Test Quality Control

Negative controls consisting of double‐distilled water were processed alongside clinical samples. Two negative controls were used: One was processed starting at the RNA extraction stage, and the other was processed from the complimentary DNA (cDNA) synthesis stage. These negative controls ensured that samples were not contaminated during either of these stages.

The assay's positive control consisted of a mix of RNAs extracted from several benign and malignant samples of thyroid formalin‐fixed, paraffin‐embedded (FFPE) resections, which were augmented with RNA extracted from whole blood to ensure detectable expression levels of all miRNAs measured by the assay. FFPE samples were used as positive controls to ensure high expression levels of all 24 assay miRNAs in these samples. In addition, resection tissue provides a high miRNA yield, facilitating use of the same positive control sample for extended periods of time. A positive control sample was run alongside each batch of clinical samples to ensure that the cDNA and polymerase chain reaction (PCR) reagents yielded consistent results. Each sample was profiled in duplicate. If repeats differed beyond an allowed range, then PCR was repeated on the sample.

The assay analyzed thyroid FNA smears to detect miRNA blood markers, thyroid epithelial markers, and malignant/benign markers. Samples that displayed high blood marker levels and low epithelial marker levels were deemed as not having enough thyroid tissue, were not classified, and were reported as failed. In addition, samples in which malignant/benign marker levels were below an allowed threshold were failed for insufficient RNA levels and were not classified.

The assay classifier measured 24 miRNAs and combined several linear discriminant analysis (linear DA) steps and a K‐nearest neighbor classifier step to differentiate between benign samples and samples that were “suspicious for malignancy by miRNA profiling”. Medullary carcinoma samples were classified by a linear discriminant analysis step based on hsa‐miR‐375 expression. Samples classified in this step received a final classification of “positive for medullary marker”. The assay's performance was based on miRNA correlations and thus did not require miRNA expression level normalization.

The assay was developed in Rosetta Genomics' research and development laboratory (Rosetta Genomics Ltd, Rehovot, Israel‐RG‐IL) and was then transferred to the Rosetta Genomics' Philadelphia laboratory (Rosetta Genomics, Inc, Philadelphia, Pa‐RGL‐US), which is certified under the Clinical Laboratory Improvement Amendments (CLIA) Act of 1988 to perform high‐complexity testing and is accredited by the College of American Pathologists. Both laboratories participated in the inter‐laboratory reproducibility studies.

The study was run on 2 types of tissue samples: FNA‐like samples (¼ of a 5‐micron FFPE resection sample diluted with whole blood; n = 8) and FNA smear slides that were cut in half, with half of the slide extracted and quantified in each laboratory (total = 27 samples).

Pearson correlation was used to compare miRNA expression profiles between samples. The chi‐square test was used for comparisons of correctness of classification under different slide characteristics, and the coefficient of determination (R²) metric was used to assess marker linearity between different RNA concentrations.

A series of 8 dilutions of positive control RNA (dilution factor = 2.5) was used to reach a final RNA quantity range of 2 × 10⁻³ ng to 1.25 ng. Assay markers were quantified in triplicate. Linearity of assay markers in the relevant cycle threshold (C_T) range was evaluated by calculating efficiency and R² values. PCR efficiency was calculated using the following formula: efficiency = (2^[−1/slope] − 1) × 100, where slope denotes the function slope of the linear fit between cycle threshold (C_T) values and log₂ (RNA quantity).