Controls for Human Papilloma Virus (HPV) DNA testing

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
Digenes 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
Number of cells Avg Ct Av Copies/assay
5,000 (red) 27.4 3,100
500 (blue) 31.0 245
50 (lavender) 34.1 29
5 (dark blue) 37.6 3
0.5 (green) gt50 0
9
Real time quantitation of SiHa and SiHaUnifected
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-1
RL-2
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 gt2 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