Transcript Name: Division: Lab Number - NIBSC

Update on the Replacement of
the HCV RNA
International Standard
Sally Baylis & Alan Heath, NIBSC
SoGAT XX, Warsaw 12-13 June 2007
Replacement of the 2nd International
Standard for HCV RNA (96/798)
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Proposal made at SoGAT 2005 to replace the HCV
RNA International Standard as requested by WHO
Agreement that HCV 1a genotype would be sourced &
would be anti-HCV negative and diluted in plasma
rather than cryosupernatant
Candidate Standards for Evaluation
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3 anti-HCV negative window period genotype 1a
donations have been obtained
The genotype of each has been confirmed by LiPA &
DNA sequencing
Absence of other viral markers confirmed in these
stocks
Material has been freeze-dried in two batches
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Batch 1 (NIBSC Code 06/100), 2085 vials, fill CV = 0.62%
Batch 2 (NIBSC code 06/102), 2100 vials, fill CV = 1.82%
Panel for Collaborative study
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Sample 1 – 2nd International Standard for HCV RNA
(96/798)
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Sample 2 - new freeze-dried preparation 06/100
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Sample 3 - new freeze-dried preparation 06/102
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Sample 4 – liquid/frozen bulk material (NIBSC code
06/118) that was used to prepare Samples 2 & 3
Collaborative Study
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Collaborative study commenced in November 2006
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Participants requested to test samples in four
independent assays
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33 laboratories from 14 countries participated in the
study
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40 data sets received; 25 from quantitative assays &
15 from qualitative assays
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NIBSC collated & analysed data
Types of Quantitative Assays Used by
Participants
Assay
Code
Assay
Number of
Data-sets
AB
Abbott RealTime HCV
7
BA
Bayer Versant HCV RNA 3.0
(bDNA)
4
CTM
COBAS Ampliprep/COBAS
TaqMan
6
HPS/COBAS TaqMan
1
In-house real-time
3
COBAS Amplicor HCV
Monitor
4
CTMH
IHRT
MO
Types of Qualitative Assays Used by
Participants
Assay
Code
A
Assay
Number of
Data-sets
Amplicor HCV 2.0
2
AC
COBAS Amplicor HCV 2.0
1
ASC
COBAS Ampliscreen HCV 2.0
3
IHQ
In-house qualitative
1
In-house real-time qualitative
3
Procleix
1*
Procleix Ultrio
1
PUT
Procleix Ultrio (TIGRIS)
1
TS
COBAS TaqScreen MPX
1
IHRTQ
P
PU
Estimated IU/ml (log10) from
Quantitative Assays
Sample
N
Mean
95% CI
SD
%CV
1
25
5.10
5.02-5.17
0.19
3.73
2
25
5.32
5.22-5.42
0.24
4.51
3
25
5.51
5.44-5.58
0.17
3.10
4
25
5.74
5.65-5.83
0.22
3.84
N - Number of laboratory estimates
SD - Standard Deviation of log10 estimates across laboratories
20
Sample 1 - Quantitative Results (IU/ml)
19
18
17
16
15
14
06
MO
13
02
MO
12
32
IHRT
11
20
IHRT
10
05
CTMH
9
29
BA
8
21
BA
7
19
BA
6
11
BA
5
26
AB
18
MO
12
AB
27
CTM
27
MO
10
AB
05
CTM
22
CTM
14
IHRT
05
AB
30
AB
13
CTM
03
CTM
01
AB
09
AB
08
CTM
4
3
2
1
0
3.0
3.5
4.0
4.5
5.0
IU/ml (log10)
5.5
6.0
6.5
7.0
20
Sample 2 - Quantitative Results (IU/ml)
19
18
17
16
15
14
13
12
11
10
9
02
MO
8
06
MO
32
IHRT
20
IHRT
13
CTM
14
IHRT
05
CTM
03
CTM
19
BA
05
CTMH
29
BA
30
AB
27
CTM
26
AB
10
AB
18
MO
21
BA
12
AB
09
AB
22
CTM
27
MO
11
BA
05
AB
01
AB
08
CTM
4.5
5.0
7
6
5
4
3
2
1
0
3.0
3.5
4.0
IU/ml (log10)
5.5
6.0
6.5
7.0
20
Sample 3 - Quantitative Results (IU/ml)
19
18
17
16
15
14
13
02
MO
12
20
IHRT
11
14
IHRT
10
05
CTMH
9
27
CTM
8
13
CTM
7
05
CTM
6
5
4
3
2
1
27
MO
03
CTM
32
IHRT
29
BA
18
MO
21
BA
26
AB
06
MO
19
BA
12
AB
22
CTM
11
BA
10
AB
09
AB
30
AB
05
AB
01
AB
08
CTM
0
3.0
3.5
4.0
4.5
5.0
IU/ml (log10)
5.5
6.0
6.5
7.0
20
Sample 4 - Quantitative Results (IU/ml)
19
18
17
16
15
14
13
12
11
27
MO
10
14
IHRT
9
27
CTM
8
13
CTM
7
05
CTM
6
5
4
3
2
1
11
BA
02
MO
30
AB
20
IHRT
26
AB
06
MO
03
CTM
12
AB
32
IHRT
29
BA
10
AB
05
CTMH
21
BA
05
AB
08
CTM
18
MO
19
BA
01
AB
09
AB
22
CTM
0
3.0
3.5
4.0
4.5
5.0
IU/ml (log10)
5.5
6.0
6.5
7.0
Estimated PCR Detectable Units/ml
(log10) from Qualitative Assays
Sample
N
Mean
95% CI
SD
%CV
1
13
5.24
5.04-5.43
0.32
6.11
2
12
5.35
5.09-5.62
0.42
7.78
3
13
5.65
5.45-5.86
0.34
6.10
4
13
6.04
5.76-6.33
0.47
7.74
N - Number of laboratory estimates
SD - Standard Deviation of log10 estimates across laboratories
22
Sample 2 - Potencies Relative to sample 1: Qualitative Assays
21
20
19
18
17
16
15
14
13
12
11
10
9
8
7
6
5
27
TS
4
28
P UT
3
23
PU
2
1
25
IHRTQ
16
IHRTQ
23
AS C
15
AC
07
IHRTQ
31
IHQ
04
A
24
A
32
AS C
33
AS C
0
3.0
3.5
4.0
4.5
5.0
IU/ml (log10)
5.5
6.0
6.5
7.0
22
Sample 3 - Potencies Relative to sample 1: Qualitative Assays
21
20
19
18
17
16
15
14
13
12
11
10
9
8
7
6
5
28
P UT
4
3
2
1
23
PU
25
IHRTQ
27
TS
17
P
16
IHRTQ
31
IHQ
07
IHRTQ
32
AS C
33
AS C
23
AS C
24
A
15
AC
04
A
0
3.0
3.5
4.0
4.5
5.0
IU/ml (log10)
5.5
6.0
6.5
7.0
22
Sample 4 - Potencies Relative to sample 1: Qualitative Assays
21
20
19
18
17
16
15
14
13
12
11
10
9
8
7
6
5
23
PU
4
17
P
3
27
TS
2
1
32
AS C
16
IHRTQ
28
P UT
31
IHQ
24
A
07
IHRTQ
15
AC
25
IHRTQ
23
AS C
04
A
33
AS C
0
3.0
3.5
4.0
4.5
5.0
IU/ml (log10)
5.5
6.0
6.5
7.0
Overall Mean Potencies (log10 IU/ml)
Relative to Sample 1
Sample
N
Mean
95% CI
SD
%CV
2
38
5.19
5.11-5.27
0.24
4.62
3
39
5.41
5.34-5.48
0.21
3.82
4
39
5.70
5.62-5.78
0.25
4.31
N - Number of laboratory estimates
SD - Standard Deviation of log10 estimates across laboratories
22
Sample 2 - Potencies Relative to sample 1
21
20
19
18
MO
18
06
MO
17
02
MO
16
20
IHRT
15
14
IHRT
14
22
CTM
13
08
CTM
12
05
CTM
11
29
BA
10
27
TS
19
BA
28
P UT
15
AC
23
PU
30
AB
27
MO
26
AB
05
CTMH
12
AB
13
CTM
10
AB
21
BA
09
AB
07
IHRTQ
16
IHRTQ
11
BA
05
AB
32
IHRT
31
IHQ
23
AS C
01
AB
03
CTM
27
CTM
04
A
24
A
32
AS C
9
8
7
6
5
4
3
2
1
25
IHRTQ
33
AS C
0
3.0
3.5
4.0
4.5
5.0
IU/ml (log10)
5.5
6.0
6.5
7.0
22
Sample 3 - Potencies Relative to sample 1
21
20
28
P UT
19
17
P
18
27
MO
17
06
MO
16
02
MO
15
20
IHRT
14
14
IHRT
13
31
IHQ
12
05
CTMH
11
27
TS
08
CTM
18
MO
05
CTM
16
IHRTQ
29
BA
07
IHRTQ
32
AS C
32
IHRT
26
AB
27
CTM
12
AB
22
CTM
10
AB
21
BA
09
AB
25
IHRTQ
19
BA
05
AB
03
CTM
13
CTM
11
BA
01
AB
33
AS C
30
AB
23
AS C
24
A
15
AC
10
9
8
7
6
5
4
3
2
1
23
PU
04
A
0
3.0
3.5
4.0
4.5
5.0
IU/ml (log10)
5.5
6.0
6.5
7.0
22
Sample 4 - Potencies Relative to sample 1
21
20
19
18
17
16
15
14
27
TS
13
28
P UT
12
02
MO
11
25
IHRTQ
06
MO
20
IHRT
14
IHRT
27
CTM
31
IHQ
23
PU
08
CTM
22
CTM
17
P
05
CTM
23
AS C
27
MO
03
CTM
26
AB
18
MO
29
BA
12
AB
32
IHRT
21
BA
10
AB
05
CTMH
16
IHRTQ
19
BA
09
AB
32
AS C
13
CTM
11
BA
05
AB
24
A
07
IHRTQ
15
AC
30
AB
01
AB
04
A
33
AS C
10
9
8
7
6
5
4
3
2
1
0
3.0
3.5
4.0
4.5
5.0
IU/ml (log10)
5.5
6.0
6.5
7.0
Accelerated Degradation Studies
Sample 2
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Incubation Time
+4°C
(10.5 months)
+20°C
(1 month)
Sample 2
0.22
0.20
Relative potencies of accelerated degradation
samples with respect to Sample 2 stored at -20ºC
(log10 drop), qPCR analysis
Stability appears similar to 96/790 and 96/798;
studies on-going
96/798 from this study has not lost potency after
>10 years storage at -20 ºC
Proposal for 3rd International Standard
for HCV RNA



Real-time & accelerated degradation data
indicate that Sample 2 (06/100) is stable &
suitable for long term use
Propose that 06/100 be established as the 3rd
International Standard for HCV RNA with a
unitage of 5.19 log10 IU/ml (4.89 log10 IU/vial)
Report will be submitted to ECBS by July
Acknowledgements
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
Nita Shah & David Padley NIBSC
Professor Ewa Brojer, Institute of Hematology & Blood
Transfusion, Warsaw
Dr Richard Smith, National Genetics Institute,
Los Angeles

Dr Vivienne James, HPA, Colindale

Dr Michael Chudy, WHO, Geneva
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Collaborative study participants
HCV RNA Collaborative Study Participants
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S Baylis, N Shah
T Cuypers M Koppelman
C Defer
L Fanning
G Gesu
A Goglio
P Grant
P Iudicone
L Jarvis, A Cleland
C Jork
S Kerby
A Klotz, M Gessner
R Madej
B Miller
S Mizusawa
C Negru
M Nübling
South Mimms, UK
Amsterdam, The Netherlands
Lille, France
Cork, Eire
Milan, Italy
Bergamo, Italy
London, UK
Rome, Italy
Edinburgh, UK
Springe, Germany
Bethesda, USA
Vienna, Austria
Pleasanton, USA
Karlsruhe, Germany
Tokyo, Japan
Timişoara , Romania
Langen, Germany
HCV RNA Collaborative Study Participants
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JM Pawlotsky
M Pecorari
G Pisani
A Portela Moreira
S Sauleda
H Schindl
G Schneider
A da Silva
R Smith
S Tassen
P Taylor
A van der Zanden
YC Yang
S Yerly
M Zaccanelli
Créteil, France
Modena, Italy
Rome, Italy
Madrid, Spain
Barcelona, Spain
Vienna, Austria
Des Plaines, USA
Lisbon, Portugal
Los Angeles, USA
Brentwood, UK
London, UK
Apeldoorn, The Netherlands
Taipei, Taiwan
Geneva, Switzerland
Treviglio, Italy
Update on the Stability of the
HAV RNA International Standard
Sally Baylis, Nita Shah & Alan Heath, NIBSC
SoGAT XX, Warsaw 12-13 June 2007
Background – Hepatitis A Virus RNA IS

The 1st International Standard for HAV RNA
(00/560) and a second batch (00/562) were prepared
in Q1, 2001

00/560 was evaluated in an international collaborative
study, with data returned from 16 laboratories

The mean log10 equivalents/ml were 5.29 (00/560) and
5.07 (00/562)

1st International Standard (00/560) was established
in 2003 with 50,000 IU/vial

ECBS noted that 00/562 may be a replacement for
00/560
Stability Data 00/560 & 00/562

Accelerated thermal degradation samples for
00/560 and 00/562, stored at +4ºC for ~6
years were tested in parallel with samples of
00/560 stored at -20 ºC

00/560 – a drop on 0.1 log10

00/562 – a drop on 1.0 log10

Conclude that 00/560 is highly stable
Proposal to Replace the
Parvovirus B19 DNA
International Standard
Sally Baylis, NIBSC
SoGAT XX, Warsaw 12-13 June
2007
Replacement of the Parvovirus
B19 DNA IS 99/800

The 1st International Standard for B19V DNA was
established by the WHO ECBS in October 2000

Estimated date of exhaustion of the IS will be
2008/9 at current rate of usage
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Materials coded AA (99/800) & BB (99/802)
showed no significant difference in potency in the
collaborative study
ECBS noted that BB (made from the same stock as
AA) could be reserved for potential future use as a
replacement standard
Candidate 2nd International Standard
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Materials coded AA (99/800) & BB (99/802) showed
no significant difference in potency in the original
collaborative study
ECBS noted that BB (prepared from the same stock
as AA) could be reserved for potential future use as a
replacement standard
Current study designed to demonstrate the
equivalence of the candidate replacement (BB) to AA
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Real-time data on samples AA & BB
Accelerated degradation data for samples AA & BB