Going deeper: fine need aspiration cytology of internal organs (Proceedings)

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 superficial sites, it often provides diagnostic information that would otherwise require surgery. Current ultrasound techniques allow guided placement into the lesion of question and visualization of the site both prior to and after aspiration which has 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' sampling technique, even with ultrasound guidance. The sample may be inadequate 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, FNA does not preserve tissue architecture.  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 FNA correlate well with histology.

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 origin of epithelial or mesenchymal cells, however discrete cell tumors can often be accurately diagnosed.  The type of inflammation and etiologic agents can be identified.

General approach to cytology of internal organs

Some types of disease processes, such as those with mesenchymal proliferation or fibrosis do not exfoliate well and repeated aspiration is not useful. Cells should be evaluated to determine if they are consistent with the expected sample. If not, the challenge is to determine if the specimen 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 common with 'blind' aspirates but occasionally happens with ultrasound guided FNA. 

Inflammatory cells are evaluated as at other sites.  Their presence should prompt a search for infectious agents at both low and high magnifications. Small lymphocytes and plasma cells are often present as part of an inflammatory response and 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. Caution should be taken when interpreting mesenchymal or epithelial pleomorphism when there is evidence of inflammation, necrosis, or hemorrhage as reactive cells can be moderately pleomorphic in appearance.

Neoplasia can develop from any of the normal cell populations that comprise an organ.  Neoplastic changes include 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. 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. 

Lung

Fine needle aspiration of the lung parenchyma is most rewarding in cases of diffuse interstitial disease or when large focal lesions can be visualized. 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. Mucus is often present in respiratory samples as ribbons of eosinophilic material.  Increased mucus is non-specific and associated with a number of pathologic processes.  Occasionally this material is found in tightly wound formations called Curschman's spirals.

In general, inflammatory cells are characterized as for other sites with the addition that in chronic disease, binucleate and multinucleate vacuolated alveolar macrophages may be seen. Blood contamination is a complication of FNA, so the presence of erythrophagia, preferably with hemosiderin, is important to distinguish pathologic hemorrhage.  Pulmonary atelectasis or collapse is suggested by large numbers of relatively normal respiratory epithelial cells.  Necrosis is characterized by abundant amounts of basophilic granular to amorphous background material.

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. Squamous metaplasia occurs in response to chronic disease and is difficult to differentiate from squamous neoplasia.

Carcinomas can arise from any level of the respiratory tract.  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 is commonly seen. Squamous cell carcinoma is distinguished by some degree of keratinization. Mesenchymal neoplasia in the lung is often metastatic as tumors arising from the pulmonary connective tissue are relatively rare.

Hemolymphatic neoplasia can appear as diffuse infiltrative disease or as discrete nodules (see Round Cell Neoplasia notes).  Pulmonary lymphomatoid granulomatosis is an uncommon, but aggressively infiltrative, pleocellular T-cell lymphoid neoplasia of dogs inconsistently associated with peripheral basophilia and canine dirofilariasis. 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. Neoplastic cells often compose the minority of the cell population present and are admixed with small lymphocytes, eosinophils, and plasma cells.

Thymus

Thymoma and lymphoma 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 found in cohesive clumps with round nuclei and abundant lightly basophilic to acidophilic cytoplasm. Hepatocytes from older animals often contain lipofuscin pigment. Small numbers of binucleate cells are normally present. 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. 

Hepatocellular hyperplasia is characterized by anisokaryosis, binucleate cells, and increased cytoplasmic basophilia and can be associated with nodular hyperplasia or as a response to hepatitis or other disease. 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.  Corticosteroids, exogenous or endogenous, cause moderate to marked glycogen accumulation resulting in a wispy appearance to the cytoplasm.  The presence of leukocytes suggests inflammation but should be differentiated from blood contamination or  extramedullary hematopoiesis (EMH). EMH in the liver is typically mild. 

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. Occasionally cells will have increased basophilia and increased binucleation.  Poorly differentiated neoplasia is readily identified by 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.  Non-hepatic neoplasia and metastasis to the liver are common.  Carcinomas, sarcomas, and discrete cell tumors may all be identified in cytologic aspirates and appear similar to primary tumors at other sties.

Spleen

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. 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. Extraskeletal myeloma and plasmacytoma may be seen in both the spleen and the liver. These are characterized by a population of variably differentiated plasma cells. Binucleate plasma cells are common.

EMH is commonly seen in splenic aspirates. Erythroid precursors and megakaryocytes are seen most frequently.  Immature cells are easily confused with lymphoblasts and can lead to the erroneous diagnosis of lymphoma. Myeloproliferative disease (MPD) 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 MPD. Concurrent evaluation of blood and bone marrow is recommended. 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 HSAs result in blood and only rare spindled cells.

Gastrointestinal tract

Scrapings of gastrointestinal tract lesions taken via endoscopy 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 are often seen with ulcerative lesions. 

Primary GI neoplasia is uncommon, however both GI epithelial and mesenchymal neoplasia are described.  Diagnosis can be challenging because of difficulty in obtaining a quality sample and because non-neoplastic epithelial and mesenchymal cells often have marked reactive responses to ulceration and inflammation.  Evaluation of the draining lymph node for atypical cells may be more definitive than evaluation of the GI mass itself.  Epithelial neoplasia can be seen at all levels of the GI tract.  Cells may be individualized or appear in clusters.  Features of malignancy are common as are granules or vacuoles suggesting a secretory nature.  Neuroendocrine tumors (APUD tumors, carcinoids) are uncommon.  These tumors tend to exfoliate well, but the cells themselves are often fragile and 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, but many have been reclassified by c-kit analysis.  Both GISTs and leiomyoma/sarcoma have 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 and are often reactive.  

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.

Pancreas

Insulinoma is the most commonly diagnosed tumor of the pancreas.  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 pleomorphism. 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 with 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, the non-neoplastic population may become reactive, and aspirates typically obtain at least small numbers of both cell types. Pheochromocytomas arise from the adrenal medulla and have a classic neuroendocrine appearance. Naked nuclei are common.  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 or malignant. Adenomas resemble normal adrenocortical cells which have abundant pale cytoplasm that often contains punctate vacuoles.  Nuclei are uniform in size, small, round to oval, granular to finely clumped chromatin, and prominent nucleoli. Adenocarcinomas frequently display numerous criteria of malignancy including moderate to marked pleomorphism, multiple nuclei, coarse chromatin, prominent multiple nucleoli, and deeply basophilic cytoplasm. However, appearance and behavior are not always correlated and some adenocarcinomas are well-differentiated. 

Kidney

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, neutrophils in a renal aspirate must be evaluated concurrently with the number seen in blood. Small lymphocytes may be markedly increased with chronic, non-suppurative inflammation and needs to be differentiated from lymphoma.  Renal 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. In urine, neutrophils become very swollen with round karyolytic nuclei and pale cytoplasm and can be confused with epithelial or mononuclear cells.Transitional cell carcinoma is the most common lower urinary tract tumor. 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 and these cells often contain eosinophilic cytoplasmic vacuoles. Occasionally these cells will undergo squamous differentiation.

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 is commonly induced secondary to inflammation and 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.Squamous metaplasia occurs in the presence of endogenous or exogenous estrogen or in cases of chronic inflammation. The cells demonstrate typical keratinization changes. Prostatic neoplasia can originate from either prostatic or transitional epithelium. Transitional cell carcinoma is similar to that seen in the bladder. Prostatic carcinomas are pleomorphic, deeply basophilic, with an increased nuclear to cytoplasm ratio, and may form acinar or tubular structures.