Yi-Chen Yang, Drug Biology DivisionBureau 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 p

ool 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 parv

ovirus B19 DNA NAT assays

Background

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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

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Product

Item

HBV DNA

National StandardHBV DNA Working

Reagent

Conc. (IU/mL) 106 103

genotype B B

Lot number BFDA lot 92-08 BFDA lot 92-08W

Volume 0.5 mL/vial 1 mL/vial

National Standards for NAT in Taiwan

Product

ItemHCV RNA National

StandardHCV RNA Working

Reagent

Conc. (IU/mL) 5.2 × 104 890

genotype 1b 1b

Lot number BFDA lot 93-09 BFDA lot 93-09W

Volume 0.5 mL/vial 1 mL/tube

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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

Objective

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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

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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

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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

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Data for the Collaborative Study National standard

Lab code Method

Result

n Mean SD CV%

1 RealArt Parvo B19 LC 3 6.200 0.065 1.05

2 RealArt Parvo B19 LC 3 6.394 0.192 3.00

3 In-house assay 3 6.247 0.073 1.17

4 Roche LC Parvo B19 3 6.273 0.069 1.11

5 In-house assay 3 6.053 0.408 6.74

6 In-house assay 3 6.408 0.196 3.05

7 COBAS TaqScreen DPx* 3 6.033 0.012 0.20

8 Nested PCR 3 6.416 0.085 1.32

9 In-house assay 3 6.612 0.199 3.01

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A In-house assay 3 6.008 0.091 1.51

B Roche LC Parvo B19 3 6.258 0.015 0.24

C RealArt Parvo B19 LC 3 6.324 0.066 1.04

* in development

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

B19 DNA Natinal Standard

012345678

1 2 3 4 5 6 7 8 9 10A 10B 10C

Lab code

Log

IU/m

L

Mean 6.269

+2SD (6.627)- 2SD (5.911)

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Results of the Collaborative Study National standard

Lab code MethodResult

n Mean SD CV%

1 RealArt Parvo B19 LC 3 6.200 0.065 1.05

2 RealArt Parvo B19 LC 3 6.394 0.192 3.00

3 In-house assay 3 6.247 0.073 1.17

4 Roche LC Parvo B19 3 6.273 0.069 1.11

5 In-house assay 3 6.053 0.408 6.74

6 In-house assay 3 6.408 0.196 3.05

7 COBAS TaqScreen DPx* 3 6.033 0.012 0.20

8 Nested PCR 3 6.416 0.085 1.32

9 In-house assay 3 6.612 0.199 3.01

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A In-house assay 3 6.008 0.091 1.51

B Roche LC Parvo B19 3 6.258 0.015 0.24

C RealArt Parvo B19 LC 3 6.324 0.066 1.04

Calculated value 　 6.269 0.179 2.86

Parvovirus B19 DNA concentration 1.9 × 106 IU/mL

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Data for the Collaborative Study Working reagent

Lab code MethodResult

n Mean SD CV%

1 RealArt Parvo B19 LC 3 4.307 0.058 1.34

2 RealArt Parvo B19 LC 3 4.362 0.079 1.81

3 In-house assay 3 4.241 0.057 1.34

4 Roche LC Parvo B19 3 4.078 0.068 1.67

5 In-house assay 3 4.446 0.092 2.08

6 In-house assay 3 4.248 0.317 7.46

7 COBAS TaqScreen DPx* 3 4.110 0.240 5.17

8 Nested PCR 3 4.645 0.063 1.53

9 In-house assay 3 4.561 0.094 2.06

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A In-house assay 3 4.025 0.274 6.81

B Roche LC Parvo B19 3 4.347 0.048 1.09

C RealArt Parvo B19 LC 3 4.336 0.123 2.83

* in development

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

B19 DNA Working Reagent

012345678

1 2 3 4 5 6 7 8 9 10A 10B 10C

Lab code

Log

IU

/mL

Mean 4.309

+2SD (4.682)

- 2SD (3.936)

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Results of the Collaborative Study Working reagent

Lab code MethodResult

n Mean SD CV%

1 RealArt Parvo B19 LC 3 4.307 0.058 1.34

2 RealArt Parvo B19 LC 3 4.362 0.079 1.81

3 In-house assay 3 4.241 0.057 1.34

4 Roche LC Parvo B19 3 4.078 0.068 1.67

5 In-house assay 3 4.446 0.092 2.08

6 In-house assay 3 4.248 0.317 7.46

7 COBAS TaqScreen DPx* 3 4.110 0.240 5.17

8 Nested PCR 3 4.645 0.063 1.53

9 In-house assay 3 4.561 0.094 2.06

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A In-house assay 3 4.025 0.274 6.81

B Roche LC Parvo B19 3 4.347 0.048 1.09

C RealArt Parvo B19 LC 3 4.336 0.123 2.83

Calculated value 　 4.309 0.186 4.33

Parvovirus B19 DNA concentration 2.0 × 104 IU/mL

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B19 DNA National Standard and Working Reagent

Product

ItemB19 DNA

National StandardB19 DNA

Working Reagent

Conc. (IU/mL) 1.9 × 106 2.0 × 104

Genotype 1 1

Lot number BFDA lot 94-08 BFDA lot 94-08W

Volume 0.5 mL/vial 1 mL/tube

Total amount 1,100 vials 1,000 tubes

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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

+56 days++24 months

+42 days++18 months

++28 days++12 months

++21 days++9 months

++14 days++6 months

++7 days+3 months

+25℃+4℃-20℃-80℃Time

TemperatureTime

Temperature

Accelerated testingLong term testing

+56 days++24 months

+42 days++18 months

++28 days++12 months

++21 days++9 months

++14 days++6 months

++7 days+3 months

+25℃+4℃-20℃-80℃Time

TemperatureTime

Temperature

Accelerated testingLong term testing

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Working reagent (25℃)

0123456789

10

0 7 14 21 28

Time (days)

Log

IU/m

L

4.309

6.269

p＞ 0.05, n.s.

p＞ 0.05, n.s.

Results of the Stability Studies ( 25℃ )

National standard (25℃)

0123456789

10

0 7 14 21 28

Time (days)

Log

IU/m

L

6.269

p＞ 0.05, n.s.

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Working reagent (4℃)

0123456789

10

0 7 14 21 28 35 42 49 56

Time (days)

Lo

g IU

/mL

National standard (4℃)

0123456789

10

0 7 14 21 28 35 42 49 56

Time (days)

Lo

g IU

/mL

p＞ 0.05, n.s.

p＞ 0.05, n.s.

4.309

6.269

Results of the Stability Studies ( 4℃ )

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Working reagent (-20℃)

0123456789

10

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

Time (months)

Log IU

/mL

National standard (-20℃)

0123456789

10

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

Time (months)

Log IU

/mL

p＞ 0.05, n.s.

4.309

6.269

Results of the Stability Studies ( -20℃ )

p＞ 0.05, n.s.

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Working reagent (-80℃)

0123456789

10

0 1 2 3 4 5 6 7 8 9 10 11 12 13

Time (months)

Log

IU

/mL

Naitonal standard (-80℃)

0123456789

10

0 1 2 3 4 5 6 7 8 9 10 11 12 13

Time (months)

Lo

g IU

/mL

p＞ 0.05, n.s.

p＞ 0.05, n.s.

4.309

6.269

Results of the Stability Studies ( -80℃ )

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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. ℃

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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. Baylis NIBSC, UK– Dr. Y. Okada NIID, Japan– Dr. M. Gessner, Dr. A. Klotz Baxter AG, Austria– Dr. D. Johnstone CSL Bioplasma, Australia– Dr. R. Smith NGI, USA– Dr. T. Grewing QIAGEN, Germany– Dr. D. Sizmann, Dr. A. Schubert Roche, Germany

– Dr. J. Saldanha Roche, USA– Dr. A. Heath NIBSC, UK

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NAT requirement in Taiwan (Dec. 19 2002 ) 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 productsOne 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 < 105 IU/ml