Transcript PowerPoint 簡報 - NIBSC

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

Diluents: pooled human plasma (negative for
(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
c) dilute into ~106 IU/mL and ~103 IU/mL
HBsAg, anti-HIV, anti-HCV and NAT of HIV, HCV, HBV, HAV, and
B19)
Collaborative Study
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Participating Labs (11 Labs)
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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
Methodology/kit
Result (IU/ml)
A
In-house real time PCR
1.4E+06
B
In-house real time PCR
2.2E+06
C
In-house qualitative PCR
2.5E+05
D
In-house real time PCR
2.3E+05
E
1. Artus RealArt HBV LC PCR
4.2E+04
2. Bayer VERSANT HBV 3.0
1.8E+06
F
HBV RealType PCR
8.4E+05
G
In-house real time PCR
2.5E+05
H
1. Cobas Amplicor HBV monitor
1.3E+06
2. In-house real time PCR
1.9E+06
1. Cobas Amplicor HBV monitor
1.3E+06
2. Cobas TAQMAN
3.2E+06
J
Artus RealArt HBV LC PCR
8.2E+05
K
Digene kit
6.6E+05
I
HBV DNA standard
10
9
8
7
Numbers
6
5
H2
4
H1
3
G
K
E2
D
I1
B
F
A
2
1
E1
C
J
I2
0
2.0
2.5
3.0
3.5
4.0
4.5
5.0
5.5
Estimated IU (log 10 /ml)
6.0
6.5
7.0
STD
Log IU/mL
10
8
+2SD
Average
5.955
6
-2SD
4
2
0
A
B
C
D
E1
E2
F
G
H1
H2
I1
I2
J
K
1st National HBV Working Reagent
Lab
Methodology/kit
Result (IU/mL)
A
In-house real time PCR
2.2E+02
B
In-house real time PCR
2.9E+03
C
In-house qualitative PCR
2.5E+02
D
In-house real time PCR
9.5E+02
E
1.Artus RealArt HBV LC PCR
ND*
2.Bayer VERSANT HBV 3.0
1.6E+03
F
HBV RealType PCR
1.4E+03
G
In-house real time PCR
4.2E+02
H
1.Cobas Amplicor HBV monitor
1.8E+03
2.In-house real time PCR
4.4E+02
1.Cobas Amplicor HBV monitor
1.4E+03
2.Cobas TAQMAN
6.4E+03
J
Artus RealArt HBV LC PCR
6.4E+02
K
Digene kit
I
*ND: not detectable
ND*
Working reagent
10
9
8
Numbers
7
6
5
I1
4
H1
3
J
F
H2
E2
I2
G
D
B
2
1
A
C
0
2.0
2.5
3.0
3.5
4.0
4.5
5.0
5.5
Estimated IU (log10/ml)
6.0
6.5
7.0
Working reagent
8
Log IU/mL
6
+2SD
4
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
1st National HBV
DNA standard
1st National
HBV working
reagent
IU/ml
106
103
Lot Number
BFDA lot 92-08
BFDA lot 92-08W
Genotype
B
B
Vol. & Amount 0.5ml/vial
500 vials
1 ml/vial
1000 vials
Storage State
Liquid Frozen
Liquid Frozen
Stability Test – HBV DNA Standard at 5 ±3oC
Log IU/mL
10
9
Cobas Amplicor
8
Real-Time PCR
7
6
5
4
0
1
3
5
7
10
days
14
21
28
42
56
Stability Test –
HBV DNA Working Reagent at 5 ±3oC
Log IU/mL
Cobas Amplicor
5
4
3
2
1
0
0
1
3
5
7
10
days
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.
 National HBV DNA standard and working
reagent are stable at 4 ℃ for 4 weeks.
HBV NAT Experience in Taiwan
Blood center
（Date collected）
Tainan
（March 2003）
Taipei
（June 2003）
Taipei
（June 2003）
Units screened
1440 ＊
1296 ＊
516 ＊＊
EIA-positive
NAT-negative
0
0
0
EIA-negative
NAT-positive
0
0
6
(1.2 %)
EIA-positive
NAT-positive
17
（1.2 %）
5
（0.4 %）
2
（0.4 %）
H
B
V
（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