Lymph node sampling and cytology is quick, easy, and usually rewarding. Cytologic samples of peripheral and/or internal lymph nodes may be collected by fine-needle aspiration biopsy or nonaspiration fine-needle biopsy techniques. Sampling can also be performed by imprints or scrapings from lymph nodes that have been surgically removed or at necropsy.
Lymph node cytology is an excellent way to evaluate a lymphadenopathy whether it is a single, multiple, or a generalized lymph node enlargement. If multiple lymph nodes are enlarged, more than one should be sampled. A lymph node away from the mouth or any site of inflammation should be aspirated as well as any lymph node close to a site of inflammation. Generally, if no lymph nodes are enlarged, lymph node cytology is generally not helpful. In addition to be unrewarding, aspiration of a nonenlarged node is difficult and usually results in aspiration of perinodal fat with little or no lymphoid tissue present. Nonetheless, normal sized lymph nodes may be aspirated on occasion to investigate the potential for metastatic disease.
Selection of a Node
The lymph nodes generally palpated in dogs and cats include the submandibular, prescapular, and popliteal lymph nodes. Popliteal and prescapular lymph nodes are preferred biopsy sites for animals with generalized lymphadenopathy. When possible, avoid submandibular lymph nodes since they are frequently reactive due to constant exposure to antigens from the oral cavity. Also, it is best to avoid extremely enlarged lymph nodes since they may yield misleading information due to the presence of necrosis or hemorrhagic tissue. A moderately enlarged lymph node is preferred.
Sample Collection and Preparation
For cutaneous lymph nodes, the skin over the node to be aspirated needs no special preparation. It is prepared as one would prepare the skin for giving 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 may be substituted for small or hard to reach nodes. When possible, insert the needle toward the periphery of the node, avoiding necrotic centers. A slight negative pressure is applied and the needle is advanced into the lesion and then redirected, if the lymph node is large enough, in a fan-like pattern until material appears in the hub of the needle. Do not pump the plunger of the syringe as this will damage the fragile lymphoid cells. During redirection of the needle, care should be taken not to withdraw the needle from the lymph node. When material appears in the hub 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. Air is then drawn into the syringe, and the needle is replaced onto the syringe. The aspirated material is then gently expelled onto a clean glass slide. A second clean slide is gently laid on top of the material, parallel to the first slide. The material is allowed to diffuse out, and the slides are gently slid apart. Slides are air-dried and are then stained using Diff-Quik or some comparable Romanowsky-type stain.
Cytological Interpretation of Lymph Node Aspirates
If the previously described guidelines are adhered to, there is generally good correlation between cytologic and histologic diagnosis. Any enlarged lymph node may be aspirated for the purpose of classifying the lesion into the following classifications:
1. Normal lymph node
2. Reactive (lymphoid hyperplasia)
3. Inflammation (lymphadenitis)
4. Lymphoid neoplasia (lymphoma)
5. Metastatic disease
6. Edema (lymphedema)
Normal Lymph Node
Normal lymph nodes contain 75–90% small, well differentiated lymphocytes. These cells measure 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 roundish to oval sometimes indented. It has dense clumps of dark chromatin and has no visible nucleolus. Normal nodes usually contain 5–10% intermediate (medium) lymphocytes (approximately 9 to 15 µm in diameter, about the same size as a neutrophil) and <5% lymphoblasts. Lymphoblasts are generally greater than 15 µm in diameter, or 2 to 5 times the size of an erythrocyte and are larger than a neutrophil. Lymphoblasts have a moderate amount of basophilic cytoplasm that may appear granular because of the dark-staining protein-rich areas and lighter staining areas of some organelles. Nuclear shape is variable, ranging from round to irregular, and generally has a stippled chromatin pattern. Single to multiple nucleoli are often visible. Plasma cells, macrophages, neutrophils and mast cells are occasionally seen in very low numbers in normal nodes.
Reactive Lymphoid Hyperplasia (RLH), or Reactive Lymph Node
In a reactive node, small, well-differentiated lymphocytes are still the predominant population, but increased numbers of intermediate lymphocytes and increased numbers of lymphoblasts is usually present, particularly in the cat. However, the lymphoblast population typically will not exceed 10 to 20% of the total lymphoid population of a reactive node. The most striking feature in reactive nodes from dogs is the presence of plasma cells. Plasma cells are medium-sized round to oval cells with a single eccentrically placed round nucleus. 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 generally have a visible Golgi apparatus appearing as a clear area located between the nucleus and greatest volume of cytoplasm.
In lymphadenitis, the predominant non-lymphoid inflammatory cell population categorizes the type of inflammation present. Suppurative inflammation is characterized by the presence of increased numbers of neutrophils beyond what may be expected from any blood contamination present. Here, greater than 5% of nucleated cells are neutrophils. This is usually the result of a bacterial infection either in the node (abscessed lymph node) or in an area being drained. Eosinophilic inflammation is characterized by an inflammatory reaction that contains an eosinophilic infiltration, usually accompanied by a mild increased numbers of neutrophils ± low numbers of macrophages. An eosinophilic lymphadenitis is most commonly caused by an allergic dermatitis, and is typically seen in the inguinal or popliteal lymph nodes. Other common causes of eosinophilic lymphadenitis include other non-dermatologic 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 an eosinophilic infiltration. Pyogranulomatous inflammation contains a significant macrophage component, with or without the presence of neutrophils. This type of inflammation typically results from fungal infections (blastomycosis, coccidioidomycosis, cryptococcosis, or sporotrichosis) protozoal infections (cytauxzoonosis, toxoplasmosis, or leishmaniasis) mycobacterial infections, Nocardia/Actinomyces, Bartonella in dogs. A mild pyogranulomatous inflammation may also be observed in lymph nodes that drain areas of chronic inflammation or neoplasia. (Figure on right is pyogranulomatous lymphadenitis with blastomycosis organisms.)
Lymphoid Neoplasia (Lymphoma)
Lymphoma is suspected whenever 30% of the cells population from a lymph node aspirate is lymphoblasts, though, typically, the lymphoid population will likely be between 50% to 90%. When >50% lymphoblast cells are present, a cytological diagnosis of lymphoma can be reliably made. Lymphomas may be classified by their tissue of origin (e.g., renal, thymic, intestinal etc.), with multicentric lymphoma being the most common type observed in dogs. However, knowing the “cytologic type” of lymphoma present may give 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 by using lymphocyte markers that will determine the subset of lymphocytes involved in the neoplastic process (e.g., B-cells, T-cells such as CD4 or CD8, or Natural Killer cells).
In canine lymphoma, the predominant cell type is the immature lymphoblast. Only rarely will the small, well-differentiated lymphocytes become neoplastic. Lymphoblasts are large cells with nuclei that vary 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 staining than in the well-differentiated lymphocyte. A variable number of distinct or indistinct nucleoli are frequently visible (Figure right, canine lymphoma).
Note: The previous administration of glucocorticoids can drastically alter the lymphocytes population within a lymph node. Lymphoblasts are very sensitive to the cytotoxic effects of glucocorticoids much more so than mature lymphocytes. This may iatrogenically decrease the differential lymphoblast count below 30% to 50% of the population therefore making a lymphoma diagnosis difficult.
The same criteria for diagnosing lymphoma are used in dogs and cats. When a lymph node aspirate or mass is aspirated and found to contain a population of lymphocytes of which 50% or more are blast cells, lymphoma can reliably be 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 more frequently composed of a population of well-differentiated lymphocytes, which is rarely observed in dogs, and 2. As mentioned previously, a condition known as “Distinctive Peripheral Lymph Node Hyperplasia” (DPLH) that clinically, cytologically and histologically may resemble multicentric lymphoma has been reported to occur in young cats (J Am Vet Med Assoc. 1987;190(2):897–899; Vet Pathol. 1986;23:286–392). In addition, 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 the internal organs occurs with relative frequency 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 metastasis. When aspirates from masses in any of organs yield a dense population of lymphoid cells, lymphoma should be suspected. When the lymphocyte population consists of primarily lymphoblasts, as is seen in many cases, the cytologic diagnosis of lymphoma can reliably be made. However, many lymphomas of liver or intestinal origin are composed of small, well-differentiated, normal-looking, neoplastic lymphocytes (Figure to right; small-cell hepatic 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 on right). It is characterized by a population of individually arranged round cells with fairly abundant cytoplasm. The cytoplasm contains a focal accumulation of azurophilic granules (resembling mast cell granules). These tumors usually involve the small intestine and are believed to be of cytotoxic T-cell or natural killer cell origin. The focal accumulation of the granules may help to distinguish this neoplasm from the intestinal form of MCT also seen in the cat. LGLs generally have less cytoplasm, fewer, larger granules, and no or few eosinophils as compared to mast cell neoplasms. LGL stain positively for lymphoid tissue markers and with PTAH (phosphotungstic acid - hematoxylin), and negative with toluidine blue, mast cell tumors stain just the opposite.
Feline Hodgkin’s-like Lymphoma
Feline-Hodgkin’s-like lymphoma resembles the condition in humans and has generally been recognized in older cats (>6 yrs). Most affected animals presented with a mass in the ventral cervical region, submandibular and/or prescapular node enlargement. As in humans, only a single node or group of nodes is generally involved with eventual contiguous nodal advancement. The cytologic diagnosis is very difficult since the neoplastic cells (Reed-Sternberg cells and their variants, binucleated cell on bottom right) only comprise 1% to 5% of the cells in the affected lymph node, the rest of the cells are non-neoplastic lymphocytes, macrophages, and granulocytes. The diagnosis needs to be confirmed histologically and various histological types of the disease exist (Vet Pathol. 2001;38:504–511).
Knowledge of the areas drained by specific lymph nodes is critical in determining 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. Since many tumors will enter the nodes through afferent, subcapsular vessels (Figure on right), or begin as focal accumulations, early metastatic disease might be missed on cytologic preparations. Metastatic disease is characterized by the presence of a homogenous cell population not normally found in a lymph node. These cells usually appear anaplastic and display obvious characteristics of malignancy. The remaining lymphoid population may appear reactive, however, the neoplasia may replace (efface) the lymph node parenchyma totally, making cyto logical identification of the swelling as a lymph node difficult. The absence of lymphadenopathy does not rule out the presence of metastatic disease. Mast cell tumors, amongst other neoplastic processes, are renowned to metastasize without creating lymphadenopathy. The presence of lymphadenopathy in a lymph node draining an area with a tumor does not automatically indicate metastasis has occurred. Lymph nodes draining an area where a tumor is located 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 cell tumors.