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Background Molecular testing of thyroid fine‐needle aspirates has demonstrated value in cases of indeterminate cytology (Bethesda categories III, IV, and V) enabling optimized individual patient management leading to better outcomes with health economic benefits. For most molecular testing modalities, including mutational panels and classifier analyses, part or all of a dedicated needle aspiration pass is required to obtain an adequate sample for testing. Our analysis, which is based on a combination approach (mutation detection and microRNA classifier status), has documented clinical validity and utility when performed on thyroid fine‐needle aspirates placed directly into RNA preservative fluid. Here we show that the combination approach can be extended to microdissected stained cytology slides provides the physician greater opportunity to resolve cytological indeterminacy. Methods Extracted nucleic acid from needle aspirate and corresponding cytology preparations of 47 thyroid nodules were analyzed using identical methodology and results were compared. Results Of 94 molecular analyses (47 mutational analyses, 47 microRNA classifier assessments based on a validated 10 marker panel) only 5 samples showed discordant results. Conclusion These findings, together with supplementary work using archival specimens shows that the combination approach can be effectively applied to both direct aspirated thyroid nodule aspirates or to nucleic acid extracted from macrodissected and microdissected cytology slide smears, with the expectation of equivalent results. The advantages of both specimen sources, direct aspirate, and cytology slide smears are discussed.

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