The Hybrid Capture 2 The Hybrid Capture 2 (hc2) System(hc2) SystemHPV DNA TestHPV DNA Test

bybyDigeneDigene

by Brenda Palaciosby Brenda Palacios

ObjectivesObjectives

• 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 State the 2 main high-risk HPV DNA

typestypes• State what type of test method is used for State what type of test method is used for

the detection of HPVthe detection of HPV• State the minimum sample volume State the minimum sample volume

required for testingrequired for testing• State what the molecular sandwich State what the molecular sandwich

consist ofconsist of

Human Papillomavirus Human Papillomavirus (HPV)(HPV)

• Primary etiological agent in cervical Primary etiological agent in cervical cancercancer

• 22ndnd most common type of cancer in most common type of cancer in women world widewomen world wide

• 33rdrd leading cause of cancer-related leading cause of cancer-related deaths in women worldwidedeaths in women worldwide

• Most common viral sexually transmitted Most common viral sexually transmitted infection, that goes undiagnosed due to infection, that goes undiagnosed due to no symptoms developedno symptoms developed

HPV CharacteristicsHPV Characteristics

• Non-enveloped double-stranded DNA Non-enveloped double-stranded DNA virusvirus

• Epitheliotrophic- has great affinity for Epitheliotrophic- has great affinity for epithelial cellsepithelial cells

• Obligatory intracellular parasites that Obligatory intracellular parasites that deliver their genome and accessory deliver their genome and accessory proteins into host cells for viral proteins into host cells for viral replicationreplication

HPV InfectionHPV Infection• E6 oncogene binds to E6 oncogene binds to p53p53 protein in protein in

host cellhost cell– p53p53 protein is a negative regulator protein is a negative regulator

• E6 protein mutates E6 protein mutates p53p53 protein protein removing its protective functionremoving its protective function

• Mutation disables Mutation disables p53p53 gene switch, gene switch, permitting cell to multiply uncontrolledpermitting cell to multiply uncontrolled

HPV TypesHPV Types

• Types 6 &11–most common low-risk Types 6 &11–most common low-risk HPV HPV – Associated with genital wartsAssociated with genital warts– Rarely found in cervical cancerRarely found in cervical cancer

• Types 16 & 18- most common high-risk Types 16 & 18- most common high-risk HPVHPV– Associated with cancers of the cervix, Associated with cancers of the cervix,

vagina, vulva, anus, and penisvagina, vulva, anus, and penis

HPV DetectionHPV Detection

• Traditional screening by Papanicolau Traditional screening by Papanicolau (pap smear) test (pap smear) test

• Used universally for initial detection Used universally for initial detection of intraepithelial abnormalitiesof intraepithelial abnormalities

• In case of atypical squamous cells of In case of atypical squamous cells of undetermined significance HPV DNA undetermined significance HPV DNA detection is recommendeddetection is recommended

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Molecular TestingMolecular Testing• Hybrid Capture 2 (hc2) System HPV Hybrid Capture 2 (hc2) System HPV

DNA TestDNA Test– Approved by FDAApproved by FDA

• Nucleic acid hybridization assayNucleic acid hybridization assay

• No target DNA amplificationNo target DNA amplification

• Single amplification using microplate Single amplification using microplate chemiluminescence for qualitative chemiluminescence for qualitative detection of 18 types of HPVdetection of 18 types of HPV

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Molecular Testing cont.Molecular Testing cont.

• Differentiates between low and high Differentiates between low and high risk HPVrisk HPV– Low-risk HPV: 6, 11, 42, 43, 44Low-risk HPV: 6, 11, 42, 43, 44– High-risk HPV: 16, 18, 31, 35, 39, 45, High-risk HPV: 16, 18, 31, 35, 39, 45,

51, 52, 56, 58, 59, 6851, 52, 56, 58, 59, 68

• Does not determine specific HPV Does not determine specific HPV genotypegenotype

Controls and ReagentsControls and Reagents• ControlsControls

– High-risk control: cloned HPV 16 DNAHigh-risk control: cloned HPV 16 DNA– Low-risk control: cloned HPV 6 DNALow-risk control: cloned HPV 6 DNA

• Negative controlNegative control– Carrier DNACarrier DNA

• Calibrators (run in triplicate)Calibrators (run in triplicate)– Low-risk Calibrator: Cloned HPV 11 DNALow-risk Calibrator: Cloned HPV 11 DNA– High-risk Calibrator: cloned HPV 16 DNAHigh-risk Calibrator: cloned HPV 16 DNA– Ensure that the reagents and calibrator Ensure that the reagents and calibrator

materials are functioning properly, for materials are functioning properly, for determination of assay cut-off valuedetermination of assay cut-off value

Controls and Reagents Controls and Reagents cont.cont.• ProbesProbes

– Low-risk Probe: HPV 6, 11, 42, 43, 44 RNA Low-risk Probe: HPV 6, 11, 42, 43, 44 RNA cocktailcocktail

– High-risk Probe: HPV 16, 18, 31, 33, 35, 39, 45, High-risk Probe: HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 58, 59, 68, RNA cocktail51, 52, 58, 59, 68, RNA cocktail

• Capture MicroplateCapture Microplate– Coated with goat polyclonal anti-RNA:DNA hybrid Coated with goat polyclonal anti-RNA:DNA hybrid

antibodiesantibodies

• Detection Reagent 1Detection Reagent 1– Alkaline phosphatase-conjugated murine Alkaline phosphatase-conjugated murine

monoclonal antibodies to RNA:DNA hybridsmonoclonal antibodies to RNA:DNA hybrids

• Detection Reagent 2Detection Reagent 2– Chemiluminescent substrateChemiluminescent substrate

Specimen RequirementsSpecimen Requirements

• Cervical specimens collected using a Cervical specimens collected using a broom type collection device placed in broom type collection device placed in PreservCyt SolutionPreservCyt Solution

• Cervical biopsies btw 2-5 mm in Digene Cervical biopsies btw 2-5 mm in Digene Specimen Transport MediumSpecimen Transport Medium

• Specimens collected with the Digene Specimens collected with the Digene Cervical Sampler, placed in SurePath Cervical Sampler, placed in SurePath Preservative FluidPreservative Fluid

• 4mL of sample needed for denaturation 4mL of sample needed for denaturation processprocess

Denaturation ProcessDenaturation Process

• Samples are mixed with Sample Samples are mixed with Sample Conversion bufferConversion buffer

• Then mixed with a 2:1 ratio of Then mixed with a 2:1 ratio of Specimen Transport Medium (STM) Specimen Transport Medium (STM) and Denaturation Reagent (DNR)and Denaturation Reagent (DNR)– STM-preservative that retards bacterial STM-preservative that retards bacterial

growth and retains DNA integritygrowth and retains DNA integrity– DNR-dilute sodium hydroxide solution, DNR-dilute sodium hydroxide solution,

which lysis cells and denatures HPV DNAwhich lysis cells and denatures HPV DNA

DetectionDetection• The Hybrid Capture 2 is a fluid-based The Hybrid Capture 2 is a fluid-based

molecular hybridization assaymolecular 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 Denatured HPV DNA is hybridized with low-risk or high-risk RNA probelow-risk or high-risk RNA probe

• Resulting hybrids are captured on Resulting hybrids are captured on microplate wells by immobilized antibodymicroplate wells by immobilized antibody

DetectionDetection• Detection is sandwich-style with a Detection is sandwich-style with a

second anti-RNA:DNA hybrid second anti-RNA:DNA hybrid conjugated to alkaline-phosphataseconjugated to alkaline-phosphatase

• Bound alkaline-phosphatase is revealed Bound alkaline-phosphatase is revealed by addition of a chemiluminescent by addition of a chemiluminescent dioxetane-based substratedioxetane-based substrate

• Substrate is cleaved by bound alkaline Substrate is cleaved by bound alkaline phosphate and emitted light is phosphate and emitted light is measured in a microplate luminometermeasured in a microplate luminometer

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Test InterpretationTest Interpretation• Emitted light is measured in Relative Emitted light is measured in Relative

Light Units (RLUs)Light Units (RLUs)– Specimens with RLU/CO ratio ≥ 1.7 with Specimens with RLU/CO ratio ≥ 1.7 with

low-risk HPV probe are considered positive low-risk HPV probe are considered positive for low-risk HPVfor low-risk HPV

– Specimens with RLU/CO ratio ≥ 1.7 with Specimens with RLU/CO ratio ≥ 1.7 with high-risk HPV probe are considered positive high-risk HPV probe are considered positive for high-risk HPVfor high-risk HPV

– Specimens with RLU/CO ratio btw 0.80-1.7 Specimens with RLU/CO ratio btw 0.80-1.7 are considered indeterminate for either low-are considered indeterminate for either low-risk or high-risk HPV and must be repeatedrisk or high-risk HPV and must be repeated

LimitationsLimitations• Significant number of false-positives (10%-Significant number of false-positives (10%-

19%) due to cross reactivity with low-risk 19%) due to cross reactivity with low-risk HPV DNAHPV DNA

• HPV DNA not amplifiedHPV DNA not amplified– Negative predicted value may be compromised in Negative predicted value may be compromised in

cases in which HPV DNA copy number is lowcases in which HPV DNA copy number is low

• No internal control used for sample No internal control used for sample sufficiencysufficiency– Not possible to determine if results are due to Not possible to determine if results are due to

insufficient DNA or true negativeinsufficient DNA or true negative

• Large number of inconclusive resultsLarge number of inconclusive results• Requires large sample volumeRequires large sample volume

Sources of ErrorSources of Error• Large concentrations of whole blood, douche, Large concentrations of whole blood, douche,

anti-fungal cream, and contraceptive jellyanti-fungal cream, and contraceptive jelly– May cause false-negativeMay cause false-negative

• Contamination of Capture Microplate and Contamination of Capture Microplate and Detection Reagent 2 with exogenous alkaline Detection Reagent 2 with exogenous alkaline phosphatasephosphatase– May cause false-positiveMay cause false-positive

• Presence of nucleases found on human skin Presence of nucleases found on human skin and materialsand materials– Causes nucleic acid degradationCauses nucleic acid degradation

• In accurate volume delivery of samples and In accurate volume delivery of samples and reagentsreagents

ConclusionConclusion• Due to various types of HPVs the Due to various types of HPVs the

most reliable method of detection is most reliable method of detection is through molecular testingthrough molecular testing

• The Hybrid Capture 2 System helps The Hybrid Capture 2 System helps differentiate btw low-risk and high-differentiate btw low-risk and high-risk HPV infectionsrisk HPV infections

• Is used in conjunction with pap Is used in conjunction with pap smears to diagnose, treat, and smears to diagnose, treat, and prevent cervical cancerprevent cervical cancer