1. The Hybrid Capture 2 (hc2) System HPV DNA Test by Digene by Brenda Palacios

2. Objectives State the 2 main low-risk HPV DNA typesState the 2 main low-risk HPV DNA types State the 2 main high-risk HPV DNA typesState the 2 main high-risk HPV DNA types State what type of test method is used for the detection of HPVState what type of test method is used for the detection of HPV State the minimum sample volume required for testingState the minimum sample volume required for testing State what the molecular sandwich consist ofState what the molecular sandwich consist of

3. Human Papillomavirus (HPV) Primary etiological agent in cervical cancerPrimary etiological agent in cervical cancer 2 nd most common type of cancer in women world wide2 nd most common type of cancer in women world wide 3 rd leading cause of cancer-related deaths in women worldwide3 rd leading cause of cancer-related deaths in women worldwide Most common viral sexually transmitted infection, that goes undiagnosed due to no symptoms developedMost common viral sexually transmitted infection, that goes undiagnosed due to no symptoms developed

4. HPV Characteristics Non-enveloped double-stranded DNA virusNon-enveloped double-stranded DNA virus Epitheliotrophic- has great affinity for epithelial cellsEpitheliotrophic- has great affinity for epithelial cells Obligatory intracellular parasites that deliver their genome and accessory proteins into host cells for viral replicationObligatory intracellular parasites that deliver their genome and accessory proteins into host cells for viral replication

5. HPV Infection E6 oncogene binds to p53 protein in host cellE6 oncogene binds to p53 protein in host cell –p53 protein is a negative regulator E6 protein mutates p53 protein removing its protective functionE6 protein mutates p53 protein removing its protective function Mutation disables p53 gene switch, permitting cell to multiply uncontrolledMutation disables p53 gene switch, permitting cell to multiply uncontrolled

6. HPV Types Types 6 &11–most common low-risk HPVTypes 6 &11–most common low-risk HPV –Associated with genital warts –Rarely found in cervical cancer Types 16 & 18- most common high-risk HPVTypes 16 & 18- most common high-risk HPV –Associated with cancers of the cervix, vagina, vulva, anus, and penis

7. HPV Detection Traditional screening by Papanicolau (pap smear) testTraditional screening by Papanicolau (pap smear) test Used universally for initial detection of intraepithelial abnormalitiesUsed universally for initial detection of intraepithelial abnormalities In case of atypical squamous cells of undetermined significance HPV DNA detection is recommendedIn case of atypical squamous cells of undetermined significance HPV DNA detection is recommended www.unsw.edu.au/.../sep/cells_image_inside.jpg

8. Molecular Testing Hybrid Capture 2 (hc2) System HPV DNA TestHybrid Capture 2 (hc2) System HPV DNA Test –Approved by FDA Nucleic acid hybridization assayNucleic acid hybridization assay No target DNA amplificationNo target DNA amplification Single amplification using microplate chemiluminescence for qualitative detection of 18 types of HPVSingle amplification using microplate chemiluminescence for qualitative detection of 18 types of HPV www.clpmag.com/graphics/mags/0409/sl03.jpg

9. Molecular Testing cont. Differentiates between low and high risk HPVDifferentiates between low and high risk HPV –Low-risk HPV: 6, 11, 42, 43, 44 –High-risk HPV: 16, 18, 31, 35, 39, 45, 51, 52, 56, 58, 59, 68 Does not determine specific HPV genotypeDoes not determine specific HPV genotype

10. Controls and Reagents ControlsControls –High-risk control: cloned HPV 16 DNA –Low-risk control: cloned HPV 6 DNA Negative controlNegative control –Carrier DNA Calibrators (run in triplicate)Calibrators (run in triplicate) –Low-risk Calibrator: Cloned HPV 11 DNA –High-risk Calibrator: cloned HPV 16 DNA –Ensure that the reagents and calibrator materials are functioning properly, for determination of assay cut-off value

11. Controls and Reagents cont. ProbesProbes –Low-risk Probe: HPV 6, 11, 42, 43, 44 RNA cocktail –High-risk Probe: HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 58, 59, 68, RNA cocktail Capture MicroplateCapture Microplate –Coated with goat polyclonal anti-RNA:DNA hybrid antibodies Detection Reagent 1Detection Reagent 1 –Alkaline phosphatase-conjugated murine monoclonal antibodies to RNA:DNA hybrids Detection Reagent 2Detection Reagent 2 –Chemiluminescent substrate

12. Specimen Requirements Cervical specimens collected using a broom type collection device placed in PreservCyt SolutionCervical specimens collected using a broom type collection device placed in PreservCyt Solution Cervical biopsies btw 2-5 mm in Digene Specimen Transport MediumCervical biopsies btw 2-5 mm in Digene Specimen Transport Medium Specimens collected with the Digene Cervical Sampler, placed in SurePath Preservative FluidSpecimens collected with the Digene Cervical Sampler, placed in SurePath Preservative Fluid 4mL of sample needed for denaturation process4mL of sample needed for denaturation process

13. Denaturation Process Samples are mixed with Sample Conversion bufferSamples are mixed with Sample Conversion buffer Then mixed with a 2:1 ratio of Specimen Transport Medium (STM) and Denaturation Reagent (DNR)Then mixed with a 2:1 ratio of Specimen Transport Medium (STM) and Denaturation Reagent (DNR) –STM-preservative that retards bacterial growth and retains DNA integrity –DNR-dilute sodium hydroxide solution, which lysis cells and denatures HPV DNA

14. Detection The Hybrid Capture 2 is a fluid-based molecular hybridization assayThe Hybrid Capture 2 is a fluid-based molecular hybridization assay Does not use HPV DNA target amplificationDoes not use HPV DNA target amplification Uses hybridized signal amplificationUses hybridized signal amplification Denatured HPV DNA is hybridized with low-risk or high-risk RNA probeDenatured HPV DNA is hybridized with low-risk or high-risk RNA probe Resulting hybrids are captured on microplate wells by immobilized antibodyResulting hybrids are captured on microplate wells by immobilized antibody

15. Detection Detection is sandwich-style with a second anti- RNA:DNA hybrid conjugated to alkaline- phosphataseDetection is sandwich-style with a second anti- RNA:DNA hybrid conjugated to alkaline- phosphatase Bound alkaline-phosphatase is revealed by addition of a chemiluminescent dioxetane-based substrateBound alkaline-phosphatase is revealed by addition of a chemiluminescent dioxetane-based substrate Substrate is cleaved by bound alkaline phosphate and emitted light is measured in a microplate luminometerSubstrate is cleaved by bound alkaline phosphate and emitted light is measured in a microplate luminometer

16. www.papillomavirus.cz/images/hybridcapture.jpg

17. Test Interpretation Emitted light is measured in Relative Light Units (RLUs)Emitted light is measured in Relative Light Units (RLUs) –Specimens with RLU/CO ratio ≥ 1.7 with low-risk HPV probe are considered positive for low-risk HPV –Specimens with RLU/CO ratio ≥ 1.7 with high-risk HPV probe are considered positive for high-risk HPV –Specimens with RLU/CO ratio btw 0.80-1.7 are considered indeterminate for either low-risk or high- risk HPV and must be repeated

18. Limitations Significant number of false-positives (10%-19%) due to cross reactivity with low-risk HPV DNASignificant number of false-positives (10%-19%) due to cross reactivity with low-risk HPV DNA HPV DNA not amplifiedHPV DNA not amplified –Negative predicted value may be compromised in cases in which HPV DNA copy number is low No internal control used for sample sufficiencyNo internal control used for sample sufficiency –Not possible to determine if results are due to insufficient DNA or true negative Large number of inconclusive resultsLarge number of inconclusive results Requires large sample volumeRequires large sample volume

19. Sources of Error Large concentrations of whole blood, douche, anti- fungal cream, and contraceptive jellyLarge concentrations of whole blood, douche, anti- fungal cream, and contraceptive jelly –May cause false-negative Contamination of Capture Microplate and Detection Reagent 2 with exogenous alkaline phosphataseContamination of Capture Microplate and Detection Reagent 2 with exogenous alkaline phosphatase –May cause false-positive Presence of nucleases found on human skin and materialsPresence of nucleases found on human skin and materials –Causes nucleic acid degradation In accurate volume delivery of samples and reagentsIn accurate volume delivery of samples and reagents

20. Conclusion Due to various types of HPVs the most reliable method of detection is through molecular testingDue to various types of HPVs the most reliable method of detection is through molecular testing The Hybrid Capture 2 System helps differentiate btw low-risk and high-risk HPV infections The Hybrid Capture 2 System helps differentiate btw low-risk and high-risk HPV infections Is used in conjunction with pap smears to diagnose, treat, and prevent cervical cancerIs used in conjunction with pap smears to diagnose, treat, and prevent cervical cancer