Thinprep

Specimen collection, specimen preparations, and results of the tests are performed, according to the instructions of the manufacturers, respectively. ThinPrep (Hologic Inc., Marlborough, Massachusetts, USA)preparations was used, and the cytological diagnosis was performed by experienced cytopathologists, according to the Bethesda system TBS 2001.

Diagnostic biopsy specimens submitted to our institution were routinely examined in the course of intraoperative consultations (frozen sections), followed by standard formalin-fixation and paraffin-embedding (FFPE) for histological workup in most cases. External cases were either received as FFPE blocks or as near-native tumour tissue submitted in SurePath^® (Becton Dickinson, USA) or ThinPrep^® preservatives (Hologic Inc., USA). For cryopreserved biopsies, one to four 70 μm thick cryosections were collected. For FFPE blocks, a series of 8 or 14 serial sections (4 μm) were collected on glass slides, and an additional H&E section in the middle of each series was used to verify tumour cell content. In samples containing significant non-neoplastic or necrotic areas on the cut surface, viable tumour cell-rich areas were manually microdissected. For native specimens, DNA was extracted with automated commercial systems: Promega Maxwell^® FFPE DNA extraction, Promega Maxwell^® blood DNA extraction, and Qiagen DNeasy^® Blood and Tissue kits on Maxwell or QiaCube^® instruments (Promega, USA; Qiagen, Germany). DNA from FFPE specimens was extracted with the Promega Maxwell^® FFPE DNA extraction, Qiagen DNeasy^®, and RecoverAll^® kits. Except for differences in average read lengths in nanopore sequencing runs for native materials, all of the above-mentioned kits produced technically valid results.

Each Pap smear sample (which are the standard samples for HPV genotyping) was evaluated by a specialized cytotechnologist and the results were confirmed by a pathologist. Samples were suspended in the liquid-based cytology (LBC) buffer (ThinPrep, Hologic, Marlborough, MA, USA). Collection of the first-void urine (FVU) samples was performed in a sterile Cell PrepPlus (Biodyne, Gyeonggi-do, Korea) urine bottle, stored at 4 °C, and processed within 3 days. For each sample, approximately 15 mL of LBC and FVU were centrifuged at 3,000 rpm for 5 min, and the supernatant was removed. The residual 800 µL of the sample suspension containing cell debris was washed and centrifuged at 8,000 rpm for 5 min. The DNA was extracted from the pellets using a DNA Prep Kit (Chaozhou Hybribio Biochemistry Ltd., Guangdong, China) and stored at −20 °C until testing.

Aliquots of each specimen were used to prepare one liquid-based (ThinPrep®) preparation according to the manufacturer's protocol (Hologic, Inc., Marlborough, MA) and one cell block preparation. Morphologic diagnosis with immunohistochemical analysis, where necessary, was performed as previously described (22 (link)). The remaining supernatant was decanted and stored at room temperature (RT) until the case was signed out, at which time it was transported to the molecular pathology laboratory. There, it underwent repeat centrifugation at 2,000 g for 30 min at 4°C, after which it was immediately stored at −80°C until DNA extraction.