Comparative Analysis of RNA Quality Between Paired Fresh-Frozen and Formalin-Fixed, Paraffin-Embedded Pancreatic Endoscopic Ultrasound-Guided Fine-Needle Biopsy Specimens: Implications for Biobanking and Molecular Diagnostics

Background: Pancreatic cancer is a highly lethal malignancy. We therefore aimed to compare RNA concentration, purity, integrity, and laboratory-assigned pre-assay quality control (QC) classifications between paired fresh-frozen (FF) and formalin-fixed, paraffin-embedded (FFPE) endoscopic ultrasound-guided fine-needle biopsy (EUS-FNB) specimens from solid pancreatic lesions. Methods: We evaluated 36 pairs of FF and FFPE EUS-FNB specimens for RNA concentration, absorbance ratios, RNA quality numbers (RQNs), and pre-assay QC classifications. FF tissues were placed in RNAlater-containing tubes, snap-frozen and transported in liquid nitrogen before storage in an ultra-low-temperature freezer at −80 °C at the biobank, whereas FFPE specimens were processed and stored according to routine institutional protocols. QC classifications were assigned by the analytical laboratory based on RNA measurements, electropherograms, and estimated assay input requirements. Results: FF specimens had significantly higher RNA concentrations, A260/A280 and A260/A230 ratios, and RQNs than paired FFPE specimens (all p < 0.001). No specimens achieved “Pass” status. All 36 FF specimens were classified as “Hold,” whereas 25 of 36 FFPE specimens (69.4%) were classified as “Fail” (p < 0.001). Archival storage duration was significantly longer in the FFPE “Fail” group than in the “Hold” group (median, 5.52 versus 4.14 months; p = 0.006). Conclusions: The FF workflow yielded significantly superior RNA quality metrics and fewer pre-assay QC failures than the FFPE workflow. Placement of specimens in tubes prefilled with RNAlater, followed by snap-freezing and storage at −80 °C, may offer a practical strategy for banking small pancreatic EUS-FNB specimens. However, as pre-assay QC does not guarantee downstream assay success, further validation of profiling performance and long-term stability is warranted.