calibration and stability of who and secondary viral … lelie - calibration...calibration and...
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Calibration and stability of WHO and secondary viral standards
Nico Lelie, Harry van Drimmelen and the International NAT Study Group
Facilities DDL Diagnostic Laboratories
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Outline • Calibration of WHO and secondary standards
– HBV
– HCV
– HIV
– Genotypes
• Stability of secondary standards
– Long term frozen stability at -70°C and -30°C
– In use stability in liquid phase at 4°C, 20°C, 37 °C
• Conclusions
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Calibration of HBV standards
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Traceability of HBV-DNA standards
Eurohep gt A
1st WHO gt A
2nd WHO gt A
Sanquin-VQC genotype A
Chimp gt A
Eurohep gt D
ISS gt D Chimp gt C
BQC gt A inactivated
WHO gt panel
3rd WHO gt A
BQC gt panel
bDNA calibrators
Dilution, lyophilization
Dilution, pasteurization
Heerman K-H et al. J Clin Microbiol 1999;37:68-73 Saldanha J et al. Vox Sang 2001;80:63-71 Grabarczyk P et al, Transfusion, in press Pisani G et al, Ann Ist Super Sanita 2007:43:69-76 Chudy M et al, J Clin Virol 2012Epub Van Drimmelen et al, unpublished
copies
IUs
ID50
Collins ML, et al. An Biochem 1995;226:120
Komiya K et al. Transfusion 2008;48:286-9
calibrations
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Calibration of native Sanquin-VQC HBV standard against WHO 97/746 standard
Experiment n
WHO n
VQC Copy/IU (95% CI)
Parallel Siemens Versant bDNA 3.0 assays (Cuijpers et al Sanquin, Amsterdam)
12 16 5.33
(5.11-5.55)
Parallel Siemens Versant bDNA 3.0 assays (Van Drimmelen, DDL, Rijswijk)
6 6 5.20
(4.61-5.80)
Multi-method WHO Collaborative study (Saldanha J et al. Vox Sang 2001;80:63-71)
46 23 4.12
Grabarczyk P et al, Transfusion, in press Data reported in Supplementary Materials accessible online on Transfusion website
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Calibration Japanese Chimpanzee infectivity plasmas against Sanquin-VQC HBV genotype A standard
Chimp plasma
Study Assays n copies/ CID50 (range)
C-246 P-57 genotype A
BQC-DDL
bDNA 3.0
6
4.0 ( 1.3-12.6)
Komiya et al
TaqMan
1
8.2 (2.6-26)
C-272 P-29 genotype C
BQC-DDL
bDNA 3.0
6
5.9 (1.8-18.5)
Komiya et al
TaqMan
1
9.5 (3.0-30)
Komiya K et al. Transfusion 2008;48:286-9 Grabarczyk P et al, Transfusion, in press (data reported in Supplementary Materials accessible online on Transfusion website)
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Native VQC HBV-DNA genotype A standard
(2.15 x 109 cps/mL, 6.8 x 108 CID50/mL)
Preparation of pasteurised BQC HBV-DNA plasma standard
100 diluted HBV-DNA standard in PBS
(2.15 x 106 cps/mL )
1:100 dilution in PBS
Pasteurized HBV-DNA standard in PBS
( ~0.6 mg/mL protein)
1:2.5 dilution in plasma
and snap freezing in liquid nitrogen
inactivated BQC HBV-DNA plasma standard
(7.23 x 106 cps/mL, <2.3 CID50/mL)
> 106 CID50
inactivation*
*Lelie PN et al J.Med.Virol. 1987:23:289-95
84%
recovery HBV-DNA
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25,000 rpm, 16h, 10°C
Banding of HBV DNA in sucrose gradient
Gerlich et al, SoGAT, Brussels 2009
WHO lyophilised ISS lyophilised BQC pasteurized VQC native
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Analytical sensitivity of Ultrio and Ultrio Plus on native and pasteurized HBV-DNA standard
data from Ultrio and Ultrio Plus validation studies in Ireland, Denmark and Poland
Standard
Native
Inactivated 8.86 (3.14-34.9)
Relative sensitivity Ultrio Plus to Ultrio
3.60 (1.45-11.0)
Assay
Ultrio
Ultrio Plus
Relative potency native to inactivated
3.78 (1.53-11.3)
1.54 (0.67-3.74)
Study standard Assay n 95% LOD (CI) 50% LOD (CI)
Ultrio 24 161 (82-378) 15.7 (8.6-28.8)
Ultrio Plus 24 44.7 (22.4-107) 4.4 (2.3-8.2)
Denmark Ultrio 58 699 (337-2301) 55 ((33-93)
Ultrio 24 607 (301-1493) 59 (32-111)
Ultrio Plus 24 68.6 (35.8-159) 6.7 (3.8-11.9)
Poland
Poland
native
inactivated
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Potency of native and pasteurized HBV-DNA standard in Ultrio Plus probit and TaqScreen Ct analysis
Assay n native
n inact
analysis Potency inactivated relative to native standard (95% CI)
Ultrio Plus 24 24 probit§ 0,65 (0.27-1.49)
TaqScreen 32 32 Ct# 0.87 (0.76-0.98)
Comparison of Ct values on 2000 cps/mL levels of both standards in parallel TaqScreen test runs performed by Dr Marco Koppelman (Sanquin, Amsterdam) #
§ Study performed by Prof. Piotr Grabarczyk (IHTM, Warsaw, Poland)
bDNA calibration n native n inact cps/mL native cps/mL inact
experiment 6 6 2.15 x 10E9 7.23 x 10E6
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Potency of 2nd WHO HBV 97/750 IS relative to 1st 97/746 IS in Ultrio validation studies
standard n Potency (95% CI)
97/746 733 reference
97/750 88 1.14 (0,57-2.35)
IU/mL
% Ultrio reactive
WHO calibration study of Baylis et al (WHO report BS/06/2034 ) showed a not significant lower potency of 0.85 in 97/750 IS relative to 97/746 IS, but unitage was kept at 10E6 IU/ampoule
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Comparison potency of HBV genotype A standards in Ultrio Plus (UP above) and TaqScreen (Tx below)
(1 IU = 5.33 copies)
HBV standard n
50% LOD (CI) cps/mL in UP
95% LOD (CI) cps/mL in UP
Potency relative to Eurohep standard
97/746 20 4.0 (2.1-7.8) 29.0 (14.9-58.9) 0.89 (0.43-1.94) 97/750 791 3.7 (3.3-4.1) 26.9 (22.9-32.3) 0.96 (0.67-1.43)
Eurohep 96 3.6 (2.7-4.7) 25.9 (18.9-36.3) 1.00 (reference) Chimp 48 3.7 (2.5-5.7) 27.2 (17.7-42.9) 0.95 (0.57-1.58)
VQC native 60 5.0 (3.4-7.2) 36.3 (24.6-54.8) 0.71 (0.45-1.14) BQC inact 24 6.6 (3.6-11.9) 48.3 (26.6-89.9) 0.54 (0.28-1.03)
HBV standard n
50% LOD (CI) cps/mL in Tx
95% LOD (CI) cps/mL in Tx
Potency relative to Eurohep standard
97/746 224 4.1 (3.6-4.7) 20.9 (17.9-25.1) 0.54 (0.31-0.98) Eurohep 12 2.6 (1.3-3.8) 11.4 (6.6-20.4) 1.00 (reference)
VQC native 12 2.8 (1.7-4.7) 14.2 (8.5-24.8) 0.80 (0.38-1.65)
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Calibration of HIV standards
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Preparation pasteurized and/or lyophilized HIV-RNA reference samples
native standard in plasma 1.05 x 108 cps/ml
1:10 dilution in PBS 2h hour 65 0C
pasteurized 1:10 in PBS
1:10 dilution in plasma
1:100 diluted 1:100 diluted pasteurized
1:100 dilution in plasma
1:100 dilution in plasma
1:10,000 diluted pasteurized 1:10,000 diluted native freeze-drying
freeze-drying
freezing freezing
regular lyophilized
1:100 dilution in plasma
pasteurized past./lyophilized
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Recovery of HIV-RNA after pasteurization and/or lyophilization
process standard Average cps/mL*
recovery
Native standard 15,542§ 100%
lyophilization 3810 24%
pasteurization 8988 56%
pasteurization & lyophilization 3634 23%
* Geometric mean values of 18 Bayer Versant bDNA assays for each process
§ cultured HIV diluted in plasma to 10,500 cps/mL as determined by 58 bDNA assays in 7 test runs
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Potency of native and pasteurized HIV-RNA standard in Ultrio probit and TaqScreen Ct analysis
Study standard n 95% LOD (CI) 50% LOD (CI)
Poland native 12 15.3 (8.8-29.2) 2.1 (1.3-3.5)
Denmark inactivated 52 22.2 (15.6-34.5) 3.1 (2.4-3.9)
Assay n native
n inact
analysis Potency inactivated relative to native standard (95% CI)
Ultrio 12 52 probit§ 0,69 (0.38-1.22)
TaqScreen 40 40 Ct# 1.04 (0.86-1.22)
Comparison of Ct values on 2000 cps/mL levels of both standards in parallel TaqScreen test runs performed by Dr Koppelman (Sanquin)
§
#
bDNA calibration n native n inact cps/mL native cps/mL inact
experiment 6 6 1.05 x 10E8 2.62 x 10E6
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Assay
N assays cps/IU on 1st WHO (97/656) standard
cps/IU on 2nd WHO (97/650) standard
1st WHO
2nd WHO
VQC mean (95%CI) mean (95%CI)
Abbott LCx
14 15 14 0.76 (0.60-0.96) 0.69 (0.56-0.86)
Roche Amplicor Monitor
125 134 112 0.70 (0.60-0.81) 0.93 (0.80-1.08)
Siemens bDNA 3.0
64 69 48 0.39 (0.34-0.44) 0.58 (0.51-0.66)
Organon Tekika NucliSens
46 51 36 0.80 (0.69-0.92) 0.43 (0.36-0.50)
Roche Amplicor Mon UltraSens
16 15 11 0.51 (0.27-0.95) 0.86 (0.49-1.51)
Calibration of VQC-Sanquin HIV-RNA subtype B standard on the first (97/656) and second (97/650) WHO HIV-1 RNA subtype B standards
calculated from raw data reported by the laboratories participating in the first WHO collaborative study
Holmes H et al, J. Virological Methods 2001, 92; 141-150 Grabarczyk P et al, Transfusion, in press ((Table reported in Supplementary Materials accessible online on Transfusion website)
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IU/mL
% TMA reactive
standard n Potency (95% CI)
97/650 718 reference
97/656 131 1.00 (0,77-1.30)
PWS 99/634 581 0.91 (0.77-1.06)
Potency of 1st HIV-1 97/656 IS and PWS 99/634 relative to 2nd 97/650 IS in TMA assay validation studies
Assay Standard n
dHIV 97/656 48
Duplex 97/656 83
Ultrio 97/650 94
Ultrio Elite 97/650 231
Ultrio Plus 97/650 393
Duplex PWS-1 99/634 48
Ultrio PWS-1 99/634 533
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Calibration of HCV standards
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Calibration 3rd HCV WHO 06/100 IS, 06/102 sample and unlyophilised bulk on 2nd 96/798 IS
Data Baylis S et al, Vox Sang 2011, 100, 409-417
quantitative assays
n labs IS 96/798 IS 06/100 06/102 unlyophilised sample 2 sample 3 sample 4
Abbott RT 7 1,00E+05 1,82E+05 2,88E+05 4,57E+05
Siemens bDNA 4 1,00E+05 1,58E+05 2,19E+05 2,95E+05
Roche CTM 6 1,00E+05 1,51E+05 2,34E+05 3,63E+05
Mean three methods
17 1,00E+05 1,63E+05 2,45E+05 3,66E+05
recovery lyophilisation 45%* 67% 100%
Overall mean six methods report
25 1,00E+05 1,55E+05 2,57E+05 5,01E+05
recovery lyophilisation 31% 51% 100%
Factor value three methods different from overall values in report
1,06 0,95 0,73
*40% in Abbott core antigen
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standard n Potency (95% CI)
96/798 594 reference
96/790 48 1.04 (0,71-1.50)
06/100 750 0.78 (0.67-0.89)*
% TMA reactive
IU/mL
Potency of 3rd 06/100 and 1st 96/790 IS relative to 2nd 96/798 IS in TMA assay validation studies
Assay standard n
Ultrio 06/100 86
Ultrio Elite 06/100 245
Ultrio Plus 06/100 419
Duplex 96/790 24
Ultrio 96/790 24
Duplex 96/798 72
Ultrio 96/798 522
* 06/100 vs 96/798, p<0.05
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Study standard n 95% LOD (CI) 50% LOD (CI)
Poland native 12 14.6 (8.3-28.6) 1.9 (1.1-3.2)
Denmark inactivated 52 28.1 (19.4-45.3) 3.6 (2.8-4.7)
Potency of native and inactivated HCV-RNA standard in Ultrio probit and TaqScreen Ct analysis
Assay n native
n inact
analysis Potency inactivated relative to native standard (95% CI)
Ultrio 12 52 probit§ 0,52 (0.27-0.92)
TaqScreen 32 32 Ct# 0.45 (0.31-0.58)
§
Comparison of Ct values on 2000 cps/mL levels of both standards in parallel test runs performed by Dr. Koppelman (Sanquin, Amsterdam, Netherlands)
#
bDNA calibration n native n inact cps/mL native cps/mL inact
1st experiment 6 12 6.30 x 10E7 6.06 x 10E7*
2nd experiment 3 3 6.30 x 10E7 4.11 x 10E7*
* 1.47 fold lower in repeat experiment
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Calibration of viral genotype standards
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Example of calibration of HBV genotype standards in multiple DNA 3.0 assays
Secondary HBV-DNA standard
bDNA 3.0 runs cps/ml 95% CI (cps/ml) 95% CI (%)
N assays
N exp
df weighted avarage
lower upper lower upper
VQC gt A 28 4 20 2.15E+09 2.11E+09 2.20E+09 98% 102% Chimp gt A 6 1 4 1.26E+06 7.30E+05 2.16E+06 58% 172%
Eurohep gt A 6 1 4 2.97E+09 1.78E+09 4.95E+09 60% 167% DDL gt B 9 3 3 1.94E+09 1.62E+09 2.33E+09 83% 120% DDL gt C 9 3 3 2.21E+09 1.87E+09 2.61E+09 85% 118%
Chimp gt C 6 1 4 1.85E+06 1.28E+06 2.67E+06 69% 144% DDL gt D 9 3 3 3.46E+09 2.95E+09 4.06E+09 85% 117%
Eurohep gt D 12 2 8 2.53E+09 2.03E+09 3.17E+09 80% 125% ISS gt D 3 1 1 3.91E+04 8.77E+03 1.75E+05 22% 446% DDL gt E 9 3 5 1.57E+09 1.42E+09 1.74E+09 90% 111% DDL gt F 9 3 5 1.43E+09 9.18E+08 2.23E+09 64% 156% DDL gt G 9 3 5 8.29E+06 6.89E+06 9.97E+06 83% 120%
Grabarczyk P et al, Transfusion, in press Data reported in Supplementary Materials accessible online on Transfusion website
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1
10
100
A B C D AE O 1 2 3 4 5 6 A B C D E F G
1
10
100
A B C D AE O 1 2 3 4 5 6 A B C D E F G
1
10
100
A B C D AE O 1 2 3 4 5 6 A B C D E F G
cp
s/m
L
cp
s/m
L
cp
s/m
L
HIV-1 subtypes HCV genotypes HBV genotypes
HIV-1 subtypes HCV genotypes HBV genotypes
Ultrio
Ultrio PLus
TaqScreen
63% detection limits in copies/mL
Adapted from Assal et al. Transfusion 2009;49:289-300
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1%
10%
100%
1000%
A B C D AE O 1 2 3 4 5 6 A B C D E F G
1%
10%
100%
1000%
A B C D AE O 1 2 3 4 5 6 A B C D E F G
1%
10%
100%
1000%
A B C D AE O 1 2 3 4 5 6 A B C D E F G
% e
ffic
ien
cy
% e
ffic
ien
cy
% e
ffic
ien
cy
HIV-1 subtypes HCV genotypes HBV genotypes
HIV-1 subtypes HCV genotypes HBV genotypes
Ultrio % detection efficiency
Ultrio Plus
TaqScreen
Fig 2
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TaqScreen Ct values on HIV-1 subtype B standard dilution series
Data set from Assal et al.Transfusion. 2009;49:301-310
low variation suitable
for run control
Large variation; Poisson distribution
Slope equals -1.0 indicating
PCR efficiency is 100 %
Below LOD
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Marker genotype Potency Ct analysis Potency probit analysis
HBV-DNA
genotype A 100 % 100 % genotype B 53 (43-65)% 38 (14-96) % genotype C 66 (54-81)% 54(21-136)% genotype D 254(230-282)% 184(85-419)% genotype E 285(254-319)% 133(61-295)% genotype F 41(76-93)% 53(24-113)% genotype G 84(76-93)% 40(17-84)%
HCV-RNA
genotype 1 100 % 100 % genotype 2 138 (130-147)% 127(64-259)% genotype 3 106(98-114)% 179(91-372)% genotype 4 22(20-23)% 34(14-69)% genotype 5 45(41-50)% 48(22-96)% genotype 6 80(73-87)% 53(25-106)%
HIV-1 RNA
subtype B 100 % 100 % subtype A 137(121-155)% 132(71-260)% subtype C 126(112-142)% 136(74-267)% CRF01_AE 69(59-80)% 134(73-261)%
Comparison of TaqScreen Ct and probit analysis
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BQC 100 and 1000 cps/mL subgenotype panels for HBV, HCV and HIV NAT assays
Panel
nr
Standard Origin HBV
genotype
Panel
nr
Standard Origin HCV
genotype
Panel
nr
Standard Origin HIV subtype
1 WHO-PEI South Afr A1 1 Sanquin-VQC Netherlands 1a,b 1 BQC Netherlands A
2 WHO-PEI Brazil A1 2 BQC-INTS Egypt 1a 2 Sanquin-VQC Netherlands B
3 Sanquin-VQC Netherl A2 3 ARC USA 1a 3 BQC Netherlands C
4 Eurohep Germany A2 4 JRC Japan 1b 4 BQC Netherlands D
5 WHO-PEI Germany A2 5 JRC Japan 1b 5 BBI Brazil F
6 BQC Indonesia B 6 JRC Japan 2a 6 BBI Romania F
7 WHO-PEI Japan B2 7 JRC Japan 2a 7 BBI Zaire G
8 WHO-PEI Japan B2 8 BQC Netherlands 2a,b 8 BBI Kenya G
9 WHO-PEI Vietnam B4 9 JRC Japan 2b 9 BBI Zaire H
10 BQC USA C 10 JRC Japan 2b 10 BQC Netherlands CRF01_AE
11 WHO-PEI Japan C2 11 BQC Netherlands 3a 11 BBI Thailand CRF01_AE
12 WHO-PEI Japan C2 12 BQC-INTS Lithuania 3a 12 BBI Ghana CRF01_AG
13 WHO-PEI Russia C2 13 BQC-INTS Lithuania 3a 13 BBI Camaroon group O
14 BQC USA D 14 BQC-INTS Thailand 3b 14 BBI Camaroon group O
15 Eurohep Germany D 15 BQC Netherlands 4 15 BBI USA group O
16 ISS Italy D 16 BQC-INTS Egypt 4a 16 BBI Spain group O
17 WHO-PEI Germany D1 17 BQC USA 4a 17 BQC Belgium HIV-2 A
18 WHO-PEI South Afr D3 18 BQC Congo 4c 18 BQC Cameroon group O
19 WHO-PEI Iran D1 19 BQC Congo 4e 19 BQC Cameroon group N
20 BQC West Afr E 20 BQC USA 5a
21 WHO-PEI West Afr E 21 BQC USA 6a
22 BQC USA F 22 BQC USA 6a
23 WHO-PEI Brazil F3 23 BQC Thailand 6n
24 BQC USA G
25 WHO-PEI Germany G
P0138 HBV genotype panel 100 cps/mL P0139 HCV genotype panel 100 cps/mL P0137 HIV subtype panel 100 cps/mL
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Long term stability of liquid frozen secondary viral standards
at -70°C and -30°C
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Stability Sanquin-VQC HBV-DNA genotype A plasma standard at -70°C
Test year n Copies/mL
average 95% CI lower
95% CI upper
bDNA 1.0
1996 9 3.73E+09
1997 2 2.38E+09
1998 4 2.94E+09 1.46E+09 5.92E+09
1999 2 2.69E+09
average 17 3.22E+09 3.20E+09 3.24E+09
bDNA 3.0
2001 16 2.15E+09 1.77E+09 2.62E+09
2006 3 2.07E+09 1.45E+09 2.96E+09
2006 3 1.40E+09 5.63E+08 3.47E+09
2008 6 2.71E+09 2.06E+09 3.57E+09
average 28 2.15E+09 2.10E+09 2.20E+09
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Stability Sanquin-VQC HCV-RNA genotype 1 plasma standard (anti-HCV+) at -70°C
Test year n Average
copies/mL 95% CI lower
95% CI upper
bDNA 1.0 1996 4 8.76E+06 raw data not available
bDNA 2.0
1997 3 1.17E+07 3.50E+05 3.89E+08
1998 4 6.31E+06 5.45E+06 7.30E+06
1999 1 7.01E+06
2000 5 9.59E+06 3.49E+06 2.63E+07
2000 30 1.06E+07 7.10E+06 1.58E+07
average 43 9.83E+06 9.41E+06 1.03E+07
bDNA 3.0
2001 8 6.21E+06 3.97E+06 9.71E+06
2003 6 6.43E+06 4.68E+06 8.84E+06
2003 3 6.77E+06 1.35E+06 3.40E+07
2004 4 8.45E+06 3.65E+06 1.96E+07
2008 6 5.00E+06 3.83E+06 6.52E+06
average 27 6.30E+06 6.13E+06 6.48E+06
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Test year n Copies/mL
average 95% CI lower
95% CI upper
bDNA 1.0 1997 13 4.71E+07
bDNA 2.0
1999 18 1.14E+08 5.79E+07 2.25E+08 1999 31 1.06E+08 6.95E+07 1.61E+08
2000 8 1.13E+08 5.06E+07 2.53E+08
Average 57 1.09E+08 1.05E+08 1.14E+08
bDNA 3.0
2001 3 8.75E+07 3.55E+07 2.16E+08
2002 6 1.63E+08 1.18E+08 2.25E+08
2002 33 9.66E+07 5.71E+07 1.64E+08
2002 2 1.66E+08
2003 6 8.99E+07 6.73E+07 1.20E+08
2004 2 4.18E+07 2008 6 1.56E+08 1.14E+08 2.14E+08
average 58 1.05E+08 1.01E+08 1.09E+08
Stability Sanquin-VQC HIV-1 RNA subtype B (cultured) plasma standard at -70°C
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Stability of native HCV and HIV-1 plasma standards at -70°C confirmed in Ultrio LODs
study year
HCV-RNA genotype 1 LOD in copies/mL
HIV-RNA subtype B LOD in copies/mL
95% (CI) 50% (CI) 95% (CI) 50% (CI)
Lelie et al1 2000 25 (19-35) 2.3 (1.8-2.9) 13 (8-22) 1.5 (1.1-2.1)
Koppelman et al2 2004 25 (14-72) 2.9 (1.9-1.8) 21 (12-52) 2.4 (1.8-3.2)
Vermeulen et al3 2009 8.3 (5-15) 1.3 (0.9-1.8)
Grabarczyk et al4 2010 15 (8-37) 2.3 (1.4-3.8) 17 (8-47) 1.9 (1.1-3.2)
1. Lelie et al. Transfusion 2002;2:27-536) 2. Koppelman et al. Transfusion 2005;45:1258-1266 3. Vermeulen et al. Transfusion 2013 Epub 4. Grabarczyk et al. Transfusion 2013 Epub
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Comparison four years stability of secondary HCV standard (a-HCV+) at -30° and -70°C
-70°C -30°C
number assays* 12 12
geomean 124,451 126,814
95% CI upper 102,332 113,107
95% CI lower 151,350 140,936
*Siemens Versant bDNA assay 3.0
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Stability of frozen HIV (cultured) plasma standards at -70°C and -30°C in EasyQ
-70°C
-30°C
Slope-0.029 (NS)
Slope-0.123
10% degradation per year at -30°C 0
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In use stability of secondary viral standards in liquid phase
at 4°C, 20°C and 37 °C
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Impact of lyophilization and pasteurization on HIV-RNA short term stability in liquid phase
Temp Time
(hours) copies/mL in bDNA 3.0 assay
liquid. lyoph. inact. lyoph.
20°C 0 15164 3299 8463 3010
20°C 8 15004 3515 9170 3591
20°C 24 14703 3096 8697 3805
4°C 0 17439 4250 9208 3380
4°C 8 16209 4307 9344 4359
4°C 24 16715 4656 9077 3801
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Temp Time
(hours) copies/mL in bDNA 3.0 assay
liquid. lyoph. inact. lyoph.
20°C 0 100% 100% 100% 100%
20°C 8 99% 107% 108% 119%
20°C 24 97% 94% 103% 126%
4°C 0 100% 100% 100% 100%
4°C 8 93% 101% 101% 129%
4°C 24 96% 110% 99% 112%
Impact of lyophilization and pasteurization on HIV-RNA stability in liquid phase
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Stability of HIV-RNA run control* at 4°C and room temperature in QT-NASBA
Time (hours)
NA
SBA
Lo
g co
pie
s/m
L
*Cultured native HIV clade B in plasma *QC sample diluted to 10,500 cps/mL in bDNA assay and 33,200 cps/mL in QT-NASBA
74% after 120h
63% after 120h
Log linear regression analysis estimated 15% degradation at 41 and 54 hours for RT and 4°C
at -70°C
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Stability native and pasteurized HBV-DNA standards in liquid phase on TaqScreen 2.0*
mean quadruplicate Ct (potency) native HBV-DNA
hours -70°C 2-8°C 20°C 37°C
8 27.97 (100%) 28.02 (97%) 27.77 (115%) 27.87 (107%)
24 28.05 (100%) 28.00 (104%) 27.88 (113%) 27.95 (107%)
48 28.17 (100%) 28.00 (113%) 28.03 (111%) 28.28 (93%)
mean quadruplicate Ct (potency) inactivated HBV-DNA
hours -70°C 2-8°C 20°C 37°C
8 28.20 (100%) 28.18 (102%) 27.98 (117%) 27.93 (121%)
24 28.22 (100%) 28.08 (111%) 27.90 (125%) 27.78 (137%)
48 28.47 (100%) 28.25 (117%) 28.25 (117%) 28.10 (130%)
*2000 cps/mL concentrations were tested in subsequent TaqScreen 2.0 test runs at storage times by Dr Marco Koppelman (Sanquin, Amsterdam, the Netherlands)
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Stability HCV gt 1 (a-HCV+) and gt 3 (a-HCV-) standards in liquid phase on TaqScreen 2.0*
mean quadruplicate Ct (potency) native HCV-RNA gt 1
hours -70°C 2-8°C 20°C 37°C
8 30.88 (100%) 30.88 (100%) 31.00 (92%) 31.48 (66%)
24 30.90 (100%) 30.88 (102%) 30.93 (98%) 32.05 (45%)
48 30.93 (100%) 31.23 (81%) 31.13 (87%) 32.38 (37%)
mean quadruplicate Ct (potency) native HCV-RNA gt 3a
hours -70°C 2-8°C 20°C 37°C
8 31.23 (100%) 31.65 (74%) 31.53 (81%) 32.33 (47%)
24 31.20 (100%) 31.55 (78%) 31.55 (78%) 31.93 (60%)
48 31.03 (100%) 31.50 (72%) 31.45 (74%) 32.13 (47%)
*2000 cps/mL concentrations were tested in subsequent TaqScreen 2.0 test runs at storage times by Dr Marco Koppelman (Sanquin, Amsterdam, the Netherlands)
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Stability native and inactivated HCV gt 3 WP standards in liquid phase on TaqScreen 2.0*
mean quadruplicate Ct (potency) native HCV-RNA gt 3a
hours -70°C 2-8°C 20°C 37°C
8 31.23 (100%) 31.65 (74%) 31.53 (81%) 32.33 (47%)
24 31.20 (100%) 31.55 (78%) 31.55 (78%) 31.93 (60%)
48 31.03 (100%) 31.50 (72%) 31.45 (74%) 32.13 (47%)
mean quadruplicate Ct (potency) inactivated HCV-RNA gt 3a
hours -70°C 2-8°C 20°C 37°C
8 32.00 (100%) 32.20 (87%) 32.38 (77%) 33.40 (38%)
24 31.83 (100%) 32.25 (74%) 32.63 (57%) 33.33 (35%)
48 31.75 (100%) 32.35 (66%) 32.13 (77%) 33.23 (36%)
*2000 cps/mL concentrations were tested in subsequent TaqScreen 2.0 test runs at storage times by Dr Marco Koppelman (Sanquin, Amsterdam, the Netherlands)
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Stability native and inactivated HIV-1 clade B standards in liquid phase on TaqScreen 2.0*
mean quadruplicate Ct (potency) native HIV-RNA clade B
hours -70°C 2-8°C 20°C 37°C
8 29.05 (100%) 29.05 (100%) 28.85 (115%) 29.03 (102%)
24 29.15 (100%) 29.05 (107%) 29.00 (111%) 29.23 (95%)
48 28.75 (100%) 29.25 (71%) 29.15 (76%) 29.80 (48%)
mean quadruplicate Ct (potency) inactivated HIV-RNA clade B
hours -70°C 2-8°C 20°C 37°C
8 28.95 (100%) 28.93 (102%) 28.68 (121%) 29.03 (95%)
24 28.90 (100%) 28.98 (95%) 28.90 (100%) 28.93 (98%)
48 28.78 (100%) 29.30 (69%) 29.20 (74%) 29.45 (63%)
*2000 cps/mL concentrations were tested in subsequent TaqScreen 2.0 test runs at storage times by Dr Marco Koppelman (Sanquin, Amsterdam, the Netherlands)
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Safety Assessment of secondary inactivated standards
for NAT blood screening
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Inactivation of viral standards for preparation of NAT blood screening run controls
BQC
standard
inactivation
method
before
inactivation
in standard after
inactivation in run controls
cps/mL ID50/
mL cps/mL ID50/mL yield cps/mL ID50/mL
HBV-
DNA
10 hours
pasteurization at
65°C, 1:100
diluted in PBS
2,15
E+09
6,80
E+08
7,23
E+06
<2,30
E+00 84% <500
<1,60
E-04
HCV-
RNA
0.14%
betapropiolactone
for 5 hours at
23°C, 18h 4°C
4,39
E+07
1,38E
+07
4,11
E+07
<4,11
E+03 94% <500
<5,00
E-02
HIV-1
RNA
2 hours
pasteurization at
65°C, 1:10 diluted
in PBS
1,05
E+08
1,66
E+06
2,62
E+06
<6,60
E-01 62% <500
1,27
E-04
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Conclusions • WHO HCV 06/100 standard when shipped at RT has lower
potency than 96/798 in TMA field studies.
• Liquid Frozen plasma standards are long term stable at -70°C, but cultured HIV standard degrades at -30°C.
• Calibration of secondary BQC standards is confirmed in NAT blood screening assays, but concentration of inactivated HCV standard needs to be reassessed.
• No indication that lyophilisation and inactivation affect integrity and stability of residual viral particles in plasma standards.
• There may be a subpopulation of less stable virus in a-HCV negative HCV-RNA plasma standards
• Sensitivity of qualitative real time PCR blood screening assays can be predicted with calibrated 1000 cps/mL genotype panels by comparing Ct values