• 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

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

• 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

• 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

• 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

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

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

• 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

• 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

• 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

• 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

• 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

• 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

• Detection is sandwich-style with a second anti-
RNA:DNA hybrid conjugated to alkalinephosphatase
• Bound 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 luminometer

www.papillomavirus.cz/images/hybridcapture.jpg

• 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 highrisk HPV and must be repeated

• 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

• Large concentrations of whole blood, douche, antifungal 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

• 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