Lymph node cytology in practice

Introduction

Lymph node fine-needle aspiration cytology (FNAC) is a common procedure in veterinary practice, and evaluation of lymph node aspirates carries many potential benefits, including detection of various neoplastic processes, inflammation, and infectious organisms. This article provides a guide for evaluation of lymph node cytology in the first opinion practice.

Reactive lymph node versus lymphoma

Lymph nodes can become reactive secondary to any source of antigenic stimulation. Cytologically, reactive lymph nodes contain a predominance of small lymphocytes with lower numbers (typically < 50%) of intermediate to large lymphocytes. Plasma cells are oftentimes increased in reactive lymph nodes, and Mott cells may also be observed (Figure 1). These are a type of plasma cell that contains discrete, light blue, cytoplasmic inclusions called Russell bodies 1. Mitotic figures can also be observed in reactive lymph nodes, but should not be seen frequently. 

In cases of large cell lymphoma, a predominance of intermediate to large lymphocytes (> 50%) is expected (Figure 2), whilst plasma cells, small lymphocytes and neutrophils are infrequent. Cytoplasmic fragments (also known as lymphoglandular bodies) may be plentiful in the background. 

While cytology is a powerful tool in the diagnosis of lymphoma, there are scenarios in which arriving at a confident diagnosis can be challenging: 

• Small cell lymphoma: small lymphocytes predominate in both reactive lymph nodes and small cell lymphoma cases, so these entities can be difficult or impossible to differentiate on cytology alone. One potential clue for when small cell lymphoma is present is the appearance of cytoplasmic pseudopods (Figure 3). These elongated cytoplasmic projections extend from the lymphocytes and are sometimes referred to as “hand-mirror cells”. This morphology is suggestive of indolent T-zone lymphoma, which has a prolonged clinical course 2. While the presence of cells with this extension is not pathognomonic for small cell lymphoma, it can certainly increase suspicion or support a presumptive diagnosis. Definitive diagnosis of small cell lymphoma often involves ancillary diagnostic testing such as PCR for antigen receptor rearrangement (PARR), flow cytometry, or histopathology. 
• Developing lymphoma: lymph nodes that have not yet become effaced by neoplastic cells can pose a diagnostic challenge. Here, if the needle has passed through both normal and neoplastic areas of the lymph node, the cytologic sample will contain a mixture of normal small lymphocytes and neoplastic large lymphocytes. In cases of developing large cell lymphoma, it may still be possible to achieve a confident diagnosis; however, in some cases the mixture of small to large lymphocytes straddles 50:50, and the interpretation must be approached with caution. Ancillary diagnostics as set out above can be useful in these cases. 
• Markedly reactive lymph nodes: such lymph nodes can in some cases contain a proportion of large lymphocytes which approach 50% of the total lymphoid population. Again, these cases often necessitate additional diagnostics to differentiate marked reactivity from lymphoma.

Inflammatory lymph nodes

Various types of inflammation can be seen in the lymph node, and identification of the inflammatory pattern can help the practitioner develop a differential list. 

• Neutrophilic inflammation: when neutrophils comprise > 5% of the cell population in a lymph node aspirate, this indicates neutrophilic inflammation (Figure 4) 3. This is a non-specific type of inflammation which can be seen with many different pathologic processes, including infectious disease, immune-mediated disease, trauma, vasculitis, and neoplasia. When neutrophils are degenerate (i.e., neutrophils which contain a swollen, hypo-segmented nucleus), a thorough search for bacteria is warranted. 
• Eosinophilic inflammation: when eosinophils comprise > 3% of the cell population in a lymph node aspirate, this indicates eosinophilic inflammation (Figure 5) 3. Low numbers of mast cells can also be seen in association with this type of inflammation. Causes vary and include allergic/hypersensitivity response, infectious disease, immune-mediated disease, and as a paraneoplastic response. This inflammatory pattern is often associated with allergic/atopic dogs or those with ectoparasites, with these cases often showing mild to moderate generalized lymphadenomegaly. Some infectious diseases can also elicit eosinophilic inflammation within lymph nodes, whilst neoplastic conditions that can elicit paraneoplastic eosinophilic inflammation include (but are not limited to) mast cell neoplasia, lymphoma, and carcinoma. It can sometimes be challenging to differentiate eosinophilic inflammation with mast cells from metastatic mast cell neoplasia with paraneoplastic eosinophilic inflammation. Differentiation between these scenarios may rely on the clinical history (i.e., does the patient have a known mast cell tumor?) and histopathology of the lymph node. 
• Histiocytic/pyogranulomatous inflammation: increased numbers of macrophages are present with histiocytic inflammation. Pyogranulomatous inflammation includes an increase in the number of neutrophils and macrophages, and multinucleated giant cells may also be present (Figure 6). This inflammatory pattern can be seen with a variety of pathologies, including systemic fungal infections, immune-mediated disease, salmon poisoning disease, and mycobacteriosis.

Infectious lymphadenopathy

Bacteria can be seen in lymph node aspirates in association with systemic or local infection. Abscessation of the lymph node will cause severe neutrophilic inflammation with degenerate neutrophils. Bacteria may or may not be seen cytologically, thus additional testing (e.g., bacterial culture or special staining) may be warranted to further investigate possible infection. Non-specific rod and cocci may be observed. However, some types of bacteria contain unique features, such as Mycobacterium spp. (non-staining rods), Actinomyces/Nocardia spp. (beaded, filamentous rods), and Yersinia pestis (bipolar coccobacilli). Observation of these features can aid in identification of the type of bacterial infection on cytology. 

Various fungal diseases can affect the lymph nodes. Blastomycosis, histoplasmosis, cryptococcosis, and coccidiomycosis are all dimorphic fungal infections which can occur in companion animals; the causal organism is inhaled from the environment and can spread to various locations in the body, including the lymph nodes. As noted above, pyogranulomatous inflammation is the classic inflammatory pattern seen with these infections. The four causal fungal organisms appear differently cytologically:

• Blastomyces dermatitidis: Yeasts are 7-15 µm in diameter and have a refractile, double-contoured cell wall (Figure 7). They exhibit broad-based budding. Yeasts can be found in any area of an infected animal’s body, including skin, organs, bones and joint fluid. 
• Histoplasma capsulatum: Yeasts are 2-4 µm in diameter and round with a thin clear halo (Figure 8). The cytoplasm is basophilic and sometimes appears as a “crescent moon” shape. The inflammatory response can be suppurative, granulomatous, or pyogranulomatous. Yeasts are often found inside macrophages and can also be seen extracellularly. 
• Cryptococcus neoformans: Yeasts vary in size from 2-20 µm and most often exhibit a thick clear “capsule” which is actually due to shrinkage of the yeast (Figure 9). Narrow-based budding is often observed. The inflammatory response is usually less robust than in other types of infectious disease, as the capsule eludes the animal’s immune system.
• Coccidioides immitis: Spherules are large, measuring between 20-200 µm. Endospores measure 2-5 µm and are contained inside the spherules until they are released. Organisms may not be prominent in aspirates from affected animals, so ancillary testing may be necessary for detection. 

Mention should also be made of Oomycetes (water molds): Pythium insidiosum and Lagenidium spp. differ from true fungi, and exhibit branching hyphae on cytology. Pyogranulomatous inflammation with eosinophils is common (Figure 10).

Metastatic neoplasia to lymph nodes

Lymph node fine-needle aspiration cytology is a common tool used in practice to assess for metastatic disease. Several studies have been published evaluating the accuracy of this method for staging, with findings showing good to excellent sensitivity for detection of mast cell neoplasia, malignant melanoma, and carcinoma 4,5. Detection of sarcoma metastasis with FNAC is not as reliable, likely because of the poor exfoliation of many sarcoma types. 

Any type of carcinoma can metastasize to the lymph node. Three specific types that may be seen are squamous cell carcinoma, urothelial carcinoma (also known as transitional cell carcinoma), and mammary carcinoma. Unique morphologic characteristics are often noted in cases of metastatic squamous cell carcinoma and urothelial carcinoma. Squamous cell carcinoma consists of a combination of atypical epithelial cells organized in clusters and individually (Figure 11). Asynchronous nuclear to cytoplasmic maturation, including inappropriate keratinization, can lead to the specific interpretation of squamous cell carcinoma. Urothelial carcinoma may not exhibit identifying characteristics, but the presence of Melamed-Wolinska bodies can serve as a clue. These are pink cytoplasmic inclusions that have been reported in urothelial carcinoma, and also in mammary carcinoma and mesothelioma (Figure 12) 6. Metastatic mammary neoplasia does not typically exhibit unique features that allow the observer to specifically identify it as mammary origin – which is why correlation with the clinical history and presence of any mass lesions is critical in these cases. 

High-grade mast cell tumors can metastasize to lymph nodes. When the metastatic process is advanced, cytologic diagnosis is typically straightforward, as large numbers of mast cells are present (Figure 13). However, when metastatic disease is in the early stages, only low numbers of mast cells may be seen. This often poses a diagnostic challenge, since low numbers of mast cells can be seen normally in lymph nodes and as part of inflammatory disease. In these cases, histopathologic evaluation and/or serial aspiration of the lymph node may be necessary for definitive assessment. 

Malignant melanomas often metastasize to both local and distant lymph nodes. Neoplastic melanocytes can exhibit rounded, polygonal, or spindle-shaped borders, and will often contain cytoplasmic melanin granules which can exhibit a range of colors, including dark yellow/brown, green, blue, and black. Cells may exhibit significant cytologic atypia, including anisocytosis, anisokaryosis, multi-nucleation, and prominent nucleoli (Figure 14). Similarly to mast cell neoplasia, a lymph node that is becoming effaced with neoplastic melanocytes lends itself to a straightforward diagnosis of metastatic disease. However, when low numbers of melanocytes are noted, these may represent a normal lymph node (Figure 15). In these cases, histopathologic evaluation and/or serial aspiration of the lymph node may be necessary for definitive assessment. 

Conclusion

Lymph node fine-needle aspiration cytology is a valuable tool for investigating causes of lymphadenomegaly and staging cancer in companion animals. While the interpretation of lymph node cytology can be complex, with practice and guidance, general practitioners can gain confidence in this skill, although the clinician should be aware that pitfalls do exist in some cases.