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Parvovirus B19 DNA Genotype Panel

Parvovirus B19 DNA Genotype Panel. Sally Baylis 1 , Alan Heath 2 , Mei- ying Yu 3 1 Viral Safety Section, PEI; 2 Informatics Laboratory, NIBSC-HPA; 3 Division of Hematology, CBER/FDA SoGAT XXI, Brussels, 28 th -29 th May 2009. Parvovirus B19 and Plasma Screening.

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Parvovirus B19 DNA Genotype Panel

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  1. Parvovirus B19 DNA Genotype Panel Sally Baylis1, Alan Heath2, Mei-ying Yu3 1Viral Safety Section, PEI; 2Informatics Laboratory, NIBSC-HPA; 3Division of Hematology, CBER/FDA SoGAT XXI, Brussels, 28th-29th May 2009

  2. Parvovirus B19 and Plasma Screening • Parvovirus B19 (B19V) viraemia can exceed 1012 copies/ml in asymptomatic blood & plasma donors • Fractionation plasma pools can contain >109 copies/ml of B19V DNA • B19V transmission has occurred in patients treated with S/D plasma, clotting factor concentrates & fibrin sealants; also blood components • For S/D plasma, no B19V seroconversion was observed in patients receiving <104 copies/ml B19V DNA, used to define threshold concentrations of B19V in start pools for certain products

  3. Regulatory Requirements for B19V DNA NAT Testing in Europe • In 2004 introduction of Ph Eur requirements for B19V DNA testing for plasma pools used in the manufacture of: • Human plasma (pooled and treated for virus inactivation) • Human anti-D immunoglobulin • Human anti-D immunoglobulin for IV administration • Human albumin/normal immunoglobulin added to anti-D immunoglobulin • Quantitative test limit of 10 IU/l

  4. Genetic Variation of Parvovirus B19 • Previously it was believed that the B19V genome varied only by 1-2% • Identification of more divergent variant viruses (V9, A6, LaLi) • Classification into three distinct genotypes, with further sub-groups identified for genotypes 1 and 3 • Overall the sequences differ by ~10%, with the promoter region differing by >20% • In the VP1/2 region variation is ~1% at the amino acid level between virus genotypes, representing a single serotype

  5. Genetic Diversity of Parvovirus B19 V9 LaLi A6 D91.1 IM81 Parsyan et al. 2007

  6. Regulatory Requirements for Detection of Variants of B19V 8th Report of the International Committee on the Taxonomy of Viruses, 2005 classified A6, LaLi and V9 as strains of B19V Now included in mandatory Ph Eur test requirement But problems of implementation Issues with specificity of commercial kits No reference materials available

  7. Assays for the Detection of Parvovirus B19 DNA • LightCycler Parvovirus B19 Quantification Kit (Roche) • No detection of gt 2 and gt 3 • RealArt Parvo B19 LC Kit (Qiagen/artus), alternative kits available for the ABI and Rotorgene platforms • Under detection of gt 3b (LC) • Under detection of gt 2 and gt 3 (TM) • In-house tests • Several are unable to detect gt 2 and gt 3

  8. Sensitivity of Detection of Different Genotypes of Parvovirus B19

  9. Batch Release - Issues The detection of different genotypes of B19V is still an ongoing issue, although presence of genotypes 2 and 3 is extremely rare in plasma from Europe and North America Published studies have reviewed the ability of different assays (in-house and commercial) to detect different genotypes Proficiency Testing Schemes (PTSs) co-ordinated by EDQM for the OMCLs and plasma fractionators highlighted discrepant results when variants of B19V have been includes in the panels Discrepant results have occurred between OMCLs and plasma fractionators – failure of batch release test

  10. Addressing the Issues • Meeting at EDQM in November 2006 to discuss commercial NAT assays for B19V • Extraordinary Meeting of SoGAT at NIBSC in March 2007 - standardization of B19V for different genotypes • Classification of B19V • Epidemiology • Suitability of test procedures • Survey of blood products • Sources of reference materials • Recommendations and way forward • Preparation of a plasma genotype panel

  11. Current Status of Parvovirus B19 NAT Screening by U.S. Manufacturers (I) • In 1999, safety of pooled plasma S/D treated correlated with those lots having <104 geq/ml of B19V DNA in manufacturing pool (a phase 4 study) • Since Sep 1999 BPAC, B19V NAT for plasma for further manufacturing is an in-process test and is reviewed under Biologics Licensing Applications (BLAs) or their supplements for plasma derivatives • Proposed B19V DNA limit: ≤104 IU/ml for manufacturing pools destined for all plasma derivatives • In-date blood components be quarantined and destroyed when possible

  12. Current Status of Parvovirus B19 NAT Screening by U.S. Manufacturers (II) • B19V NAT methods for testing minipools and manufacturing pools • In-house procedures, mostly quantitative NAT • Minipool testing (384 – 512 donations) & sensitivity for excluding original donations of ~106 IU/ml • The need to detect all 3 genotypes • Detection is indirectly based upon sequence alignment analysis of primers and probes with known isolates • Both WHO 1st /2nd International Standards and CBER working standard for B19V DNA are all genotype 1 • Validation as an analytical procedure according to ICH and OMCL guidelines

  13. Current Status of Parvovirus B19 NAT Screening by U.S. Manufacturers (III) • Currently several weeks can elapse between donation collection & identification of B19V NAT-positive donation. Hence, no meaningful notification or retrieval is feasible within the dating period of any cellular blood component intended for use in transfusion • FDA encourages steps to shorten this time lapse • When a donor management procedure is in place, such as identification & deferral of individual B19V-reactive Whole Blood (WB) donors, it is no longer considered in-process testing. The threshold level to withhold WB donations or components from use for transfusion needs to be evaluated in clinical trials

  14. WHO Collaborative Study 1st International Standard for B19V DNA * All are genotype 1 B19V 99/800=WHO 1st IS (5 x 105 IU/vial) established in Oct 2000 (Saldanha et al, Vox Sang 2002) 99/802=WHO 2nd IS in Oct 2008 CC = CBER working standard, liquid frozen stored at ≤−70 ºC since 1999

  15. Genotype Panel Preparation (I) • 3 window-period plasma donations for preparing 3 positive members • Gt 1: same originating plasma (NIBSC-UK-ENG-3QG), ~1012 IU/ml, for WHO 1st IS and 2nd IS for B19V DNA from NIBSC • Gt 2: ~1011 IU/ml, IM-81 strain (GenBank AY903437) from Baxter BioScience under MTA [Blümel et al, J Virol 2005] • Gt 3: ~5 x 1011 IU/ml (P1, GenBank FJ265736) from Talecris under MTA [Rinckel et al, Transfusion, in press]

  16. Genotype Panel Preparation (II) • One negative member derived from pooled plasma (also used as diluent for viral stocks) • Formulation of a defibrinated negative human plasma pool (Basematrix, Lot 113985, SeraCare) derived from 25 screened Source Plasma units (~20 L in total) kindly provided by National Genetics Institute (NGI). All donations were found negative for the following markers: • Anti-HIV 1/2, anti-HCV, HBsAg, anti-HBc (IgG and IgM, Abbott Corzyme), & anti-B19V (IgG and IgM, Biotrin) • ID-NAT: HIV-1, HCV, HBV, B19V, HAV, & WNV • Pooled plasma testing • ID-NAT for HIV-1, HCV, HBV, HAV, & B19V by both NGI and Talecris; negative for all viral markers

  17. Genotype Panel Preparation (III) • Formulation of intermediate viral stocks, ~1010 & ~108 IU/ml, for all 3 genotypes • Quantification of 3 intermediate stocks, ~108 IU/ml, by 4 laboratories (PEI, NGI, Talecris, and CBER) • 6 quantitative NAT methods • Formulation of 3 final bulks targeted to contain ~106IU/ml • Each bulk monitored before fill by CBER-TaqMan • Filling of 4-member genotype panel • 3000 vials filled per member (1.1 ml fill per vial) per week and stored at ≤−70 ºC (filled under contract by SeraCare)

  18. Testing Summary of Intermediate Viral Stocks, ~108 IU/ml, for 3 Genotypes B19V DNA, IU/ml Assay Gt 1 Gt 2 Gt 3 2.8 x 107 PEI/Artus kit 8.6 x 107 2.0 x 108 PEI/ LC 1.2 x 108 4.7 x 108 3.4 x 108 PEI/ TaqMan 1.3 x 108 6.0 x 108 3.1 x 108 7.0 x 108 NGI* 1.3 x 108 2.2 x 108 4.1 x 108 Talecris 1.2 x 108 1.4 x 108 CBER/TaqMan 9.8 x 107 3.5 x 108 1.7 x 108 GeoMean 1.1 x 108 4.5 x 108 2.2 x 108 * Average of two runs, removing the outlier results

  19. B19V Genotype Panel Collaborative Study • The plasma panel (Members 1-4) has been evaluated in parallel with the 2nd WHO IS (genotype 1) 99/802 • Member 1 – gt 1 • Member 2 – gt 2 • Member 3 – gt 3 • Member 4 – negative plasma • The collaborative study commenced in October 2008 • Participants were requested to use assays targeting conserved sequences of B19V and to test samples in four independent assays

  20. B19V Genotype Panel Collaborative Study contd. • 35 laboratories, from 13 different countries participated in the collaborative study • 33 sets of data from quantitative assays and 9 sets from qualitative assays were returned for analysis • The majority of data sets are from in-house real-time PCR assays (quantitative) • Variety of different extraction methods; assays cover different regions of the B19V genome • The negative panel member (Member 4) has been consistently reported as non-reactive

  21. Estimated IU/ml (log10) from Quantitative Assays N - Number of laboratory estimates

  22. Absolute Estimates of 2nd IS 99/802

  23. Absolute Estimates of Panel Member M1

  24. Absolute Estimates of Panel Member M2

  25. Absolute Estimates of Panel Member M3 Labs 02 & 34A found M3 negative. Lab 19 found M3 positive, but unable to quantify the B19V DNA content. Results excluded.

  26. Overall Means of Potencies (log10 IU/ml) Relative to Concurrently Tested IS for Quantitative and Qualitative Assays N - Number of laboratory estimates

  27. Potency of Panel Member M1 Relative to the IS

  28. Potency of Panel Member M2 Relative to the IS

  29. Potency of Panel Member M3 Relative to the IS

  30. Overall Means of Potencies (log10 IU/ml) Relative to Concurrently Tested IS for Quantitative assays Data are shown for quantitative assays (excluding Labs 9 and 28 for M2)

  31. Stability Studies • Stability studies are in progress reviewing the real-time stability of the panel members, to date there has been no evidence for loss of titre of the panel members (~12 months at recommended storage conditions i.e. ≤−70 ºC) • A review of the potency of CC and DD from the original WHO Collaborative Study has been performed on samples stored at ≤−70 ºC for > 9 years, there is no evidence for loss of potency of these preparations • Conclude the B19V plasma positive samples are stable at recommended storage conditions

  32. Conclusions and Recommendations • Assays used by participants have, on the whole performed extremely well with the different genotypes, the majority being in-house assays • Propose that the M1-M4 be established as the 1st International Reference Panel for B19V DNA, NIBSC code 09/110 • Whilst no unitage will be assigned to M1-M3, it is proposed to include in the “instructions for use” the results of the quantitative assay absolute estimates and cite the range of reported values from the collaborative study • Study report is currently with participants for review prior to submission to ECBS by July

  33. Genetic Diversity of Parvovirus B19 V9 LaLi A6 D91.1 IM81 Parsyan et al. 2007

  34. B19V DNA Collaborative Study Participants

  35. B19V DNA Collaborative Study Participants contd.

  36. Acknowledgements • NGI, CSL Behring, Baxter BioScience & Talecris Biotherapeutics for materials used in the studies • Collaborative study participants • David Padley, NIBSC • Li Ma, CBER/FDA

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