mature. Pre-B-cells, which are defined as the stage of maturation before heavy and light chain antigen receptor rearrangement, express CD79a and IgM in the cytoplasm. As cytoplasmic IgM is exported to the surface, pre-B-cells transition into immature B-cells. CD21 and surface IgM are first found on immature, naive B-cells as they enter circulation. Following antigen stimulation, B-cells will mature and express surface IgG. CD79a. CD79a is the transmembrane signal transduction portion of the B cell receptor. It is found on all stages of B cells. However, the commercially available antibody for use in dogs detects the intracellular portion limiting its utility in flow cytometry (cells need to be permeabilized for detection). CD79a also has aberrant binding patterns which are best seen by immunohistochemistry or immunocytochemistry. Nuclear staining is a common artifact and does not indicate B-cell origin. Visualization of cytoplasmic positivity is necessary to confirm B-cell lineage. CD21. CD21 is one of the complement receptors. With surface Ig, CD79a, CD79b, CD19, and CD35, it complexes with the B-cell antigen receptor for signaling. CD21 is expressed on mature B cells , however it may also be expressed on follicular dendritic cells in the lymph node germinal center. Surface Ig. Surface IgM is found on immature B-cells while surface IgG is found on mature B cells. CD1c. There are five CD1 genes (A-E). CD1 molecules are related to MHC class I and present lipid, glycolipd, and some peptide antigens. CD1 molecules are often thought of as dendritic cell markers, however CD1 molecules are expressed on a number of hematopoietic cells. CD1c may be expressed on subpopulations of B-cells. See also CD172. T-cell markers Like B-cells, T-cells also express a variety of markers as they mature. Cells from the thymus may give rise to thymocytes, several subsets of T-cells, natural killer (NK) cells, and dendritic cells. Early T-cell progenitors in the thymus (thymocytes) express CD3, CD2, CD5, CD7, and CD1a. T-cell precursors undergo rearrangement of the T-cell receptor (TCR) and begin to express both CD4 and CD8 (double positive cells). As the cells mature further, expression of only CD4 or CD8 is maintained (single positive cells). CD3. CD3 is a complex transmembrane protein expressed on early thymocytes and maintained throughout lymphocyte maturation. Therefore, CD3 is the most useful and consistent marker for detecting T cells. However some antibodies used in flow cytometry detect part of the surface chains of CD3, while those used in IHC/ICC detect the intracellular portion of the CD3 chains. This can lead to discrepancies in results if the neoplastic cells do not maintain the CD3/TCR complex on the cell surface. CD3 is closely associated with the TCR and is critical for cellular activation. The TCR exists as two forms, an alpha/beta TCR or a gamma/delta TCR. Detection of CD3 does not distinguish whether cells are expressing alpha/beta or gamma/delta TCRs. In the dog, monoclonal antibodies to differentiate alpha/beta and gamma/delta TCR are available. CD5. Although CD5 is typically considered a T-cell marker, CD5 is expressed on mature T-cells, thymocytes, and a subset of B cells. CD5 helps to modulate signaling through both the TCR and B-cell receptor complex by acting as a co-stimulatory molecules. CD5 is useful in humans, because the subpopulation of B-cells that express CD5 are frequently the same cells that develop into CLL. However, in dogs and cats, CLL is a T-cell disease and the majority of cells (both normal and neoplastic) found in blood, bone marrow, spleen, and lymph nodes that express CD5 are T-cells. We have found several cases where only CD3 or CD5 is expressed on the surface of neoplastic T-cell populations submitted for flow cytometric analysis and routinely run both antibodies (in the same tube as CD21) to detect the overall cell phenotype. CD1a. See also description of CD1c under B-cells. CD1a is expressed on cortical thymocytes but is lost as the cells mature. Thy1. Thy1 or CD90 was first described as a thymic antigen. Expressed by most lymphocytes, but is also expressed by monocytes and macrophages. CD4. CD4 is expressed on helper T-cells of dogs and cats. However, CD4 expression is also seen on canine and feline neutrophils (depending on the antibody clone used) which is very different from that seen in people. The CD4 seen on neutrophils is very bright while that detected on lymphocytes is intermediate to bright. CD4 expression can also be upregulated on antigen presenting cells (monocytes, macrophages, dendritic cells). In these cases, expression tends to be dim to intermediate. CD8. CD8 is expressed on cytotoxic and suppressor T-cells. The CD8 molecule is comprised of two chains. Alpha/beta heterodimers predominate in both dogs and cats, however alpha/alpha homodimers may also be detected in both species and may expand in certain disease. A subset of NK cells also expresses CD8 (alpha/alpha). CD4CD8 double positive cells. The presence of double positive cells is uncommon in dogs and cats although small numbers may be seen with certain inflammatory/infectious diseases (e.g. FIV of cats is the best described). Large numbers of double positive cells suggests expansion of thymic intermediates, such as would be seen with a thymoma. NK markers CD56 and CD57. There are no definitive markers of NK cells in dogs. In cats, antibodies against CD56 and CD57 can identify NK cells, although CD57 needs to be used in conjunction with CD5 and CD8 to differentiate CD8 subsets. Myelo-monocytic markers CD11/CD18. CD18 complexes with one of four different alpha subunits to form the beta2 integrins, one type of leukocytes adhesion molecules. CD18 is found on most leukocytes, however the CD11 subunits are specific to different cell types. CD11a/CD18 is found on all leukocytes. CD11b/CD18 is found on granulocytes, monocytes, and some macrophages. CD11c/CD18 is found on granulocytes, monocytes, and dendritic cells. CD11d/CD18 is found on macrophages, splenic red pulp T-cells, and large granular lymphocytes. CD14. CD14 labels monocytes and macrophages. Not all neoplastic histiocytes label with CD14. Mac387. Mac387 is an anti-human macrophage antibody that reacts in dogs and cats. It stains macrophages and monocytes, but also labels canine neutrophils. Myeloperoxidase (MPO). Cross reactive antibodies for canine (and perhaps feline) MPO have recently been identified. In dogs, MPO labels neutrophils but also a small proportion of monocytes. Mast cells See CD117. Other MHC II is expressed on monocytes and macrophages and other APC as expected. However, MHC II is also expressed on many canine and feline lymphocytes, especially activated lymphocytes. Diagnosis based on clonality There are no specific phenotypic markers that identify a clonal T-cell population in humans or veterinary species. In humans, the use of kappa and lambda light chain markers can help to identify clonal B-cell populations when the populations uniformly express one or the other light chain. However, there are currently no antibodies for detection of canine or feline light chains by flow cytometry. In addition, lambda light chains predominate over kappa chains in dogs, reducing the utility of light chain detection. Therefore, identification of T-cell or B-cell clonality in dogs and cats requires detection of receptor gene rearrangement. This is most typically done by PCR and is referred to as PCR for antigen rearrangement (PARR). As part of their development, T-cells undergo rearrangement of genes encoding the T-cell receptor (TCR) while B-cells undergo rearrangement of genes encoding the immunoglobulin (Ig) receptor. The result is that nearly every lymphocyte in the body has a unique TCR or Ig receptor. PARR of normal tissue detects a smear or ladder of PCR products representing the diversity of the normal receptors. Because neoplastic transformation typically occurs after the cells have undergone receptor rearrangement, all malignant daughter cells will have the same antigen receptor gene. This is detected on PCR as a single band and represents a monoclonal population. Occasionally bi or tri-clonal populations may also be detected. CLL Canine CLL. In dogs, CLL appears to be primarily a T-cell disease although B-cell CLL has also been reported (3:1 ratio of T:B). The cytologic morphology of T-cell CLL in dogs is typically of granular lymphocytes. The granular cells primarily express CD3, CD8, and CD11d. Detection of the alpha/beta TCR is more common although about a third of the reported cases express the gamma/delta TCR. Non-granular T-cell CLL more commonly express the alpha/beta TCR but may be either CD4 or CD8 positive. B-cell CLL in dogs express CD21 and/or CD79a. The majority (~95% of those examined), also express CD1c. CD5 (which is commonly seen in human B-cell CLL) is not detected on canine B-cell CLL. B-cell CLL and T-cell CLL appear to have somewhat different patterns of disease progression. B-cell CLL affects the bone marrow early in disease and may be considered a primary bone marrow disease. T-cell CLL typically does not affect the bone marrow until late in the disease and may spread from the marrow after splenic involvement. Feline CLL. Similar to that seen in dogs, feline CLL is primarily a T-cell disease. However, unlike that seen in dogs, feline CLL is primarily a result of CD4 or helper T-cell proliferation although occasional cases of CD8+, CD4CD8 double positive, and CD4CD8 double negative CLL have also been reported. Acute leukemia While several markers are useful for the diagnosis of AML in people, AML in dogs and cats is difficult to diagnose through flow cytometry immunophenotyping as the available antibody panels do not provide lineage specific myeloid or myelomonocytic markers. Combinations of antibodies, coupled with flow scatter patterns can be used to help characterize myeloid precursors, no currently available panel of antibodies can consistently identify myelomonocytic leukemia or AML. The best marker for AML is myeloperoxidase. Currently, this is detected by most labs through cytochemistry on cytology or histology slides. Acute leukemia in the dog appears slightly more likely to be of myeloid than lymphoid origin, and about 10% of acute leukemias lack identifiable differentiation markers (acute undifferentiated leukemia). Acute lymphoid leukemia in the dog may be comprised of cells of B, T, or NK origin. When examined, CD34 is often detected on AML and ALL, but is rarely (or not) detected on lymphoma and therefore serves as a useful marker to differentiate ALL with tissue involvement from lymphoma with marked leukemia. |
