Application of Liquid-Based Cytology Using the ThinPrep System to Thyroid Fine-Needle Aspiration Samples

Fine-needle aspiration (FNA) is the gold-standard diagnostic procedure for the initial evaluation of thyroid nodules because it is minimally invasive, cost-effective, and highly accurate for distinguishing benign from malignant lesions. However, conventional smear preparation is frequently affected by operator-dependent variability, uneven cell distribution, blood contamination, and air-drying artifacts, all of which may compromise specimen adequacy and diagnostic performance. Liquid-based cytology (LBC) has emerged as an effective alternative, offering standardized specimen processing, improved cellular preservation, and enhanced slide quality. Among the available LBC platforms, the ThinPrep system is one of the most extensively validated technologies for thyroid cytopathology and is increasingly integrated into routine clinical practice and molecular diagnostic workflows. This protocol provides a comprehensive, standardized workflow for processing thyroid FNA specimens using the ThinPrep liquid-based cytology system. The protocol describes specimen collection, transportation, fixation, processing with the ThinPrep 5000 processor, slide preparation, staining, and quality control procedures to ensure optimal cytomorphological evaluation. Recommendations for acceptable specimen preservation, including transport in PreservCyt solution or alternative fixation methods, are also provided. In addition, the protocol includes procedures for recovering the residual cellular material preserved in the ThinPrep vial for paraffin block processing, which allows for complementary studies, e.g., immunohistochemistry, fluorescence in situ hybridization (FISH), DNA and RNA extraction, and next-generation sequencing (NGS), thus expanding the diagnostic utility of thyroid cytology beyond conventional microscopic examination. When properly executed, this protocol generates high-quality monolayer cytology preparations suitable for standardized interpretation according to The Bethesda System for Reporting Thyroid Cytopathology (TBSRTC) while preserving residual material for downstream molecular analyses. Compared with conventional smear cytology, the ThinPrep system improves specimen reproducibility, reduces background artifacts and inadequate samples, facilitates laboratory standardization, and supports precision medicine applications. This protocol provides a practical and reproducible approach for both clinical diagnostic laboratories and translational research. © 2026 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol: Simultaneous preparation of ThinPrep cytology slides and cell blocks from thyroid fine-needle aspiration specimens using liquid-based cytology Alternate Protocol: Alternative ThinPrep FNA processing using CytoLyt and PreservCyt.