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

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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 Presentation

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Page 1: 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 XVIIParis, FranceMay 27, 2004

Page 2: Controls for  Human Papilloma Virus  (HPV) DNA testing

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

Page 3: Controls for  Human Papilloma Virus  (HPV) DNA testing

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

Page 4: Controls for  Human Papilloma Virus  (HPV) DNA testing

HPV Sample Collection and Testing

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

Page 5: Controls for  Human Papilloma Virus  (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

Page 6: Controls for  Human Papilloma Virus  (HPV) DNA testing

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

Page 7: Controls for  Human Papilloma Virus  (HPV) DNA testing

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

Page 8: Controls for  Human Papilloma Virus  (HPV) DNA testing

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

Page 9: Controls for  Human Papilloma Virus  (HPV) DNA testing

Real time quantitation of SiHa and SiHa+Unifected cells

Red: SiHa cells - 8,480 copies/ml.Blue: SiHa & H9 cells – 8,370 copies/ml.

Page 10: Controls for  Human Papilloma Virus  (HPV) DNA testing

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

Page 11: Controls for  Human Papilloma Virus  (HPV) DNA testing

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

Page 12: Controls for  Human Papilloma Virus  (HPV) DNA testing

0

3

6

9

12

15

Reference Lab

RL

U/C

OField Study Results

RL

-1

RL

-2

RL

-3

Page 13: Controls for  Human Papilloma Virus  (HPV) DNA testing

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

Page 14: Controls for  Human Papilloma Virus  (HPV) DNA testing

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

Page 15: Controls for  Human Papilloma Virus  (HPV) DNA testing

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.

Page 16: Controls for  Human Papilloma Virus  (HPV) DNA testing

Acknowledgements

BBI Diagnostics– Alan Doty– Barbara Weiblen– Harmesh Sharma

BBI Biotech– Janet Lathey– Yiping Zhang– Ron Meixell

Digene – Iwona Mielznska

Cleveland Clinic – Susan Schindler