The objective of the HPV DNA Project was to establish the best material and preparation method for specimens to issue to laboratories as a means of assessing laboratory testing performance for HPV DNA.
Two Pilot Studies were conducted with the support of a QUPP Grant (Quality Use of Pathology Program), Department of Health and Ageing, Australia to ensure that changes/adjustments to the material preparation were satisfactory and appropriate for evaluating testing performance of participating laboratories using a variety of assays.
Twenty participants from Australia and five from New Zealand participated in the project; 15/20 (75%) using the Hybrid Capture Method and the remaining 5/20 (25%) using Roche Amplicor (n=2), Genera Paptype (n=1) and In-house Method (n=2). All participated via the internet, using the RCPA Serology QAP (SQAP) website to print questionnaires and reports and enter results by a specified due date. Improvements and changes were made to both the questionnaire format and the website data entry screens in response to participant feedback, reporting requirements and testing protocols after each of the Pilot Studies.
A panel of eight PreservCyt specimens were sent to all 20 participants and a panel of eight dry swab specimens were also sent to 10 participants for Pilot Study One. For Pilot Study Two, all 20 participants received identical panels of six specimens; four PreservCyt and two dry swab specimens. All specimens were tested prior to dispatch by an in-house HPV assay, Hybrid Capture, Roche HPV Amplicor and Roche Linear Assay.Top of page
Results from Pilot Study One indicated that the specimens using PreservCyt worked well for all methodologies whereas results for the dry swab specimens using Hybrid Capture methodology showed reactivity for 3/5 participants in only one of the specimens (highest copy number of HPV 16). The limit of detection of Hybrid Capture is 5000 copies and it was clear that in order to utilise dry swabs for Hybrid Capture, a 100 fold more HPV DNA needed to be seeded onto the swabs for Pilot Study Two. One participant using Hybrid Capture methodology did not detect 4 of 6 positive samples while other participants showed variation with the duplicate specimens and detection of low-risk type virus.
Results for the PreservCyt specimens using alternative methods showed 2/5 participants correctly identified all of the samples in the panel. Two participants identified one of duplicate negative samples as positive, which could reflect contamination at the testing laboratory. One site did not detect HPV in either of the low copy number HPV 16 and 35 duplicates and another only detected one of two.
Results for the Dry Swab specimens using alternative methods showed one site correctly identified all specimens. Another participant returned negative results for two samples containing low copy number HPV 16 and one participant detected a weak positive in the negative sample. Three participants detected HR-HPV in a sample that only contained HPV 6.
Results overall improved in Pilot Study Two, partly due to the improvements and changes in specimen preparation but also due to improvement in participant testing. Specimens with HPV medium copy high-risk types were included in duplicate to assess assay reproducibility.Top of page
All participants using the Hybrid Capture method correctly identified PreservCyt Specimen 2A, 2B, 2C & 2D and dry swab Specimen 2E. 11/14 (79%) participants returned positive results for dry swab Specimen 2F. All participants who returned positive (correct) results for Specimens (2A, 2C, 2D & 2F) identified them as High Risk (HR).
All participants using the Hybrid Capture method returned positive (correct) results for duplicates of medium copy High Risk (HR) positive Specimens (2A and 2C) mimicking HPV 31, 52, 54 and 56 mixed infection. 11/15 (73%) participants had values for the duplicate samples within 25% of each other.
All results returned by participants using alternative methods to Hybrid Capture identified the PreservCyt Specimens 2A, 2B & 2C and dry swab specimens 2E & 2F correctly. 4/5 (80%) returned positive results for (PreservCyt) Specimen 2D. All participants who returned positive (correct) results for Specimens (2A, 2C, 2D & 2F) identified them as High Risk (HR).
Overall, results were pleasing with specimen preparation working well for all participants using a range of methodologies. Testing performance was greatly improved in Pilot Study Two and it is anticipated that this improvement will be ongoing. HPV DNA is now included in the Molecular Diagnostics Module in the RCPA Serology QA Program.
Herein, please find the Milestone 4 Overall Report for the HPV DNA QA Project; including results from Pilot Studies One and Two.Top of page