Preanalytic variables in quality and quantity of nucleic acids extracted from FNA specimens of thyroid gland nodules collected in CytoLyt: Cellularity and storage time.

TitlePreanalytic variables in quality and quantity of nucleic acids extracted from FNA specimens of thyroid gland nodules collected in CytoLyt: Cellularity and storage time.
Publication TypeJournal Article
Year of Publication2020
AuthorsHeymann JJ, Yoxtheimer LM, Park HJin, Fernandez EM, Facey KE, Alperstein SA, Tran HV, Baek I, Scognamiglio T, Rennert H, Siddiqui MT, Song W
JournalCancer Cytopathol
Volume128
Issue9
Pagination656-672
Date Published2020 09
ISSN1934-6638
KeywordsAdult, Aged, Aged, 80 and over, Biopsy, Fine-Needle, Cytodiagnosis, Female, Follow-Up Studies, Genetic Testing, Humans, Male, Middle Aged, Mutation, Nucleic Acids, Prognosis, Retrospective Studies, Specimen Handling, Thyroid Gland, Thyroid Neoplasms, Thyroid Nodule, Young Adult
Abstract

BACKGROUND: Morphologic and genetic analysis of thyroid nodules may be performed from a single vial. Preanalytic variables that affect nucleic acid extracted from a single vial are evaluated.

METHODS: Thyroid fine-needle aspiration (FNA) specimens collected in CytoLyt were evaluated. A ThinPrep slide was prepared. Extracted nucleic acids were analyzed using Oncomine Comprehensive Panel, version 2, after Ion AmpliSeq library preparation. A pathologist and a cytotechnologist enumerated specimen cellularity.

RESULTS: Fifty-six samples were collected from 55 nodules in 53 patients. Bethesda category correlated with cellularity (P = .01), and storage time (median, 43 days; range, 7-77 days) was longer for specimens in categories II and III than for those in categories IV and VI (P = .01). The mean specimen DNA concentration was 4.5 ng/µL (range, 0-23.8 ng/µL), and 25 (45%) had concentrations >3.3 ng/µL. The mean specimen RNA concentration was 4.8 ng/µL (range, 0-42.4 ng/µL), and 31 (55%) had concentrations >1.4 ng/µL. Nucleic acid quantity increased with epithelial cellularity. Storage time weakly correlated with the quantity of extracted DNA, independent of cellularity, but not extracted RNA. Greater proportions of cell-free DNA and lesser proportions of long, intact RNA fragments were extracted from a subset of samples with longer storage time. Among 15 single nucleotide variants, the median mutant allelic fraction was 15.1%. One false-negative result was identified. Five specimens subsequently determined to harbor a genetic alteration failed quality metrics.

CONCLUSIONS: Cellularity and storage time affect the quantity and quality of nucleic acid extracted from thyroid FNA specimens collected in CytoLyt. Further investigation will serve to quantify the magnitude of such effects and to elucidate other contributing factors.

DOI10.1002/cncy.22270
Alternate JournalCancer Cytopathol
PubMed ID32267620
Related Faculty: 
Jonas Heymann, M.D. Momin Siddiqui, M.D. Theresa Scognamiglio, M.D.

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