The effect of preanalytical processes on nucleic acid quality in biomarker analysis with next-generation sequencing

Abstract

Preanalytical processes—including sample collection, fixation, and handling—significantly influence the integrity of nucleic acids, which in turn impacts the accuracy of next-generation sequencing (NGS)-based biomarker analyses. Though commonly utilized, cytological specimens remain underrepresented in studies evaluating preanalytical quality. Methods We retrospectively analyzed 1048 NGS tests performed between May 2019 and December 2022, encompassing cytological materials (smear slides and cell blocks), small/core biopsies, and resection specimens. Quality control (QC) was assessed using pre-sequencing cycle threshold (Ct) values and post-sequencing metrics such as fragment length, on-target unique fragments, deduplication ratio, and unique start. Results Overall, 22.9% of samples did not meet manufacturer Ct thresholds. The final QC failure rates were 12.4% for RNA and 3.5% for DNA. RNA QC failures were significantly more frequent in cytology and resection specimens compared to biopsies. However, post-sequencing analysis revealed that smear samples exhibited significantly higher median DNA and RNA fragment lengths compared to FFPE specimens, indicating superior nucleic acid preservation in samples that passed QC. Conclusion Cytological specimens are viable for NGS-based molecular testing, particularly when preanalytical variables are standardized and optimized. DNA quality from smears may even exceed that of FFPE samples, while RNA integrity remains more sensitive to preparatory conditions. These findings support the broader use of cytological materials in molecular diagnostics and highlight the need for tailored preanalytical workflows.