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J Clin Endocrinol Metab. 2012 Dec;97(12):E2297-306. doi: 10.1210/jc.2012-1923. Epub 2012 Oct 18.

Analytical performance verification of a molecular diagnostic for cytology-indeterminate thyroid nodules.

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Veracyte, Inc., South San Francisco, California 94080, USA.



Our objective was to verify the analytical performance of the Afirma gene expression classifier (GEC) in the classification of cytologically indeterminate thyroid nodule fine-needle aspirates (FNAs).


Analytical performance studies were designed to characterize the stability of RNA in FNAs during collection and shipment, analytical sensitivity as applied to input RNA concentration and malignant/benign FNA mixtures, analytical specificity (i.e. potentially interfering substances) as tested on blood and genomic DNA, and assay performance studies including intra-nodule, intraassay, inter-assay, and inter-laboratory reproducibility.


RNA content within FNAs preserved in FNAProtect is stable for up to 6 d at room temperature with no changes in RNA yield (P = 0.58) or quality (P = 0.56). FNA storage and shipping temperatures were found to have no significant effect on GEC scores (P = 0.55) or calls (100% concordance). Analytical sensitivity studies demonstrated tolerance to variation in RNA input (5-25 ng) and to the dilution of malignant FNA material down to 20%. Analytical specificity studies using malignant samples mixed with blood (up to 83%) and genomic DNA (up to 30%) demonstrated negligible assay interference with respect to false-negative calls, although benign FNA samples mixed with relatively high proportions of blood demonstrated a potential for false-positive calls. The test is reproducible from extraction through GEC result, including variation across operators, runs, reagent lots, and laboratories (sd of 0.158 for scores on a >6 unit scale).


Analytical sensitivity, analytical specificity, robustness, and quality control of the GEC were successfully verified, indicating its suitability for clinical use.

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