Human Papillomavirus detection by The Hybrid Capture 2 (hc2

The Hybrid Capture 2 (hc2)
by Brenda Palacios
• 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
• State what the molecular sandwich consist of
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
HPV Characteristics
• Non-enveloped double-stranded DNA virus
• Epitheliotrophic- has great affinity for epithelial
• Obligatory intracellular parasites that deliver
their genome and accessory proteins into host
cells for viral replication
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
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
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
Molecular Testing
• Hybrid Capture 2 (hc2) System HPV DNA Test
– Approved by FDA
• Nucleic acid hybridization assay
• No target DNA amplification
• Single amplification using microplate
chemiluminescence for qualitative detection of 18
types of HPV
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
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
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
• Detection Reagent 1
– Alkaline phosphatase-conjugated murine monoclonal
antibodies to RNA:DNA hybrids
• Detection Reagent 2
– Chemiluminescent substrate
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
Denaturation Process
• Samples are mixed with Sample Conversion
• 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 antiRNA: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
Test Interpretation
• Emitted light is measured in Relative Light Units
– 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
Sources of Error
• 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
– 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
• The Hybrid Capture 2 System helps
differentiate btw low-risk and high-risk HPV
• Is used in conjunction with pap smears to
diagnose, treat, and prevent cervical cancer

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