Human papillomavirus laboratory manual - WHO

1 Human papillomaviruslaboratory manualFirst edition, 2009 WHO/ ORIGINAL: ENGLISHI mmunization, Vaccines and BiologicalsHuman papillomaviruslaboratory manualFirst edition, 2009 WHO/ ORIGINAL: ENGLISHI mmunization, Vaccines and BiologicalsiiThe Department of Immunization, Vaccines and Biologicals thanks the donors whose unspecified financial support has made the production of this document document was produced by the Quality, Safety and Standards (QSS) teamof the Department of Immunization, Vaccines and Biologicals Ordering code: WHO/ Printed: November 2010 This publication is available on the Internet at: of this document as well as additional materials on immunization, vaccines and biologicals may be requested from: World Health Organization Department of Immunization, Vaccines and Biologicals CH-1211 Geneva 27, Switzerland Fax: + 41 22 791 4227 Email: World Health Organization 2010 All rights reserved.

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4 In no event shall the World Health Organization be liable for damages arising from its use. The named authors alone are responsible for the views expressed in this by the WHO Document Production Services, Geneva, SwitzerlandiiiContentsAbbreviations and Acronyms ..vAcknowledgements ..ix1. Introduction .. Purpose and target audience of the manual .. Biology and natural history of HPV .. The burden of the disease caused by HPV .. References ..62. Role of the laboratory in HPV surveillance and vaccine impact monitoring .. WHO position on HPV vaccines .. The WHO HPV LabNet .. HPV vaccine surveillance .. References ..133. laboratory quality assurance .. The basis of laboratory quality assurance .. Standard operating procedures.

5 Documentation .. Equipment and instruments .. Supplies .. laboratory safety .. Audits .. References ..254. Collection and handling of specimens for HPV testing .. Intro duction .. Serum .. Cervical exfoliated cell samples .. Other samples for HPV testing .. Shipping specimens .. Receiving specimens .. References ..345. DNA extraction and HPV DNA testing .. Principles .. Sample extraction/lysis .. Overview of molecular methods for detection of HPV DNA .. HPV detection with PGMY09/11 PCR .. HPV typing with type-specific hybridization and chemiluminescent detection .. Quality control of PGMY PCR and RBH .. References ..63iv6. Assay validation .. Introduction .. Quality assurance specific for HPV DNA testing.

6 Issues to be considered during performance of an assay .. Test material .. Validation of an assay .. References ..717. HPV serology ELISA assay .. Introduction .. Principle .. Equipment, supplies and reagents .. Test procedure .. Quality control of VLPs .. Determination of coating concentration of VLPs .. Control of sensitivity and specificity performance of a new batch of VLP ..838. HPV neutralization assay .. Introduction .. Equipment, supplies and reagents .. Procedure .. Data analysis and interpretation of results .. References ..949. International standards and secondary standards .. General concepts .. Secondary (or working) standards .. ISs for HPV DNA and antibodies .. Preparation of secondary standards for HPV DNA and their calibration in IU.

7 Preparation of secondary standards for HPV antibodies and their calibration in IU .. Storage and stability of secondary standards .. Dispatch of the secondary standards to end-users .. Batch replacement .. References ..10410. Data management .. Introduction .. Recording laboratory results .. Reporting laboratory activity and results ..108 Annex ..109vAbbreviations and AcronymsABTS 2,2 -azino-bis(3-ethylbenzthiazoline-6-sulph onic acid)AIS adenocarcinoma in situ bp base pairBSL basic biosafety levelBPV bovine papillomavirusC centigradeCIN cervical intraepithelial neoplasiacm centimetreCt cycle thresholdDNA deoxyribonucleic aciddNTPs deoxynucleoside triphosphates DMEM Dulbecco s Modified Eagle MediumDTT dithiothreitolECBS Expert Committee on Biological StandardizationECL enhanced chemiluminescent substrateEDAC N-Ethyl-N -(3-dimethylaminopropyl) carbodiimide hydrochlorideEDQM European Directorate for the Quality of Medicines & HealthCareEDTA ethylene-diamine-tetra-acetic acidE1-E7 early proteins of HPV numbered E1 E7 (however no E3)

8 EIA enzyme immunoassayELISA enzyme-linked immunosorbent assayFCS fetal calf serumg gravityGE genome equivalentsg gramGRL WHO global reference laboratoryh hourHBV hepatitis B virusHC2 hybrid capture 2 (Digene , Qiagen)HCV hepatitis C virusHIV Human immunodeficiency virusviHLA histocompatibility leukocyte antigenHPLC high-performance liquid chromatography HPV Human papillomavirusHPV LabNet WHO HPV laboratory NetworkHR high riskHRP horseradish peroxidaseHS horse serumHS-PBS 10% HS in PBSHSIL high grade squamous intraepithelial lesionIARC International Agency for Research on CancerICH International Committee on HarmonizationIg immunoglobulinIgG immunoglobulin GIS international standardIU international unitkb kilobaseskbp kilobase pairskDa kilodaltonL litreL1, L2 late proteins of HPV, major (L1) and minor (L2)

9 Proteins of capsidlab laboratoryLQA laboratory quality assuranceLR low riskLSIL low grade squamous intraepithelial lesionM molarMab monoclonal antibodymAmp milliamperemg milligrammin minutemL millilitremM millimetre L microlitreNAT nucleic-acid amplification techniqueNB neutralization bufferNEAA non-essential amino acidsNIBSC National Institute for Biological Standards and ControlNIH National Institutes of Health nm nanometer (10-9 metre)NTC no template controlviiOD optical densityORF open reading framePBS phosphate buffered salinePBS-T Tween 20 PCR polymerase chain reactionpg picogramPLL parallel linePsV pseudovirionPTS Proficiency Testing Scheme (WHO LabNet)QA quality assuranceQC quality controlRBH reverse blotting hybridizationRLU relative light unitsRNA ribonucleic acidRRL WHO regional reference laboratoryRT room temperatureRUO research use onlySDS sodium dodecylsulphateSEAP secreted alkaline phosphatases second(s)SOP standard operating procedureSTM specimen transport media (Qiagen) SSPE saline sodium phosphate EDTATBE Tris-Borate-EDTATC tissue cultureTex no template extraction controlURR upstream regulatory regionUSA United States of AmericaUV ultravioletV voltVLP virus-like particleWHO World Health OrganizationviiiixThis document was produced under the auspices of the World Health Organization (WHO) HPV laboratory Network (HPV LabNet) (see Annex in this document)*.

10 The editors were Dr Elizabeth R Unger, Global WHO HPV Reference laboratory (GRL), Centers for Disease Control and Prevention, USA and Professor Joakim Dillner, GRL, laboratory Medicine Sk ne, Malm University Hospital, following authors contributed greatly to this document: Dr Morag Ferguson (former Principal Scientist) and Dr Dianna Wilkinson, WHO International laboratory for Biological Standards, Division of Virology, National Institute for Biological Standards and Control (NIBSC), United Kingdom; Dr Denise Nardelli Haefliger and Dr Roland Sahli, Regional WHO HPV Reference laboratory (RRL), Switzerland; Ms Carina Eklund and Ms Eva Hedvall, GRL, Sweden; Dr Gitika Panicker, GRL, USA.