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Controls for Human Papilloma Virus (HPV) DNA testing

Controls for Human Papilloma Virus (HPV) DNA testing. Mark Manak, Ph.D. Boston Biomedica, Inc. West Bridgewater, MA. SoGAT XVII Paris, France May 27, 2004. Human Papilloma Virus Infections. One of most common sexually transmitted viruses

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Controls for Human Papilloma Virus (HPV) DNA testing

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  1. Controls for Human Papilloma Virus (HPV) DNA testing Mark Manak, Ph.D. Boston Biomedica, Inc. West Bridgewater, MA SoGAT XVII Paris, France May 27, 2004

  2. Human Papilloma Virus Infections One of most common sexually transmitted viruses • Most common cause of mortality from gynecological malignancy world wide • Between 1- 5.5 million new infections in US per year • 15,000 women in US get cervical cancer - 5,000 die 80% of population will be infected with HPV in their lifetime. • 28-46% by age 25. • Most infections spontaneously cleared, • 5-10% of women over 30 are persistent carriers of HPV Fully preventable if precancerous cell changes detected and treated early Detected by PAP smear and HPV DNA

  3. Cervical Cytology Cell Morphology %HPV DNA + • Normal 10% • ASC-US 20-40% Atypical squamous cells of undetermined origin • LSIL 60-85% Low grade squamous intraepithelial lesions • HSIL 80-90% High grade squamous intraepithelial lesions • Squamous Cell Carcinoma 99% FDA approved combination of cervical cytology and HPV DNA testing for primary screening for women aged 30 yr and older

  4. HPV Sample Collection and Testing • Cervical Swabs • PreservCyt (buffered methanol), or • Sure Path (ethanol based preservative) • Used for both cytology and HPV DNA testing

  5. Digene’s Hybrid Capture 2 HPV DNA Test • Uses two RNA probe cocktails to differentiate between carcinogenic and low risk HPV types • Hybrid Capture of DNA-RNA hybrids • High Risk Subtypes • 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68 • Low Risk Subtypes • 6, 11, 42, 43 44

  6. Design of HPV DNA Control • 50,000 HPV positive Cells Plus 450,000 uninfected cells • In 4 ml PreservCyt or SurePath • Quantitated by Cell Count, Cell staining, Digene HC2 and HPV TaqMan • Convenient Liquid Cytology “Liquid Pap Smear” Sample

  7. Accurun 370: HPV DNA Control Processing identical to Digene Clinical Sample • Packaged to enter directly into the Digene Sample handling procedure • Provided in PreservCyt (buffered methanol) or • SurePath (ethanol solution) • In Centrifuge tube as recommended in Digene package insert Provides challenging, yet consistent low level HPV Clinical Sample • Cultured human epithelial diluted with excess non-infected human cells • Positive cells contain 2 copies of HPV Type 16 DNA per cell • HPV-16 most common high risk subtype in the US Mimics patient sample as required by CLIA • Low copy HPV DNA in a high concentration of cells • Ease of use and ability to monitor long term assay performance • Extended refrigerator storage

  8. Real time detection of Serial Dilutions of SiHa cells

  9. Real time quantitation of SiHa and SiHa+Unifected cells Red: SiHa cells - 8,480 copies/ml. Blue: SiHa & H9 cells – 8,370 copies/ml.

  10. Stability of HPV DNA Controls

  11. Validation Study Design • 1 lot of control material • 4 testing sites • 3 clinical, 1 IVD manufacturer • Results reported as RLU/cutoff ratio • Standard Digene Protocol • liquid Pap smear samples • 52 replicates • 8 assay runs • 4 assay runs manual equipment • 4 assays automated “Rapid Capture” • 2 assay reagent lots

  12. Field Study Results RL-2 RL-1 RL-3

  13. Distribution of RLU/Cutoff Values N = 52 Mean = 7.7 Median = 7.8 SD = 1.08

  14. Conclusion • Convenient format compatible with Digene specimen collection • Mimics Low Level human PAP sample • Well accepted by technologists • Consistent, well developed cell button formation • Controls for entire testing process • Minimal training for use and interpretation • Stable, consitent product • Stable up to 65 days at 22-250 C • Reflects >2 years of refrigerated storage

  15. Conclusion BBI Accurun 370 is a convenient to use low level HPV DNA control Specifically designed to be used with the Digene HC2 HPV DNA Test Provides stable, efficient positive external control material for HPV DNA testing in clinical laboratories.

  16. Acknowledgements BBI Diagnostics • Alan Doty • Barbara Weiblen • Harmesh Sharma BBI Biotech • Janet Lathey • Yiping Zhang • Ron Meixell • Digene • Iwona Mielznska • Cleveland Clinic • Susan Schindler

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