The impact of cytological preparation techniques on RNA quality: a comparative study on smear samples

Abstract

Background: High-quality RNA is essential for accurate molecular testing. This study evaluates the impact of cytological preparation techniques (May–Grünwald–Giemsa [MGG], Papanicolaou [PAP], Diff-Quik, and air-dried) on RNA quality in smear slides. Methods: A total of 182 smears were prepared from fresh surgical specimens of 26 patients using seven different techniques. RNA was isolated, reverse-transcribed, and analyzed using quantitative polymerase chain reaction (qPCR). RNA quality was assessed using ΔCt (ΔCt = 45 – Ct, cycle threshold), where higher ΔCt indicates better RNA quality. Results: RNA quality, measured by ΔCt, showed clear differences (p <.001) in-between preparation methods, whereas RNA concentration did not differ significantly among smear types (p =.07). MGG-stained smears (both film- and coverslip-mounted) demonstrated the highest and most consistent ΔCt values. PAP-stained smears yielded the lowest ΔCt values, indicating the poorest RNA quality. Air-dried unstained smears showed highly variable ΔCt values and frequent amplification failures. Diff-Quik preparations had intermediate performance. Mounting method (film vs. coverslip) did not significantly affect RNA quality. Conclusion: Among cytology smear techniques, MGG provided the best RNA preservation, PAP the worst, and air-dried slides yielded inconsistent results. These findings highlight the critical role of smear preparation in preserving RNA for molecular testing, especially RNA-based next-generation sequencing. © 2025 American Cancer Society.