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

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