Report for Human Papilloma Virus DNA Quality Assurance Pilot Studies

20th June 2008 – Milestone 3 Report – Progress and Outcomes of Pilot Study One

Page last updated: 01 November 2008

Current Status of the Project

  • Specimens and questionnaires were dispatched at the end of March with 18 days to test. This date was extended 1 week due to several participant requests (revised due date 18/04/08). This is an unusual situation but as this was a pilot study the SQAP considered it important that all participants return results to ensure more complete analysis of results and feedback for future studies.
  • On survey close, the data submitted by each participant was downloaded from the website (21/04/08), into an Excel file and the information collated into a draft report. The data was also manipulated into graphical form for clear presentation of results.
  • The draft report was forwarded to Assoc. Prof. Sepehr Tabrizi (Senior Research Scientist, Department of Microbiology, The Royal Women's Hospital, VIC; responsible for preparation of the samples) for review and an opportunity for comment on testing or results obtained by participants. This review included alterations to the graphical presentations and discussion of results.
  • The Preliminary Report was then prepared which included all participant raw data (confidentiality of each participant is maintained with the use of participant numbers, as discussed in a previous report). The complete Preliminary Report was then forwarded to Prof William Rawlinson (SQAP Chair) for approval prior to distribution (as per all RCPA SQAP reports).
  • The Preliminary Human Papillomavirus Report for Pilot Study One was issued via email on the 27th May, 2008 to participants and the members of the HPV committee for comments and suggestions prior to issue of the final report (due date for comments: 6th June, 2008).
  • Several suggestions were made by Prof. Ian Frazer, including the addition of an executive summary. Two participants noted transcription errors with results submitted. (This was due to the issue of multiple participant numbers especially for the pilot study – this is a unique situation for the pilot study only). These alterations to the report were made and reviewed by Assoc. Prof. Sepehr Tabrizi.
  • The Final Human Papillomavirus Report for Pilot Study One was issued on the 12th June, 2008. The results of this survey are discussed below; see attached for the full report.
  • A number of presentations will be taking place in June and July to discuss the results of the pilot study with participants and interested parties to establish the way forward for Pilot Study Two.

Results from HPV DNA Pilot Study One

Website Data Entry

All participants were enrolled as website participants; results and related data are entered by the participant at the website rather than completing a paperwork questionnaire.

During the direct data entry of results several of the new participants (ie participants not usually enrolled in the SQAP and consequently, unfamiliar with website data entry of results) required a tutorial through the data entry screens. (The tutorial has been available for several years on the SQAP website and was designed to assist new participants with website entry of results). Once the tutorial was performed, participants seemed pleased with the screens and happy to use website data entry for both Pilot Studies.
  • Feedback for the website data entry included the following comments;
  • the number of digits for entering numbers needed to be extended – this was fixed immediately
  • there was some confusion with the unit options available and that being used by participants – this was reflected in the results downloaded at the conclusion of the survey and will be addressed for Pilot Study 2
  • the use of multiple Participant numbers for the those receiving both the PreservCyt and Dry swab specimens caused some confusion – this is a special occurrence for the pilot studies only and will be simplified once the specimen type has been decided and implemented after Pilot Study One.

Specimen Details

Specimens were prepared and preliminary testing by Assoc. Prof. Sepehr Tabrizi at The Royal Women's Hospital, VIC. Pre-issue testing was performed at The Royal Women's Hospital, VIC and Symbion Laverty Pathology, North Ryde, NSW. A full description of the specimen details and expected results are listed in the final report (attached). A number of duplicate samples were included to further assess assay performance. Participants were provided with instructions on how to process each type of specimen.

Participant Results

20 participants returned results for this first HPV Pilot Study.

PreservCyt Solution
15/20 (75%) participants used the Digene Hybrid Capture kit for the PreservCyt Solutions.

5/20 (25%) participants used alternative methods for the PreservCyt Solutions; three participants used in-house methods and two participants used Roche Magnapure / Amplicor HPV.

Dry Swab
5/20 (25%) participants used the Digene Hybrid Capture kit for the dry swabs.

5/20 (25%) participants used alternative methods for the dry swabs.

Analysis of Results

As mentioned above there appeared to be inconsistency with the value units reported, i.e. some participants reported the RLU levels while others provided RLU/Cut-off (S/CO). Therefore, only assessment of positivity for high-risk HPV was assessed.

Participants were given multiple options for assigning a result to the value for each result provided; these were negative, equivocal, positive, low risk and high risk; an array of these results were returned. For analysis equivocal results were considered positive. Positive and high-risk results were considered to be the same.

Results for the PreservCyt specimens using Hybrid Capture methodology showed 11/15 (73%) sites correctly identified all samples in the panel. One participant did not detect 4 of 6 positive samples. Other participants showed variation with the duplicate specimens and detection of low-risk type virus. All participants returned negative results for the duplicate negative specimens. Of the duplicate low copy positive specimens, 93% participants (specimen mimicking HPV 16 and 35 mixed infections) and 87% participant (specimen mimicking HPV 16 infection) reported results in agreement.

Results for the PreservCyt specimens using alternative methods show 2/5 sites 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. The number of laboratories using tests other than Hybrid Capture in this survey was significantly lower, however results demonstrate that there could be higher variability for these other tests. One possible explanation could be due to different specimen processing and extraction protocols used.

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). These results were expected, as the limit of detection of Hybrid Capture is 5000 copies. It appears in order to utilise the Dry Swab for Hybrid Capture a 100 fold more HPV DNA should be seeded on the swabs.

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 laboratories detected HR-HPV in a sample that only contained HPV 6 which indicates specificity of the assays utilised would need to be addressed by relative laboratories. Detection of HR types in this as well as in the negative specimens suggests contamination as a possibility.

The number of participants testing the Dry Swab panel was lower than for the PreservCyt specimens and direct comparison between testing these samples may not be appropriate. The returned results suggest that there is greater variability among participants using amplification methods than those using Hybrid Capture. This is most probably due to the use of different assays with variability in DNA extraction and volume as well as employing assays that target different amplification target, size as well as detection methodology. These panels however can assist laboratories to improve their methodology for more accurate diagnostics.

Timeline Progress

Due to several delays, involving the acquisition of information regarding specimen preparation the original projected timeline is behind schedule. However, the progress made has resulted in good feedback and a definite direction for future surveys.

Stability studies will be ongoing to confirm that shipping specimens at ambient temperature does not affect the integrity of the specimens.

Imminent Processes

  • Discussions regarding the second pilot study are to take place following Assoc. Prof. Sepehr Tabrizi’s presentation at the ASM in July. The discussion of results and participant feedback will be paramount and changes implemented (if required) for the second Pilot Study.
  • Several updates to the website will be made prior to the establishment of ‘Automated Personalised Assessment Reports’ generated through the website for HPV DNA. Participant feedback will guide development.


Overall, this pilot study showed the utility of a quality assurance program to the testing of HPV DNA. The formulation of a second pilot study should continue taking into account the copy number evaluated and inconsistencies noted between participants with values and units reported for the Hybrid Capture methodology.