Ethics approval and consent to participate
This study was conducted with the approval of the Beijing Obstetrics and Gynecology hospital, Capital Medical University, Institutional Review Board (IRB) Committee on Human Research in the Medical Sciences (CHRMS). A written informed patient consent was signed by each patient before joining this study project. All agrees to provide specimens and their data to be further published as part of the study results.
Patient population and sample preparations
115 adult female outpatients ranging in age from 23 to 71 years were included in this study. The cervical samples were collected between Dec 2018 and March 2019 followed the procedures described below. After exposure cervical entrance, its surface was scratched two circles to collect cervical scraped cells using two different TCT sample collection brushes, respectively. For most patients, first circle of scraped cells was sent for HPV PCR tests, whereas the second circle of scraped cells was tested for RNAscope assays. For the latter collected ones, the TCT sample collection brush was cut and the tip with cells were kept in a 50ml tube with 10% neutral buffered formalin (NBF). The tubes were kept at 4ºC overnight then the cell samples on the brush tips were physically scraped down from the brush into 10% NBF. The tubes were centrifuged at 800rmp for 10min to collect cells. Cell pellet of each sample were transferred into 2ml EP tube and washed by PBS once. 1.5ml 70% ethanol was used to resuspend the cells and stored at room temperature (RT) for 2hr. The cells in 2ml EP tubes were then centrifuged at 8000-10000 rpm for 5min and the supernatant was discarded. The cell pellets were regarded as a chunk and went through 70%, 80%, 95% and 100% ethanol, respectively, at RT for 10min. After 100% ethanol, the cell pellet chunk floated and were transferred into a filter paper to totally try. Melt CellGel (Beijing Pursuit Bio Co., ltd.) was dropped onto hydrophobic paper (parafilm paper). The dried cell pellet chunk was embedded in the CellGel, solidified with the CellGel and became a bigger block. The latter one was transferred into tissue processing histology Cassette and went through 85% ethanol for 45 min; 95% ethanol for 30min, 100% ethanol I for 30min, 100% ethanol II for 30min, 100% ethanol III for 45 min, xylene I for 30min, xylene II for 30min, xylene III for 45min, Wax I for 30min, Wax II for 30min, Wax III for 30min and Wax IV for 30min, then embedded in paraffin to become Formalin-fixed paraffin-embedding (FFPE) blocks. Each FFPE block carried a patient cervical cell pellet were sectioned of 5μm for RNA in situ hybridization tests.
RNA Chromogenic in situ Hybridization
RNA in situ hybridization was performed on FFPE cell pellet sections (5 μm) using the RNAscope 2.5 HD assay-Red (Advanced Cell Diagnostics, Inc.) and the RNAscope Probes (Advanced Cell Diagnostics, Inc.) including HPV-HR18 (pool of 18 individual high-risk human papillomavirus subtype E6/E7 mRNA probes: HPV 16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68, 73, and 82), HPV-LR6 (pool of 6 individual low-risk HPV subtype E6/E7 mRNA probes 6, 11, 40, 42, 43 and 44) and Hs-TERC probe. A negative probe targeting diaminopimelate B (DapB) and a positive RNA probe targeting human ubiquitin C (Hs-UBC), were used to evaluate each sample quality. Samples with no signal from DapB, and score >= 2 by UBC were counted as quality control (QC) passed. The RNAscope 2.5 HD-Red manual assays was followed per the manufacturer’s instructions. Each sample was tested for RNA quality control (QC) firstly (Hs-UBC and DapB). The QC passed ones were further studied using HPV-HR18, HPV-LR6 and Hs-TERC probes, respectively.
qPCR Analysis of HPV DNA
Human Papillomavirus Polymerase Chain Reaction HR-HPV PCR was performed using the 23 HPV Genotyping Real-time PCR Kit (Hybribio, China) containing 17 high risk HPV types: 16, 18, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68, 73 and 82, and 6 low risk HPV types: 6, 11, 42, 43, 44 and 81.
Interpreting Results
RNAscope stained FFPE cell sections were scanned using Leica AT2 scanner (Leica, US). Whole sections were examined at 40× magnification. RNAscope results of HPV were recorded based on signal location and positive cells. For probe-HPV-HR and probe-HPV-LR results, the signal locations of cells and the positive cell numbers in each sample, <3, ≧3≦10 or >10, were recorded. Besides classic RNAscope signals in cytoplasm or nuclear, there are HPV RNAs gathered as clusters above one or more cells, which were recorded as well. For TERC results, RNA signals in nucleus were recorded. Two gynecologists (Z. H. and H. Y.) evaluated the scanned sections independently. If a disagreement occurred, they reviewed the case together and reached the final agreement. The interpretation was generally straightforward; therefore, no significant disagreements lead to incompatibility.