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Hybrid Capture 2 (hc2) System: Efficient HPV DNA Testing

The Hybrid Capture 2 (hc2) System is a FDA-approved molecular hybridization assay that detects 18 types of HPV without DNA amplification. The test differentiates between low-risk and high-risk HPV, enabling early detection of cervical cancer. With no target DNA amplification, this test uses a unique sandwich-style detection method for accurate results. The test interpretation is based on Relative Light Units (RLUs), categorizing samples as positive for low-risk or high-risk HPV based on specific RLU/CO ratios. Although effective, the test may have limitations such as false positives due to cross-reactivity with low-risk HPV DNA. The system requires a minimum sample volume for testing, making it an efficient and reliable option for HPV detection worldwide.

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Hybrid Capture 2 (hc2) System: Efficient HPV DNA Testing

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  1. The Hybrid Capture 2 (hc2) SystemHPV DNA TestbyDigeneby Brenda Palacios

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

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

  4. HPV Characteristics • Non-enveloped double-stranded DNA virus • Epitheliotrophic- has great affinity for epithelial cells • Obligatory 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 cell • p53 protein is a negative regulator • E6 protein mutates p53 protein removing its protective function • Mutation disables p53 gene switch, permitting cell to multiply uncontrolled

  6. HPV Types • Types 6 &11–most common low-risk HPV • Associated with genital warts • Rarely found in cervical cancer • Types 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) test • Used universally for initial detection of intraepithelial abnormalities • In 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 Test • Approved by FDA • Nucleic acid hybridization assay • No target DNA amplification • Single amplification using microplatechemiluminescence 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 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 genotype

  10. Controls and Reagents • Controls • High-risk control: cloned HPV 16 DNA • Low-risk control: cloned HPV 6 DNA • Negative control • Carrier DNA • 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. • Probes • 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 Microplate • Coated with goat polyclonal anti-RNA:DNA hybrid antibodies • Detection Reagent 1 • Alkaline phosphatase-conjugated murine monoclonal antibodies to RNA:DNA hybrids • Detection Reagent 2 • Chemiluminescent substrate

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

  13. Denaturation Process • Samples are mixed with Sample Conversion buffer • 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 assay • Does not use HPV DNA target amplification • Uses hybridized signal amplification • Denatured HPV DNA is hybridized with low-risk or high-risk RNA probe • Resulting 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-phosphatase • Bound alkaline-phosphatase is revealed by addition of a chemiluminescentdioxetane-based substrate • Substrate is cleaved by bound alkaline phosphate and emitted light is measured in a microplateluminometer

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

  17. Test Interpretation • 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 DNA • HPV 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 sufficiency • Not possible to determine if results are due to insufficient DNA or true negative • Large number of inconclusive results • Requires large sample volume

  19. Sources of Error • Large 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 phosphatase • May cause false-positive • Presence of nucleases found on human skin and materials • Causes nucleic acid degradation • In accurate volume delivery of samples and reagents

  20. Conclusion • Due 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 • Is used in conjunction with pap smears to diagnose, treat, and prevent cervical cancer

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