Cytology in evaluation of lymphoid tissue in the dog and cat (Proceedings)

aspiration procedure

For cutaneous lymph nodes, the skin over the node to be aspirated needs no special preparation. it is prepared as the skin would be prepared for an injection. the aspiration technique requires the use of a 22-gauge needle and a 6 or 12 cc syringe. a 22-gauge butterfly catheter can be substituted for small or hard-to-reach nodes. when possible, insert the needle toward the periphery of the node, avoiding necrotic centers. slight negative pressure is applied and the needle is advanced towards the lesion and then redirected, if the lymph node is large enough, in a fan pattern until material appears in the center of the needle. do not pump the plunger of the syringe as this will damage the fragile lymphoid cells. during needle redirection, care must be taken not to withdraw the needle from the lymph node. when material appears in the center of the needle, the plunger is released and the needle is withdrawn from the node and skin. the needle should be removed from the syringe. then air is drawn into the syringe and the needle is put back on the syringe. the aspirated material is then gently expelled onto a clean glass slide. a second clean slide is gently placed on the material, parallel to the first slide. The material is allowed to diffuse and the slides are gently separated. Slides are air dried and then stained with diff quik or a comparable Romanowsky-type stain.

Reading: Reactive lymphoid hyperplasia in dogs

cytologic interpretation of lymph node aspirates

If the guidelines described above are followed, there is generally a good correlation between cytological and histological diagnosis. any enlarged lymph node may be aspirated in order to classify the lesion into the following classifications:

1. Normal lymph node.

2. reactive (lymphoid hyperplasia).

3. inflammation (lymphadenitis).

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4. lymphoid neoplasm (lymphoma).

5. metastatic disease.

6. edema (lymphedema).

normal lymph node

Normal lymph nodes contain 75% to 90% small, well-differentiated lymphocytes. these cells are 7 to 10 μm or 1 to 1.5 times the size of erythrocytes. they contain a thin rim of cytoplasm and the nucleus is round or oval, sometimes indented. it has dense clumps of dark chromatin and no visible nucleoli. normal nodules typically contain 5 to 10% intermediate (medium) lymphocytes (approximately 9 to 15 μm in diameter, about the same size as a neutrophil) and <5% lymphoblasts. lymphoblasts are usually more than 15 μm in diameter, or 2 to 5 times the size of an erythrocyte, and larger than a neutrophil. lymphoblasts have a moderate amount of basophilic cytoplasm that may appear granular due to dark-staining protein-rich areas and lighter-staining areas of some organelles. the nuclear shape is variable, from rounded to irregular, and generally has a stippled chromatin pattern. single or multiple nucleoli are often visible. Plasma cells, macrophages, neutrophils, and mast cells are occasionally seen in very low numbers in normal ganglia.

reactive lymphoid hyperplasia (rlh) or reactive lymph node

In a reactive node, small, well-differentiated lymphocytes remain the predominant population, but there is usually a greater number of intermediate lymphocytes and a greater number of lymphoblasts, especially in the cat. however, the lymphoblast population will typically not exceed 10% to 20% of the total lymphoid population of a reactive nodule. The most striking feature of reactive ganglia in dogs is the presence of plasma cells. plasma cells are medium-sized round or oval cells with a single round nucleus placed eccentrically. the nucleus of a mature plasma cell is the same size and color as a small lymphocyte but the cytoplasm is much more abundant. the cytoplasm is deeply basophilic and usually has a visible golgi apparatus that appears as a clear area located between the nucleus and the larger volume of cytoplasm.

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inflammation (lymphadenitis)

In lymphadenitis, the predominant population of non-lymphoid inflammatory cells classifies the type of inflammation present. suppurative inflammation is characterized by the presence of increased numbers of neutrophils beyond what might be expected from any blood contamination present. here, more than 5% of the nucleated cells are neutrophils. this is usually the result of a bacterial infection in the node (abscessed lymph node) or in an area that is draining. eosinophilic inflammation is characterized by an inflammatory reaction containing an eosinophilic infiltration, generally accompanied by a slight increase in the number of neutrophils +/- low number of macrophages. Eosinophilic lymphadenitis is most commonly caused by allergic dermatitis and is typically seen in the inguinal or popliteal lymph nodes. Other common causes of eosinophilic lymphadenitis include other nondermatologic allergic/hypersensitivity reactions, eosinophilic granuloma complex, parasitic diseases, eosinophilic gastroenteritis, hypereosinophilic syndrome, and mast cell tumors. in rare cases, lymphoma cells may secrete chemotactic factors that result in eosinophilic infiltration. Pyogranulomatous inflammation contains an important macrophage component, with or without the presence of neutrophils. this type of inflammation usually results from fungal infections (blastomycosis, coccidioidomycosis, cryptococcosis, or sporotrichosis), protozoal infections (cytauxzoonosis, toxoplasmosis, or leishmaniasis), mycobacterial infections, nocardia/actinomyces, bartonella in dogs. Mild pyogranulomatous swelling may also be seen in lymph nodes draining areas of chronic inflammation or neoplasia. (the figure on the right is pyogranulomatous lymphadenitis with blastomycosis organisms)

lymphoid neoplasm (lymphoma)

Lymphoma is suspected whenever 30% of the cell population in a lymph node aspirate is lymphoblasts, although typically the lymphoid population will likely be between 50% and 90%. when there are more than 50% lymphoblastic cells, a reliable cytological diagnosis of lymphoma can be made. lymphomas can be classified by their tissue of origin (eg renal, thymic, intestinal, etc.), with multicentric lymphoma being the most common type seen in dogs. however, knowing the “cytologic type” of lymphoma present may provide some indication of the grade of malignancy, the potential for response to chemotherapy, and the potential or explanation for paraneoplastic syndromes such as hypercalcemia. The most accurate means of typing lymphoma is through the use of lymphocyte markers that will determine the subset of lymphocytes involved in the neoplastic process (eg, B cells, T cells such as CD4 or CD8, or natural killer cells).

canine lymphoma

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In canine lymphoma, the predominant cell type is the immature lymphoblast. only rarely do small, well-differentiated lymphocytes become neoplastic. lymphoblasts are large cells with nuclei ranging in size from 2 to 5 times the size of erythrocytes with a deeply basophilic cytoplasm that is more abundant than that of small or intermediate lymphocytes. the chromatin pattern is more diffuse and paler in staining than in the well-differentiated lymphocyte. a variable number of distinct or indistinct nucleoli are frequently visible. (figure below, canine lymphoma).

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Note: Prior administration of glucocorticoids can dramatically alter the population of lymphocytes within a lymph node. lymphoblasts are highly sensitive to the cytotoxic effects of glucocorticoids, much more so than mature lymphocytes. this can iatrogenically lower the differential lymphoblast count below 30% to 50% of the population, making the diagnosis of lymphoma difficult.

feline lymphoma

The same criteria are used for the diagnosis of lymphoma in dogs and cats. when a lymph node or mass is aspirated and found to contain a population of lymphocytes of which 50% or more are blast cells, lymphoma can be reliably diagnosed. however, two complicating factors make the diagnosis of lymphoma in the cat more difficult than in the dog: 1. Lymphomas in the cat are most often composed of a population of well-differentiated lymphocytes, which is rarely seen in dogs. dogs, and 2. as mentioned above, a condition known as “distinctive peripheral lymph node hyperplasia” (dplh) has been reported to occur in young cats and may clinically, cytologically, and histologically resemble multicentric lymphoma (j. amer.vet.med.assoc.190(2):897-899, 1987 and vet.pathol, 23:286-392, 1986). furthermore, multicentric lymphoma, involving only the peripheral lymph nodes, is common in the dog but rare in the cat. therefore, a diagnosis of lymphoma cannot be made when evaluating aspirates taken from cats with only generalized peripheral lymphadenopathy.

anatomic forms of feline lymphoma

Lymphoma involving internal organs occurs relatively frequently in the cat. various forms may include mediastinal, hepatic, alimentary, renal, ocular, and primary CNS lymphoma. There may be a relationship between alimentary and renal lymphoma and with renal lymphoma and CNS metastases. when aspirates of masses in any of the organs show a dense population of lymphoid cells, lymphoma should be suspected. When the lymphocyte population is composed primarily of lymphoblasts, as is seen in many cases, the cytological diagnosis of lymphoma can be made with confidence. however, many lymphomas of hepatic or intestinal origin are composed of small, well-differentiated, normal-appearing neoplastic lymphocytes (figure below; hepatic small cell lymphoma). Many lymphomas in the cat are composed of T cells transformed by the felv virus, but most arising from the gastrointestinal tract are felv-negative b-cell lymphomas.

An unusual form of alimentary lymphoma classified as large granular lymphoma (lgl) is also reported in the cat (figure below). it is characterized by a population of individually arranged round cells with a fairly abundant cytoplasm. the cytoplasm contains a focal accumulation of azurophilic granules (resembling mast cell granules). These tumors generally involve the small intestine and are believed to originate from cytotoxic T cells or natural killer cells. the focal accumulation of granules may help distinguish this neoplasm from the intestinal form of mct that is also seen in the cat. lgls generally have less cytoplasm, fewer larger granules, and no or few eosinophils compared with mast cell neoplasms. lgl stains positively for lymphoid tissue markers and with ptah (phosphotungstic acid-hematoxylin), and negatively with toluidine blue, staining for mast cell tumors just the opposite.

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feline hodgkin lymphoma

Feline Hodgkin-like lymphoma resembles the condition in humans and has generally been recognized in older cats (≥ 6 years). Most of the affected animals presented a mass in the ventral cervical region, enlargement of the submandibular and/or prescapular ganglia. As in humans, only a single node or group of nodes is usually involved in the eventual advancement of contiguous nodes. Cytological diagnosis is very difficult since neoplastic cells (Reed-Sternberg cells and their variants) only comprise 1% to 5% of the cells of the affected node, the rest of the cells are non-neoplastic lymphocytes, macrophages and granulocytes. the diagnosis needs to be confirmed histologically and there are several histological types of the disease (vet. pathol. 38:504-511, 2001).

metastatic disease

Knowledge of the areas drained by specific lymph nodes is essential to determine the presence of metastatic disease. It is also important to remember that the absence of obvious metastatic disease in a cytology specimen does not rule out the possibility of early metastasis. Because many tumors enter the nodes through subcapsular afferent vessels (figure below) or begin as focal collections, early metastatic disease may be missed on cytology preparations. Metastatic disease is characterized by the presence of a homogeneous cell population not normally found in a lymph node. these cells usually have an anaplastic appearance and show obvious features of malignancy. the remaining lymphoid population may appear reactive, however, the neoplasm may replace (erase) the lymph node parenchyma entirely, making it difficult to identify the swelling cytologically as a lymph node. the absence of lymphadenopathy does not rule out the presence of metastatic disease. mast cells, among other neoplastic processes, are known to metastasize without creating adenopathies. the presence of lymphadenopathy in a lymph node draining an area with a tumor does not automatically indicate that metastasis has occurred. lymph nodes draining an area where a tumor is found often become reactive in response to the regional inflammatory process induced by the neoplasm. in addition, many lymph nodes may be normal in size and have significant metastatic disease. this is particularly true of metastatic mast cells.

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