Molecular testing was performed blinded for cytology and histology outcomes at the Department of Pathology of Amsterdam UMC. The QIAscreen HPV PCR Test (Qiagen) was used for high-risk HPV testing with separate genotyping for HPV16, HPV18, and a pool of 13 other high-risk HPV types (HPV 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 67, and 68). The QIAsure Methylation Test (Qiagen) was used for FAM19A4/miR124-2 methylation testing on bisulfite-converted DNA. The methylation test result was labeled positive if the QIAsure Methylation Test result exceeded the preset threshold for methylation positivity. Baseline clinician-collected samples stored in SurePath (n = 11) were not tested for FAM19A4/miR124-2 methylation because of insufficient DNA quality after DNA isolation.
Qiascreen hpv pcr test
Manufactured by Qiagen
Sourced in Germany
The QIAscreen HPV PCR Test is a qualitative in vitro diagnostic test that detects and identifies human papillomavirus (HPV) DNA in clinical samples. The test utilizes real-time PCR technology to detect the presence of HPV DNA.
Automatically generated - may contain errors
5 protocols using qiascreen hpv pcr test
1
HPV and Methylation Testing Protocol
2
Immunostaining and HPV Testing Protocol
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Immunostaining of p16INK4a was performed with mouse monoclonal antibodies against the p16INK4a antigen (clone E6H4; Roche, Basel, Switzerland), using the Optiview detection kit with the automated BenchMark ULTRA IHC/ISH system (Roche). p16INK4A immunohistochemistry was scored positive when diffuse or block staining was observed and negative with a negative or patchy staining pattern.22 High‐risk HPV DNA‐testing was performed using the QIAscreen HPV PCR Test (Qiagen), as described previously for use on FFPE biopsy specimens.23 The assay is directed against the E7 gene of 15 (probably) high‐risk HPV genotypes, that is, 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 67 and 68, with partial genotype information (HPV16 and −18).24 Beta‐globin served as internal quality control. Samples were considered invalid for PCR testing when the cycle threshold (Ct) >30 for beta‐globin and no HPV was found.
HPV status was determined in all VIN and VSCC and not in controls. HPV status was considered positive when p16INK4A and/or HPV PCR were positive, and negative when p16INK4A was negative and HPV PCR was negative or invalid.
HPV status was determined in all VIN and VSCC and not in controls. HPV status was considered positive when p16INK4A and/or HPV PCR were positive, and negative when p16INK4A was negative and HPV PCR was negative or invalid.
3
High-Risk HPV DNA Detection
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The QIAscreen® HPV PCR Test (QIAgen, Hilden, Germany) was used to perform high-risk HPV DNA-testing, according to the manufacturer’s instructions. Analysis was directed against the E7 gene of the following high-risk HPV genotypes, i.e., 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 67, and 68, with partial genotype information (HPV16 and 18) [25 (link)]. β-Globin was used for internal quality control.
4
High-risk HPV Detection Protocols
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Samples were tested for hrHPV as described previously [3 (link),9 (link),10 (link)] using either the SPF10 DNA enzyme immunoassay (DEIA) followed by Line Probe Assay25 (Labo Biomedical Products B.V., Rijswijk, The Netherlands), the GP5+/6+- PCR EIA followed by Luminex typing, or the QIAscreen HPV PCR Test (Qiagen, Hilden, Germany).
5
Evaluating WGS for HPV and EBV
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WGS based detection of presence of high-risk Human Papillomavirus (HPV) and/or Epstein-Barr virus (EBV) DNA was compared against routine pathological testing (Netherlands Cancer Institute) using the QIAscreen HPV PCR Test (Qiagen) for HPV and EBER IHC for detection of presence of EBV in the tumor (both according to standard protocols). If available, results of routine testing for HPV and/or EBV were used for comparison with WGS. If not available, HPV status was determined retrospectively using an aliquot of the DNA (20 ng) that was used for WGS.
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