Assessment of cellularity, genomic DNA yields, and technical platforms for BRAF mutational testing in thyroid fine‐needle aspirate samples

Abstract

BACKGROUND: BRAF mutation V600E (substitution Val600Glu) is a molecular signature for papillary thyroid carcinoma (PTC). Testing for BRAF mutation is clinically useful in providing prognostic prediction and facilitating accurate diagnosis of PTC in thyroid fine-needle aspirate (FNA) samples. METHODS: This study assessed the correlation of cellularity with DNA yield and compared 2 technical platforms with different sensitivities in detection of BRAF mutation in cytologic specimens. Cellularity was evaluated based on groups of 101 cells on a ThinPrep slide: 11 (1-5 groups), 21 (6-10 groups), 31 (11-20 groups), and 41 (> 20 groups). Genomic DNA was extracted from residual materials of thyroid FNAs after cytologic diagnosis. RESULTS: Approximately 49% of thyroid FNA samples had low cellularity (1-21). DNA yield is proportionate with increased cellularity and increased nearly 4-fold from 11 to 41 cellularity in cytologic samples. When applied to BRAF mutational assay, using a cutoff of 6 groups of follicular cells with 101 cells per group, 96.7% of cases yielded enough DNA for at least one testing for BRAF mutation. Five specimens (11.6%) with lower cellularity did not yield sufficient DNA for duplicate testing. Comparison of Sanger sequencing to allele-specific polymerase chain reaction methods shows the latter confers better sensitivity in detection of BRAF mutation, especially in limited cytologic specimens with a lower percentage of malignant cells. CONCLUSIONS: This study demonstrates that by using 6 groups of 101 follicular cells as a cutoff, nearly 97% of thyroid FNA samples contain enough DNA for BRAF mutational assay. Careful selection of a molecular testing system with high sensitivity facilitates the successful conduction of molecular testing in limited cytologic specimens.

Lymph Node Fine-Needle Cytology: Beyond Flow Cytometry

“…Considering that 40 ng of DNA/RNA may be obtained from 4 × 10 6 cells, a couple of additional passes from LN FNC may be sufficient to obtain genetic material sufficient for any procedure. Considering the technical support, genetic material has been successfully obtained from different cytological supports, such as smears, cell blocks or cryopreseved cells [7,36,102,103]. This variability in technical supports is due to the usage of archival material or FNC performed by clinicians for routine diagnostic purposes that is only subsequently utilized for retrospective molecular studies.…”

High-Resolution Melting Is a Sensitive, Cost-Effective, Time-Saving Technique for BRAF V600E Detection in Thyroid FNAB Washing Liquid: A Prospective Cohort Study

“…Differently from previous reports [32], in our hands HRM is a valid methodology also in samples characterized by poor cellularity (<6 clusters of 10 cells/group).…”

Cytomorphologic Evaluation of the Thin Prep for Fine Needle Aspiration of the Fresh Thyroidectomy Specimens

“…The best quality in specimens may be obtained because blood and debris may be avoided from the floor and it is less-invasive [8]. It allows protection of nuclear details and decreased scan times with cytopathology, immunohistochemistry and molecular diagnostics [3,[9][10][11]. In some cases, the cell block has importance because of its pivotal role in diagnosis of preparation samples [11].…”