Yi-Chen Yang, Drug Biology Division Bureau of Food and Drug Analysis Department of Health, Taiwan Collaborative study for establishment of the first national standard for Parvovirus B19 DNA NAT assays SoGAT XX June 2007

2 NAT requirements in Taiwan ( 2002 ) –NAT test for Parvovirus B19 is suggested on plasma pool or mini-pool the cut-off limit of B19 DNA should be < 10 5 IU/mL WHO International standard for B19 virus DNA (99/800) European Pharmacopoeia Biological Reference Preparation (BRP) for B19 Virus DNA Testing of Plasma Pools by NAT To facilitate the implementation of the policy in Taiwan –national standard and working reagent for human parvovirus B19 DNA NAT assays Background

3 National Standards for NAT Reasons for Preparation –Difference genotypes among countries and regions (HBV, HCV) –Limited vials of international standards –One of BFDA’s task: supply of reference standards Intended use  National standard : as a laboratory standard or reference material  Working reagent : as a run control for routine NAT assays Blood/cell/tissure donor screening by NAT assays Plasma pool screening by NAT assays Testing for Class III IVD marketing approval Quality control of IVD Post marketing surveillance of IVD Research However it is for the user to establish suitability of purpose

4 Product Item HBV DNA National Standard HBV DNA Working Reagent Conc. (IU/mL) genotypeBB Lot number BFDA lot 92-08BFDA lot 92-08W Volume0.5 mL/vial1 mL/vial National Standards for NAT in Taiwan Product Item HCV RNA National Standard HCV RNA Working Reagent Conc. (IU/mL)5.2 × genotype1b Lot number BFDA lot 93-09BFDA lot 93-09W Volume0.5 mL/vial1 mL/tube

5  To establish the B19 DNA national standard  10 6 IU/mL, 0.5 mL/vial  around 1,000 vials  To prepare the B19 DNA working reagent  10 4 IU/mL, 1 mL/vial  around 1,000 vials Objective

6 Flow Chart of NAT Standard and Working Reagent Preparation High titer positive plasma Dilute positive plasma to suitable concentration Calibrate the titers of candidates against the international standard by a collaborative study Pooled cryosupernatant Stability study

7 Preparation of B19 DNA National Standard and Working Reagent High titer positive plasma –Screening for other viruses Anti-HIV 1/2, HBsAg, Anti-HCV by EIA: (-) HAV RNA, HBV DNA, HCV RNA & HIV-1 RNA by NAT: (-) –Quantitative analysis of B19 DNA –DNA sequencing/ Nucleotide-nucleotide BLAST (NCBI) Diluent : Pooled human cryosupernatant Anti-HIV 1/2, HBsAg, Anti-HCV by EIA: All (-) HAV RNA, HBV DNA, HCV RNA, HIV-1 RNA & B19 DNA by NAT : All (-) Anti-B19 IgM, Anti-B19 IgG by EIA: All (-) Check the titers of preparations in 3 different assay methods –LightCycler Parvovirus B19 Quantification Kit –RealArt Parvo B19 LC kit –In-house assay

8 International Collaborative Study for B19 DNA Standards Participating Labs including: 10 Labs from 7 countries –Official Medicine Control Laboratories (OMCL) –NAT testing laboratory –Manufacturers of plasma products –Manufacturers of in vitro diagnostics Each Lab received 3 vials of each sample, and 1 vial of WHO B19 DNA IS (code: 99/800) –Perform 3 independent assays for each sample –Calibrate the candidates against the IS

9 Data for the Collaborative Study National standard Lab codeMethod Result nMeanSDCV% 1RealArt Parvo B19 LC RealArt Parvo B19 LC In-house assay Roche LC Parvo B In-house assay In-house assay COBAS TaqScreen DPx* Nested PCR In-house assay AIn-house assay BRoche LC Parvo B CRealArt Parvo B19 LC * in development

10  All data were within the mean ± 2 SD for national standard, showed that all laboratories are in good agreement with the results. Mean SD (6.627) - 2SD (5.911)

11 Results of the Collaborative Study National standard Lab codeMethod Result nMeanSDCV% 1RealArt Parvo B19 LC RealArt Parvo B19 LC In-house assay Roche LC Parvo B In-house assay In-house assay COBAS TaqScreen DPx* Nested PCR In-house assay AIn-house assay BRoche LC Parvo B CRealArt Parvo B19 LC Calculated value Parvovirus B19 DNA concentration1.9 × 10 6 IU/mL

12 Data for the Collaborative Study Working reagent Lab codeMethod Result nMeanSDCV% 1RealArt Parvo B19 LC RealArt Parvo B19 LC In-house assay Roche LC Parvo B In-house assay In-house assay COBAS TaqScreen DPx* Nested PCR In-house assay AIn-house assay BRoche LC Parvo B CRealArt Parvo B19 LC * in development

13  All data were within the mean ± 2 SD for working reagent, showed that all laboratories are in good agreement with the results. Mean SD (4.682) - 2SD (3.936)

14 Results of the Collaborative Study Working reagent Lab codeMethod Result nMeanSDCV% 1RealArt Parvo B19 LC RealArt Parvo B19 LC In-house assay Roche LC Parvo B In-house assay In-house assay COBAS TaqScreen DPx* Nested PCR In-house assay AIn-house assay BRoche LC Parvo B CRealArt Parvo B19 LC Calculated value Parvovirus B19 DNA concentration2.0 × 10 4 IU/mL

15 B19 DNA National Standard and Working Reagent Product Item B19 DNA National Standard B19 DNA Working Reagent Conc. (IU/mL)1.9 × × 10 4 Genotype11 Lot number BFDA lot 94-08BFDA lot 94-08W Volume0.5 mL/vial1 mL/tube Total amount1,100 vials1,000 tubes

16 Stability Study for B19 DNA Standards Check the titers in 2 different assay methods –RealArt Parvo B19 LC kit –In-house assay Performed 3 independent assays for each method

p ＞ 0.05, n.s. Results of the Stability Studies ( 25 ℃ ) p ＞ 0.05, n.s.

18 p ＞ 0.05, n.s Results of the Stability Studies ( 4 ℃ )

19 p ＞ 0.05, n.s Results of the Stability Studies ( -20 ℃ ) p ＞ 0.05, n.s.

20 p ＞ 0.05, n.s Results of the Stability Studies ( -80 ℃ )

21 Summary In this international collaborative study, a high level of agreement between results was obtained from different laboratories. The first national standard and working reagent for B19 DNA NAT assays with an assigned potency of 1.9 × 10 6 IU/mL and 2.0 × 10 4 IU/mL, respectively, were established. The national standard and working reagent were stable at 25 ℃ for 4 weeks, 4 ℃ for 8 weeks, -20 ℃ and -80 ℃ for at least 12 months.

22 Thank you for your attention Acknowledgements thanks to all participants of the collaborative study group –Dr. M. Y. Yu CBER, USA –Dr. C. M. Nübling, Dr. M. Chudy PEI, Germany –Dr. S. BaylisNIBSC, UK –Dr. Y. OkadaNIID, Japan –Dr. M. Gessner, Dr. A. KlotzBaxter AG, Austria –Dr. D. JohnstoneCSL Bioplasma, Australia –Dr. R. SmithNGI, USA –Dr. T. GrewingQIAGEN, Germany –Dr. D. Sizmann, Dr. A. SchubertRoche, Germany –Dr. J. SaldanhaRoche, USA –Dr. A. HeathNIBSC, UK

23 NAT requirement in Taiwan (Dec ) to improve the safety of blood products 1.NAT tests on plasma pool are required: negative for HIV, HBV, HCV 2.Virus inactivation/removal steps for enveloped and non- enveloped viruses: two steps or one step ( shown to be reliably effective ) 3.For S/D treated blood products One additional step should be performed e.g. monoclonal purification or nanofiltration ( at least 4 log reduction of HAV ) or The plasma pool should be HAV NAT(-) before the manufacturing process 4.NAT test for Parvovirus B19 is suggested on plasma pool or mini-pool the cut-off limit of B19 DNA should be < 10 5 IU/ml