Optimizing spin‑column elution parameters for efficient HPV DNA recovery from preservative liquid‑based cytology cervical scrape samples

Optimizing DNA extraction protocols is critical for improving downstream molecular analyses, particularly for low‑cellularity clinical samples, such as HPV ThinPrep liquid‑based cytology specimens. Variations in pre‑analytical extraction parameters substantially influence DNA yield, purity and integrity; yet, these factors remain underexplored beyond default manufacturer guidelines. The present study systematically evaluated the effects of lysis incubation duration (30, 40, 50, 60 and 80 min), elution volume (ranging from 30 to 75 µl) and the number of elution cycles on DNA recovery using a silica spin‑column extraction method. The extraction performance was assessed using fluorometry and spectrophotometry, alongside DNA integrity number (DIN) evaluation using a TapeStation. To account for between‑sample biological variations and storage temperature confounders, a linear mixed‑effects model (LMM) was employed for DNA yield, while a generalized linear mixed model (GLMM) evaluated purity. Response surface methodology was subsequently applied to determine non‑linear parameter effects and global optima. The LMM identified the number of elution cycles and its interaction with incubation duration as critical determinants of DNA yield. Response surface methodology optimization identified an individual optimum for maximizing yield, and for optimal purity. A compromise operational point was established to simultaneously satisfy both responses. Furthermore, the first elution cycle consistently recovered the vast majority of DNA mass, rendering a second cycle largely redundant. Quality control analysis demonstrated that an elevated DNA yield does not inherently guarantee a high DNA integrity, emphasizing the compounding impact of storage temperature. On the whole, these findings demonstrate that tailoring extraction parameters and utilizing a single‑elution workflow can significantly enhance the quality and quantity of DNA recovered from low‑biomass clinical specimens for advanced molecular diagnostics.