Collaborative study for establishment of the first national standard for Parvovirus B19 DNA NAT assa...
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Collaborative study for establishment of the first national standard for Parvovirus B19 DNA NAT assays. Yi-Chen Yang, Drug Biology Division Bureau of Food and Drug Analysis Department of Health, Taiwan. SoGAT XX June 2007. Background. NAT requirements in Taiwan ( 2002 )

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Yi chen yang drug biology division bureau of food and drug analysis

Collaborative study for establishment of the first national standard for Parvovirus B19 DNA NAT assays

Yi-Chen Yang, Drug Biology Division

Bureau of Food and Drug Analysis

Department of Health, Taiwan

SoGAT XX June 2007


Background

Background

  • 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 < 105 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


National standards for nat

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


Yi chen yang drug biology division bureau of food and drug analysis

National Standards for NAT in Taiwan


Objective

Objective

  • To establish the B19 DNA national standard

    • 106 IU/mL, 0.5 mL/vial

    • around 1,000 vials

  • To prepare the B19 DNA working reagent

    • 104 IU/mL, 1 mL/vial

    • around 1,000 vials


Yi chen yang drug biology division bureau of food and drug analysis

Pooled cryosupernatant

Dilute positive plasma to suitable concentration

Calibrate the titers of candidates against the international standard

by a collaborative study

Stability study

Flow Chart of NAT Standard and

Working Reagent Preparation

High titer positive plasma


Preparation of b19 dna national standard and working reagent

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


International collaborative study for b19 dna standards

International Collaborative Studyfor 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


Data for the collaborative study national standard

Data for the Collaborative Study National standard

* in development


Yi chen yang drug biology division bureau of food and drug analysis

Mean 6.269

+2SD (6.627)

- 2SD (5.911)

  • All data were within the mean ± 2 SD for national standard, showed that all laboratories are in good agreement with the results.


Results of the collaborative study national standard

Results of the Collaborative Study National standard


Yi chen yang drug biology division bureau of food and drug analysis

Data for the Collaborative Study Working reagent

* in development


Yi chen yang drug biology division bureau of food and drug analysis

Mean 4.309

+2SD (4.682)

- 2SD (3.936)

  • All data were within the mean ± 2 SD for working reagent, showed that all laboratories are in good agreement with the results.


Yi chen yang drug biology division bureau of food and drug analysis

Results of the Collaborative Study Working reagent


B19 dna national standard and working reagent

B19 DNA National Standard and Working Reagent


Stability study for b19 dna standards

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


Results of the stability studies 25

6.269

p>0.05, n.s.

Results of the Stability Studies ( 25℃ )

6.269

p>0.05, n.s.

4.309

p>0.05, n.s.


Results of the stability studies 4

Results of the Stability Studies ( 4℃ )

6.269

p>0.05, n.s.

4.309

p>0.05, n.s.


Results of the stability studies 20

Results of the Stability Studies ( -20℃ )

6.269

p>0.05, n.s.

4.309

p>0.05, n.s.


Results of the stability studies 80

Results of the Stability Studies ( -80℃ )

6.269

p>0.05, n.s.

4.309

p>0.05, n.s.


Summary

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 × 106 IU/mL and 2.0 × 104 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.


Acknowledgements

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

Thank you for your attention


Nat requirement in taiwan dec 19 2002 to improve the safety of blood products

NAT requirement in Taiwan (Dec. 19 2002 ) to improve the safety of blood products

  • NAT tests on plasma pool are required:

    negative for HIV, HBV, HCV

  • Virus inactivation/removal steps for enveloped and non-enveloped viruses: two steps or one step ( shown to be reliably effective )

  • 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

  • NAT test for Parvovirus B19 is suggested on plasma pool or mini-pool

    the cut-off limit of B19 DNA should be < 105 IU/ml


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