Preparation of HBV DNA reference standards and the experience of HBV NAT in Taiwan
Dr. Hwei-Fang Cheng Department of Health, Taiwan
HBV DNA Reference Standards
National HBV DNA standard ：106 IU/mL, 0.5 mL/vial (relative to NIBSC HBV NAT standard)
National HBV DNA working reagent ：103 IU/mL, 1 mL/vial (relative to NIBSC HBV NAT working reagent)
Intended use for HBV NAT testing of blood and pooled plasma and biological research
High titer (~109 IU/ml) HBV positive plasma
(negative for anti-HIV,anti-HCV and NAT of HIV, HCV , HAV , and B19) a) genotype B b) virus inactivation: 60 oC for 1 hr 6 3 c) dilute into ~10 IU/mL and ~10 IU/mL
Diluents: pooled human plasma (negative for
HBsAg, anti-HIV, anti-HCV and NAT of HIV, HCV, HBV, HAV, and B19)
Collaborative Study
Participating Labs (11 Labs)
US CBER/FDA National Institute of Infectious Diseases (NIID) ,Japan Canadian Blood Services National Institute for Biological Standards and Control (NIBSC) Victorian Infectious Diseases Reference Laboratory (VDRL), Australia NTUH Hepatitis Research Center, Taiwan Center of Disease Control, Taiwan BFDA, Taiwan Roche Diagnostics Artus GmbH Digene Diagnostic Technology
1st National HBV DNA Standard
Lab
A
B C
Methodology/kit
In-house real time PCR
In-house real time PCR In-house qualitative PCR
Result (IU/ml)
1.4E+06
2.2E+06 2.5E+05
D E
F
In-house real time PCR 1. Artus RealArt HBV LC PCR
2. Bayer VERSANT HBV 3.0 HBV RealType PCR
2.3E+05 4.2E+04
1.8E+06 8.4E+05
G
H
In-house real time PCR
1. Cobas Amplicor HBV monitor 2. In-house real time PCR
2.5E+05
1.3E+06 1.9E+06
I
J K
1. Cobas Amplicor HBV monitor 2. Cobas TAQMAN
Artus RealArt HBV LC PCR Digene kit
1.3E+06 3.2E+06
8.2E+05 6.6E+05
HBV DNA standard
10 9
8
7
6
Numbers
5
H2
4
H1
3
G
2
K
I1 J F
6.0
E2
B A
6.5
D
1
E1
0
C
5.0 5.5
I2
7.0
2.0
2.5
3.0
3.5
4.0
4.5
Estimated IU (log 10 /ml)
STD
10
Log IU/mL
8
+2SD
6
Average 5.955
-2SD
4
2
0 A B C D E1 E2 F G H1 H2 I1 I2 J K
1st National HBV Working Reagent
Lab
A
B C
Methodology/kit
In-house real time PCR
In-house real time PCR In-house qualitative PCR
Result (IU/mL)
2.2E+02
2.9E+03 2.5E+02
D E
F
In-house real time PCR 1.Artus RealArt HBV LC PCR
2.Bayer VERSANT HBV 3.0 HBV RealType PCR
9.5E+02 ND*
1.6E+03 1.4E+03
G
H
In-house real time PCR
1.Cobas Amplicor HBV monitor 2.In-house real time PCR
4.2E+02
1.8E+03 4.4E+02
I
J K
1.Cobas Amplicor HBV monitor
2.Cobas TAQMAN Artus RealArt HBV LC PCR Digene kit
1.4E+03
6.4E+03 6.4E+02 ND*
*ND: not detectable
Working reagent
10
9
8
7
Numbers
6
5
I1
4
H1
3
J
2
F
H2
1
E2
D
3.0
I2
B
3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0
A
0
2.0
C
2.5
G
Estimated IU (log10/ml)
Working reagent
8
6
Log IU/mL
4
+2SD Average 2.953
2
-2SD
0 A B C D E2 F G H1 H2 I1 I2 J
Summary of National HBV DNA Standard and Working Reagent
product item IU/ml
Lot Number
1st National HBV DNA standard
106 BFDA lot 92-08
1st National HBV working reagent
103 BFDA lot 92-08W
Genotype B Vol. & Amount 0.5ml/vial 500 vials Storage State Liquid Frozen
B 1 ml/vial 1000 vials Liquid Frozen
Stability Test – HBV DNA Standard at 5 ±3oC
Log IU/mL
10 9 8 7 6 5 4 Real-Time PCR Cobas Amplicor
0
1
3
5
7
10
14
21
28
42
56
days
Stability Test – HBV DNA Working Reagent at 5 ±3oC
Log IU/mL
5 4 3 2 1 0 0 1 3 5 7
Cobas Amplicor
days
10
14
21
28
42
56
Summary
The assigned potency of HBV DNA
standard and HBV working reagent are 106 IU/ml and 103 IU/ml, respectively.
Genotype of national HBV DNA standard
and working reagents is genotype B; and the serotype is adw. reagent are stable at 4 ℃ for 4 weeks.
National HBV DNA standard and working
HBV NAT Experience in Taiwan
Blood center （Date collected）
Tainan （March 2003）
Taipei （June 2003）
Taipei （June 2003）
Units screened
EIA-positive NAT-negative
1440 ＊
0
1296 ＊
0
516 ＊＊
0
H B V
EIA-negative NAT-positive EIA-positive NAT-positive
0
0
6 (1.2 %) 2 （0.4 %）
17 （1.2 %）
5 （0.4 %）
（1）EIA：Murex HBsAg version 3.0 kit; Sensitivity: <0.2 ng （2）NAT ＊ Pooled units test (12 units/pool); Roch High Pure Viral Nucleic Acid Kit (0.2 ml of extraction volume); In house nested PCR; Sensitivity of 22 IU/ml. ＊＊ Single unit test Ampliscreen HBV/Roch (1 ml of extraction volume) ; In house nested PCR； Sensitivity of 12 IU/ml