Triage strategies and colposcopy referrals in HPV-positive women with low-grade cytology 131 6 several categories: normal (no dysplasia; CIN0), LSIL/CIN1, HSIL/CIN2 and HSIL/CIN3, which are hereafter referred to as CIN1, CIN2 and CIN3, respectively 22. All participants provided informed consent for additional follow-up research. Ethical approval for the current study was obtained from the Medical Ethics Committee of Amsterdam UMC, Vrije Universiteit Amsterdam (Amsterdam, The Netherlands; METC 2018/09, TcB 2018.106). HPV GENOTYPING, FAM19A4/MIR124-2 METHYLATION AND ASCL1/LHX8 METHYLATION For this study, we used existing data for HPV genotyping and ASCL1/LHX8 methylation analysis 13. HPV DNA testing was performed using the GP5+/6+ PCR enzyme immunoassay (EIA; Labo Biomedical Products BV, Rijswijk, Netherlands). This assay detects 14 high-risk HPV types, i.e., HPV16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68. GP5+/6+ PCR amplicons were genotyped using the LMNX Genotyping kit HPV GP HR (Diassay, QIAgen, Hilden, Germany) according to the instructions of the manufacturer. Methylation analysis of ASCL1/LHX8 was conducted as previously described 13, 16, 23 using quantitative methylation-specific PCR (qMSP) on bisulphite-converted DNA. The ASCL1/LHX8 methylation status was labelled positive if the result exceeded the predefined predicted probability threshold 12. FAM19A4/miR124-2 methylation analysis was performed using the QIAsure Methylation Test® (Qiagen, Hilden, Germany) on the same bisulphite-converted DNA samples according to the instructions of the manufacturer. The FAM19A4/miR124-2 methylation status was labelled positive if the result surpassed the predefined ΔΔCq value threshold, in accordance with the manufacturer’s instructions. Only samples with a valid test results for all assays were included in this study. DATA AND STATISTICAL ANALYSIS The study evaluated the performance of twelve single and combined triage strategies utilising FAM19A4/miR124-2 methylation, ASCL1/LHX8 methylation, HPV16/18 genotyping and/or HPV16/18/31/33/45 genotyping. A triage result was labelled positive when test positivity was shown for (I) FAM19A4/miR124-2 methylation, (II) ASCL1/LHX8 methylation, (III) HPV16/18 genotyping, (IV) HPV16/18/31/33/45 genotyping, (V) HPV16/18 genotyping AND FAM19A4/miR124-2 methylation, (VI) HPV16/18 genotyping OR FAM19A4/miR124-2 methylation, (VII) HPV16/18 genotyping AND ASCL1/LHX8 methylation, (VIII) HPV16/18 genotyping OR ASCL1/LHX8 methylation, (IX) HPV16/18/31/33/45 genotyping AND FAM19A4/miR124-2 methylation, (X) HPV16/18/31/33/45 genotyping OR FAM19A4/ miR124-2 methylation, (XI) HPV16/18/31/33/45 genotyping AND ASCL1/LHX8 methylation and (XII) HPV16/18/31/33/45 genotyping OR ASCL1/LHX8 methylation.
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