Detection of HAV RNA reference materials
using different methods
SoGAT
24th Blood Virology and 4th Clinical Diagnostics Meeting
Ljubljana, 8 - 9 May 2013
Michael Chudy
Section of Molecular Virolgy
Department of Virology
E-Mail: [email protected]
1
1st IS for HAV RNA
 Established in 2003
 Developed by NIBSC
 #00/560 (100,000 IU/ml)
 1st IS stocks likely to be exhausted in ~1 year
 Second freeze-dried batch from the same bulk (#00/562) is not
a suitable replacement material due to stability issues
(WHO/BS/07.2056)
 New candidate material is needed
 Collaborative study to evaluate the candidate for 2nd IS is
ongoing (NIBSC)
©L3
1st IS for HAV RNA (WHO/BS/03.1959)
5.0 log10 IU/ml
3.69 log10 IU/ml
©L3
6.15 log10 IU/ml
BRP for HAV RNA needed (EDQM)
 Revised monograph (1646) for human plasma (pooled
and treated for virus inactivation) requires tests for
HAV RNA by NAT
 A positive control at 100 IU/mL of HAV RNA must be
included and react positively
 Project was endorsed by the SC in January 2008
 Project leader PEI
 Secondary reference preparation
©L3
BRP for HAV RNA
 Evaluation of different candidate materials from the
plasma industry
 PEI: reference NAT assay
- QIAamp DSP Virus Kit / artus HAV LC RT-PCR Kit
- Qiagen, Hilden, Germany
- quant, CE marked
©L3
N6116
PEI No
Lab A
Lab B
PEI
Lab C
Lab D
Lab E
in-house
in-house
artus LC
artus TM
Roche LC
astra
in-house
Roche
TaqScreen
DPX Test
SuperQuant
TaqMan
Hybr.
TaqMan
Hybr.
TaqMan
TaqMan
TaqMan
?
5'NCR
VP1
VP1
?
5'NCR
5'NCR
?
quant
quant
quant
quant
quant
qual
qual
qual (HAV RNA)
IU/ml
IU/ml
IU/ml
HAV RNA
copies/ml
N6116
©L3
IU/ml
8'000'000 2'000'000
IU/ml
3'307
IU/ml
4'438
IU/ml
86'078
540'000
300'000
500'000
N6116 / N6223
PEI No
Lab A
Lab B
PEI
Lab C
Lab D
Lab E
in-house
in-house
artus LC
artus TM
Roche LC
astra
in-house
Roche
TaqScreen
DPX Test
SuperQuant
TaqMan
Hybr.
TaqMan
Hybr.
TaqMan
TaqMan
TaqMan
?
5'NCR
VP1
VP1
?
5'NCR
5'NCR
?
quant
quant
quant
quant
quant
qual
qual
qual (HAV RNA)
IU/ml
IU/ml
IU/ml
HAV RNA
copies/ml
N6116
N6223
©L3
IU/ml
8'000'000 2'000'000
100'000
IU/ml
3'307
756
IU/ml
4'438
IU/ml
86'078
3'293
540'000
300'000
500'000
N6116 / N6223 / N6227 and N228
PEI No
Lab A
Lab B
PEI
Lab C
Lab D
Lab E
in-house
in-house
artus LC
artus TM
Roche LC
astra
in-house
Roche
TaqScreen
DPX Test
SuperQuant
TaqMan
Hybr.
TaqMan
Hybr.
TaqMan
TaqMan
TaqMan
?
5'NCR
VP1
VP1
?
5'NCR
5'NCR
?
quant
quant
quant
quant
quant
qual
qual
qual (HAV RNA)
IU/ml
IU/ml
IU/ml
HAV RNA
copies/ml
N6116
N6223
IU/ml
8'000'000 2'000'000
100'000
IU/ml
3'307
4'438
IU/ml
86'078
756
3'293
192'754
N6227
1'330'000
86'459
N6228
1'780'000
208‘436
©L3
IU/ml
540'000
321‘239 13'000'000
300'000
500'000
Genetic relationship between different HAV strains
CR326
IA
MBB
N6223
IB
HM175
attHM175
HMH
SLF88
IIIA
VII
st
WHO 1 IS
FG
GBM
IA
HAS15
LA
N6116
N6227
N6228 EDQM BRP
0.1
The dendogram derived from ClustalW
illustrates the comparison of nucleotide
sequences within a 304-base segment at the
VP1/2A junction.
BRP for HAV RNA
 Candidate material (N6228)
 HAV RNA 208.436 IU/ml (QIAamp DSP Virus Kit / artus HAV LC RT-PCR
Kit)
 Dilution 1:10 with neg plasma pool
 Volume 2.5 L
 final concentration about 20.000 IU/ml
 Testing of pre-lyo and post-lyo material in parallel with IS
BSP liqu
10
BSP lyo
<
mean
37.153
corr.
3.387
WHO
220.511 1.102.920
19.993
100.000
?
Test configurations / combinations
 HAV samples (liqu vs lyo)
 sec BRP / 1st IS / cand 2nd IS
 Sample preparation kits
 QIAamp DSP Virus Kit (Qiagen); CE marked
 QIAamp Viral RNA Mini Kit (Qiagen); non-CE marked
 High Pure Viral Nucleic Acid Kit (Roche); non-CE marked
 HAV NATs
 artus HAV LC RT-PCR Kit (Qiagen); quant, CE marked
 RealStar HAV RT-PCR Kit 1.0 (altona Diagnostics); qual, CE marked
pending
 LC HAV Quantification Kit (Roche); quant; non-CE marked
 Nine different test combinations
11
28
28
26
26
24
24
QIAamp DSP Virus Kit / artus HAV LC RT-PCR Kit
0,01
IS liqu IS lyo
0,1
BSP liqulog
BSP
lyo 2IS liqu 2IS lyo
dilution
1
0,01
40
40
38
38
36
36
34
34
Cp-values
Ct-values
1
32
30
32
30
28
28
Lyo > liqu
26
26
1st IS>2nd IS=BRP
24
1
24
0,01
0,1
log dilution
12
1
0,01
IS liqu IS lyo
altona
artus HAV
RealStar
LC RT-PCR
HAV RT-PCR
40
38
3838
3838
38
3636
3636
36
3434
3434
28
26
0,01
0,1
1
3030
3030
2828
2828
2626
2626
2424
0,01
0,01
log dilution
34
32
30
28
26
24
0,01
0,1
Cp-values
32
30
1st IS>2nd IS>BRP
28
26
2424
0,01
0,01
24
0,1
0,1
11
0
log
logdilution
dilution
4040
40
3838
3838
38
3636
3636
36
3434
3434
34
3232
3030
3030
2828
2828
2626
2626
2424
0,01
0,01
liqu > lyo
0,10,1
1 1
1st
IS>2nd
32
30
IS=BRP
28
26
2424
0,01
0,01
24
0,1
0,1
loglog
dilution
dilution
11
0
log
logdilution
dilution
4040
4040
40
38
3838
3838
38
36
3636
3636
36
3434
3434
30
28
24
0,1
log dilution
1
34
3232
3030
3030
2828
2828
2626
2626
2424
0,01
0,01
liqu < lyo
Cp-values
3232
26
0,01
Ct-values
Cp-values
32
Ct-values
Ct-values
QIAGEN DSP Virus Kit
40
34
Ct-values
11
3232
1
34
4040
log dilution
QIAGEN DSP Virus Kit
0,10,1
logdilution
dilution
log
Ct-values
Cp-values
36
Roche High Pure Viral Nucleic Acid Kit
38
Ct-values
Roche High Pure Viral Nucleic Acid Kit
40
liqu = lyo
3232
Cp-values
30
3232
Cp-values
Cp-values
32
Ct-values
Cp-values
34
Cp-values
Cp-values
4040
QIAamp Viral RNA Mini Kit
4040
24
13
Roche
artus
LC HAV
HAV LC
Quantification
RT-PCR
Kit
40
36
Ct-values
QIAamp Viral RNA Mini Kit
altona RealStar HAV RT-PCR
BSP liqu BSP lyo 2IS liqu 2IS lyo
0,1
0,1
logdilution
dilution
log
11
2424
0,01
0,01
1st
IS>2nd
32
30
IS=BRP
28
26
24
0,10,1
loglog
dilution
dilution
1 1
0
IS liqu IS lyo
artus HAV LC RT-PCR
40
38
38
38
36
36
36
34
34
34
32
Cp-values
40
32
30
30
28
28
28
26
26
26
24
0,01
0,1
0,01
1
24
1
0,01
38
38
38
36
36
36
34
34
34
Cp-values
40
32
32
30
30
28
28
28
26
26
26
24
0,1
1
0,01
log dilution
liqu > lyo
0,1
24
1
0,01
log dilution
38
38
38
36
36
36
34
34
34
Cp-values
40
Ct-values
40
32
30
30
28
28
28
26
26
26
24
0,01
0,1
log dilution
1
0,01
liqu < lyo
0,1
log dilution
1
32
30
24
0,1
log dilution
40
32
1
32
30
0,01
0,1
log dilution
40
24
Ct-values
0,1
40
Cp-values
Ct-values
Roche High Pure Viral Nucleic Acid Kit
liqu = lyo
log dilution
log dilution
QIAGEN DSP Virus Kit
32
30
24
14
Roche LC HAV Quantification Kit
40
Cp-values
Ct-values
QIAamp Viral RNA Mini Kit
altona RealStar HAV RT-PCR
BSP liqu BSP lyo 2IS liqu 2IS lyo
24
1
0,01
0,1
log dilution
1
Conclusion
 Secondary BRP (post lyo)
 Contains HAV RNA, probably in sufficient concentration
 Consistency between vials is shown
 WHO 1st IS / candidate 2nd IS / secondary BRP
 Commutability of pre-lyo samples vs post-lyo samples?
 Commutability of standards or question of assay design?
 Detection of 100 IU/ml (EP)
 How to design the collaborative study?
 Usefulness of QIAamp DSP Virus Kit and QIAamp DSP Virus Kit/artus
HAV LC RT-PCR Kit?
15
Special thanks to
Angele Costanzo and Karl-Heinz Buchheit, EDQM
Jacqueline Fryer, NIBSC
all colleagues from PEI in particular to…
Christine Pfannkuch
16