B-392 Standardization of Nucleic acid Extraction Method From Thyroid Fine Needle Aspiration (FNA) Cells on Stained and Fixed Slides for Next-Generation Sequencing Protocols
Authors: Martins L A , Domingues R S , de Moraes G M , Penna G C , Zauli D A , de Lima A B 
Published: September 2023

Abstract

Background
Thyroid Fine Needle Aspiration (FNA) cytopathology is the best method for histological classification of thyroid nodules. However, between 20%–30% of thyroid nodules show indeterminate classification and are often referred for surgery due to the malignancy risk. Post-surgical histological reports indicate that 84% of these cases are benign, highlighting a high rate of unnecessary surgeries. In this sense, molecular approaches using next-generation sequencing (NGS) have been useful to confirm benignity or malignancy. As important as the choice of a powerful methodology to evaluate different genes simultaneously such as NGS, is the standardization of nucleic acid extraction methods to allow sufficient quality recovery of these molecules. Pre-analytical steps involved in FNA slide preparation include fixation and stain methods that contribute to the low quality and poor recovery of nucleic acid, representing a great challenge to NGS analysis. Thus, this work aims to describe a standardization of nucleic acid extraction method from FNA slides for NGS protocols.

Methodology
Nucleic acid isolation from 30 FNA samples collected between 2016–2022 were performed by Trizol or RecoverAll kits, according to the manufacturer’s instructions (ThermoScientific). Initially, cells’ slides were counted in an optical microscope, considering cell clusters &gt;10 (equivalent to 1 counter unit). Before extractions with Trizol, the slides were submerged in xylene for 15 min. For RecoverAll extractions, the varnished slides were previously exposed to dry ice for 5 min, while the slides containing coverslip were also exposed to dry ice and submerged in xylene for 15 min. In all cases, the slides were hydrated before scraping and then extracted. Subsequently, the extraction of whole slides and split slides was tested. To split slides, one-half of the slide was separated for RNA extraction, while the remaining half was for DNA extraction. Then, the DNA/RNA were quantified using the Qubit™ 4 Fluorometer equipment (ThermoScientific) and concentrated using Amicon® Ultra filters (Millipore).

Results
The DNA and RNA quantifications range from 0.0720–1.77 ng/µL and 0.508–7.43 ng/µL, respectively. Among samples, 12% (DNA) and 62.5% (RNA) presented concentration below the quantification limit (too low). Four samples extracted with Trizol did not obtain recovery of nucleic acid and this methodology was discontinued. The use of Amicon® filter improved all samples’ yield. The number of thyroid cell clusters was not proportional to the amount of DNA/RNA recovered. The split slides showed worst RNA/DNA recovery, being ideal to use at least two slides for each patient since co-extraction using the RecoverAll kit was not possible.

Conclusion
The isolations of nucleic acid from FNA slides are essential to performing subsequent molecular tests, such as NGS. However, specific protocols for nucleic acid extraction from FNA slides are hardly found in the literature. This work showed the combination of methodologies that allowed the scraping of material from FNA slides by exposure to dry ice in samples with varnish or coverslip. The nucleic acid recovery capacity described for this type of sample indicates a way of possibilities to improve the FNA extraction methodology.