Fluorescence in situ hybridisation BRAF-mutation analysis on pre-stained smears from thyroid fine-needle aspiration cytology

Abstract

Background: PTC is a differentiated thyroid carcinoma and the most common (70-80%) among all thyroid cancers. BRAFV600E-mutation is the most prevalent in PTC and a highly sensitive marker. Ultrasoundguided fine-needle aspiration cytology (FNAC) as a fundamental tool is highly sensitive for diagnosing thyroid nodules. However, ~25 % are of indeterminate lesions. Hence, the purpose is to develop a FISH BRAFV600E-mutation analysis on pre-operative thyroid FNAC smears stained with MGG or DiffQuick to eliminate such indeterminate lesions. Methods: MGG or DQ-stained FNAC smears of pre-operative thyroid nodules histologically verified as PTC were used to work out a FISH BRAF-mutation analysis. ~50 smears were tested, where different parameters on the FISH assay were combined for an optimal protocol. After protocol modification, 28 smears from 14 individuals, where 14 smears were stained with a BRAF break-apart translocation probe and the rest 14 with BRAF amplification probe, were tested. Results: Optimal results with a combination of a TE buffer (pH 9), pepsin digestion and post-fixation of smears in formalin 10 %, were obtained. Signals of BRAF break-apart probe and amplification probe were detected using a Zeiss confocal microscope. Conclusion: A modified FISH method on air-dried and Giemsa/DQ stained smears from thyroid FNAC is suitable on areas with least bloodstain on smears avoiding an autofluorescence. A combination of pretreatment buffer TE at pH 9, pepsin digestion and a 5 mins post-fixation in formalin 10 % are highly beneficial steps in developing this method.