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Why Anti-HBc?

Establishment of the 1st WHO International Standard for Detection of Antibodies to Hepatitis B Virus Core Antigen (anti-HBc) SoGAT XXI 29 May 2009 Dr. Heiner Scheiblauer Paul-Ehrlich-Institut, Langen/Germany PEI-IVD. Why Anti-HBc?.

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Why Anti-HBc?

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  1. Establishment of the 1st WHO International Standard for Detection of Antibodies to Hepatitis B Virus Core Antigen (anti-HBc)SoGAT XXI29 May 2009Dr. Heiner ScheiblauerPaul-Ehrlich-Institut, Langen/GermanyPEI-IVD

  2. Why Anti-HBc? • Anti-HBc are the first antibodies after Hepatitis B virus infection and persist trough out life • Anti-HBc can detect a persistent HBV viraemia, that is not easy detectable by HBsAg or HBV DNA • Anti-HBc can detect HBV infections with HBV escape mutants or genotypes which are not detectable by certain HBsAg or HBV DNA test kits • Anti-HBc screening can prevent transfusion-transmitted HBV infections • Anti-HBc has a regulatory impact: requirement for obligatory screening in some countries worldwide

  3. WHO Anti-HBc StandardDemand • Estimation of the analytical sensitivity for anti-HBc tests • Calibration of anti-HBc test systems by manufacturers • Accuracy and reproducibility verification in batch testing • Current national PEI anti-HBc standard (no 82, serum, 100 PEI U/ml) has been used widely • Sensitivity of assays and anti-HBc content of samples expressed as PEI-U/ml • PEI anti-HBc standard stocks declined, replacement preparation needed

  4. WHO Anti-HBc Collaborative StudySamples Tested Candidate Material

  5. WHO Anti-HBc Collaborative StudyStudy design • Analytical sensitivity of Samples A (NIBSC 95/522) and B (PEI 82) at the intercept with the assay´s cut-off (endpoint titer, detection limit in U/ml) • Potency of NIBSC 95/522 relative to PEI 82 (U/ml) • GMV ratio of the detection limit • Parallel line model • Correlation between analytical sensitivity for NIBSC 95/522 and anti-HBc detection in Samples C (PEI 108166) and D (CBER Panel #11) • Variability in results between assay methods (repeatability), and between laboratories (reproducibility)  Geometric coefficient of variation (GCV%) • Statistical significance of differences between methods and laboratories • Stability

  6. WHO Anti-HBc Collaborative StudyParticipants • Hemocentro de Sao Paulo, Brazil • CBER, USA • INTS, France • Abbott Laboratories, USA • KFDA, Seoul, Republic of Korea • American Red Cross, Gaithersburg, USA • National Institute of Infectious Diseases, Tokyo, Japan • NIBSC, Hertsfordshire, UK • AFSSAPS, France • National Institute for the Control of Pharmaceutical and Biological Products Beijing Zhongyuan, China • NRL, Australia • VQC Laboratory Sanquin-CLB Diagnostics, Netherlands • Paul-Ehrlich-Institut, Germany

  7. ADVIA Centaur HBcT Architect Anti-HBc AxSYM Core Bioelisa anti-HBc Corzyme Elecsys Anti-HBc Enzygnost Anti-HBc monoclonal Genedia Anti-HBc ELISA Plus Hepanostika anti-HBc Uniform Immulite 2000 anti-HBc Immuncomb II HBc IgG IMx Core Anti-HBc EIA Lumipuls Presto HBc Ab-N Monolisa anti-HBc Plus Murex anti-HBc Ortho anti-HBc PRISM HBCore ST AIA-Pack HBcAb Architect Anti-HBc II (ex-US) / Architect Core (US) WHO Anti-HBc Collaborative StudyTest kits

  8. WHO Anti-HBc Collaborative StudySummary of test kits characteristics

  9. Collaborative Study - Suitability for variety of tests, mfcts, labs, geography • 20 anti-HBc test kits included in the study from 15 different manufacturers and from 7 different countries • Tested in 10 different countries (Australia, Brazil, China, France, Japan, Germany, Korea, Netherlands, UK, USA) • Including the current available anti-HBc test technologies • Competitive test format (n=14) • Indirect test format (n=5) • Sandwich test format (n=1) • Reductant pre-treatment (e.g. DTT) with (n=5) or without (n=15) • Manually conducted (n=9) • Automated (n=10) • Rapid assay (n=1) • All anti-HBc test kits coated with recombinant HBc antigen

  10. - PEI-U/ml NIBSC 95/522 0.08 0.16 0.31 0.63 1.25 2.50 5.00 10 9 9 8 PEI-82 NIBSC 95/522 8 7 7 6 6 5 5 S/Co S/Co 4 4 3 3 2 2 1 1 0 0 0.03 0.06 0.13 0.25 0.50 1.00 2.00 - PEI-U/ml PEI 82 WHO Anti-HBc Collaborative StudyPotency of NIBSC 95/522 relative to PEI 82 Dilution ranges for NIBSC 95/522 (sample A) was in the dynamic measuring range of assays Within an individual assay, Sample A and B (PEI 82) gave comparable dose responses

  11. Dilution equivalent to cut-off – NIBSC 95/522 1/1024 Endpoint dilution 1/512 1/256 1/128 1/64 1/32 7 12 1 0 12 13 1 7 12 12 1 4 7 12 9 12 7 5 12 2 6 12 10 8 2 5 Laboratory 7 11 12 5 9 12 4 12 1 3 4 12 8 1 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 Assay WHO Anti-HBc Collaborative Study Endpoint titers

  12. WHO Anti-HBc Collaborative Study Overall Potency • Overall potency was 49.8 U/ml (95%-CI 44.2 - 56.1 U/m) • Assay 1 had a potency of 22.8 U/ml (mean of 2 labs) • Assay 8 had a potency of 73.3 U/ml • Overall potency excluding assays 1 and 8 was 51.1 U/ml (95%-CI 45.3 - 57.5 U/ml)

  13. WHO Anti-HBc Collaborative Study Commutability Analytical sensitivity (low detection limits) in Sample A (NIBSC 95/522) correlated with positive score for anti-HBc in Samples C (PEI 108166) and D (CBER panel #11) One assay of the study did not follow this correlation

  14. NIBSC 95/522 Stability after 4 years tested with Architect anti-HBc 90 fresh +4°C +20°C 37°C 45°C 80 70 60 50 Endpoint titer 40 30 20 10 0 0 1 2 3 4 5 6 7 8 9 10 Replicates WHO Anti-HBc Collaborative Study Stability • The proposed anti-HBc standard NIBSC 95/522 is likely to be highly stable when stored at the recommended temperature of -20°C

  15. 1st International Anti-HBc Standard Conclusions • NIBSC 95/522 1st International Standard (IS) for detection of anti-HBc • Assigned unitage of 50 IU per ampoule (ml) • The IU provides continuity with the currently used PEI units/ml • Analytical sensitivity for anti-HBc was effective for estimation of sensitivity performance  the lower the detection limit with the IS, the higher the positive score for anti-HBc concentration in other samples • The proposed International anti-HBc Standard was found suitable for • estimation of analytical sensitivity for anti-HBc detection • calibration of anti-HBc test kit sensitivity by manufacturers • to calibrate secondary standards • for quality control procedures, e.g. in batch release testing

  16. 1st International Anti-HBc Standard Acknowledgements • NIBSC: Dr. Morag Ferguson • CBER: Dr. Robin Biswas • PEI: Dr. Peter Volkers Dr. Sigrid Nick Dr. Micha Nübling • WHO: Dr. Ana Padilla

  17. Thank you for your attention!

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