Parvovirus B19 DNA Genotype Panel
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Transcript of Parvovirus B19 DNA Genotype Panel
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
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
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
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
Parsyan et al. 2007
D91.1
V9
A6
Genetic Diversity of Parvovirus B19
IM81
LaLi
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
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
Sensitivity of Detection of Different Genotypes of Parvovirus
B19
0
1
2
3
4
5
6
7
8
Roche Artus
Lo
g C
on
cen
tra
tio
n I
U/m
l
Genotype 1 (B19)
Genotype 2 (A6)
Genotype 3 (V9)
Genotype 3 (D91.1)
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
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
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
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
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
WHO Collaborative Study 1st International Standard for B19V DNA
Candidate Preparation*
Log10 geq/ml Log10 IU/ml Anti-B19
AA (lyophilized)
NIBSC 99/800
5.76 6.00 Pos
BB (lyophilized)
NIBSC 99/802
5.73 5.96 Pos
CC (liquid)CBER
5.82 6.06 Neg
DD (liquid)CLB
7.70 7.94 Neg
* All are genotype 1 B19V99/800=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 2008CC = CBER working standard, liquid frozen stored at ≤−70 ºC since 1999
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]
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
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 ~106 IU/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)
Testing Summary of Intermediate Viral Stocks, ~108 IU/ml, for 3
Genotypes
B19V DNA, IU/mlAssayGt 1 Gt 2 Gt 3
Talecris
8.6 x 107 2.8 x 107
2.0 x 108
PEI/ LC
PEI/ TaqMan
NGI*
PEI/Artus kit
CBER/TaqMan
GeoMean
1.2 x 108 4.7 x 108 3.4 x 108
1.3 x 108 6.0 x 108 3.1 x 108
1.3 x 108 7.0 x 108 2.2 x 108
1.2 x 108 4.1 x 1081.4 x 108
9.8 x 107 3.5 x 108 1.7 x 108
1.1 x 108 4.5 x 108 2.2 x 108
* Average of two runs, removing the outlier results
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
B19V Genotype Panel Collaborative Study
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
Estimated IU/ml (log10) from Quantitative Assays
Sample N Mean 95% CI Min.-Max. Range
IS 32 5.99 5.95-6.03 5.75-6.22 0.47
M1 33 5.98 5.92-6.03 5.61-6.32 0.71
M2 31 5.94 5.85-6.04 5.43-6.52 1.09
M3 30 5.97 5.86-6.08 5.21-6.54 1.33
N - Number of laboratory estimates
Absolute Estimates of IS 99/802
0123456789
101112131415161718192021222324252627282930
Estimated IU (log10/ml)
4.00 4.25 4.50 4.75 5.00 5.25 5.50 5.75 6.00 6.25 6.50 6.75 7.00 7.25 7.50 7.75 8.00
09
29A
29B
34A
34B
01A
01B1
01B2
02
03
05
06
07
10
12
18
19
20
21A
21B
21C
22
23
24
27
28
32
35
08
16
30
Absolute Estimates of 2nd IS 99/802
Absolute Estimates of Panel Member M1
0123456789
101112131415161718192021222324252627282930
Estimated IU (log10/ml)
4.00 4.25 4.50 4.75 5.00 5.25 5.50 5.75 6.00 6.25 6.50 6.75 7.00 7.25 7.50 7.75 8.00
09 01A
01B1
13
19
22
29A
29B
01B2
03
05
10
12
18
20
21A
21B
21C
23
24
26
27
28
32
34A
34B
35
02
06
07
08
16
30
Absolute Estimates of Panel Member M1
Absolute Estimates of Panel Member M2
0123456789
101112131415161718192021222324252627282930
Estimated IU (log10/ml)
4.00 4.25 4.50 4.75 5.00 5.25 5.50 5.75 6.00 6.25 6.50 6.75 7.00 7.25 7.50 7.75 8.00
28 09 01A
02
18
19
29A
29B
34A
01B1
32
34B
01B2
03
05
08
10
13
20
21A
21B
22
24
26
35
06
07
12
21C
23
27
30
16
Absolute Estimates of Panel Member M2
Absolute Estimates of Panel Member M3
0123456789
101112131415161718192021222324252627282930
Estimated IU (log10/ml)
4.00 4.25 4.50 4.75 5.00 5.25 5.50 5.75 6.00 6.25 6.50 6.75 7.00 7.25 7.50 7.75 8.00
18 09
22
01A
01B1
03
08
13
28
29A
34B
01B2
05
07
10
20
21A
24
27
29B
30
32
35
12
21B
23
26
06
16
21C
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.
Overall Means of Potencies (log10 IU/ml) Relative to Concurrently Tested IS for Quantitative and Qualitative Assays
Sample AssayType
NOverallMean
log10 IU/ml
95% CIlog10 IU/ml Min.-Max. Range
M1Qual. 10 5.95 5.68 6.22 5.10-6.57 1.47
Quant. 34 5.98 5.94 6.02 5.74-6.20 0.46
M2Qual. 10 5.84 5.26 6.41 3.78-6.68 2.90
Quant. 34 5.87 5.74 5.99 4.52-6.36 1.83
M3Qual. 9 5.47 4.75 6.18 3.69-6.32 2.63
Quant. 31 5.97 5.87 6.07 5.18-6.58 1.40
N - Number of laboratory estimates
Potency of Panel Member M1 Relative to the IS
Potency of Panel Member M1 Relative to IS
0123456789
1011121314151617181920212223242526272829303132
IU (log10/ml)
3.50 3.75 4.00 4.25 4.50 4.75 5.00 5.25 5.50 5.75 6.00 6.25 6.50 6.75 7.00 7.25 7.50
11 01A
01B1
09
13
19
25
01B2
01C2
02
03
05
07
08
10
12
14
15
17
18
20
21A
21B
21C
22
23
24
26
27
28
29A
29B
30
32
33
34B
34C
35
01C1
06
16
31
34A
04
Potency of Panel Member M2 Relative to the IS
Potency of Panel Member M2 Relative to IS
0123456789
1011121314151617181920212223242526272829303132
IU (log10/ml)
3.50 3.75 4.00 4.25 4.50 4.75 5.00 5.25 5.50 5.75 6.00 6.25 6.50 6.75 7.00 7.25 7.50
04 28 09 01A
02
15
18
19
01B1
08
11
29A
29B
32
34A
34C
01B2
03
05
07
10
12
13
14
20
21A
21B
22
23
24
26
27
30
31
33
34B
35
01C2
06
16
17
21C
25 01C1
Potency of Panel Member M3 Relative to the IS
Potency of Panel Member M3 Relative to IS
0123456789
1011121314151617181920212223242526272829303132
IU (log10/ml)
3.50 3.75 4.00 4.25 4.50 4.75 5.00 5.25 5.50 5.75 6.00 6.25 6.50 6.75 7.00 7.25 7.50
11 15 25 18 01A
08
01B1
07
09
13
14
17
22
28
01B2
03
05
10
20
21A
23
24
27
29A
30
31
32
33
34B
34C
35
01C1
01C2
12
21B
26
29B
06
16
21C
Overall Means of Potencies (log10 IU/ml) Relative to Concurrently Tested IS for
Quantitative assays
Sample NOverall Mean
log10 IU/ml95% CI
log10 IU/ml
M1 34 5.98 5.94 6.02
M2 32 5.94 5.86 6.02
M3 31 5.97 5.87 6.07
Data are shown for quantitative assays (excluding Labs 9 and 28 for M2)
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
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
Parsyan et al. 2007
D91.1
V9
A6
Genetic Diversity of Parvovirus B19
IM81
LaLi
B19V DNA Collaborative Study Participants
Dr Sally Baylis Langen, Germany
Dr Johannes Blümel Langen, Germany
Dr Joan Bodiya/Maria Noedel Tempe, USA
Dr Kevin E Brown London, UK
Dr Daniel Candotti Cambridge, UK
Dr Michael Chudy/ Dr Micha Nübling Langen, Germany
Dr Theo Cuypers/ Dr Marco Koppelman Amsterdam, The Netherlands
Prof. Dr Anna-Maria Eis-Hübinger Bonn, Germany
Dr Giorgio Gallinella Bologna, Italy
Dr Thomas Gärtner Frankfurt am Main, Germany
Dr Thomas Grewing/Alke Heitmann Hamburg, Germany
Dr Yiu-Lian Fong Emeryville, USA
Dr Christoph Jochum Dreieich, Germany
Dr Marta José Barcelona, Spain
Dr Harkanwal Halait/Dr Yosh Ohhashi Pleasanton, USA
Dr Andreas Klotz/Dr Sandra Rieger Vienna, Austria
Dr Thomas Laue/Sabine Raith Hamburg, Germany
Dr Tzong-Hae Lee/Lani Montalvo San Francisco, USA
B19V DNA Collaborative Study Participants contd.
Dr German Leparc/Benjamin Reynolds St.Petersburg, USA
Dr Jeffrey M. Linnen San Diego, USA
Dr Eliza Moretti Lucca, Italy
Prof. Dr. Susanne Modrow/Dr Jürgen Wenzel Regensburg, Germany
Dr Yoshiaki Okada Tokyo, Japan
Dr Matthias Opp Luxembourg
Dr Takashi Owada Hokkaido, Japan
Mr David Padley Potters Bar, UK
Dr Lutz Pichl Hagen, Germany
Dr Giulio Pisani/Dr Francesco Marino Rome, Italy
Dr Lori Rinckel/Joshua Marley Raleigh, USA
Dr Gunnar Schalasta/Dr Martin Enders Stuttgart, Germany
Dr Annabelle Servant-Delmas Paris, France
Dr Richard Smith/Dr Jeffery Albrecht Los Angeles, USA
Dr Maria Söderlund-Venermo/Dr Maija Lappalainen
Helsinki, Finland
Dr Martin Stolz Bern, Switzerland
Dr Mei-ying W. Yu/Dr Li Ma Bethesda, USA
Ms Yi-Chen Yang Taipei, Taiwan
Acknowledgements
NGI, CSL Behring, Baxter BioScience & Talecris Biotherapeutics for materials used in the studies
Collaborative study participants
David Padley, NIBSC
Li Ma, CBER/FDA