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Cytopathology of Neoplasia

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CYTOPATHOLOGY OF NEOPLASIA
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CYTOPATHOLOGY of NEOPLASIA Mary Anna Thrall, DVM, MS, DACVP Colorado State University Ross University Introduction Cytology is the examination of individual cells without regard to the architectural structure of the tissue. Although cytologic biopsy does not always replace excisional biopsy and histopathologic examination, it can provide a rapid, inexpensive aid in establishing a diagnosis and is superior to histopathology when examining some tissues, such as bone marrow and lymph nodes. Examples of common sources of cytologic specimens include cutaneous and subcutaneous masses, body cavity fluids, lymph nodes, liver, spleen, pancreas, prostate gland, bone marrow, conjunctiva, respiratory tract, ear swabs, vaginal and rectal mucosa, and urine. The body fluid most commonly examined cytologically is venous blood. Many of the cells seen in other fluids and tissues are those commonly seen in blood. This discussion will emphasize cytopathology of neoplasia and is directed toward anatomic pathologists who have an interest in cytopathology. Stain Most veterinary cytopathologists prefer using Romanowsky type hematologic stains such as Wrights stain, Wrights-Giemsa, Diff-Quik, and other modifications Sample acquisition Cells may spontaneously exfoliate, as they do in body cavities or inflammatory exudates, or they may be mechanically removed by techniques such as aspirating, scraping or washing. Fluids Complete fluid analysis should include a total nucleated cell count, protein content estimation, and cytology, and color, clarity and odor should be recorded. If the cell concentration is low (<5 to 10,000cells/µl), the cells should be concentrated by centrifugation. If the fluid consists of predominantly blood, 1
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Page 1: Cytopathology of Neoplasia

CYTOPATHOLOGY of NEOPLASIAMary Anna Thrall, DVM, MS, DACVP

Colorado State University Ross University

IntroductionCytology is the examination of individual cells without regard to the architectural

structure of the tissue. Although cytologic biopsy does not always replace excisional biopsy and histopathologic examination, it can provide a rapid, inexpensive aid in establishing a diagnosis and is superior to histopathology when examining some tissues, such as bone marrow and lymph nodes. Examples of common sources of cytologic specimens include cutaneous and subcutaneous masses, body cavity fluids, lymph nodes, liver, spleen, pancreas, prostate gland, bone marrow, conjunctiva, respiratory tract, ear swabs, vaginal and rectal mucosa, and urine. The body fluid most commonly examined cytologically is venous blood. Many of the cells seen in other fluids and tissues are those commonly seen in blood. This discussion will emphasize cytopathology of neoplasia and is directed toward anatomic pathologists who have an interest in cytopathology.

StainMost veterinary cytopathologists prefer using Romanowsky type hematologic stains such

as Wrights stain, Wrights-Giemsa, Diff-Quik, and other modifications

Sample acquisition Cells may spontaneously exfoliate, as they do in body cavities or inflammatory exudates,

or they may be mechanically removed by techniques such as aspirating, scraping or washing.

Fluids Complete fluid analysis should include a total nucleated cell count, protein content

estimation, and cytology, and color, clarity and odor should be recorded. If the cell concentration is low (<5 to 10,000cells/µl), the cells should be concentrated by centrifugation. If the fluid consists of predominantly blood, a packed cell volume or red blood cell count should be performed. When blood is present, buffy coat preparations may be useful for detecting neoplastic cells.

Aspirates. Aspiration biopsies are useful for obtaining cells from masses, including lymph nodes

and internal organs, and are performed using a 21 to 25 gauge needle, and a 12 ml syringe. Skin is prepared as for a venipuncture, unless a body cavity or joint is being invaded, in which case a surgical preparation should be performed. The mass is stabilized with one hand to aid in penetration of the mass and the needle is directed by the other hand. When the mass is penetrated, negative pressure is applied to the syringe. One may redirect the needle and sample various portions of large masses while maintaining negative pressure. The negative pressure should be released prior to removing the needle from the tissue. The needle is removed from the syringe and air is aspirated into the syringe; the contents are then expelled onto the middle of a glass slide. Alternatively, excellent aspirates can be obtained using the capillary method described below. The needle is not attached to a syringe, but is guided by holding the hub of the

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needle; the vigorous back-and-forth motion of the needle and the capillary pressure are adequate to detach cells without applying negative pressure. The needle is then attached to a syringe containing air, and the contents of the needle are expelled onto a slide as described above. Advantages of this method include less hemodilution of sample and increased cellularity. If enough material is present, several preparations should be made. Films are made by placing a spreader slide on the surface of the drop of material, allowing the material to spread from the weight of the top slide, then gently sliding the two slides apart. The sample is allowed to dry in the air, then stained.

Scrapings. Scrapings are occasionally useful to obtain cells from firm surfaces. Examples include

conjunctival scrapings, or scrapings of firm cutaneous lesions. Scalpel blades or spatulas (commercially available for this purpose) may be used. Cells procured are then gently spread on a glass slide. This method commonly results in numerous broken cells, but areas of intact cells can usually be found.

Swabs. Swabs may be used to obtain cells from mucosal surfaces, such as the vagina, rectum, or

nose. After sample collection, the swab is gently rolled along the surface of a microscope slide.

Imprints. Imprints can be made of superficial cutaneous lesions or from tissues removed at surgery

or necropsy. Imprints of superficial lesions may be diagnostic, but often only reflect surface inflammation and contamination. When taking imprints of other tissues, the freshly cut surface is blotted with absorbent paper such as paper towel to remove excess blood and tissue fluid. Gently touch a glass microscope slide to the surface of the tissue, taking care not to smear the tissue on the slide, as the shearing forces will rupture cell and nuclear membranes.

Disadvantages of CytologyMany of the specimens are nondiagnostic, and may take longer to examine. In addition,

many slides may be submitted from one aspiration. (We have considered charging by weight!) Cytology specimens of non-diagnostic quality may be caused by poor technique, but some are due to inherent problems with cytologic sampling. The latter include preparations that contain few cells because the tissue is difficult to sample, samples that consist of only blood because the lesion is vascular, samples in which the cells obtained are not necessarily representative of the lesion, tissue types in which it is difficult to distinguish normal from neoplastic, and samples in which the architecture of the tissue, rather than cell type, is critical for the diagnosis.

Poor techniqueProblems related to poor technique include samples that are of low cellularity due to

having missed the lesion, samples that are too thick, preparations in which all of the cells are broken, samples that consist of only blood due to the large needle size, slides with formalin fume artifact, samples that are over a week old when stained, slides that have had the cells removed when they were wiped to clean the oil, and samples in which the cells are not representative of the lesion, often because an ulcerated tumor is imprinted rather than aspirated,. Cytology can be

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successful only if cells are present on the slide, are one cell layer thick, are not broken, and are adequately stained.

Low Cellularity Slides may contain few or no nucleated cells because the target tissue was missed, the

tissue was difficult to sample, or the sample was made up of fat cells that disappeared in the alcohol during the staining process. It is inherently difficult to obtain cells from mesenchymal (connective) tissue. Some mesenchymal tumors may be of relatively low cellularity due to the presence of a large amount of matrix that is produced by the connective tissue and surrounds the cells. The more benign the connective tissue tumor, the more matrix and fewer cells it will have. Aspirates of lipomas usually contain very few cells, and what few cells are aspirated may wash off when dipped into alcohol, the first step in most of the rapid stains. One should examine the slide prior to staining, and make a note if the slide appears greasy or oily. Aspirated subcutaneous fat appears very similar to fat aspirated from a lipoma. Finally, it is common to aspirate perinodal fat when attempting to aspirate lymph nodes, especially popliteal lymph nodes.

Blood contaminationBlood contamination commonly occurs if the needle used for aspirating is larger than 20

gauge. If platelets are present in the sample, one may assume that the blood present represents contamination. Aspirates of some abdominal organs, such as spleen and liver, tend to contain a large amount of blood. If macrophages are present that have phagocytized erythrocytes or contain hemosiderin, a red blood cell breakdown product composed of iron, previous hemorrhage has likely occurred in the lesion. For example, hematomas usually contain large numbers of macrophages that are filled with erythrocytes. Hemangiosarcomas, a type of mesenchymal tumor, are filled with blood, and may yield few to no cells when aspirated. While erythrocyte morphology is usually insignificant in cytologic preparations, an exception is the presence of acanthocytes in aspirates from hemangiosarcomas. This red blood cell shape change is very typically seen in the blood of patients with hemangiosarcoma. Finally, imprints of excised material must first be blotted with absorbent paper prior to their being imprinted on the glass slide, or the imprint will consist of only blood or tissue fluid.

Cells obtained are not related to the primary lesionInflammatory cells may surround a tumor, and sometimes only the inflammatory cells are

aspirated. One should redirect the needle in lesions so as to obtain cells from various parts of the lesion. This is particularly true of enlarged lymph nodes in which metastatic neoplasia is suspected. Lesions that are usually diffuse, such as lymphoma, are easier to sample than are focal lesions, from which cells may not be obtained. A common example of obtaining cells that do not typify the primary lesion is cytologic preparations of nasal discharge. Typically only neutrophils are present because nasal tumors do not tend to exfoliate cells into the discharge. The nasal mass itself usually must be aspirated or imprinted. If a cutaneous or subcutaneous mass is ulcerated, imprints of the ulcerated surface usually yield only neutrophils and bacteria, rather than cells from the underlying mass. Attempts to aspirate submandibular lymph nodes often result in obtaining only normal salivary gland epithelial cells, which should not be confused with a carcinoma. Another example of the presence of confusing epithelial cells is putative thoracic aspirates that contain liver cells.

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Misleading cytologic appearance of the tissueTransitional epithelial cells almost always appear malignant, even when normal, because

they replicate rapidly and thus exhibit some of the features that are considered criteria of malignancy. Rapidly dividing fibroblasts in an inflammatory (usually pyogranulomatous or granulomatous) lesion may have many of the features common to malignant cells. Mesothelial cells in body cavity effusions are easily mistaken for carcinoma cells. Epithelioid macrophages in inflammatory lesions can appear very similar to epithelial cells. Nasal carcinomas, on the other hand, often appear quite benign, as the cells are often small and uniform. Neoplasms of endocrine tissue, such as thyroid or perirectal apocrine gland adenocarcinomas (which behave like endocrine tumors, producing parathyroid hormone-related protein) also often appear quite benign, even though their behavior is usually malignant (1). Osteoblasts from well-differentiated osteosarcomas may appear similar to osteoblasts from normal or reactive bone.

Preparation is too thickCytology is successful only when cells are one layer thick and not broken. Some tissues,

such as lymph nodes, bone marrow and some tumors, yield many cells and thus the preparation may be inherently thick. Usually in these situations one can go to the edge of the smear and find areas that are adequately thin enough to examine. Other causes of thick preparations are "squirt" preparations, in which the cells are squirted onto the slide from the syringe, then not spread thinly.

Sample is inadequately stainedInadequate staining may simply be a result of not dipping for long enough in the stain, or

not changing the stain often enough. Stain should be changed weekly, not only because it eventually stains less intensely, but because many of the quick Wrights-Giemsa type stains, such as Diff-Quik and Quick-Dip, support the growth of bacteria and fungi. These contaminants may be mistaken for pathogens when examining the preparation. Other reasons for inadequate staining include samples that are too thick, contamination of the sample with formalin fumes, and not staining the sample within a week of collection. Formalin fume contamination is a common problem because samples are often prepared next to the formalin jar or samples are shipped in the same box as bottles containing formalin. Cells that have been fixed by formalin fumes will not take up the stain and appear as light blue structures without any nuclear detail. Stain precipitate is usually not a problem when "dip" type stains are used but can sometimes form in aged stain. Stain precipitate usually appears as clumps of purple dots and may be mistaken for bacteria.

Cells are too degenerate or broken to identifyDegenerating cells may be unavoidable when necrotic tissue is aspirated. More

commonly, cells are broken during the preparation process. Certain types of cells such as lymphoblasts are quite fragile. Cells must be distributed on the slide quite gently by placing one slide over the slide with the aspirated cells, allowing the material to spread out, then pulling the apart in a gentle manner. If these types of preparations are made like blood films, using a push type approach, cells will often break. Cells also break as a result of being incorporated in clots. Aspirated material should be placed on a slide and spread out within approximately 30 seconds of collection. If this cannot be accomplished, one can place a few drops of EDTA from a purple

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top vacutainer tube into the syringe into which the cytology sample is collected. Broken nuclear material usually streams across the slide, and may be mistaken for fibrin or spindle-shaped cells. Other causes of degenerate cells include the environment to which they are exposed. For example, cells from airway washings come into contact with saline, and are often degenerate, as are cells in urine. Aging of a sample prior to slide preparation will result in degeneration of cells. If the aspirated material is of a fluid nature, films should be made within 30 minutes of collection to prevent degeneration of cells. Finally, cells can be broken or even removed after staining and examination of the slide by attempting to wipe the immersion oil from the slide. Instead, a solvent such as "Americlear" or "Safe Clear" should be used to remove oil.

Lack of ArchitectureA definitive diagnosis of tumor type is often not possible. Types of sarcomas and

carcinomas cannot usually be differentiated. In addition, tumors cannot be graded and margins cannot be evaluated.

Advantages of cytology over histopathologyBecause of the lack of cell shrinkage, cellular detail is better in cytologic samples. This

is of particular advantage when examining hematopoietic cells and round discrete cells, such as lymphoid cells. Mast cell granules stain much better with Romanowsky (Wright’s type) stains, and microorganisms can be easily seen and identified.

Logical and systematic approach to the specimen Preparations should be examined initially using a low power objective (10x). At this

magnification one can evaluate cell density, observe different cell types that are present and thus recognize multiple processes, such as inflammation and neoplasia. Perhaps most importantly, while examining at low power, one can find a suitable area to examine at higher magnification. Unlike blood films, cytologic preparations do not have one area to examine that is preferable over another. It is necessary to scan the entire preparation in order to not miss diagnostic cells, and also to find an area that is not too thick, is stained adequately, and contains intact cells. Then using the 50x or 100x oil objective, one can positively identify various cell types, examine cellular detail such as the presence of nucleoli and search for small microorganisms. The ability to recognize the appearance of normal cells is important. Examples include the presence of normal hepatocytes in thoracic fluid aspirates when the liver is inadvertently aspirated, normal salivary gland epithelial cells when one attempts to aspirate submandibular lymph node and salivary gland is mistakenly aspirated, and normal cells from various organs such as urinary bladder, liver, spleen, pancreas, etc. It is also important to recognize artifacts, such as talc crystals, stain sediment, and ultrasound gel. The examination is then followed by a complete description and interpretation of the preparation The description should contain information regarding the degree of cellularity, the predominant cell type, and the morphologic appearance of various cells and other structures, including microorganisms.

An interpretation of these findings is then provided. Interpretations usually include whether the preparation is indicative of inflammation, and if so, what type of inflammation is present, if microorganisms are present, and whether the sample is indicative of neoplasia, and if so what type of neoplasia is present. The diagnosis of neoplasia is based on the presence of cells with malignant characteristics or the presence of cells that are foreign to the tissue source. For example, epithelial cells should not be in a lymph node aspirate. If the cell morphology suggests

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that the specimen is neoplastic, the tumor can usually be classified as a discrete (round) cell tumor, a tumor of epithelial origin, or a tumor of connective tissue origin. Neoplasms, especially squamous cell carcinoma, may induce an inflammatory response, so that the interpretation may be both inflammatory and neoplastic.

Criteria of malignancy. Nuclear criteria. Nuclear changes in malignant cells are a reflection of increased nuclear activity or

replication. Some nuclear criteria are found in cells undergoing hyperplasia, or controlled growth (for example, fibroblasts in granulation tissue). Common nuclear criteria include variable nuclear size (anisokaryosis), variable and usually increased nucleus to cytoplasm ratio, abnormally clumped chromatin patterns, and large, multiple, and irregularly shaped nucleoli. Other nuclear changes sometimes observed include abnormal mitoses, micronuclei, and nuclear molding, which is the presence of nuclei in multinucleated cells in which one nucleus conforms to the shape of another nucleus. While multinucleation and normal-appearing mitotic figures are commonly observed in neoplastic cells, they are also observed in normal cells such as fibroblasts, macrophages and mesothelial cells.

Cytoplasmic criteriaCytoplasmic criteria of malignancy are less important than nuclear criteria and include

increased basophilia and vacuolation. Both of these features are more commonly seen in tumors of epithelial origin. Vacuoles in tumor cells are sometimes simply a reflection of rapid growth and degeneration. In adenocarcinoma cells, however, characteristic perinuclear vacuolation representative of the secretory and packaging role of the cell is commonly observed. When large secretory droplets compress the nucleus to the side of the cell, the cell is termed a "signet ring" cell. Normal cells that imbibe fluids can also have this appearance. Neoplastic cells are sometimes phagocytic or cannibalistic, and will phagocytize erythrocytes or other tumor cells.

Structural criteria Certain types of neoplastic cells attain a very large size. Neoplastic epithelial cells will

sometimes replicate without dividing, resulting in long chains of attached cells. Epithelial cells have intercellular connections and large clumps of connected cells can often be observed. In aspirates of neoplastic glandular epithelium, acini can sometimes be seen. Cellular crowding (piling) is a very common feature of aspirates of malignant epithelial tissue.

Classification of neoplasms Discrete round cell tumors. Cells from round cell tumors are small, discrete (individualized) and round. Lymphoma,

plasma cell tumors, mast cell tumors, histiocytomas, transmissible venereal tumors, and malignant histiocytosis are typically classified as discrete cell tumors.

Lymphoma Lymphoma can be found in virtually any organ (2-4). While neoplastic lymphoid tissue

may consist of either small lymphocytes or large lymphoblasts, cells are more commonly lymphoblastic. When lymphoblastic, the cytologic diagnosis is often relatively simple. Lymphoblasts are the predominant cell type and are approximately twice the size of neutrophils,

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with scant cytoplasm that is light to medium blue and is occasionally vacuolated. Nuclei are usually indented with fine to stippled chromatin and generally contain one to three prominent nucleoli. Types of lymphoblastic lymphoma can be further subdivided according to cellular morphology, histopathologic appearance, and whether the lymphoblasts are of B or T cell origin. The diagnosis of lymphoma in tissues other than lymph nodes is usually quite simple because lymphoblasts should not be present in large numbers in the skin, bone marrow, liver, intestine, or other non-lymphoid organs. Lymphoblasts are fragile and are commonly broken during preparation. Cytoplasmic fragments are often abundant and are sometimes referred to as lymphoglandular bodies.

If neoplastic lymphoid cells are small they usually have abnormal features such as retained nucleoli or increased cytoplasm, often with projections or pseudopodia. Polymerase chain reaction (PCR) or histopathologic examination may be required to confirm the diagnosis of small cell lymphoma (5). Because clonality is the hallmark of malignancy, an assay has been developed that uses PCR to amplify the variable regions of immunoglobulin genes and T-cell receptor genes in order to detect the presence of a clonal lymphocyte population. Gene rearrangement is appropriate for the immunophenotype (immunoglobulin gene rearrangement in B-cell lymphoma, and T-cell receptor gene rearrangement in T-cell lymphoma). The clonal rearrangement can be detected when very small amounts of the DNA are derived from neoplastic cells. Because all lymphomas are clonal expansions of lymphocytes, each particular neoplasm contains DNA regions that are unique in both length and sequence. The CDR3 region of both immunoglobulin and T-cell receptor (TCR) genes encodes the antigen-binding region of the respective receptor and contains the majority of this unique sequence. While it is assumed that malignancy is always clonal, all clonal expansions of lymphocytes are not necessarily malignancies, and it is important to consider this possibility if samples have clonal rearrangements but no other evidence of lymphoma: Aspirates of lymph nodes (or other organs) can be submitted in approximately 1 ml of physiologic saline. Two to three aspirates, with rinsing the syringe in the saline each time, provides adequate material. Alternatively, stained or unstained cytologic preparations can be scraped and PCR performed on the scraped cells. Samples may be sent to Colorado State University Diagnostic Laboratory, Attention: Dr. Anne Avery, Fort Collins CO 80523.

Plasma cell neoplasiaPlasmacytomas are solitary plasma cell tumors that may occur subcutaneously

and in the oral cavity. These tumors consist of plasma cells that may be well-differentiated or somewhat immature or poorly differentiated. They have small nuclei with stippled chromatin, abundant basophilic cytoplasm and often a clear perinuclear golgi area. Numerous bi- or tri-nucleated plasma cells are often present. Poorly differentiated plasma cell tumors may appear similar to malignant histiocytosis cells or amelanotic melanoma cells. Plasma cell myeloma (multiple myeloma), on the other hand, is a relatively rare lymphoproliferative neoplasm in which plasma cells or their precursors proliferate abnormally. As implied by the term "multiple myeloma", neoplastic plasma cells proliferate in the bone marrow at multiple sites, and are also commonly present in the spleen and liver; they are rarely within lymph nodes. While markedly increased plasma cell concentration in the bone marrow (greater than 20 percent of all nucleated cells) is usually due to plasma cell neoplasia, plasma cell

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proliferation may also occur secondary to chronic antigenic stimulation. Neoplastic plasma cells within the bone marrow are often seen in large aggregates, and sometimes appear slightly abnormal or immature, with occasional multinucleated plasma cells present. However, they may appear very well differentiated and are then difficult to distinguish from normal plasma cells. An important diagnostic and clinical manifestation of plasma cell myeloma is the presence of a monoclonal or biclonal gammopathy, usually IgG or IgA but occasionally IgM.

Histiocytic neoplasmsHistiocytomas

While histiocytomas are typically considered tumors of young dogs, they are also seen in middle aged and old dogs. They are benign in behavior and appearance and usually regress spontaneously. The cells have a moderate amount of pale blue cytoplasm, and round nuclei with fine chromatin and indistinct nucleoli and are epithelial dendritic cells (Epidermal Langerhans cells). As the tumors spontaneously regress, they are infiltrated with small lymphocytes that may be in higher concentration than the histiocytoma cells. The background fluid in aspirates is often darker than the cytoplasm of the cells, giving the cytoplasm a pale appearance.

Histiocytic SarcomasHistiocytic sarcomas may be localized or systemic. Systemic histiocytic sarcoma is

usually referred to as malignant histiocytosis. Malignant histiocytosis cells may be derived from interstitial dendritic cells or from macrophages (25). If they are derived from macrophages, they are usually very phagocytic, and accompanied by extramedullary hematopoiesis. Malignant histiocytosis (systemic histiocytic sarcoma) is a rapidly progressive, ultimately fatal, proliferative disorder of the mononuclear phagocyte system that has been described in adult dogs, including Bernese mountain dogs and other breeds, and is relatively rare in cats. An increased incidence of the disorder has been suggested in the golden retriever and flat coated retriever breeds (6). Malignant histiocytosis is often characterized by the systemic proliferation of large, pleomorphic, single and multinucleated histiocytes with marked cellular atypia and phagocytosis of erythrocytes and leukocytes. The spleen, liver, and marrow are almost always involved. Lung, lymph nodes, and central nervous system may also be involved. Positive reactivity of neoplastic cells to histiocytic markers, such as lysozyme and alpha-1-antitrypsin, can be demonstrated by immunohistochemistry, which aids differentiation of neoplastic histiocytic cells from lymphoid and epithelial neoplasms, and is helpful in making the definitive diagnosis of the neoplasm. Non-phagocytic systemic histiocytic sarcoma cells are derived from interstitial dendritic cells and thus originate in lymphoid organs such as spleen and lymph nodes. Dendritic cells and macrophages express cell surface molecules that serve as markers to identify their origin (25).

Neoplastic histiocytes are pleomorphic, large, discrete, markedly atypical mononuclear cells; nuclei are round to oval or reniform. Features of malignancy include marked anisocytosis and anisokaryosis, prominent nucleoli, bizarre mitotic figures, and abundant, lightly basophilic, often vacuolated cytoplasm. The presence of multinucleated giant cells also supports the diagnosis. The cells are thought to arise from dendritic antigen-presenting cells. Aspirates of malignant histiocytosis tumors can appear somewhat similar to malignant fibrous histiocytomas. They are usually quite easy to differentiate from aspirates of granulomatous inflammatory lesions.

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Mast Cell Tumors Mast cell tumors consist predominantly of mast cells, which are round cells that are

usually slightly larger than lymphoblasts. They have variable numbers of distinctive small, purple staining granules within the cytoplasm that may obscure the nucleus. The nucleus is round to oval and usually stains somewhat pale, presumably because the granules take up the stain. The cells are somewhat fragile and free granules are usually present. Eosinophils are often present in aspirates of mast cell tumors. Some mast cell tumors have large numbers of mesenchymal cells scattered throughout the aspirate, and pink amorphous material, probably degenerated collagen, may also be seen. The granules in a small percentage of mast cell tumors do not stain with some of the quick stains such as Diff-Quik. Mast cell neoplasms diagnosed by cytology should be widely excised, submitted for histopathology and graded to help ascertain a prognosis.

Transmissible Venereal Tumors Transmissible venereal tumors (TVTs) are sexually transmitted tumors of dogs. They are

often malignant in behavior, but respond well to chemotherapy and radiation therapy. They consist of discrete cells with a moderate amount of light blue cytoplasm that is sometimes filled with numerous small distinct clear vacuoles. The nuclei are round with coarse chromatin and large prominent nucleoli. They are usually found on mucous membranes such as the penis, vagina, and nose but can be found in many other areas to which they have metastasized.

Tumors of Epithelial Origin Neoplastic epithelial cells are usually much larger and have more cytoplasm than discrete

tumor cells and they typically exhibit many of the criteria of malignancy discussed above. Epithelial cells tend to adhere to each other and thus clusters or clumps of cells are usually seen in aspirates. Cell to cell relationships are usually apparent. Nuclear chromatin may be fine to coarse, and nuclei contain one or more nucleoli that may be quite large and prominent when cells are malignant. Nuclear molding may be present in multinucleated cells. Various types of epithelial tumors such as thyroid carcinomas, basal cell tumors, perianal gland adenomas, squamous cell carcinomas, and sebaceous cell adenomas have specific characteristics that aid in identification.

Basal Cell TumorsBasal cell tumors are relative common tumors in dogs and cats. They are similar to other

epithelial cell tumors, but the cells are small and quite uniform and may be mistaken for those of a round cell tumor because the cells may appear as individual cells with a high nucleus to cytoplasm ratio. However, they typically appear in rows and clusters. The nucleus is small with somewhat dense chromatin and a small indistinct nucleolus. Cytoplasm is scant and lightly basophilic to gray. Basal epithelial cells may be pigmented (contain melanin), and can be confused with melanomas, although malignant melanoma cells almost always exhibit more features of malignancy. Some basal cells may be seen that are differentiating into sebaceous cells (see sebaceous adenoma).

Sebaceous Cell tumors

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Sebaceous cell adenomas are the most common benign skin tumor in dogs, especially in cocker spaniels and poodles, and they also occur in cats. These neoplasms are often on the head and neck and may have a wart-like appearance on gross examination. They are benign lesions. Sebaceous cells are characterized by the presence of cells with numerous small relatively uniform clear vacuoles within the abundant cytoplasm. The nucleus is usually small, round and dark. These cells can be distinguished from macrophages by the lack of phagocytic activity and the uniform size and shape of the vacuoles. Basal epithelial cells may also be present. Sebaceous cell carcinomas are uncommon. These cells exhibit criteria of malignancy including variability in nuclear and cell size and large nucleoli. They can sometimes be confused with liposarcoma cells due to the numerous clear vacuoles within the cytoplasm. This distinction can generally be made by the fact that carcinoma cells are usually in groups and adherent to each other.

Squamous cell carcinomasSquamous cell carcinomas are common tumors of older domestic animals that may occur

on the skin, oral cavity, stomach, respiratory tract and reproductive tract. They can usually be identified by the presence of very large superficial squamous epithelial cells, often keratinized with retained nuclei. Epithelial cells exhibit criteria of malignancy including the presence of nuclei and large nucleoli. Well-differentiated squamous cell carcinomas may be difficult to differentiate from benign lesions that contain superficial epithelial cells and keratin, such as epithelial inclusion cysts and cornifying epitheliomas. However, cysts and epitheliomas do not contain epithelial cells that have large nuclei or nucleoli. Aspirates from squamous cell carcinomas often contain inflammatory cells, possibly because the keratin elicits an inflammatory reaction.

Perianal gland tumorsCells from perianal gland adenomas and adenocarcinomas have a very characteristic

hepatoid appearance, in that the cells closely resemble hepatocytes. The cytoplasm is abundant, and light blue; nuclei are centrally located, round and uniform and usually contain one distinct nucleolus. Basal epithelial cells may be present. Carcinomas are difficult to distinguish from adenomas based on cytology. However, if a large amount of cellular variability is present and the nucleus to cytoplasm ratio is smaller than usual, a carcinoma should be suspected.

Apocrine gland adenocarcinomas of anal sacs Cells from perirectal apocrine gland adenocarcinomas exfoliate easily but the cells are

usually broken, and therefore many nuclei are present with no visible cytoplasm. Typically, some cells will be in clusters, indicating that the cells are epithelial in origin. Intact cells have a small nucleus to cytoplasm ratio and the small amount of cytoplasm is usually light blue in color. Nuclei are uniform, round to oval, and the chromatin pattern is usually fine with small nucleoli present. Although the cells appear relatively benign on cytology, their behavior is very malignant and many of these tumors have metastasized by the time of initial diagnosis. Many of these patients are hypercalcemic, as the tumors produce parathyroid hormone-related protein (PTH-rp).

Mammary gland tumors

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Aspirates of mammary gland tumors are very variable in appearance. Neoplasia can usually be differentiated from inflammation or hyperplasia. However, benign and malignant tumors can be difficult to differentiate unless cellular criteria of malignancy are obvious (7,8). As with other carcinomas, criteria of malignancy include cellular piling, and variability in cell size and shape, nuclear size, and large prominent nucleoli. Adenocarcinoma cells may have secretory product within the cytoplasm.

Thyroid gland tumors Aspirates of thyroid gland tumors, usually located in the ventral cervical region, are

similar to aspirates of other endocrine tissue (1). They usually contain few to moderate numbers of relatively small uniform appearing epithelial cells, many of which are broken . Numerous free nuclei are usually present and the aspirates often contain many erythrocytes. The cytoplasm of some cells may contain blue-black granules, thought to be tyrosine. The nuclei are usually centrally located with clumped chromatin. Criteria of malignancy are often not present, even in carcinomas, making adenomas difficult to distinguish from carcinoma. Tumors with more aggressive cytologic features are characterized by some of the above observations but coupled with moderate to marked cellular and nuclear variability, multinucleation, high nuclear to cytoplasm ratio and cellular piling. Most thyroid tumors in dogs are carcinomas, and almost all thyroid tumors in cats are adenomas. Parathyroid gland tumors and C cell tumors of the thyroid gland are similar in appearance to thyroid gland tumors.

Neuroendocrine tumorsNeuroendocrine tumors are similar in appearance to thyroid tumors. They typically

consist of large numbers of broken cells, similar to thyroid tumors and apocrine gland adenocarcinomas of anal sacs. The intact cells are usually individualized and rarely form aggregates or clumps of cells, and are derived from neuroectoderm. Intact cells have central round to oval nuclei of uniform size, light chromatin, with usually a single nucleolus and lightly basophilic cytoplasm. Neuroendocrine tumors include carotid body tumors (chemodectomas) that are near the angle of the jaw, aortic body tumors (heart base tumors), and pheochromocytomas, which are found in the adrenal glands. Brachycephalic breeds tend to have more of these tumors than do other breeds.

Tumors of Connective Tissue Origin (Sarcomas). Spindle-cell tumors are similar in appearance to normal connective tissue cells, but

exhibit variable criteria of malignancy. The cytoplasm is light blue to gray or lightly eosinophilic, and follows the contour of the nucleus and is elongated, oval shaped or streams at both ends to form tail-like points. Sometimes the cells are plump with an angular, pointed cellular end. Nuclear chromatin arrangement and number of nucleoli vary considerably. More cellular tumors tend to have multiple nuclei, multiple nucleoli and varying shapes and sizes of nuclei and nucleoli. Multinucleation is a characteristic of more aggressive tumors. More malignant, less well-differentiated cells are usually less spindled in appearance. Cells usually appear individually rather than in clusters but may be very close together, making them difficult to distinguish from epithelial tumors. Cells have indistinct cytoplasmic borders, pale blue cytoplasm that may contain a few vacuoles, and large oval nuclei with prominent, often multiple nucleoli. Tumors of mesenchymal origin may be difficult to definitively diagnose as to tissue origin. Osteosarcomas usually have a distinctive appearance in that the cells appear similar to

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giant plasma cells, with an eccentric nucleus appearing to protrude from one end of the cell. The cytoplasm usually contains small eosinophilic granules. Background pink-colored matrix is typically present. Chondrosarcomas and myxosarcomas usually have abundant background matrix (glycosaminoglycans). Liposarcomas have a distinctive appearance, in that the cytoplasm contains multiple small fat vacuoles. Cells from lipomas appear identical to normal fat cells as described above. Other spindle cell tumors include fibrosarcomas, hemangiosarcomas, hemangiopericytomas, neurofibromas (peripheral nerve sheath tumors), leiomyosarcomas, rhabdomyosarcomas and vaccine-induced sarcomas. Selected connective tissue tumors are described in more detail below.

FibrosarcomasFibrosarcomas produce cellular preparations consisting of numerous spindle cells with

moderate to marked cellular and nuclear variability, including multinucleation. Eosinophilic to purple granules may be seen in a few cells. A small amount of extracellular amorphous pink to eosinophilic material that resembles osteoid and interpreted to be collagen is usually present.

Malignant fibrous histiocytomasMalignant fibrous histiocytomas (MFH) have a more distinctive appearance cytologically

in that they consist of multinucleated giant cells in addition to neoplastic appearing spindled cells. When the cells from MFH lesion appear more round than spindled, the aspirates can resemble those from malignant histiocytosis.

HemangiosarcomasAspirates of hemangiosarcomas often yield only peripheral blood. Cells are spindle

shaped but often quite plump with multiple criteria of malignancy. Nuclei usually contain prominent, sometimes multiple nucleoli and a few cells may be multinucleated. Cytoplasm is light to moderately basophilic and sometimes vacuolated.

OsteosarcomasOsteoblasts are similar in appearance to plasma cells but are larger, with the nucleus

appearing to protrude from one end of the cell. The cytoplasm usually contains small eosinophilic to magenta-colored granules. Multinucleated cells are commonly present. Background pink-colored matrix (osteoid) is typically present. Cells vary in shape from oval to spindle-shaped.

Chondrosarcomas and myxosarcomasAspirates of these tumors contain abundant background matrix (glycosaminoglycans) that

is light blue to eosinophilic and sometimes stippled, similar to background glycoasminoglycans in joint fluid. The cellularity is usually low and the cells may not spread into a monolayer due to the glycosaminoglycan that adheres to the cells. This material may cause the cells to align in rows as they do in other viscous fluids.

Synovial cell sarcomasThese appear similar to other mesenchymal tumors but the cellularity is usually quite

high and criteria of malignancy can be variable. They are usually distinguished by their location

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and the presence of glycoasminoglycans in the background. Well differentiated synovial cell sarcomas can be difficult to distinguish from synovial hyperplasia.

LiposarcomasLiposarcomas have a distinctive appearance in that the cytoplasm contains multiple small

fat vacuoles and the nuclei are much larger than those of adipocytes. They exhibit criteria of malignancy, and can sometimes appear similar to sebaceous cell carcinomas.

Lipomas Lipomas consist of adipocytes that appear as very large clear round cells with very small

dense nuclei. They often appear in clusters and may wash off in the alcohol fixative. Numerous free fat droplets may be present. Aspirates of subcutaneous fat and perinodal fat can appear identical to aspirates of lipomas.

Vaccine induced sarcomas These lesions, located at typical vaccine sites in cats, are initially inflammatory,

and appear similar to other pyogranulomatous inflammatory lesions. However, the distinctive appearance of vaccine adjuvant, which is seen within macrophages as well as between cells, is very suggestive of a vaccine-induced lesion. This material is blue to magenta in color and is quite amorphous, similar to ultrasound gel. As these lesions transform into sarcomas, more mesenchymal cells appear, and eventually these cells exhibit many criteria of malignancy including very large cell, nuclear, and nucleolar size. Numerous multinucleated giant cells may be present.

Hemangiopericytomas and nerve sheath tumorsAspirates of these common tumors of dogs are typically very high in cellularity and

consist of distinctly spindle shaped cells with long “tails” of cytoplasm that extend on both ends of the cells. They may appear singly or in clusters or whorls. Nuclei are usually oval to elongated, with prominent nucleoli. Cytoplasm is lightly basophilic and may contain small discrete clear vacuoles. These are commonly found on the limbs of dogs and have a tendency to recur following excision.

RhabdomyosarcomasAspirates of these tumors tend to exhibit spindle cells in rows. Only rarely can cells be

seen in which the cross striations are still visible, whereas aspiration of normal skeletal muscle will yield muscle fibers in which cross striations are very apparent.

MelanomasMelanomas are classified as mesenchymal tumors but their appearance is distinctive in

that some cells appear similar to epithelial cells, some are spindled, and yet others appear to be round and discrete. Malignant melanocytes typically have large single “owl’s eye” nucleoli within the nuclei. If the cytoplasm of the cells contains melanin granules (usually fine gray dust-like particles), they are readily diagnosable. However, many are non-pigmented (amelanotic).

Neoplasia of body cavity effusionsNeoplastic effusions may be modified transudates or inflammatory exudates and are

characterized by the presence of neoplastic carcinoma or lymphoma cells, since sarcomas do not

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readily exfoliate cells. The total protein is variable, generally ranging from 3 to 4.5 g/dl, and the cell concentration is also variable, usually ranging from 3000/µl to 30,000/µl. Fluid that has accumulated secondary to a carcinoma may also contain variable numbers of inflammatory cells. Some degree of expertise is required to differentiate carcinoma cells from mesothelial cells. Mesothelial cells line the body cavities, and when fluid accumulates, these cells tend to proliferate and exfoliate into the fluid where they may continue to replicate. They may appear singly or in clusters of 2, 4, 8, or 16 cells. They are large (12 - 30 µ), have light to dark basophilic cytoplasm and have single or multiple round to oval nuclei with one or more nucleoli. Cells in mitosis may be seen. The cytoplasmic border may appear to have a pink "fringe" around it. Lymphoma is usually less difficult to cytologically diagnose than is carcinoma, as the tumors typically shed large numbers of lymphoblasts that are not easily confused with other types of cells.

Neoplasia of lower respiratory tractCytopathology of the lower respiratory tractNeoplasia of the lower respiratory tract can occasionally be diagnosed by tracheal

washings or bronchoalveolar lavage, but direct aspiration of lung lesions previously identified by imaging are typically much more rewarding. The cytologic appearance of the various types of neoplastic cells is as described above. Lung tumors that may be diagnosed by cytopathology include lymphoma, malignant histiocytosis, pulmonary adenocarcinoma, squamous cell carcinoma, metastatic mammary carcinoma, osteosarcoma and other types of metastatic sarcoma. Of these, carcinomas are the most common. If epithelial cells have cilia on their surface, it is very unlikely that they are malignant. Malignant histiocytosis is sometimes confused with anaplastic carcinoma because both tumor types may have multinucleated giant cells and abundant cytoplasm. However, the anaplastic carcinomas typically have cells that adhere to each other. Hyperplasia of respiratory epithelium is uncommon in dogs and therefore the diagnosis of neoplasia is usually apparent. However, hyperplasia of type II pneumocytes is a common response to interstitial pneumonia in cats, making the distinction between hyperplasia and neoplasia more difficult.

Neoplasia of intra-abdominal organsCytologic material that has been aspirated from enlarged abdominal organs and

abnormal masses within the abdominal cavity is commonly used as a diagnostic aid. While aspiration can often be accomplished by palpation, ultrasonography and magnetic resonance imaging are extremely helpful aids in localizing specific organs and masses. Samples obtained by ultrasonography may contain ultrasound gel on the slide, which is amorphous and stains a dark magenta color. This material can be so abundant on the slide that it interferes with evaluation. Therefore alcohol, rather than ultrasound gel, is recommended when obtaining samples for cytology. If this is not possible it is important to keep the biopsy site and fingers free of gel.

The general principles of cytology apply to the interpretation when examining material from any abdominal mass or organ (9). One is usually attempting to determine if the tissue is normal, and if not, if inflammation or neoplasia is present. One additional complication to the interpretation of aspirates from the abdominal cavity is that normal structures, usually of epithelial origin, may be aspirated. These normal epithelial cells

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must then be distinguished from neoplastic cells. The cytologic appearance of cells and classification of neoplasms are the same as those discussed above. Some specific types of intra-abdominal neoplasms are discussed briefly below.

Liver Hepatomegaly or liver areas of differing echogenicity are the primary reasons for liver

aspiration (10-12). Other indications include the suspicion of neoplasia or inflammation within the liver. Nodular lesions can also be aspirated with the aid of imaging procedures such as ultrasonography. The only contraindications are abnormal hemostasis, caused by either thrombocytopenia (<30,000/µl) or decreased coagulation factor activity, and the suspicion of hemangiosarcoma, which might rupture if lacerated during aspiration. Good technique is critical for obtaining diagnostic liver aspirates.

Excellent samples can be obtained using the method of Menard and Papageorges (12). Briefly, a 22 gauge 1.5 to 3.5-inch needle is connected to a 12 ml syringe via an 84-cm flexible extension set for intravenous lines, and the syringe is pre-filled with air. The needle hub is held like a pen to allow precise manipulations, positioned within the liver, and the tip is then moved rapidly back and forth 8 to 10 times within the same path. The needle is then withdrawn and the biopsy material is expelled immediately onto a glass slide using the syringe that was pre-filled with air. A gentle squash preparation technique is then used to make the films. This technique keeps the liver cells at the center and displaces blood to the periphery.

Normal hepatocytes are uniform large round to oval cells with abundant basophilic, somewhat granular cytoplasm. Cells contain one or two round, centrally located nuclei with a single prominent pale blue nucleolus . Normal hepatocytes often contain a small amount of dark blue-black pigment. This pigment may be a type of bile pigment or occasionally may be lipofuscin, which is commonly seen in old cats and is a more blue color than is bile pigment. Hemosiderin may also be seen within hepatocytes and is usually a more golden brown color. Hepatocytes may occur singly or in clusters. Very infrequently, rectangular crystalline clear inclusions may be seen in the nuclei of some hepatocytes and are of no known significance (13). Biliary epithelial cells may also be seen in normal aspirates and are small cells that are uniform in size with round nuclei and relatively small amount of pale blue cytoplasm. Other cells occasionally observed in small numbers in aspirates from normal livers include mast cells, macrophages (Kupffer cells), lymphocytes and neutrophils. Mesothelial cells from the surface of the liver are frequently seen in liver aspirates and should not be confused with neoplastic epithelial cells, fibroblasts, or biliary epithelium. Non-neoplastic abnormalities that can be diagnosed by cytology include cholestasis, hepatic lipidosis, increased glycogen storage, hepatocellular degeneration, various types of inflammation, extramedullary hematopoiesis, copper-associated hepatopathy, and lysosomal storage disorders (15).

Primary neoplasia of the liverPrimary neoplasms of the liver that may be diagnosed by cytology include hepatocellular

adenomas, hepatocellular carcinomas, bile duct carcinomas and neuroendocrine tumors. Neoplastic hepatocytes (hepatocellular carcinoma) may resemble normal hepatocytes to some extent, depending on their degree of differentiation. Unless they are very well differentiated they exhibit features of malignancy such as cellular crowding, multiple nucleoli, variation in nuclear

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size, increased nucleus to cytoplasm ratio and variation in cell size, Malignant hepatocytes are usually distinguishable from hyperplastic hepatocytes. Occasionally hepatocellular carcinomas are so poorly differentiated that they cannot be distinguished from metastatic carcinomas. In some aspirates of hepatic tumors, numerous capillaries can be seen amongst the neoplastic cells.

Malignant biliary epithelial cells (bile duct carcinoma cells) are similar to any other malignant epithelial cell, with cytologic features of malignancy. Biliary cells are smaller than hepatocytes and have less cytoplasm.

Neuroendocrine tumors or carcinoids are derived from the APUD (amine precursor uptake and decarboxylation) cells of the biliary system. The cells are similar to other neuroendocrine tumors and are usually quite fragile, thus numerous naked nuclei are seen. Intact cells are small with round central nuclei. Chromatin is usually condensed, nucleoli are indistinct, and the cytoplasm is moderate to abundant and usually pale in color. Cytoplasmic vacuoles may be present.

Metastatic neoplasia of the liverMetastatic carcinomas have malignant features as described earlier, including large size,

cell to cell relationships (cohesiveness), variability in cell size, variability in nuclear size, prominent nucleoli, basophilic cytoplasm and perinuclear cytoplasmic vacuoles, especially in secretory cells. Commonly the tissue of origin cannot be determined. Metastatic endocrine and neuroendocrine tumors appear as described above.

Although sarcomas may originate in the liver, they are more commonly metastatic. Sarcoma cells are spindle-shaped with oval nuclei and prominent nucleoli. The cytoplasm is basophilic and may be vacuolated and the cells are not cohesive. They may be uniform or variable in size and shape. Sarcoma cells do not exfoliate as readily as carcinoma cells but they are readily observed on imprints of surgical biopsies. These cells should not be confused with fibroblasts, which may be present in animals with hepatic fibrosis. Hemangiosarcomas are the most common sarcomas and have usually metastasized from the spleen. Evidence of previous hemorrhage (erythrophagocytosis and hemosiderin laden macrophages) and polychromatophilic erythrocytes may be observed, as well as abnormally shaped erythrocytes (acanthocytes) commonly observed in blood films from dogs with hemangiosarcoma.

Discrete cell neoplasms are often found in the liver. Lymphoma, plasma cell tumors, mast cell tumors and malignant histiocytosis are relatively common neoplasms of the liver and are usually relatively easy to diagnose, as neoplastic cells from these types of tumors are usually diffusely distributed. Liver aspirates containing large numbers of small lymphocytes are more difficult to interpret because the lymphoid population may be representative of either an inflammatory process or small cell lymphoma. Surgical biopsy and histopathology, as well as blood, bone marrow, lymph node cytology, and PCR can be used to distinguish the two processes.

Myeloproliferative disease may be present in the liver and spleen. If they are somewhat differentiated, recognizable progranulocytes with pink cytoplasmic granules will aid in the diagnosis. If they are undifferentiated, the immature cells may resemble lymphoblasts, and PCR, special cytochemical stains, bone marrow or blood examination will be necessary to distinguish between the two. This disorder is sometimes misdiagnosed as extramedullary hematopoiesis or inflammation.

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PancreasThe pancreas is typically aspirated only if pancreatic masses are detected. As with other

organs, inflammation, neoplasia, and cystic structures can usually be differentiated based on cytologic findings. Neoplasms of the exocrine pancreas (pancreatic adenocarcinoma) have characteristics similar to those of other adenocarcinomas including variability in cell size, variability in nuclear size, prominent nucleoli, high nucleus to cytoplasm ratio, and basophilic vacuolated cytoplasm.

Neoplasms of the endocrine pancreas are usually tumors of the beta islet cells, and are referred to as insulinomas, beta cell tumors, or islet cell tumors. They are most common in large breed older dogs and ferrets and are rare in cats. Their appearance is similar to that of other endocrine tumors such as thyroid gland tumors. Aspirates typically consist of numerous naked nuclei in a background of blue cytoplasm. However, unlike thyroid tumors, the cytoplasm of intact cells is commonly filled with small discrete clear vacuoles (1). Nuclei usually contain a single prominent nucleolus. The cells are typically quite uniform although some variability in cell and nuclear size may be present. Most of these tumors in dogs are malignant in behavior and metastasize to regional lymph nodes and liver. Most are functional and secrete excess insulin, resulting in hypoglycemia that may be quite severe (less than 50 mg/dl).

Adrenal GlandAdrenal gland tumors may arise from the adrenal cortex or the medulla. Tumors of the

cortex produce excess gluococorticosteroids, and result in hyperadrenocorticism. Tumors of the medulla produce excess catecholamines. Adrenocortical enlargement is usually secondary to pituitary tumors in dogs, in which case it is bilateral. However, adrenocortical tumors occur in less than fifty percent of dogs with hyperadrenocorticism, with approximately equal numbers of adenomas and adenocarcinomas. Aspiration of these masses is usually accomplished using ultrasonography. The cells from adrenal cortical adenomas are similar to normal or hyperplastic secretory cells of the adrenal gland. They are also similar to aspirates of other endocrine organs, with most cells appearing as naked nuclei. The cytoplasm is usually moderately basophilic and often contains numerous small discrete vacuoles. Nuclei are round, uniform, and typically contain a single prominent nucleolus. Cells from adenocarcinomas may be more variable in size with more prominent multiple nucleoli. However, cells may also appear similar to cells from adenomas.

Tumors of the adrenal medulla (pheochromocytomas or chromaffin cell tumors) are rare and may be aspirated with the aid of ultrasonography. Cells from these tumors are similar to those from other neuroendocrine tumors and consist of clusters of fairly uniform cells with numerous naked nuclei. The cytoplasm is light blue and may contain pale basophilic granules. The nuclei are round with a single small nucleolus. Significant criteria of malignancy are not typically present.

Gastrointestinal tract Although cytology of the gastrointestinal tract is often obtained via endoscopy, large

masses involving the GI tract may be palpated and aspirated through the abdominal wall, with or without the aid of ultrasound imaging. Intestinal epithelial cells are normally quite uniform with round nuclei and basophilic cytoplasm that may appear confluent. Mucus producing goblet cells may also be seen. Goblet cells contain large clear vacuoles within the cytoplasm and may contain magenta-colored mucin granules. Normal appearing lymphocytes may be aspirated from

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lymphoid follicles, or may be present in animals with lymphocytic enteritis. A few large granular lymphocytes may also be observed. Inflammatory lesions may also be present, with variable numbers of neutrophils, macrophages, mast cells, lymphocytes and eosinophils.

Neoplasia of the GI tract can often be diagnosed using aspiration. Lymphoma is typically characterized by large numbers of lymphoblasts. Adenocarcinoma of the GI tract is similar in appearance to other types of adenocarcinoma. Adenocarcinoma of the GI tract may elicit a scirrous reaction in which numerous fibroblasts are also present. Sarcomas such as leiomyosarcoma are occasionally diagnosed by cytology.

KidneyKidneys are almost always aspirated because they are large or abnormally shaped.

(15,16). Neoplastic kidneys, especially those with lymphoma, are likely to provide diagnostic material. Renal tubular epithelial cells are usually present in the sample and are approximately 20 µm in diameter, have abundant light blue cytoplasm and round nuclei with a single distinct nucleolus. The cytoplasm may contain dark granules and is often vacuolated in cats due to the presence of lipid. Blood contamination is often present and platelet clusters may be seen.

SpleenAspirates of the spleen are typically performed in patients with splenomegaly, and

cytopathology of the spleen has been described in detail elsewhere (17-21). Common causes of splenomegaly include hyperplasia, inflammation, extramedullary hematopoiesis, and neoplasia. Common types of neoplasia include lymphoma, myeloproliferative disorders, mast cell tumors, malignant histiocytosis and hemangiosarcoma. Fibrosarcoma, leiomyosarcoma, and metastatic neoplasia may also be seen. Splenic enlargement may be diffuse and symmetric or localized and asymmetric. One of the contraindications of splenic aspirate is hemangiosarcoma which is usually asymmetric; the tumor may rupture and metastasize when aspirated. Normal cytologic preparations from the spleen consist of blood, normal splenic stromal cells (fibrocytes) and small lymphocytes.

Prostate Normal prostatic cells are cuboidal to columnar and uniform, varying from approximately

10 to 15 µm in diameter. The nuclei are round to oval and are basilar in columnar cells. Nucleoli are small and inconspicuous. The cytoplasm is finely granular and basophilic. These cells are easily differentiated from transitional epithelial cells, which are larger with lighter staining cytoplasm. Cells from hyperplastic prostates almost always appear very similar to those from normal prostates. Numerous neutrophils and variable numbers of macrophages are present in material from inflamed prostates. Lymphocytes and plasma cells are seen infrequently. Many of the neutrophils exhibit karyolysis and cytoplasmic vacuolation in the presence of bacteria.

Large (20 to 80 µm) pleomorphic cells are usually present in aspirates of prostatic tumors. The nuclei are large, round to oval, and contain large, prominent usually multiple nucleoli. The cytoplasm is frequently vacuolated and often contains eosinophilic areas that represent secretory material. Mitotic figures and multinucleate cells are frequently observed. Other criteria of malignancy, such as nuclear molding, abnormal mitoses, abnormal chromatin structure, giant cell formation, free nuclear fragments, increased nuclear to cytoplasmic ratio, and incomplete separation of cells after division are often evident. Transitional cell carcinoma cells are cytologically similar to prostatic adenocarcinoma cells and the two types of carcinomas are

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often not differentiated based on cytologic examination (22). Prostatic tumors may be missed during aspiration, unless the gland is diffusely involved or imaging is used.

Cytopathology of bone marrow neoplasia.Bone marrow neoplasia is beyond the scope of this discussion, and is discussed in

detail elsewhere (23). Briefly, leukemia, a neoplastic proliferation of hematopoietic cells within the bone marrow, is defined by the presence of neoplastic blood cells in the peripheral blood or bone marrow, and is classified broadly into myeloproliferative and lymphoproliferative disorders. Diagnosis of these disorders is based on finding characteristic blast cells in the blood and/or bone marrow, and associated hematologic abnormalities. Specific cell types are identified by their morphologic appearance in Wright's stained blood and bone marrow films, cytochemical staining properties, electron microscopic appearance, and monoclonal antibody binding to surface antigens. In some cases, cells may appear so morphologically undifferentiated that classifying the disorder into either the myeloproliferative or lymphoproliferative category may be difficult. Myeloproliferative leukemias include neoplastic proliferation of ertyhrocytes, granulocytes, monocytes, and megakaryocytes. Multiple cell lines may be neoplastic if the affected stem cell is multipotential; an example is myelomonocytic leukemia, in which both neutrophils and monocytes have been neoplastically transformed. Lymphoproliferative disorders of bone marrow include acute lymphoblastic leukemia, chronic lymphocytic leukemia and multiple myeloma.

Leukemias are further classified as acute or chronic, based primarily on the maturity or degree of differentiation of the neoplastic cells, as well as by the clinical course. The neoplastic cells in acute leukemias are immature (blasts) and the patient survival time is usually quite short. By definition, the presence of 30 percent or more blast cells in the marrow is diagnostic of acute myeloid leukemia. The percentage of blast cells in the blood, on the other hand, is quite variable in these patients. Chronic leukemias are characterized by the predominance of mature, more well-differentiated, cells in the blood and marrow, and the patient survival time is usually longer. Neoplastic cells can commonly be found in organs other than the marrow in patients with leukemia. The spleen is commonly involved; liver and lymph nodes may also contain neoplastic cells. Other neoplastic disorders involving bone marrow include mast cell neoplasia, malignant histiocytosis, and rarely, metastatic carcinoma and sarcoma.

Metastatic bone marrow neoplasia (Carcinomas and Sarcomas) Epithelial and mesenchymal tumors may rarely metastasize to the bone marrow.

Epithelial tumors (carcinomas) tend to form groups of cohesive cells that are easy to distinguish from normal hematopoietic cells. Metastatic sarcomas are more difficult to diagnose, and are characterized by the presence of large discrete spindle-shaped cells with multiple criteria of malignancy. These cells must by distinguished from fibroblasts that may be observed in myelofibrosis.

References.

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1. Alleman AR. Endocrine System. In Raskin RE, Meyer DJ, editors: Atlas of Canine and Feline Cytology. Philadelphia, 2001, WB Saunders Co.

2. Duncan JR. The lymph nodes. In Cowell RI, Tyler RD, editors: Diagnostic Cytology and Hematology of the Dog and Cat, 2nd edition, St. Louis, 1999, Mosby, Inc.

3. Raskin R. Lymphoid system. In Raskin RE, Meyer DJ, editors: Atlas of Canine and Feline Cytology. Philadelphia, 2001, WB Saunders Co.

4. Thrall MA. Cytology of Lymphoid Tissue, Comp Cont Educ 9:104-112, 1987.

5. Burnett RC, Vernau W, Modiano, et al:. Diagnosis of Canine Lymphoid Neoplasia Using Clonal Rearrangements of Antigen Receptor Genes. Vet Pathol 40:32-41, 2003.

6. Brown DE, Thrall MA, Getzy DM, et al. Cytology of canine malignant histiocytosis. Vet Clin Pathol 23:118-122, 1994.

7. Griffiths GL, Lumsden JH, Valli VEO: Fine needle aspiration cytology and histologic correlation in canine tumors. Vet Clin Pathol 13:13-17, 1984.

8. Allen SW, Prasse KW, Mahaffey EA: Cytologic differentiation of benign from malignant canine mammary tumors. Vet Pathol 23:649-655, 1986.

9. Burkhard MJ, Meyer DJ: Invasive cytology of internal organs. Cytology of the thorax and abdomen. Vet Clin N Am Small Anim Prac 26:1203-1222, 1996.

10. Blue JT, French TW, Meyer DJ: The Liver. In Cowell RI, Tyler RD, editors: Diagnostic Cytology and Hematology of the Dog and Cat, 2nd edition, St. Louis, 1999, Mosby, Inc.

11. Meyer DJ: The Liver. In Raskin RE, Meyer DJ, editors: Atlas of Canine and Feline Cytology. Philadelphia, 2001, WB Saunders Co.

12. Menard M, Papageorges M: Cytologic evaluation of fine-needle liver biopsies. In August JR, editor: Consultations in Feline internal Medicine, 4th edition, Philadelphia, 2001, WB Saunders Co.

13. Richter WR. Stein RJ. Rdzok EJ. et al. Ultrastructural studies of intranuclear crystalline inclusions in the liver of the dog. Am J Path 47: 587-599, 1965.

14. Brown DE, Thrall MA, Walkley SU, et al. Feline Niemann-Pick disease type C. Am J Pathol 144:1412-1415, 1994.

15. Meinkoth JH, Cowell RL, Tyler RD: The Renal Parenchyma. In Cowell RI, Tyler RD, editors: Diagnostic Cytology and Hematology of the Dog and Cat, 2nd edition, St. Louis, 1999, Mosby, Inc.

16. Borjesson D: Urinary Tract. In Raskin RE, Meyer DJ, editors: Atlas of Canine and Feline Cytology. Philadelphia, 2001, WB Saunders Co.

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17. MacWilliams PS. The Splenic Parenchyma. In Cowell RI, Tyler RD, editors: Diagnostic Cytology and Hematology of the Dog and Cat, 2nd edition, St. Louis, 1999, Mosby, Inc.

18. O’Keefe DA, Couto CG. Fine-needle aspiration of the spleen as an aid in the diagnosis of splenomegaly. J Vet Int Med 1:102-109, 1987.

19. Raskin RE. Lymphoid System. In Raskin RE, Meyer DJ, editors: Atlas of Canine and Feline Cytology. Philadelphia, 2001, WB Saunders Co.

20. Spangler WL, Culbertson MR. Prevalence and type of splenic diseases in cats: 455 cases (1985-1991). J Am Vet Med Assoc 201:773-776, 1992.

21. Spangler WL, Culbertson MR. Prevalence, type and importance of splenic diseases in dogs: 1,480 cases (1985-1989). J Am Vet Med Assoc 200:829-834, 1992.

22. Thrall MA, Olson PN, Freemyer FG: Cytologic Diagnosis of Canine Prostatic Disease. J Am Anim Hosp Assoc 21:95-102, 1985.

23. Zinkl JG, Feldman BF: The Male Reproductive Tract: Prostate, Testes, Semen. In Cowell R, Tyler R, editors: Diagnostic Cytology of the Dog and Cat. St. Louis, 1989, American Veterinary Publications, Inc.

24. Thrall MA, Weiser MG, Jain N: Laboratory Evaluation of Bone Marrow. In Thrall MA , Baker D, Campbell T, DeNicola D, Fettman MG, Lassen ED, Rebar AH, Weiser MG, Veterinary Hematology and Clinical Chemistry, Baltimore, 2004,.Lippincott, Williams and Wilkins.

25. Moore PF, Affolter VK, Vernau, W: Canine hemophagocytic histiocytic sarcoma: A proliferative disorder of CD11d+ macrophages. Vet Pathol 43:632-645, 2006.

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