Lymph node fine-needle aspiration and cytology is a noninvasive, rapid, and cost-effective diagnostic tool. This article highlights a step-by-step approach to lymph node cytology after sample acquisition, explains the most common cytologic findings, and describes how to recognize nondiagnostic and poor-quality specimens.<\/p>\n
A thin, highly cellular smear with predominantly intact cells is crucial for accurate interpretation; making an interpretation from a thick smear or one with predominantly ruptured cells can be misleading. Every smear should be screened on low power to evaluate if the sample is adequate, to recognize unevenly distributed cells (i.e., metastatic cells), and to identify the area of interest for detailed high-power microscopy.<\/p>\n
Take-Home Points<\/strong><\/p>\n Lymph node cytology submissions account for a large proportion of all cytology submissions to a veterinary pathologist. Lymph node cytology can also be performed in-house to evaluate the quality of a smear before sending it to a pathologist for final interpretation or to make a preliminary diagnosis if rapid therapeutic intervention is indicated. Common clinical indications to perform lymph node cytology include1,2<\/sup>:<\/span><\/p>\n A detailed description of how to obtain a good sample for cytology, including supplies, collection methods, smear preparation techniques, and processing, has been published elsewhere<\/a>.3<\/sup> Every cytology interpretation starts with a good-quality diagnostic specimen that, ideally, is a thin, highly cellular preparation of intact cells. Smears can be made from an FNA sample and\/or a touch imprint.3-6<\/sup> Lymphoid cells generally exfoliate well. Techniques vary, and aspiration or nonaspiration techniques can be used. The nonaspiration technique may be preferred for vascular and\/or firm lesions in which aspiration tends to produce hemodiluted specimens (e.g., metastatic lymph node) and especially with delicate cell populations such as lymphocytes.3-6<\/sup> <\/span><\/p>\n To make a good-quality smear, gentle pressure should be used to spread cells; applying too much pressure can cause excessive cell damage. For lymph node aspirates, especially lymphoma, the \u201croll preparation\u201d technique has been successful at the author\u2019s institution in minimizing cell lysis that can occur with the traditional smear technique.3<\/sup> If FNA is performed using ultrasound guidance, the ultrasound gel must be wiped from the surface prior to inserting the needle through the skin. Even small amounts of ultrasound gel, like lubricant, can adhere to cells, causing damage and poor cell staining and resulting in nondiagnostic or suboptimal slides.7<\/sup> <\/span><\/p>\n Smears should be air dried, not heat fixed\/dried, and stained with a Romanowsky-type stain (e.g., Wright-Giemsa). The water-based Romanowsky-type stains (Diff-Quik) are inexpensive, fast, and easy to use and work very well for lymphoid cells. Compared with alcohol-based Romanowsky-type stains, the nuclear chromatin can appear slightly coarser with Diff-Quik (<\/strong><\/span>FIGURE 1<\/span><\/strong>)<\/strong>. The sample should be fixed properly in the fixation solution to ensure adequate staining. Thick, highly cellular smears may require more time for adequate fixation.<\/span><\/p>\n Figure 1A. Lymph node aspirate from a dog with lymphoma with predominantly large lymphocytes. (A) Aqueous Romanowsky stain (Diff-Quik), 100\u00d7 objective. Note the coarser texture of the chromatin and prominent nucleoli (arrows) compared with B.<\/p><\/div>\n Figure 1B. Lymph node aspirate from a dog with lymphoma with predominantly large lymphocytes. Alcohol-based Romanowsky stain (modified Wright-Giemsa) from the same aspirate, 100\u00d7 objective. The nuclear chromatin texture is finely stippled and smooth. Nucleoli (arrows) are visible but not as prominent as in A.<\/p><\/div>\n Step 1:<\/b> Determine whether the sample is adequate and highly cellular. <\/span><\/p>\n Screening multiple areas on the slide using low power is essential in this stage of the evaluation. Do not rush to oil. Slide examination should begin by focusing on low power (4\u00d7 and 10\u00d7 objectives). The distribution of certain cell types, such as metastatic cells, can be patchy, and such cells can be easily missed when starting with the 40\u00d7 objective and rushing to oil (50\u00d7 or 100\u00d7). If metastatic neoplasia is suspected, a thorough low-power screen of all slides is necessary. <\/span><\/p>\n If the sample is not adequate (e.g., acellular, poorly cellular, all cells are broken, only blood is present), resampling or performing a different test (i.e., surgical biopsy) is recommended.<\/span><\/p>\n Step 2:<\/b> Identify the area of interest for high-power microscopy. Avoid examining poorly stained or overtly thick regions. Thick or poorly stained areas can make cell size difficult to judge (<\/strong><\/span>FIGURES 2A AND 2B<\/span><\/strong>)<\/strong>. Additionally, pale staining results in pale blue nuclei that lack sharp chromatin detail and nucleoli that can appear very prominent; therefore, an understained lymph node sample can mimic lymphoma.<\/span><\/p>\n Figure 2A. Identifying the most appropriate area for microscopic evaluation of a lymph node aspirate is crucial. (A AND B) Areas that are too thick will be understained. In A, red blood cells appear in dense, thick aggregates (arrows) and are piled on top of each other.<\/p><\/div>\n Figure 2B. Identifying the most appropriate area for microscopic evaluation of a lymph node aspirate is crucial. (A AND B) Areas that are too thick will be understained.<\/p><\/div>\n When moving to high-power microscopy, if only a 40\u00d7 objective (no 50\u00d7) is available, a thin coverslip should be placed on the stained smear. A smear without a coverslip will look blurry (<\/strong><\/span>FIGURE 3<\/span><\/strong>)<\/strong>. Oil should never be used with the 40\u00d7 objective.<\/span><\/p>\n Figure 3A. Lymph node aspirate from a cat. Without a coverslip, the field appears blurry, which prevents evaluation of cellular details.<\/p><\/div>\n Figure 3B. Same area as A, with a coverslip on top of the slide. Cellular details are clearly visible. Wright-Giemsa stain, 40\u00d7 objective.<\/p><\/div>\n Step 3:<\/b> <\/span>Confirm cells are intact (FIGURES 2C AND 2D<\/span><\/strong>)<\/strong>.<\/span><\/p>\n Figure 2C. Many ruptured cells (asterisks) are present along with a few small, intact lymphocytes and a neutrophil. Ruptured cells should not be falsely interpreted as large lymphocytes.<\/p><\/div>\n Figure 2D. Pink streaming nuclear material from ruptured cells. Rare intact lymphocytes (arrows) are present.<\/p><\/div>\n Step 4:<\/b> Size the lymphocytes. A thin area with intact cells should be used. Thin areas can be identified by red blood cells in the background of the slide that are not overtly piled up. <\/span><\/p>\n There are different ways to size lymphocytes; a helpful measuring stick is the neutrophil. A small lymphocyte will be smaller than a neutrophil and will have coarse, dark chromatin and just a small amount of cytoplasm, often visible on only one side of the cell. Intermediate-sized lymphocytes are approximately neutrophil-sized. They have a little more cytoplasm, and the chromatin is slightly more \u201copen\u201d (dispersed, stains paler, and is less clumped). Large lymphocytes are larger than a neutrophil (up to 4 times the diameter of a red blood cell); have pale, lacy chromatin and a thin rim of cytoplasm; and may or may not have visible nucleoli. <\/span><\/p>\n Step 5:<\/b> Evaluate the relative percentages of small, intermediate, and large lymphocytes (<\/strong><\/span>FIGURES 2E AND 2F<\/span><\/strong>)<\/strong>. Also look for plasma cells, histiocytes\/macrophages, granulocytes, mast cells, and other cell types (e.g., atypical cells). The proportion of nondegenerate neutrophils should be interpreted in light of the amount of hemodilution as peripheral blood contamination can increase the number of leukocytes, especially neutrophils, if\u00a0 <\/span>leukocytosis is present.<\/span><\/p>\n Figure 2E. Markedly hemodiluted lymph node aspirate. Too few lymphocytes are present for interpretation.<\/p><\/div>\n Figure 2F. Adequate area characterized by a thin monolayer with predominantly intact cells that are nicely spread out. All Figure 2 images, Wright-Giemsa stain; A-E, 100\u00d7 objective; F, 50\u00d7 objective.<\/p><\/div>\n Step 6:<\/b> Interpret findings.<\/span><\/p>\n The 3 basic findings for lymph tissue samples are (1) normal or unremarkable, (2) reactive\/hyperplastic, and (3) lymphoma. Additional lymph node findings include inflammation\/infection, metastatic neoplasia, and nondiagnostic specimens (e.g., acellular, aspiration of perinodal fat, salivary tissue).8<\/sup><\/span><\/p>\n In a normal lymph node, small, mature lymphocytes compose the majority of cells (approximately 75% to 95%; <\/span>FIGURE 4A<\/span><\/strong>).8,9<\/sup> The remaining cells are primarily intermediate lymphocytes and a few large lymphocytes. Rare plasma cells may be seen; these cells have moderate amounts of deeply basophilic cytoplasm with a perinuclear clear zone (Golgi zone) and a round, eccentrically located nucleus with very coarse chromatin. Mott cells are plasma cells distended with large vacuoles of immunoglobulin (Russell bodies). Rare histiocytes\/macrophages and\/or mast cells may be seen; however, mast cell granules may not stain with Diff-Quik. Granulocytes should be proportional to the degree of hemodilution. Small basophilic fragments of lymphocyte cytoplasm (lymphoglandular bodies) are often present in the background. They are highly characteristic of lymphoid tissue but are not specific for lymphoma.<\/span><\/p>\n Figure 4A. Lymph node aspirate from a cytologically unremarkable lymph node. Small lymphocytes predominate and are smaller in diameter than a neutrophil (arrow). Wright-Giemsa stain, 50\u00d7\u00a0objective.<\/p><\/div>\n A \u201cnormal\u201d cytologic appearance in a lymph node that is grossly increased in size can be consistent with either hyperplasia (reactive) or small-cell lymphoma. Small-cell lymphoma usually requires additional testing for diagnosis, such as histopathology, flow cytometry, and\/or polymerase chain reaction for antigen receptor rearrangement. Discussion of these advanced diagnostics can be found elsewhere.10,11<\/sup> <\/span><\/p>\n A reactive, hyperplastic lymph node has a mixed appearance on low magnification, with significant variation in cell size and chromatin texture and\/or color (dark and coarsely clumped to pale and open). In contrast, \u201cnormal\u201d and \u201clymphoma\u201d nodes appear more homogenous. Small lymphocytes still predominate in a reactive lymph node, but more intermediate and large lymphocytes are present (FIGURE<\/span><\/strong><\/span>\u00a04B<\/span><\/strong>)<\/strong>, and there may be an increased number of plasma cells and\/or Mott cells (FIGURE<\/span><\/strong><\/span>\u00a04C<\/span><\/strong>)<\/strong>. Increases in other cell types (macrophages, mast cells) are variable.8,9<\/sup> Neutrophils and eosinophils may also be increased but represent less than 5% and 3% of the nucleated cell count, respectively (if greater, then the node is inflamed).<\/span><\/p>\n Figure 4B. Lymph node aspirate from a reactive lymph node. Small lymphocytes (black arrow) still predominate, but the lymphoid population is mixed, with intermediate (orange arrow) and large lymphocytes (asterisk) also present. Wright-Giemsa stain, 100\u00d7 objective.<\/p><\/div>\n Figure 4C. A group of plasma cells, which is a common finding in reactive lymph nodes. Wright-Giemsa stain, 100\u00d7 objective.<\/p><\/div>\n A single reactive lymph node or region of nodes can be due to any source of inflammation and\/or antigenic stimulation in the draining area, such as skin disease, an abscess, injury, or an inflamed neoplasm. Generalized reactions affecting most or all peripheral and\/or internal lymph nodes can be seen with systemic infections (e.g., Rocky Mountain spotted fever, ehrlichiosis\/anaplasmosis<\/a>, feline leukemia virus<\/a>, feline immunodeficiency virus), immune-mediated disease, or any other systemic inflammatory condition (e.g., atopic dermatitis).<\/span><\/p>\n Lymphoma is the top differential in any dog with generalized lymphadenopathy. Lymphoma of the lymph node (large-cell lymphoma) can be diagnosed when large lymphocytes represent more than 50% of the cell population, but most cases have more than 80% to 90% of these cells (FIGURES<\/span><\/strong><\/span> 1 AND 5<\/span><\/strong>)<\/strong>.8,9<\/sup> Unlike many malignant neoplastic populations, lymphoma tends to be relatively uniform on cytology (less anisocytosis and anisokaryosis). Lymphoglandular bodies are often numerous in the background, and mitotic figures may be seen. However, the last 2\u00a0findings can also be observed in reactive lymph nodes. Other, less common, variants than large-cell lymphoma also exist (e.g., T-zone lymphoma, small-\/intermediate-cell lymphoma, large granular lymphocyte lymphoma, lymphoma with a mixed-cell appearance).8,12<\/sup><\/span><\/p>\n Figure 5. Aspirate from an enlarged lymph node from a dog with lymphoma. Large lymphocytes predominate throughout the smear. A neutrophil is shown at the bottom for size comparison. Lymphocytes have small amounts of deeply basophilic cytoplasm and a nucleus with fine chromatin and multiple visible nucleoli. Small, deeply basophilic cytoplasmic fragments (lymphoglandular bodies) (arrows) are scattered in the background. Wright-Giemsa stain, 100\u00d7 objective.<\/p><\/div>\n In cats, peripheral lymphadenopathy is much more commonly reactive than lymphoma. An in-house diagnosis of lymphoma in the peripheral lymph node of a cat should always be confirmed by a pathologist.<\/span><\/p>\n Lymphadenitis<\/strong><\/p>\n Inflammation within the lymph node is characterized by increased numbers of neutrophils (>5%), eosinophils (>3%), macrophages, or multinucleated giant cells. Reactive hyperplasia is usually also present. Neutrophilic or pyogranulomatous inflammation should trigger a thorough screening for etiologic agents (e.g., bacteria, protozoa, fungi, algae; <\/span>FIGURE 6A<\/span><\/strong>). Some neoplasms can induce neutrophilic inflammation if metastatic to the lymph node (e.g., squamous cell carcinoma).8,9<\/sup><\/span><\/p>\n Figure 6A. Lymph node aspirate from a dog with fever, cough, and mild peripheral lymphadenopathy. A group of 3 Blastomyces species yeast cells is present in the center of the image, surrounded by mixed lymphocytes, some macrophages (asterisks), and mildly increased neutrophils (arrows). Wright-Giemsa stain, 100\u00d7 objective.<\/p><\/div>\n Metastatic Neoplasia<\/strong><\/p>\n This is characterized by the presence of a foreign cell population (e.g., epithelial cells in metastatic carcinoma) or high numbers of a cell type that should only be present in very low numbers (e.g., mast cells, melanocytes; FIGURES<\/span><\/strong><\/span>\u00a06B AND 6C<\/span><\/strong>).8,9<\/sup> The absence of metastatic cells on cytology does not exclude the possibility of metastatic disease, due to sample collection bias (e.g., minimally sampled metastatic lymph node, sample collected during early metastasis from area of a metastatic lymph node that did not contain metastatic cells).<\/span><\/p>\n Figure 6B. Submandibular lymph node aspirate from a dog with chronic nasal discharge and recent onset of epistaxis. Rare small clusters of large cells (black box) are identified on low-power examination. Wright-Giemsa stain, 10\u00d7 objective.<\/p><\/div>\n Figure 6C. Same case as B. On high-power examination, these cells are pleomorphic epithelial cells surrounded by mixed lymphocytes. The diagnosis was metastatic carcinoma. The primary tumor was a nasal adenocarcinoma. The metastatic cells in the lymph node could have been easily missed without a thorough low-power screen. Wright-Giemsa stain, 100\u00d7 objective.<\/p><\/div>\n Adipose Tissue<\/strong><\/p>\n Perinodal adipose tissue appears similar to normal subcutaneous fat. Popliteal lymph nodes are often surrounded by prominent adipose tissue (FIGURE<\/span><\/strong><\/span>\u00a07<\/span><\/strong>)<\/strong>.<\/span><\/p>\n\n
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Lymph Node Cytology Slide Preparation<\/h2>\n
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<\/a>Approach to Lymph Node Sample Evaluation<\/h2>\n
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<\/a>POSSIBLE LYMPH NODE CYTOLOGY FINDINGS<\/h2>\n
Normal Lymph Node<\/h3>\n
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<\/a>Hyperplastic\/Reactive Lymph Node<\/h3>\n
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<\/a>Lymphoma<\/h3>\n
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<\/a>Other Important Findings<\/h3>\n
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