BACKGROUND: The majority of thyroid nodules are diagnosed using fine-needle aspiration (FNA) biopsies. The authors recently described the clinical validation of a molecular microRNA-based assay, RosettaGX Reveal, which can diagnose thyroid nodules as benign or suspicious using a single stained FNA smear. This paper describes the analytical validation of the assay. METHODS: More than 800 FNA slides were tested, including slides stained with Romanowsky-type and Papanicolaou stains. The assay was examined for the following features: intranodule concordance, effect of stain type, minimal acceptable RNA amounts, performance on low numbers of thyroid cells, effect of time since sampling, and analytical sensitivity, specificity, and reproducibility. RESULTS: The assay can be run on FNA slides for which as little as 1% of the cells are thyroid epithelial cells or from which only 5 ng of RNA have been extracted. Samples composed entirely of blood failed quality control and were not classified. Stain type did not affect performance. All slides were stored at room temperature. However, the length of time between FNA sampling and processing did not affect assay performance. There was a high level of concordance between laboratories (96%), and the concordance for slides created from the same FNA pass was 93%. CONCLUSIONS: The microRNA-based assay was robust to various physical processing conditions and to differing sample characteristics. Given the assay's performance, robustness, and use of routinely prepared FNA slides, it has the potential to provide valuable aid for physicians in the diagnosis of thyroid nodules. Cancer Cytopathol 2016;124:711-21. © 2016 Rosetta Genomics. Cancer Cytopathology published by Wiley Periodicals, Inc. on behalf of American Cancer Society.

Department of Surgery and Translational Medicine Pathology University of Milano Bicocca Monza Italy

Institute of Clinical and Molecular Pathology and Institute of Molecular and Translational Medicine Faculty of Medicine and Dentistry Palacky University and University Hospital of Olomouc Olomouc Czech Republic

Pathology Institute Tel Aviv Sourasky Medical Center Tel Aviv Israel

Rosetta Genomics Inc Philadelphia Pennsylvania

Rosetta Genomics Ltd Rehovot Israel

The Johns Hopkins University School of Medicine Baltimore Maryland

The Sackler School of Medicine Tel Aviv University Tel Aviv Israel

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Molecular markers in well-differentiated thyroid cancer

Multicentre validation of a microRNA-based assay for diagnosing indeterminate thyroid nodules utilising fine needle aspirate smears