The Critical Role of Ancillary Testing in Fine-Needle Aspiration of Lymph Nodes, Thymus, and Spleen

Background: Fine-needle aspiration cytology (FNAC) is a minimally invasive and reliable technique for sampling lymph nodes, thymus, and spleen. However, morphological interpretation alone is often insufficient due to overlapping features among reactive, infectious, and neoplastic processes. Objective: To review the essential role of ancillary testing in enhancing the diagnostic accuracy of FNAC in lymphoid and mediastinal organs. Methods: A narrative review of the literature focusing on the applications, strengths, and limitations of flow cytometry, immunocytochemistry, in situ hybridization, and molecular testing in FNAC samples from lymph nodes, thymus, and spleen. Results: Ancillary studies significantly increase the diagnostic precision of FNAC, enabling lineage assignment, clonality determination, detection of defining genetic alterations, and identification of therapeutic biomarkers. These techniques are particularly valuable in paucicellular aspirates, mediastinal lesions, splenic lymphomas, and cases with overlapping morphological patterns. Conclusion: Integration of ancillary techniques with cytomorphology aligns FNAC with modern WHO and IAC–IARC–WHO diagnostic frameworks, transforming it into a powerful multiparametric tool that supports accurate diagnosis, subclassification, prognostication, and treatment planning.