1. Preparation of HBV DNA reference standards and the experience of HBV NAT in Taiwan Dr. Hwei-Fang Cheng Department of Health, Taiwan

2. HBV DNA Reference Standards  National HBV DNA standard ： 10 6 IU/mL, 0.5 mL/vial (relative to NIBSC HBV NAT standard)  National HBV DNA working reagent ： 10 3 IU/mL, 1 mL/vial (relative to NIBSC HBV NAT working reagent)  Intended use for HBV NAT testing of blood and pooled plasma and biological research

3. High titer (~10 9 IU/ml) HBV positive plasma (negative for anti-HIV,anti-HCV and NAT of HIV, HCV, HAV, and B19) a) genotype B b) virus inactivation: 60 o C for 1 hr c) dilute into ~10 6 IU/mL and ~10 3 IU/mL Diluents: pooled human plasma (negative for HBsAg, anti-HIV, anti-HCV and NAT of HIV, HCV, HBV, HAV, and B19)

4. Collaborative Study Participating Labs (11 Labs) US CBER/FDA National Institute of Infectious Diseases (NIID),Japan Canadian Blood Services National Institute for Biological Standards and Control (NIBSC) Victorian Infectious Diseases Reference Laboratory (VDRL), Australia NTUH Hepatitis Research Center, Taiwan Center of Disease Control, Taiwan BFDA, Taiwan Roche Diagnostics Artus GmbH Digene Diagnostic Technology

5. LabMethodology/kitResult (IU/ml) AIn-house real time PCR1.4E+06 BIn-house real time PCR2.2E+06 CIn-house qualitative PCR2.5E+05 DIn-house real time PCR2.3E+05 E1. Artus RealArt HBV LC PCR4.2E+04 2. Bayer VERSANT HBV 3.01.8E+06 FHBV RealType PCR8.4E+05 GIn-house real time PCR2.5E+05 H1. Cobas Amplicor HBV monitor1.3E+06 2. In-house real time PCR1.9E+06 I1. Cobas Amplicor HBV monitor1.3E+06 2. Cobas TAQMAN3.2E+06 JArtus RealArt HBV LC PCR8.2E+05 KDigene kit6.6E+05 1 st National HBV DNA Standard

6. 0 1 2 3 4 5 6 7 8 9 10 2.02.53.03.54.04.55.05.56.06.57.0 E1C D G JF I1 K A B E2 H1 H2 I2 HBV DNA standard Estimated IU (log 10 /ml) Numbers

7. +2SD -2SD Average 5.955

8. 1 st National HBV Working Reagent Lab Methodology/kitResult (IU/mL) AIn-house real time PCR2.2E+02 BIn-house real time PCR2.9E+03 CIn-house qualitative PCR2.5E+02 DIn-house real time PCR9.5E+02 E1.Artus RealArt HBV LC PCRND* 2.Bayer VERSANT HBV 3.01.6E+03 FHBV RealType PCR1.4E+03 GIn-house real time PCR4.2E+02 H1.Cobas Amplicor HBV monitor1.8E+03 2.In-house real time PCR4.4E+02 I1.Cobas Amplicor HBV monitor1.4E+03 2.Cobas TAQMAN6.4E+03 JArtus RealArt HBV LC PCR6.4E+02 KDigene kitND* *ND: not detectable

9. 0 1 2 3 4 5 6 7 8 9 10 Estimated IU (log10/ml) 2.02.53.03.54.04.55.05.56.06.57.0 ACG H2 J D E2 F H1 I1 B I2 Working reagent Numbers

10. +2SD -2SD Average 2.953

11. Summary of National HBV DNA Standard and Working Reagent product item 1 st National HBV DNA standard 1 st National HBV working reagent IU/ml 10 6 10 3 Lot Number BFDA lot 92-08BFDA lot 92-08W GenotypeBB Vol. & Amount0.5ml/vial 500 vials 1 ml/vial 1000 vials Storage StateLiquid Frozen

12. Stability Test – HBV DNA Standard at 5 ±3 o C days

13. Stability Test – HBV DNA Working Reagent at 5 ±3 o C Cobas Amplicor days

14. Summary  The assigned potency of HBV DNA standard and HBV working reagent are 10 6 IU/ml and 10 3 IU/ml, respectively.  Genotype of national HBV DNA standard and working reagents is genotype B; and the serotype is adw.  National HBV DNA standard and working reagent are stable at 4 ℃ for 4 weeks.

15. HBV NAT Experience in Taiwan

16. Blood center （ Date collected ） Tainan （ March 2003 ） Taipei （ June 2003 ） Taipei （ June 2003 ） Units screened 1440 ＊ 1296 ＊ 516 ＊＊ HBVHBV EIA-positive NAT-negative 000 EIA-negative NAT-positive 006 (1.2 %) EIA-positive NAT-positive 17 （ 1.2 % ） 5 （ 0.4 % ） 2 （ 0.4 % ） （ 1 ） EIA ： Murex HBsAg version 3.0 kit; Sensitivity: <0.2 ng （ 2 ） NAT ＊ Pooled units test (12 units/pool); Roch High Pure Viral Nucleic Acid Kit (0.2 ml of extraction volume); In house nested PCR; Sensitivity of 22 IU/ml. ＊＊ Single unit test Ampliscreen HBV/Roch (1 ml of extraction volume) ; In house nested PCR ； Sensitivity of 12 IU/ml