Optimization of RNAscope for HPV mRNA detection in liquid-based cervical cytology samples

Purpose To establish an RNAscope-based joint detection scheme compatible with liquid-based cytology (LBC) slides and staining systems, enabling simultaneous observation of cytological phenotype and HPV E6/E7 mRNA expression in cervical cancer and precancerous lesions. Methods From March 2023 to February 2024, 115 LBC samples were collected at the Affiliated Obstetrics and Gynecology Hospital of Fudan University. Histopathological diagnosis included 30 LSIL, 33 HSIL, 29 HPV-related SCC, 8 HPV-related adenocarcinoma and 15 negative for cervical intraepithelial lesions cases. RNAscope technology, originally applied to paraffin samples, was adapted for LBC by modifying slide type selection, extending target retrieval, and enhancing permeabilization. The improved RNAscope in situ hybridization was then employed to detect high-risk HPV E6/E7 mRNA. Positive rates, diagnostic concordance with histopathology and, expression pattern were statistically analyzed. Results RNAscope combined with LBC showed high positive rates, 93.33% for LSIL, 96.97% for HSIL, 100.00% for SCC, and 87.50% for HPV-related adenocarcinoma. Compared with HPV PCR testing, the diagnostic concordance rate reached 95.65% (P<0.001). Figures illustrate RNAscope positive expression patterns in LSIL, HSIL, SCC and, HPV-related adenocarcinoma. Conclusion The adaptation of RNAscope technology, originally designed for paraffin samples, enables accurate diagnosis in cervical LBC, enhances consistency with histopathology, and provides a novel diagnostic approach for cervical lesions.