controls for human papilloma virus (hpv) dna testing
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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 - PowerPoint PPT PresentationTRANSCRIPT
Controls for Human Papilloma Virus
(HPV) DNA testing
Mark Manak, Ph.D.Boston Biomedica, Inc.West Bridgewater, MA
SoGAT XVIIParis, FranceMay 27, 2004
Human Papilloma Virus InfectionsOne 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
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
HPV Sample Collection and Testing
• Cervical Swabs• PreservCyt (buffered methanol), or• Sure Path (ethanol based preservative) • Used for both cytology and HPV DNA testing
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
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
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
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) >50 0
Real time detection of Serial Dilutions of SiHa cells
Real time quantitation of SiHa and SiHa+Unifected cells
Red: SiHa cells - 8,480 copies/ml.Blue: SiHa & H9 cells – 8,370 copies/ml.
Stability of HPV DNA ControlsTitration of S iHa Cells
0.1
1.0
10.0
100.0
1 10 100Number of SiHA Cells (x 103)
HPV Stability Study Test (37o C)
0.1
1.0
10.0
100.0
0 5 10 15 20Days
RL
U/C
O
HPV Freeze-Thaw
0.1
1.0
10.0
100.0
0 1 2 3 4
Number of Freeze-Thaws
RL
U/C
OHPV Stability Study (22-25o C)
0.1
1.0
10.0
100.0
0 20 40 60 80
Days
RL
U/C
O
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
0
3
6
9
12
15
Reference Lab
RL
U/C
OField Study Results
RL
-1
RL
-2
RL
-3
Distribution of RLU/Cutoff Values
0
2
4
6
8
10
12
14
RLU/CO
Frequency
N = 52
Mean = 7.7
Median = 7.8
SD = 1.08
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
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.
Acknowledgements
BBI Diagnostics– Alan Doty– Barbara Weiblen– Harmesh Sharma
BBI Biotech– Janet Lathey– Yiping Zhang– Ron Meixell
Digene – Iwona Mielznska
Cleveland Clinic – Susan Schindler