Lymph Node Assessment in Veterinary Practice: A Comprehensive Guide to Cytological Evaluation

Lymph node assessment constitutes a fundamental diagnostic modality in veterinary medicine, providing critical pathological data for neoplastic staging, inflammatory disease characterization, and infectious agent identification. Fine-needle aspiration cytology has established itself as a minimally invasive, cost-effective diagnostic technique enabling rapid cytomorphological evaluation of lymphadenopathy. This review examines contemporary best practices in lymph node assessment, emphasizing optimal sampling methodologies, systematic cytological interpretation, and integration with advanced diagnostic platforms including digital pathology systems such as Pathview. Recent technological advances in sentinel lymph node mapping and digital cytology have transformed lymph node evaluation protocols, facilitating enhanced precision in staging algorithms and therapeutic decision matrices.

Introduction

Lymph node cytology represents one of the most frequently utilized diagnostic procedures in veterinary clinical pathology, comprising a substantial proportion of cytological specimens submitted for pathological evaluation (Meichner et al., 2023). This diagnostic modality offers significant advantages over histopathological examination, including rapid turnaround time, minimal invasiveness, and cost-effectiveness, establishing it as an indispensable component of routine diagnostic protocols (Raskin & Meyer, 2010).

The clinical significance of precise lymph node assessment has been amplified by developments in precision oncology and targeted immunotherapy. Contemporary therapeutic protocols increasingly require detailed staging data, rendering lymph node evaluation essential for optimal patient management algorithms (Beer et al., 2018). Furthermore, integration of digital pathology platforms has enhanced accessibility and standardization of lymph node cytological interpretation, enabling telepathology consultation and standardized educational resources (Piccione et al., 2025).

Anatomy and Physiology of Lymph Nodes

Normal Lymph Node Structure

Lymph nodes serve as specialized filtration organs within the lymphatic system, designed to trap antigens and facilitate immune responses. Understanding normal lymph node architecture is essential for recognizing pathological changes during cytological evaluation.

Normal lymph nodes contain a diverse population of cells, with small lymphocytes comprising 80-90% of the cellular population (Raskin & Meyer, 2010). These cells are characterized by high nuclear-to-cytoplasmic ratios, condensed chromatin, and minimal cytoplasm. The remaining cellular population consists of intermediate lymphocytes (10-15%), large lymphocytes or lymphoblasts (2-5%), plasma cells (<5%), and scattered macrophages and dendritic cells.

Lymphatic Drainage Patterns

Recent advances in sentinel lymph node mapping have highlighted the complexity of lymphatic drainage patterns, demonstrating that regional lymph nodes may not always correspond to the actual draining lymph node (Beer et al., 2018). Computed tomography lymphography has emerged as a valuable alternative to lymphoscintigraphy for sentinel lymph node identification, particularly in cases where radiotracer availability is limited (Ferraris et al., 2023).

Indications for Lymph Node Assessment

Neoplastic Staging Requirements

Lymph node assessment is critical for staging various neoplasms, particularly those demonstrating high metastatic potential. Mast cell tumors, melanomas, histiocytic sarcomas, and most carcinomas require comprehensive lymph node evaluation, even in the absence of palpable lymphadenomegaly (Krick et al., 2009). The sensitivity and specificity of fine-needle aspiration for detecting metastatic disease demonstrate significant variation depending on tumor histotype and sampling methodology.

Lymphadenopathy Evaluation

Lymphadenopathy may result from reactive hyperplasia, neoplasia (primary or metastatic), inflammatory processes, or infectious etiologies. Systematic cytological evaluation enables differentiation between these pathological entities, directing appropriate therapeutic interventions.

Sampling Techniques and Preparation

Fine-Needle Aspiration Technique

Optimal sampling technique is essential for obtaining diagnostically adequate specimens. The decision between aspiration and non-aspiration techniques depends on the specific characteristics of the target lymph node and the operator's preference. See our article outlining this here.

Sample Preparation and Staining Protocols

Rapid preparation of high-quality cytological preparations is essential for optimal diagnostic interpretation. The squash preparation technique utilizing two glass slides typically provides optimal cellular morphology and distribution. Romanowsky-type stains (Wright-Giemsa) remain the standard for routine lymph node cytology, providing excellent nuclear chromatin detail and cytoplasmic characteristics.

When submitting specimens for external evaluation, inclusion of both stained and unstained slides is essential, as individual pathologists may prefer specific staining protocols for optimal interpretation.

Cytological Interpretation

Normal Cytological Findings

Normal lymph nodes demonstrate a predominance of small lymphocytes with occasional intermediate and large lymphocytes. The presence of tingible body macrophages, which contain phagocytosed nuclear debris, indicates active cellular turnover and is considered a normal finding.

Plasma cells may be present in small numbers (<10%) in normal lymph nodes, particularly in nodes draining areas exposed to environmental antigens. The identification of Russell bodies (Mott cells) within plasma cells suggests active antibody production and is not inherently pathological.

Reactive Hyperplasia

Reactive hyperplasia represents the most common cause of lymphadenopathy in dogs and cats. Cytologically, reactive lymph nodes maintain a mixed population of lymphocytes but show increased numbers of intermediate and large lymphocytes (typically <50% combined). Increased mitotic activity may be observed, but atypical mitotic figures should not be present.

The presence of increased plasma cells, particularly those containing Russell bodies, supports a diagnosis of reactive hyperplasia. Tingible body macrophages are often increased in reactive lymph nodes, reflecting enhanced cellular turnover.

Inflammatory Patterns

Neutrophilic Inflammation

When neutrophils comprise >5% of the cellular population, neutrophilic inflammation is present (Baker, 2023; Meichner et al., 2023). This pattern can be associated with bacterial infections, immune-mediated diseases, trauma, or neoplasia. The presence of degenerate neutrophils warrants careful examination for bacterial organisms.

Eosinophilic Inflammation

Eosinophils comprising >3% of the cellular population indicate eosinophilic inflammation (Baker, 2023; Meichner et al., 2023). This pattern can be associated with hypersensitivity reactions, parasitic infections, or paraneoplastic responses to certain neoplasms, including mast cell tumors and lymphomas.

Granulomatous Inflammation

The presence of increased macrophages and multinucleated giant cells suggests granulomatous inflammation (Baker, 2023). This pattern often indicates fungal infections, immune-mediated diseases, or foreign body reactions. Careful examination for infectious organisms is essential when this pattern is identified.

Neoplastic Changes

Primary Lymphoid Neoplasia

Lymphoma diagnosis requires identification of a monomorphic population of lymphocytes, typically with increased numbers of large lymphocytes or lymphoblasts. The traditional "rule of 20" suggests that >20% large lymphocytes in a lymph node aspirate supports a diagnosis of lymphoma, though this guideline has limitations.

Recent studies have emphasized the importance of phenotyping for accurate lymphoma diagnosis and prognosis. B-cell lymphomas generally have better response rates and longer survival times compared to T-cell lymphomas when treated with conventional chemotherapy protocols.

Metastatic Neoplasia

The identification of non-lymphoid cells in lymph node aspirates suggests the presence of metastatic disease. Careful attention to cellular morphology, including nuclear characteristics, cytoplasmic features, and cellular arrangements, is essential for identifying metastatic cells.

Round cell tumors, including mast cell tumors, plasma cell tumors, and histiocytic neoplasms, can be particularly challenging to differentiate from primary lymphoid neoplasms. The use of cytochemical stains and immunocytochemistry can aid in definitive diagnosis.

Advanced Diagnostic Modalities

Flow Cytometry

Flow cytometry provides valuable information regarding lymphocyte populations and can aid in lymphoma diagnosis and phenotyping. This technique is particularly useful when cytological findings are equivocal or when specific prognostic information is required.

Sample preparation for flow cytometry requires special handling, including suspension in appropriate media and rapid processing. Coordination with the testing laboratory is essential to ensure optimal sample quality.

Digital Pathology and PathView Integration

The integration of digital pathology platforms has revolutionized lymph node cytology education and consultation. PathView and similar platforms enable high-resolution imaging of cytological preparations, facilitating remote consultation and standardized case review.

Digital cytology offers several advantages, including improved image quality, enhanced collaboration capabilities, and the ability to create comprehensive case databases for educational purposes. Recent validation studies have demonstrated excellent concordance between digital microscopy and traditional light microscopy for lymph node cytology interpretation (Bonsembiante et al., 2019).

Molecular Diagnostics

Advanced molecular techniques, including PCR-based assays and next-generation sequencing, are increasingly being applied to lymph node samples. These techniques can provide valuable information regarding clonality, specific genetic mutations, and therapeutic targets.

The K9 lymphoma assay represents a significant advancement in canine lymphoma diagnostics, enabling comprehensive genetic profiling of lymphoid neoplasms. This 31-gene panel can identify actionable mutations and provide prognostic information that complements traditional morphological assessment (Marconato et al., 2024).

Quality Assurance and Standardization

Sample Quality Assessment

Adequate sample quality is essential for accurate cytological interpretation. Factors affecting sample quality include proper sampling technique, rapid slide preparation, appropriate staining, and timely evaluation. The development of standardized quality metrics has improved the reliability of lymph node cytology.

Inter-observer Variability

Studies evaluating inter-observer agreement in lymph node cytology have highlighted the importance of standardized criteria and ongoing education. The use of digital pathology platforms can facilitate standardized training and quality assurance programs.

Integration with Clinical Findings

Lymph node cytology should always be interpreted in the context of clinical findings, including signalment, history, physical examination, and other diagnostic test results. Close communication between clinicians and pathologists is essential for optimal diagnostic accuracy.

Limitations and Pitfalls

Sampling Limitations

Despite its many advantages, fine-needle aspiration cytology has inherent limitations. The technique samples only a small portion of the lymph node, potentially missing focal lesions or providing unrepresentative samples. In cases where cytological findings are inconsistent with clinical suspicion, histopathological examination should be considered.

Interpretation Challenges

Certain conditions can be challenging to diagnose cytologically, including indolent lymphomas (marginal zone lymphoma, T-zone lymphoma), early metastatic disease, and specific infectious agents. The development of standardized diagnostic criteria and the integration of ancillary testing can help address these challenges.

Species-Specific Considerations

Important species differences exist in lymph node cytology interpretation. Feline lymph nodes are more commonly reactive than neoplastic, and in-house diagnosis of feline lymphoma should always be confirmed by a pathologist (Watson et al., 2015). Additionally, breed-specific variations in lymphocyte populations have been reported and should be considered during interpretation (Bexfield et al., 2008).

Conclusion

Lymph node assessment remains a fundamental component of veterinary diagnostic medicine, providing critical information for disease staging, diagnosis, and treatment planning. The integration of advanced diagnostic modalities, including digital pathology platforms like PathView, has enhanced the accuracy and accessibility of lymph node cytology.

Successful lymph node assessment requires a thorough understanding of normal lymph node biology, proper sampling techniques, systematic cytological interpretation, and integration with clinical findings. Ongoing education and quality assurance programs are essential for maintaining diagnostic excellence in this rapidly evolving field.

References

Baker, K. (2023). Interpreting lymph node aspirates of cats and dogs. VetFocus, 33(2), 28-35.

Beer, P., Pozzi, A., Rohrer Bley, C., Bacon, N., Pfammatter, N. S., & Venzin, C. (2018). The role of sentinel lymph node mapping in small animal veterinary medicine: a comparison with current approaches in human medicine. Veterinary and Comparative Oncology, 16(2), 178-187.

Bexfield, N. H., Heiene, R., Gerritsen, R. J., Risoen, U., Eliassen, K. A., Herrtage, M. E., & German, A. J. (2008). Breed-specific hematological phenotypes in the dog: a natural resource for the genetic dissection of hematological parameters in a mammalian species. PLoS One, 3(9), e3080.

Bonsembiante, F., Bonfanti, U., Cian, F., Cavicchioli, L., Zattoni, B., & Gelain, M. E. (2019). Diagnostic validation of a whole-slide imaging scanner in cytological samples: diagnostic accuracy and comparison with light microscopy. Veterinary Pathology, 56(3), 429-434.

Ferraris, E. I., Olimpo, M., Giacobino, D., Manassero, L., Iussich, S., Lardone, E., ... & Morello, E. M. (2023). Sentinel lymph node mapping with computed tomography lymphography for mast cell tumours and a comparison between regional and sentinel lymph node histological status: sixty-two cases. Veterinary and Comparative Oncology, 21(2), 208-220.

Krick, E. L., Billings, A. P., Shofer, F. S., Watanabe, S., & Sorenmo, K. U. (2009). Cytological lymph node evaluation in dogs with mast cell tumours: association with grade and survival. Veterinary and Comparative Oncology, 7(2), 130-138.

Marconato, L., Martini, V., Aresu, L., Sampaio, M., Valentini, F., Rinaldi, V., ... & Comazzi, S. (2024). The K9 lymphoma assay allows a genetic subgrouping of canine lymphomas with improved risk classification. Scientific Reports, 14, Article 16193.

Meichner, K., Watson, V. E., & Sharkey, L. C. (2023). Small animal lymph node cytology. Today's Veterinary Practice, 33(3), 42-49.

Piccione, J., Anderson, S. F., Neal, S. V., & Varvil, M. S. (2025). Digital pathology in veterinary clinical pathology: A review. Veterinary Pathology, 62(1), 45-58.

Raskin, R., & Meyer, D. (2010). Canine and feline cytology: A color atlas and interpretation guide (2nd ed.). Saunders Elsevier.

Watson, V. E., Hobbs, S., Comazzi, S., May, C., & Gelain, M. E. (2015). The diagnostic utility of lymph node cytology samples in dogs and cats. Journal of Small Animal Practice, 56(2), 125-129.

Corresponding Author: Dr. [Author Name], DVM, DACVP Department of Veterinary Pathology [Institution Name] Email: [email]

Keywords: lymph node cytology, veterinary pathology, digital pathology, PathView, sentinel lymph node mapping, canine lymphoma, metastatic disease