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Image Gallery: Lymph Node Cytology

Maxey Wellman, DVM, MS, PhD, DACVP (Clinical Pathology), The Ohio State University

M. Judith Radin, DVM, PhD, DACVP (Clinical Pathology), The Ohio State University

Clinical Pathology

|January 2017|Peer Reviewed|Web-Exclusive

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Lymphadenopathy (ie, lymphadenomegaly) can be caused by lymphoid and plasma cell hyperplasia, inflammation, or lymphoid and metastatic neoplasia. Cytology of lymph node aspirates is easily performed in general practice while being relatively noninvasive and inexpensive. Depending on clinician experience, results can provide a quick, accurate diagnosis when coupled with clinical history and physical findings.

Lymph Node Aspiration Procedure

Choosing a sample site. If multiple lymph nodes are enlarged, sampling of mandibular lymph nodes should be avoided because they often are hyperplastic and mildly inflamed. Other peripheral lymph nodes that are easily sampled include popliteal and prescapular lymph nodes. Extremely enlarged lymph nodes also should be avoided because necrosis or hemorrhage may preclude adequate sampling and evaluation.
Sampling technique. Lymph node samples should be collected with a 21- or 25-gauge needle using an aspiration or nonaspiration technique. For the aspiration technique, a 21-25-gauge needle coupled to a 12-20 ml syringe is used, whereas for the non-aspiration technique, only the needle is used to prick the lymphoid tissue, using the cutting action of the needle to collect the sample. The needle can then be attached to a syringe prior to making the smears. If only a small amount of material has been collected, the needle is detached, air is aspirated into the syringe, the needle is replaced, and a small amount of material is carefully expelled onto several clean glass slides.

Lymph Node Cytology Submandibular Lymphadenopathy: The Cytologist’s Challenge

Redirecting the needle to several locations within the lymph node is recommended to ensure that a representative aspirate has been obtained. Only a small amount of material is needed.
Sample handling.

After capturing the sample, the contents should be sprayed on to a clean slide using a clean, empty syringe. A spreader slide should be used to gently disperse the cells, creating a thin monolayer of cells.

Common errors during slide preparation include:

Failure to disperse the cells on the slide: results in smears that are too thick, and
Applying too much pressure with the spreader slide, resulting in broken cells. Broken cells may not be reliably evaluated.

Smears should be clearly labeled with pencil or a marker specifically designated for sample labeling. Smears should be dried in ambient air before staining.
Slides should not be stored in a refrigerator or exposed to formalin because water condensation from refrigeration can lyse cells and formalin fumes can prohibit adequate staining.

Slide Staining. Because additional staining, immunophenotyping by flow cytometry, and clonality testing sometimes can be helpful to detect causative agents, determine cell lineage, or support a diagnosis of neoplasia, saving several unstained slides is recommended.

Romanowsky-type stains (eg, Wright's, Wright-Giemsa, Diff-Quik or comparable commercial quick stain) provide good color contrast and acceptable cytoplasmic and nuclear detail, plus can stain most infectious agents.

If slides are being sent to a reference laboratory for interpretation, patient signalment, relevant clinical and laboratory findings, and the degree and extent of lymph node enlargement should be described in detail.

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FIGURE 1 Normal lymph node.

Most (85%-95%) of the cells (gray arrow) are small lymphocytes (ie, smaller than a neutrophil [blue arrow]), with round nuclei, condensed chromatin; inconspicuous nucleoli; and scant, pale cytoplasm, resulting in a high nuclear:cytoplasmic (N:C) ratio. Occasional intermediate lymphocytes (red arrow), which are similar in size to neutrophils, have less condensed chromatin and slightly more cytoplasm. Occasional large lymphocytes, sometimes called lymphoblasts (black arrow), are larger than neutrophils, and have finely stippled chromatin and nucleoli, along with moderate amounts of basophilic cytoplasm. Numerous RBCs are consistent with blood contamination. (Wright-Giemsa stain, magnification 1000×)

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FIGURE 2 Aspirate of perinodal fat.

Simultaneous aspiration of fat can result in rupture of lymphocytes and a nondiagnostic preparation as shown. Adipocytes are large cells with a small oval nucleus (black arrow), condensed chromatin, and abundant clear cytoplasm. Ruptured lymphocytes appear as smudged pink-to-purple chromatin or as bare nuclei (red arrows). Evaluation of the lymphocytes is not possible without intact cytoplasmic and nuclear membranes. Obtaining aspirates of other enlarged nodes or a different area of the affected node is recommended when the slide contains primarily adipose tissue. (Wright-Giemsa stain, magnification 200×)

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FIGURE 3 Aspirate of salivary gland.

Due to close proximity, the mandibular salivary gland can be mistakenly aspirated when attempting to collect a sample from the mandibular lymph node. In this sample, note how the RBCs are arranged in a linear array (windrowing) from the viscosity of the mucus. The salivary gland epithelial cells (black arrows) occur individually or in small clusters and are characterized by abundant, vacuolated basophilic cytoplasm and round nuclei with condensed chromatin. Notice how these epithelial cells are much larger than the neutrophil in the lower right corner (red arrow). (Wright-Giemsa stain, magnification 200×)

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FIGURE 4 Aspirate showing plasma cell hyperplasia and macrophagic inflammation.

Three macrophages (black arrows) are characterized by abundant, vacuolated cytoplasm and irregularly shaped nuclei, with moderately condensed chromatin. Four plasma cells (red arrows), similar in size to the neutrophil (blue arrow), have eccentric nuclei, condensed chromatin, and abundant deeply basophilic cytoplasm that often contains a perinuclear clear area (Golgi region). In addition, numerous small lymphocytes, an intermediate lymphocyte (yellow arrow), and several RBCs are present. Plasma cell hyperplasia can develop with antigenic stimulation. Macrophagic inflammation can be attributed to infectious agents (e.g., fungal, protozoal, mycobacterial, or algal infections), vasculitis, chronic inflammation, and hemorrhage or hemolysis. (Wright-Giemsa stain, magnification 1000×)

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FIGURE 5 Aspirate showing lymphoid and plasma cell hyperplasia.

Note the size of 2 large lymphocytes (black arrows), as compared with the neutrophils (gray arrow). Note that the neutrophil granules stain more eosinophilic in this preparation, but are not as prominent as eosinophil granules (see Figures 7 and 10). Two of the several plasma cells contain numerous distinct clear inclusions (red arrows); these plasma cells are referred to as Mott cells, which have inclusions (ie, Russell bodies) packed in the cytoplasm. Russell bodies are typically round but can vary in size, shape, and color and can result from dilation of the rough endoplasmic reticulum during antibody production. Also present are numerous broken lymphocytes and RBCs. The sharply demarcated refractile areas in the RBCs are a common artifact. (Wright-Giemsa stain, magnification 1000×)

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FIGURE 6 Aspirate of marked mixed inflammation attributed to blastomycosis.

Most of the cells are neutrophils (red arrows), which are difficult to identify because they are degenerate and slightly out of focus to more clearly show the yeast form of Blastomyces dermatitidis (gray arrows). These large fungal organisms have characteristic features on routine staining: large size (diameter of 10-20 μm), blue color (very basophilic), and broad-based budding (as shown here), with a clear, thick cell wall. Several macrophages (black arrows) are also present. Mixed inflammation characterized by neutrophils and macrophages is typical of blastomycosis. (Wright-Giemsa stain, magnification 1000×)

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FIGURE 7 Aspirate showing neutrophilic and eosinophilic inflammation with plasma cell hyperplasia.

Increased numbers of neutrophils and eosinophils can indicate inflammation. Presence of a high proportion of eosinophils can be associated with hypersensitivity reactions, parasitic infection, neoplasia (e.g., mast cell tumor), or a paraneoplastic syndrome. Inflammation can be accompanied by lymphoid and plasma cell hyperplasia, as shown here, depending on the type of antigenic stimulation. Numerous small lymphocytes, several intermediate lymphocytes (red arrows), and a macrophage (black arrow) also are present. Several plasma cells (white arrows) and eosinophils (yellow arrows) are seen in the field. (Wright-Giemsa stain, magnification 500×)

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FIGURE 8 Aspirate of lymphoma.

Diffuse large cell lymphoma, the most common form of lymphoma in dogs, is characterized by lymphocytes larger in diameter than a neutrophil (yellow arrow). The chromatin pattern is fine, and one to multiple prominent nucleoli are evident. A mitotic figure is found in the upper right corner (black arrow). These large cells are often fragile, and cytoplasmic fragments (sometimes referred to as lymphoglandular bodies) may be present in the background (red arrows). Diagnosis of lymphoma may be confounded by superimposition of the hyperplastic response. The cytologic diagnosis of lymphoma involving small or intermediate lymphocytes can be more challenging and often necessitates biopsy, immunophenotyping by flow cytometry, or DNA based testing for confirmation. (Wright-Giemsa stain magnification 1000×)

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FIGURE 9 Aspirate of metastatic carcinoma.

Aspiration of regional lymph nodes may be helpful in determining whether a neoplasm has metastasized, as seen with this carcinoma. The neoplastic cells (red arrows) form cohesive clusters typical of cells of epithelial origin and exhibit many characteristics of malignancy: the nuclei are round and exhibit moderate anisokaryosis (variation in size of nuclei beyond what is normal for the tissue), chromatin is stippled, and a single prominent nucleolus is seen. These cells have a moderate amount of gray-blue cytoplasm with clear, punctate vacuoles. A population of primarily small lymphocytes and occasional intermediate and large lymphocytes confirms that this aspirate was obtained from a lymph node. When a node is effaced by neoplastic cells, lymphocytes may not be present. (Modified Wright-Giemsa stain, original magnification 200x)

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FIGURE 10 Aspirate of metastatic mast cell tumor obtained from a lymph node.

Aspiration of regional lymph nodes is often included as part of tumor staging. While mast cells may be found in low numbers in normal and hyperplastic lymph nodes, large numbers of mast cells, atypical mast cells, or mast cells in aggregates support a diagnosis of metastatic disease. Note that the mast cells (red arrows) are large and have a single nucleus with fine-to-stippled chromatin. Prominent nucleoli are evident in some of the mast cells. The amount of basophilic cytoplasm is moderate, with variable numbers of fine-to-coarse purple granules. In metastatic mast cell tumors, eosinophils (black arrows) can be found within lymph nodes because mast cells release chemoattractants for eosinophils. Free pink and purple granules present in the background are due to breakage of eosinophils and mast cells. A population of normal small lymphocytes (gray arrow) is present. It is important to remember that some mast cells stain poorly with commercial quick stains (eg, Diff-Quik), which makes the detection of metastatic mast cell disease much more difficult. (Modified Wright-Giemsa stain, original magnification 500×)