Hbv genotype Panel Background

HBV Genotype Panel

Background

• During the ‘WHO Consultation on Global Measurement Standards and their use in the in vitro Biological Diagnostic Field’ in June 2004 concern was raised that HBsAg and HBV NAT test kits might be less sensitive for some HBV genotypes other than A2, which is represented in the current WHO International Standard preparations

• During the ECBS* meeting in October 2005 the PEI proposed a project to establish WHO International Reference Panels representing different HBV genotypes/HBsAg subtypes

• This project was assigned as high priority by ECBS

International reference panels for HBV genotypes

• HBV genotype panel (NAT tests)

• HBV genotype panel (HBsAg tests)

HBV genotype panels

• Efforts to collect plasma units worldwide

• HBV DNA/HBsAg high titre plasma samples with sufficient volume

• Characterization of 215 potential candidate materials

• quant HBV DNA, quant HBsAg
• Sequencing of entire S ORF,
• genotyping, escape mutations, HBsAg subtyping

• HBV genotypes A – G (H)

• One genotype H sample received recently
• This sample could not be considered for the NAT panel

• HBV genotype panel (NAT tests): 15 panel members

• HBV genotype panel (HBsAg tests): 16 panel members

• 12 samples are member in both panels

• Project in close cooperation with Prof Gerlich (Institute of Virology, University Giessen)

HBV genotype panel (NAT tests) Final characterization of the panel members

HBV genotype panel (NAT tests) Final characterization of the panel members

HBV genotype panel (NAT tests) Dilution of panel members, filling and lyophilisation

• Dilution matrix for panel members: Negative plasma pool

• Testing of 117 negative pre-screened plasma units
• HBV serology: anti-HBs; anti-HBc
• HBV/HCVHIV NAT: cobas Taqscreen MPX Test; Procleix Ultrio Assay
• Pooling of negative plasma units

• If possible, dilution to a HBV DNA concentration of about 106 IU/ml (12 / 15 samples)

• Dilution to a volume of 1.2 litre per sample

• 15 x 2,000 vials

• Filling volume 0.5 ml per sample

• Filling and lyo by a certified Swiss company

HBV genotype panel (NAT tests) Characterization of the final product

• Pre-study (PEI): Control of HBV DNA concentration before and after lyo

• no loss of HBV DNA and HBsAg concentration

• Stability testing programme

• Residual moisture content: 0.82% (SD ± 0.03%) (method acc. EP)

• Collaborative study

HBV genotype panel (NAT tests) – collaborative study

• Study purpose: evaluation of the HBV genotype panel using different NAT assays; parallel testing of the 2nd HBV DNA IS (97/750)

• Invited 23 labs

• Reply from 18 labs

• 19 participants (incl. PEI)

• 5 NCLs: CBER, ISS, NIBSC, NIID Japan, PEI,
• 6 special labs (special diagnostic expertise in viral hepatology): Argentina, Brazil, Germany, South Africa, Spain, USA
• 8 kit manufacturers: Germany, Korea, Sweden, Switzerland, Taiwan, USA (3)

• HBV NAT: quant 15 labs (17 tests), qual 3 labs

• HBV DNA sequencing and genotyping: 1 lab

• Statistical analysis

• Draft report for circulating

• Report to ECBS in 2009

HBV genotype panel (NAT tests) – collaborative study Preliminary results

HBV genotype panel (NAT tests) – collaborative study Preliminary results

Blast HBV genotyping tool – sequence comparison

HBV genotype panel (HBsAg tests)

• Prof. Gerlich, Univ. Giessen

• Cloning and sequencing of entire S ORF

• Genosubtype, HBsAg subtype, escape mutations

• Determination of HBs antigen concentration by

• Laurell immune electrophoresis (PEI-units)
• qCLIA (Architect, IU)

• Determination of HBsAg protein by

• UV photometry after purification (ng)

• Removal of virions from the HBsAg subviral particles by ultracentrifugation over sucrose cushion (decrease of infectivity about 99%)

• Determination of HBsAg content in the supernatant by qCLIA (IU/ml)

• PEI

• Residual HBV-DNA by quant NATs

• Dilution in negative plasma pool (HBsAg concentration about 30 IU/ml)

• Pilot study to investigate the effects of lyophilisation on the consistency of HBsAg detection

• Filling and lyophilisation

• Collaborative study

• Report for establishment to ECBS 2010

International reference panels for HBV genotypes Conclusion

• Two panels (NAT tests and HBsAg tests)

• Well characterized panels

• serological and molecular characterization, protein characterization
• NAT: 16 different assays (13 quant; 3 qual), overall good correlation in detecting most of the genotypes, no unitage of panel members
• HBsAg: results from pilot studies provide hints for different detection efficiency

• Intended use

• Assay validation
• Assay comparison
• BV testing (NCLs, manufacturers)

• Establishment by ECBS

• NAT test panel probably 2009
• HBsAg panel 2010

• Global availability

Acknowledgements

• Prof Gerlich, Institute of Virology, University Giessen

• Prof Yoshizawa & Prof Tanaka, Hiroshima University, Japan

• Biotest AG, Dreieich

• Federal Blood Center, Moscow, Russia

• Fundação Pró-Sangue Homocentro de São Paulo, Brazil

• German Red Cross Frankfurt/Main

• Iranian Blood Transfusion Organization, Tehran, Iran

• South African National Blood Service

• WHO Collaborative Study Group

• Dr Ana Padilla, WHO, HSS / EMP / QSM, Geneva

• NIBSC

• People from PEI

• Section of Molecular Virology and IVD-Section
• Administrative staff

• Thank you for your attention!