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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

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

Calibration of HBV standards

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in press Pisani G et al, Ann Ist Super Sanita 2007:43:69-76 Chudy M et al, J Clin Virol 2012Epub Van Drimmelen et al, unpublished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

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

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)

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

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

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

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

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

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)

Calibration of HIV standards

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



1:10,000 diluted pasteurized 1:10,000 diluted native freeze-drying

freeze-drying

freezing freezing

regular lyophilized


pasteurized past./lyophilized

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

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


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)

§

#


experiment 6 6 1.05 x 10E8 2.62 x 10E6

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)

IU/mL

% TMA reactive



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

Calibration of HCV standards

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



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

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


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)

#


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

Calibration of viral genotype standards

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

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


Ultrio

Ultrio PLus

TaqScreen

63% detection limits in copies/mL

Adapted from Assal et al. Transfusion 2009;49:289-300

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



Ultrio % detection efficiency

Ultrio Plus

TaqScreen

Fig 2

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

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

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

Long term stability of liquid frozen secondary viral standards

at -70°C and -30°C

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

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

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

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

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

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

In use stability of secondary viral standards in liquid phase

at 4°C, 20°C and 37 °C

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

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

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

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)

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%)


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%)


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%)


Safety Assessment of secondary inactivated standards

for NAT blood screening

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

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

calibration and stability of who and secondary viral … lelie - calibration...calibration and...

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