The Cytological Grey Zone: Mapping Concealed Morphological Signatures in Indeterminate Thyroid Fine-Needle Aspiration Cytology

Background
Thyroid nodules rank as one of the most prevalent endocrine discoveries, with fine-needle aspiration cytology (FNAC) serving as the primary minimally invasive method for their assessment. Despite the recent 2023 revision of the Bethesda System for Reporting Thyroid Cytopathology (TBSRTC), which has established standardized terminology and malignancy-risk assessments, a significant number of aspirates continue to be classified as indeterminate specifically atypia of undetermined significance (Bethesda III), follicular neoplasm (Bethesda IV), and suspicious for malignancy (Bethesda V). These classifications possess intersecting nuclear, architectural, cytoplasmic, and contextual characteristics that constrain the distinguishing capability of any individual morphological standard.
Aim
The goal is to propose a structured methodology for detecting and categorizing nuanced, composite ("hidden") morphological indicators in indeterminate thyroid FNAC, as well as to explain how these indicators may be linked to histopathology, supplementary/molecular assessments, and malignancy risk, thereby improving diagnostic differentiation within the cytological grey area.
Methods
A systematic narrative synthesis of the 2023 TBSRTC and current literature regarding nuclear, architectural, cytoplasmic, and background characteristics in Bethesda III–V thyroid FNAC was conducted, enhanced by data on cytohistological correlation, supplementary immunocytochemistry, molecular classifiers, and AI-assisted digital cytology. A five-domain conceptual framework known as the Cytological Grey-Zone Morphological Signature (CGZ-MS) was developed from this synthesis, categorising characteristics into Nuclear, Architectural, Cytoplasmic, and Background domains that culminate in an Integrated Risk domain.
Results
Composite cytomorphological patterns, as opposed to singular cytological anomalies, seem to be more reliably linked with malignant or borderline histology in Bethesda III–V thyroid tumours. The simultaneous occurrence of nuclear atypia, structural disarray, modified cytoplasmic differentiation, and a conducive cytological environment like psammomatous calcification and little colloid might offer enhanced diagnostic significance compared to depending solely on isolated characteristics. Incorporating immunocytochemical indicators such as thyroglobulin, TTF-1, PAX8, and calcitonin alongside genetic mutations and gene-expression classifiers such as BRAF, RAS, RET/PTC, and TERT, as well as Afirma and ThyroSeq platforms, could enhance risk categorization. The advent of AI-enhanced digital cytology may offer an extra dimension of objective analysis; yet, these methods should be viewed as supplementary to, rather than substitutes for, professional cytomorphological examination.
Conclusion
Systematic, multi-domain mapping of concealed morphological signatures may convert descriptive atypia into reproducible morphological phenotypes that complement, rather than replace, the 2023 Bethesda System. The proposed CGZ-MS framework is a conceptual, hypothesis-generating model that requires prospective, multicentre validation against histopathological and molecular outcomes before any diagnostic application.

Thyroid FNAC; Bethesda System 2023; cytological grey zone; atypia of undetermined significance; follicular neoplasm; morphological signature; molecular cytopathology; digital pathology; artificial intelligence.

1. Juhlin, C. C., & Baloch, Z. W. (2024). Pitfalls In Thyroid Fine-Needle Aspiration Cytopathology: An Approach To Atypical Findings. Acta Cytologica, 68(3), 179-193. [Google Scholar] [Crossref]

2. Canberk, S., & Baloch, Z. W. (2026). The Current Status Of Thyroid Cytology In The Era Of New Classifications, Molecular Profiling, And Clinical Risk Stratification. Acta Cytologica. [Google Scholar] [Crossref]

3. Saini, T., Kundu, R., Rohilla, M., Gupta, P., Gupta, N., Srinivasan, R., ... & Dey, P. (2024). Gray Zone Bethesda Category Iii–Atypia Of Undetermined Significance Lesions Of The Thyroid: Potential Diagnostic Issues And Image Morphometry As A Useful Adjunct To Cytomorphology. Cytojournal, 21, 38. [Google Scholar] [Crossref]

4. Piga, I., L’imperio, V., Capitoli, G., Denti, V., Smith, A., Magni, F., & Pagni, F. (2023). Paving The Path Toward Multi-Omics Approaches In The Diagnostic Challenges Faced In Thyroid Pathology. Expert Review Of Proteomics, 20(12), 419-437. [Google Scholar] [Crossref]

5. Guo, Y., Zhao, T., Zhang, L., Liu, Y., Liu, K., Han, B., ... & Zhao, L. (2026). Artificial Intelligence In Thyroid Ultrasound: Clinical Applications And Perspectives. Frontiers In Endocrinology, 17, 1877780. [Google Scholar] [Crossref]

6. Bilookyi, O. V., Hovornyan, S. L., & Bilookyi, V. V. (2026). Possibilities And Prospects Of Application Of Modern Methods In The Diagnostics Of Thyroid Diseases. International Journal Of Endocrinology (Ukraine), 22(5), 537-546. [Google Scholar] [Crossref]

7. Rossi, E. D., Piermattei, A., Cianfrini, F., Cappoli, N., Mulè, A., & Pantanowitz, L. (2026). Thyroid Cytology: Practical Tricks And Pitfalls. Virchows Archiv, 488(1), 129-146. [Google Scholar] [Crossref]

8. Tralongo, P., Ballato, M., Fiorentino, V., Zuccalà, V., Pizzimenti, C., Pepe, L. R., ... & Fadda, G. (2026). The Role Of Artificial Intelligence In Thyroid Cytology Of Indeterminate Nodules: From Digital Cytology To Multimodal Precision Triage. Frontiers In Endocrinology, 17, 1800918. [Google Scholar] [Crossref]

9. Ribeiro, M., Canberk, S., & Bongiovanni, M. (2026). The Role Of Cytology, Histology And Molecular Pathology In The Diagnostic Process Of Thyroid Nodules. Cancers, 18(11), 1814. [Google Scholar] [Crossref]

10. Bordoni, M., Aboud, N., Silvetti, F., Taccaliti, A., Balercia, G., & Salvio, G. (2025). Towards An Integrated Multi-Omic Approach To Improve The Diagnostic Accuracy Of Fine-Needle Aspiration In Thyroid Nodules With Indeterminate Cytology. Diagnostics, 15(12), 1506. [Google Scholar] [Crossref]

11. Capitoli, G., Piga, I., L’imperio, V., Clerici, F., Leni, D., Garancini, M., ... & Pagni, F. (2022). Cytomolecular Classification Of Thyroid Nodules Using Fine-Needle Washes Aspiration Biopsies. International Journal Of Molecular Sciences, 23(8), 4156. [Google Scholar] [Crossref]

12. Monia, B., Nairus, A., Francesca, S., Augusto, T., Giancarlo, B., & Gianmaria, S. (2025). Towards An Integrated Multi-Omic Approach To Improve The Diagnostic Accuracy Of Fine-Needle Aspiration In Thyroid Nodules With Indeterminate Cytology. Diagnostics, 15(12), 1506. [Google Scholar] [Crossref]

13. Suo, W., Guo, C., Zhang, X., Li, H., Lai, W., Zhang, J., ... & Zhang, J. (2026). Radiomics In Thyroid Nodule Assessment. Bmc Medical Imaging. [Google Scholar] [Crossref]

14. Braga, E. R., Braga Júnior, R. M., Braga, M. S., Cerutti, J. M., & Salcedo, W. J. (2026). Portable Multispectral Optoelectronic System For Thyroid Cancer Detection. Sensors, 26(14), 4448. [Google Scholar] [Crossref]

15. Kumar, S., Dutta, A., Singh, B. P., Vallur, S., & Singh, S. (2026). Ai-Driven Early Diagnosis And Management Of Head-And-Neck Malignancies: Current Insights And Future Directions. Indian Journal Of Otolaryngology And Head & Neck Surgery, 78(6), 3543-3556. [Google Scholar] [Crossref]

16. Li, M., Yang, D., Liu, T., Liu, S., Sun, W., Cai, P., ... & Li, J. (2026). Label-Free Molecular Profiling Of Cancer Using Raman Spectroscopy: From Fundamentals To Clinical Applications. Frontiers In Oncology, 16, 1822181. [Google Scholar] [Crossref]

17. Radhachandran, A. (2026). Deep Learning For Ultrasound Imaging: From Task-Specific Architectures To Foundation Models (Doctoral Dissertation, University Of California, Los Angeles). [Google Scholar] [Crossref]

18. Singh, A. P., Siddiqui, J., & Singh, V. (2026). Advanced Tissue Processing: Automation And Emerging Technologies. Indian Journal Of Pathology: Research And Practice, 15(1). [Google Scholar] [Crossref]

19. Verma, K., Kumar, N., Singh, R., & Singh, A. P. Path Lab-Ai: An Autonomous Framework For Error-Free Histopathology Slide Interpretation. Research & Reviews: A Journal Of Bioinformatics. 2026; 13 (1): 19–30p. Stm Journals 2026. All Rights Reserved, 20, 2. [Google Scholar] [Crossref]

20. Singh, A. P., Siddiqui, J., & Singh, V. (2026). Safety And Ethics In The Histopathology Laboratory. Indian Journal Of Pathology: Research And Practice, 15(1), 31-45. [Google Scholar] [Crossref]

21. Semwal, S., Mishra, P., Kumar, A., & Singh, A. P. (2026). Integrated Assessment Of Haematological, Inflammatory And Hormonal Biomarkers In Women With Polycystic Ovary Syndrome Aged 15–40 Years. Biochemical & Cellular Archives, 26(1), 951. [Google Scholar] [Crossref]

22. Singh, R., Verma, K., Kumar, N., & Singh, A. P. (2026). Beyond The H&E Slide: Uncovering Hidden Histopathological Signatures With Spatial Omics And Artificial Intelligence. Journal Of Basic And Applied Research International, 32(5), 75-91. [Google Scholar] [Crossref]

23. Singh, A. P., Saxena, R., & Saxena, S. (2023). Cytopathology: An Important Aspect Of Medical Diagnosis. Research & Reviews: Journal Of Oncology And Hematology. 2023; 12 (3): 13–18p. Cytopathology: An Important Aspect Of Medical Diagnosis Singh Et Al. Stm Journals, 2. [Google Scholar] [Crossref]

24. Rahman, H. A., & Singh, A. P. (2024). Section Cutting In Histopathology: An Update. Research & Reviews: A Journal Of Health Professions. 2024; 14 (1): 29–34p. Section Cutting In Histopathology: An Update Rahman And Singh Stm Journals, 2. [Google Scholar] [Crossref]

25. Cruz, S., & Murugan, J. (2026). Comprehensive Review Of Fuzzy Systems In Cancer Research. Archives Of Computational Methods In Engineering, 1-45. [Google Scholar] [Crossref]

• Measuring Waste of Patient Time in Health Care at Non-Digitized Hospital: An Observational Study in Bangabandhu Sheikh Mujib Medical University, Bangladesh
• Reaffirming Clinical Confidence in Atorvastatin Therapy: A Digital Outreach Case Study from Tamil Nadu, India
• Clinical Manifestations and Therapeutic Response in a Patient with Hypothyroidism: A Case Report
• Eranda (Ricinus Communis) In Gridhrasi (Sciatica): Classical Rationale, Pharmacology and Clinical Evidence- A Narrative Literature Review
• Magnetotherapy in Pain Management: Mechanisms, Clinical Applications, and Future Perspectives – A Review