Indications for Performing Lymph Node Fine-Needle Biopsy

1.  Lymphadenomegaly: Enlargement of a single or multiple lymph nodes.

2.  Evaluation of lymphoma: Definitive classification ultimately requires immunophenotyping. However, cytology may help guide initial therapy while further diagnostic results are pending. Some molecular techniques such as PCR for receptor antigen rearrangement (PARR) can be performed on submitted slides. Cytological evaluation of lymph nodes and other tissues can aid in lymphoma staging.

3.  Evaluation of metastatic disease: Lymph node fine-needle biopsy is commonly utilized to evaluate for metastatic disease. When sampling for this reason, one must consider normal lymphatic drainage patterns.

Pearls Regarding Sampling and Submission

• When sampling very large lymph nodes, avoid sampling the centre. The central sinus areas may contain myriads of neoplastic lymphocytes yet may also be spared, containing few. Additionally, with tumor expansion, the possibility of central necrotic areas or hemorrhagic tissue exists, potentially leading to non-diagnostic samples.
• With generalized peripheral lymphadenomegaly, the prescapular and popliteal lymph nodes are preferred sampling sites. The mandibular lymph node is constantly exposed to oral cavity antigens and ideally should never be the solely sampled lymph node when generalized lymphadenomegaly is present. Close proximity to salivary glands also creates the potential for incidental salivary gland collection and non-diagnostic samples.
• Use a non-aspiratory technique. Lymphocytes tend to be fragile and aspiration sampling techniques creates enough negative pressure to readily rupture them. A non-aspiratory technique using multiple, quick, stabbing needle motions helps prevent cell rupture as well as excess blood contamination.
• Use gentle smear spreading techniques. Neoplastic lymphoid cells are easily ruptured even when employing gentle smearing methods. Only use the surface tension that is created between two glass slides to prepare smears. Rupturing is enhanced with corticosteroid administration (even a single dose); collect samples prior to administering therapeutics whenever possible.
• Avoid formalin fumes. Formalin exposure, including its fumes, to cytology smears will prematurely fix cells resulting in poor staining and non-diagnostic results. Cytologic and histologic samples should be stored and shipped separately to avoid formalin artifact.

Use a systematic approach to cytologic review. Before enthusiastically applying oil to view cells using an oil immersion lens, take a few moments to scan the entire slide at lower magnifications (40x, 100x). Review a systematic checklist to assess whether slides warrant further evaluation—see Basic Cytology notes. Assuming cellularity, cell preservation, cellular detail and staining are adequate, continue with a systematic approach now focused on meaningful details.

1.  Scan the entire slide at low magnification (40x, 100x) taking note of:

a.  Cellular arrangement—are cells found solely as individual cells, present in tight cohesive clusters, loose clumps or other arrangements? Lymphoid cells are typically found singly or in clumps of variable size and density. Finding cohesive clusters may serve as a clue for potential metastatic epithelial cells.

b.  Metastatic cells—although low magnification scanning does not allow cellular details to be seen, it is often easier to identify metastatic cells in a sea of lymphoid cells than at 1000x. Never be hasty and move to the oil immersion lens too quickly!

c.  Best areas for higher magnification—much of our time as cytologists is spent looking for areas on slides worthy to invest more time on at higher magnification. Distinguishing these areas saves time as we move from low to high magnification.

2.  Is the lymphoid population uniform or heterogeneous? Similar to metastatic cells, assessing the overall lymphoid population for uniformity (suggestive of lymphoma) or heterogeneity (suggestive of benign hyperplasia/reactivity) is often easier at lower magnification. I tend to utilize 200x magnification for this purpose.

3.  Categorize lymphoid and, if present, non-lymphoid populations. At higher magnification, perform lymphoid cell counts, recording the proportions of small, medium and large lymphocytes, lymphoblasts and plasma cells. If present, evaluation of inflammatory cells, metastatic cells or any infectious organisms is also performed.

Cytological evaluation of lymph nodes requires experience. Fortunately, lymph node cytologic interpretations are generally limited to five major categories:

1.  Normal

• A normal lymph node is not grossly enlarged and is predominated by small lymphocytes (>90% of total cellularity) with fewer medium and large lymphocytes (collectively ∼8–10%). Occasional immature lymphocytes (lymphoblasts) and/or plasma cells may be present (∼<2–3% collectively). Macrophages and rare mast cells are occasionally present.
• Leukocyte numbers are proportional to the amount of blood present.
• The size of lymphocytes is conventionally determined by comparing the nucleus diameter to an erythrocyte (RBC).
• Small = 1–1.5x an RBC. Small lymphocytes have scant volumes of cytoplasm and dark clumped chromatin in the nuclei.
• Medium = 2–2.5x an RBC. Low cytoplasmic volumes and finely stippled to clumped chromatin.
• Large = >2.5x an RBC. Similar cytoplasm/chromatin as medium lymphocytes.

2.  Hyperplastic or Reactive

• Lymphocyte proportions in a hyperplastic lymph node are often left shifted (increased numbers of immature lymphocytes present). However, proportions may be similar to that of a normal lymph node, yet the lymph node is grossly enlarged. Finding normal lymphoid proportions in an enlarged lymph node should raise suspicion for a hyperplastic lymph node or possibly a small cell lymphoma.
• Proportions of medium and large lymphocytes increase but are typically and collectively less than 50%. Increased mitotic figures may or may not be seen.
• Plasma cell numbers may or may not be increased. When increased, this supports reactivity. Mott cells, which contain multiple spherical to linear, pale blue structures representing immunoglobulin secretions (Russell bodies) may be seen.
• Macrophages, neutrophils, eosinophils and mast cells may also mildly increase in response to antigen stimulation, but numbers are lower than expected in lymphadenitis.
• Look for a source of antigenic stimulation such as metastatic cells or organisms. The lymph node(s) may instead be responding to systemic antigenic stimulation or are draining an area of local stimulation such as a nearby abscess.

3.  Lymphadenitis

• Proportions of inflammatory cells are greater than expected with any concurrent blood contamination in the sample. Proportions vary depending on blood cell counts, but guidelines for diagnosing neutrophilic and eosinophilic lymphadenitis exist:
• Neutrophilic: Neutrophils comprise >5% of total cellularity
• Eosinophilic: Eosinophils comprise >3% of total cellularity
• Neutrophilic lymphadenitis is due to various infectious and non-infectious causes, including bacterial infections as well as neoplastic and immune-mediated conditions.
• Eosinophilic lymphadenitis is commonly associated with flea bite hypersensitivity, feline eosinophilic skin disease, hypereosinophilic syndrome and as a paraneoplastic syndrome with mast cell tumors, certain lymphomas and infrequently with carcinomas and sarcomas.
• Histiocytic/macrophagic lymphadenitis is often associated with systemic fungal disease and other infectious diseases, including mycobacteriosis, leishmaniasis, protothecosis, pythiosis and salmon fluke poisoning disease.
• Combinations of inflammatory cells may be present.

4.  Lymphoma

• Diagnosis of lymphoma is straightforward when a uniform population of lymphoblasts or medium to large lymphocytes predominates (>50%, often >90% of total cellularity).
• Diagnosis of small cell lymphomas can be challenging. Small lymphocytes comprising these types of lymphomas often have less clumped chromatin compared to normal lymphocytes. They also tend to have mildly increased volumes of cytoplasm localized to a rounded point, giving cells a hand-held mirror or tadpole appearance. The pointing direction these cytoplasmic tails is random, making a smearing effect unlikely. This morphology has been reported with T-cell lymphomas but is by no means confirmatory for neoplasia as it also occurs with normal and hyperplastic lymph nodes.
• Importantly, there is a strong uniformity to overall cell morphology, regardless of cell size. This is sometimes better appreciated at 200x or 500x than at 1000x magnification.
• Increased numbers of mitotic figures may or may not be seen.
• Concurrent hyperplasia/reactivity may be present, complicating interpretation.

5.  Metastatic disease

• Finding metastatic cells depends on how much of the lymph node is affected by the tumor and if the needle happened to enter an affected area during sampling. Otherwise, cytology is as accurate as histology in identifying metastatic disease. Collecting multiple samples helps increase odds of identifying metastatic cells.
• Entire slides need to be examined. A lack of metastatic cells does not completely rule out metastasis.
• Metastatic cells are easily identified when large or present in cohesive clusters, sheets, acini or other arrangements atypical of lymphocytes.
• Hyperplasia/reactivity and/or lymphadenitis may be concurrently present.
• Histiocytes and epithelioid macrophages can easily be misinterpreted as neoplastic cells. Foamy vacuolated macrophages with evidence of cytophagia and presence of other inflammatory cells favours inflammation rather than metastasis.
• Occasional melanophages and melanocytes may be present in normal or hyperplastic/reactive lymph nodes draining areas of dermatitis, especially in dark pigmented patients. While potentially suspicious for metastatic melanoma, occasional widely dispersed pigmented-laden cells are insufficient to confirm it. Hemosiderin must be differentiated from melanin using special stains. High numbers of pigmented cells, especially when found in groups or clusters, and/or with sufficient malignant criteria, are required for a confident cytologic diagnosis of metastatic melanoma.
• Low numbers of mast cells are present in normal and hyperplastic/reactive lymph nodes, especially if draining an area affected by hypersensitivity or parasitic disease. This is also the case for gastrointestinal lymph nodes, which often have more inflammatory cells, including plasma cells and mast cells. Similar to diagnosing metastatic melanoma, confidence of metastatic mast cell neoplasia is much greater when mast cells are numerous, present in clusters and display numerous criteria of malignancy.

References

1.  Barger AM, MacNeill A. Small Animal Cytologic Diagnosis. 1st ed. Boca Raton, FL: CRC Press; 2017.

2.  Cian F, Freeman, K. Veterinary Cytology. 2nd ed. Boca Raton, FL: CRC Press; 2017.

3.  Raskin RE, Meyer D. Canine and Feline Cytology: A Color Atlas and Interpretation Guide. 3rd ed. St. Louis, MO: Elsevier; 2016.

4.  Valenciano AC, Cowell RL. Cowell and Tyler’s Diagnostic Cytology and Hematology of the Dog and Cat. 4th ed. St. Louis, MO: Elsevier; 2013.

5.  Szladovits B. Cytology in Practice. Proceedings of the World Small Animal Veterinary Association. 2014. [internet]. Serbia: WSAVA; 2014. Available from: www.sasap.org.rs/download/pf_1423495947_fe22.pdf.