Calibration and stability of WHO and secondary viral standards

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1 Calibration and stability of WHO and secondary viral standards Nico Lelie, Harry van Drimmelen and the International NAT Study Group Facilities DDL Diagnostic Laboratories

2 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

3 Calibration of HBV standards

4 Traceability of HBV-DNA standards bdna calibrators copies Eurohep gt A Sanquin-VQC genotype A Eurohep gt D 1 st WHO gt A Dilution, lyophilization IUs Dilution, pasteurization Chimp gt A Chimp gt C ID 50 ISS gt D 2nd WHO gt A 3rd WHO gt A BQC gt A inactivated BQC gt panel calibrations 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in press Pisani G et al, Ann Ist Super Sanita 2007:43:69-76 Chudy M et al, J Clin Virol 2012Epub Van Drimmelen et al, unpublished WHO gt panel Collins ML, et al. An Biochem 1995;226:120 Komiya K et al. Transfusion 2008;48:286-9

5 Calibration of native Sanquin-VQC HBV standard against WHO 97/746 standard Experiment Parallel Siemens Versant bdna 3.0 assays (Cuijpers et al Sanquin, Amsterdam) Parallel Siemens Versant bdna 3.0 assays (Van Drimmelen, DDL, Rijswijk) Multi-method WHO Collaborative study (Saldanha J et al. Vox Sang 2001;80:63-71) n WHO n VQC Copy/IU (95% CI) 5.33 ( ) 5.20 ( ) Grabarczyk P et al, Transfusion, in press Data reported in Supplementary Materials accessible online on Transfusion website

6 Calibration Japanese Chimpanzee infectivity plasmas against Sanquin-VQC HBV genotype A standard Chimp plasma C-246 P-57 genotype A C-272 P-29 genotype C Study Assays n copies/ CID 50 (range) BQC-DDL bdna ( ) Komiya et al TaqMan (2.6-26) BQC-DDL bdna ( ) Komiya et al TaqMan (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)

7 Preparation of pasteurised BQC HBV-DNA plasma standard Native VQC HBV-DNA genotype A standard (2.15 x 10 9 cps/ml, 6.8 x 10 8 CID 50 /ml) 1:100 dilution in PBS > 10 6 CID 50 inactivation* 100 diluted HBV-DNA standard in PBS (2.15 x 10 6 cps/ml ) 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 10 6 cps/ml, <2.3 CID 50 /ml) 84% recovery HBV-DNA *Lelie PN et al J.Med.Virol. 1987:23:289-95

8 Banding of HBV DNA in sucrose gradient WHO lyophilised ISS lyophilised BQC pasteurized VQC native 25,000 rpm, 16h, 10 C Gerlich et al, SoGAT, Brussels 2009

9 Analytical sensitivity of Ultrio and Ultrio Plus on native and pasteurized HBV-DNA standard Study standard Assay n 95% LOD (CI) 50% LOD (CI) Ultrio (82-378) 15.7 ( ) Poland native Ultrio Plus ( ) 4.4 ( ) Denmark Ultrio ( ) 55 ((33-93) inactivated Ultrio ( ) 59 (32-111) Poland Ultrio Plus ( ) 6.7 ( ) Standard Relative sensitivity Ultrio Plus to Ultrio Native 3.60 ( ) Inactivated 8.86 ( ) Assay Ultrio Ultrio Plus Relative potency native to inactivated 3.78 ( ) 1.54 ( ) data from Ultrio and Ultrio Plus validation studies in Ireland, Denmark and Poland

10 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 probit 0,65 ( ) TaqScreen Ct# 0.87 ( ) Study performed by Prof. Piotr Grabarczyk (IHTM, Warsaw, Poland) # Comparison of Ct values on 2000 cps/ml levels of both standards in parallel TaqScreen test runs performed by Dr Marco Koppelman (Sanquin, Amsterdam) bdna calibration n native n inact cps/ml native cps/ml inact experiment x 10E x 10E6

Potency of 2 nd WHO HBV 97/750 IS relative to 1 st 97/746 IS in Ultrio validation studies % Ultrio reactive standard n Potency (95% CI) 97/746 733 reference 97/750 88 1.14 (0,57-2.

11 Potency of 2 nd WHO HBV 97/750 IS relative to 1 st 97/746 IS in Ultrio validation studies % Ultrio reactive standard n Potency (95% CI) 97/ reference 97/ (0, ) IU/mL 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

12 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/ ( ) 29.0 ( ) 0.89 ( ) 97/ ( ) 26.9 ( ) 0.96 ( ) Eurohep ( ) 25.9 ( ) 1.00 (reference) Chimp ( ) 27.2 ( ) 0.95 ( ) VQC native ( ) 36.3 ( ) 0.71 ( ) BQC inact ( ) 48.3 ( ) 0.54 ( ) HBV standard n 50% LOD (CI) cps/ml in Tx 95% LOD (CI) cps/ml in Tx Potency relative to Eurohep standard 97/ ( ) 20.9 ( ) 0.54 ( ) Eurohep ( ) 11.4 ( ) 1.00 (reference) VQC native ( ) 14.2 ( ) 0.80 ( )

13 Calibration of HIV standards

14 Preparation pasteurized and/or lyophilized HIV- RNA reference samples native standard in plasma 1.05 x 10 8 cps/ml 1:100 dilution in plasma 1:10 dilution in PBS 2h hour 65 0 C pasteurized 1:10 in PBS 1:10 dilution in plasma 1:100 diluted 1:100 diluted pasteurized 1:100 dilution in plasma 1:10,000 diluted native freezing regular freezedrying freezedrying lyophilized 1:100 dilution in plasma 1:10,000 diluted pasteurized freezing pasteurized past./lyophilized

15 Recovery of HIV-RNA after pasteurization process standard and/or lyophilization Average cps/ml* recovery Native standard 15, % lyophilization % pasteurization % pasteurization & lyophilization % * 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

16 Potency of native and pasteurized HIV-RNA standard in Ultrio probit and TaqScreen Ct analysis Assay n native n inact analysis Potency inactivated relative to native standard (95% CI) Ultrio probit 0,69 ( ) TaqScreen Ct# 1.04 ( ) # Study standard n 95% LOD (CI) 50% LOD (CI) Poland native ( ) 2.1 ( ) Denmark inactivated ( ) 3.1 ( ) 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 x 10E x 10E6

17 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 Assay Abbott LCx Roche Amplicor Monitor Siemens bdna 3.0 Organon Tekika NucliSens Roche Amplicor Mon UltraSens 1st WHO N assays 2nd WHO cps/iu on 1 st WHO (97/656) standard cps/iu on 2nd WHO (97/650) standard VQC mean (95%CI) mean (95%CI) ( ) 0.69 ( ) ( ) 0.93 ( ) ( ) 0.58 ( ) ( ) 0.43 ( ) ( ) 0.86 ( ) calculated from raw data reported by the laboratories participating in the first WHO collaborative study Holmes H et al, J. Virological Methods 2001, 92; Grabarczyk P et al, Transfusion, in press ((Table reported in Supplementary Materials accessible online on Transfusion website)

Potency of 1 st HIV-1 97/656 IS and PWS 99/634 relative to 2nd 97/650 IS in TMA assay validation studies % TMA reactive standard n Potency (95% CI) 97/650 718 reference 97/656 131 1.00 (0,77-1.

18 Potency of 1 st HIV-1 97/656 IS and PWS 99/634 relative to 2nd 97/650 IS in TMA assay validation studies % TMA reactive standard n Potency (95% CI) 97/ reference 97/ (0, ) PWS 99/ ( ) IU/mL Assay Standard n dhiv 97/ Duplex 97/ Ultrio 97/ Ultrio Elite 97/ Ultrio Plus 97/ Duplex PWS-1 99/ Ultrio PWS-1 99/

19 Calibration of HCV standards

20 Calibration 3 rd HCV WHO 06/100 IS, 06/102 sample and unlyophilised bulk on 2 nd 96/798 IS quantitative assays n labs IS 96/798 Data Baylis S et al, Vox Sang 2011, 100, 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 17 1,00E+05 1,63E+05 2,45E+05 3,66E+05 methods 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

Potency of 3rd 06/100 and 1 st 96/790 IS relative to 2nd 96/798 IS in TMA assay validation studies % TMA reactive standard n Potency (95% CI) 96/798 594 reference 96/790 48 1.04 (0,71-1.

21 Potency of 3rd 06/100 and 1 st 96/790 IS relative to 2nd 96/798 IS in TMA assay validation studies % TMA reactive standard n Potency (95% CI) 96/ reference 96/ (0, ) 06/ ( )* * 06/100 vs 96/798, p<0.05 IU/mL Assay standard n Ultrio 06/ Ultrio Elite 06/ Ultrio Plus 06/ Duplex 96/ Ultrio 96/ Duplex 96/ Ultrio 96/

22 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 probit 0,52 ( ) TaqScreen Ct# 0.45 ( ) # Study standard n 95% LOD (CI) 50% LOD (CI) Poland native ( ) 1.9 ( ) Denmark inactivated ( ) 3.6 ( ) 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 1 st experiment x 10E x 10E7* 2 nd experiment x 10E x 10E7* * 1.47 fold lower in repeat experiment

23 Calibration of viral genotype standards

24 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 E E E+09 98% 102% Chimp gt A E E E+06 58% 172% Eurohep gt A E E E+09 60% 167% DDL gt B E E E+09 83% 120% DDL gt C E E E+09 85% 118% Chimp gt C E E E+06 69% 144% DDL gt D E E E+09 85% 117% Eurohep gt D E E E+09 80% 125% ISS gt D E E E+05 22% 446% DDL gt E E E E+09 90% 111% DDL gt F E E E+09 64% 156% DDL gt G E E E+06 83% 120% Grabarczyk P et al, Transfusion, in press Data reported in Supplementary Materials accessible online on Transfusion website

25 cps/ml cps/ml cps/ml 100 HIV-1 subtypes HCV genotypes HBV genotypes 10 Ultrio 63% detection limits in copies/ml A B C D AE O A B C D E F G Adapted from Assal et al. Transfusion 2009;49: Ultrio PLus 1 A B C D AE O A B C D E F G TaqScreen 1 A B C D AE O A B C D E F G HIV-1 subtypes HCV genotypes HBV genotypes

26 % efficiency % efficiency % efficiency 1000% HIV-1 subtypes HCV genotypes HBV genotypes 100% 10% 1% 1000% Ultrio % detection efficiency A B C D AE O A B C D E F G 100% 10% 1% 1000% Ultrio Plus A B C D AE O A B C D E F G 100% Fig 2 10% 1% TaqScreen A B C D AE O A B C D E F G HIV-1 subtypes HCV genotypes HBV genotypes

suitable for run control Slope equals -1.

27 TaqScreen Ct values on HIV-1 subtype B standard dilution series Below LOD Large variation; Poisson distribution low variation suitable for run control Slope equals -1.0 indicating PCR efficiency is 100 % Data set from Assal et al.transfusion. 2009;49:

28 Comparison of TaqScreen Ct and probit analysis Marker genotype Potency Ct analysis Potency probit analysis HBV-DNA HCV-RNA HIV-1 RNA genotype A 100 % 100 % genotype B 53 (43-65)% 38 (14-96) % genotype C 66 (54-81)% 54(21-136)% genotype D 254( )% 184(85-419)% genotype E 285( )% 133(61-295)% genotype F 41(76-93)% 53(24-113)% genotype G 84(76-93)% 40(17-84)% genotype % 100 % genotype ( )% 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)% subtype B 100 % 100 % subtype A 137( )% 132(71-260)% subtype C 126( )% 136(74-267)% CRF01_AE 69(59-80)% 134(73-261)%

29 BQC 100 and 1000 cps/ml subgenotype panels for HBV, HCV and HIV NAT assays P0138 HBV genotype panel 100 cps/ml P0139 HCV genotype panel 100 cps/ml P0137 HIV subtype panel 100 cps/ml 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

30 Long term stability of liquid frozen secondary viral standards at -70 C and -30 C

31 Stability Sanquin-VQC HBV-DNA genotype A plasma standard at -70 C Test year n Copies/mL average E E+09 95% CI lower 95% CI upper bdna E E E E+09 average E E E E E E E E E+09 bdna E E E E E E+09 average E E E+09

32 Stability Sanquin-VQC HCV-RNA genotype 1 plasma standard (anti-hcv+) at -70 C Test year n Average 95% CI 95% CI copies/ml lower upper bdna E+06 raw data not available E E E E E E+06 bdna E E E E E E E+07 average E E E E E E E E E+06 bdna E E E E E E E E E+06 average E E E+06

33 Stability Sanquin-VQC HIV-1 RNA subtype B Test year n Copies/mL average bdna E+07 bdna 2.0 bdna 3.0 (cultured) plasma standard at -70 C 95% CI lower 95% CI upper E E E E E E E E E+08 Average E E E E E E E E E E E E E E E E E E E E+08 average E E E+08

34 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 al (19-35) 2.3 ( ) 13 (8-22) 1.5 ( ) Koppelman et al (14-72) 2.9 ( ) 21 (12-52) 2.4 ( ) Vermeulen et al (5-15) 1.3 ( ) Grabarczyk et al (8-37) 2.3 ( ) 17 (8-47) 1.9 ( ) 1. Lelie et al. Transfusion 2002;2:27-536) 2. Koppelman et al. Transfusion 2005;45: Vermeulen et al. Transfusion 2013 Epub 4. Grabarczyk et al. Transfusion 2013 Epub

35 Comparison four years stability of secondary HCV standard (a-hcv+) at -30 and -70 C -70 C -30 C number assays* geomean 124, ,814 95% CI upper 102, ,107 95% CI lower 151, ,936 *Siemens Versant bdna assay 3.0

36 Stability of frozen HIV (cultured) plasma standards at -70 C and -30 C in EasyQ Slope (NS) Slope C -30 C 10% degradation per year at -30 C 0

37 In use stability of secondary viral standards in liquid phase at 4 C, 20 C and 37 C

38 Impact of lyophilization and pasteurization on HIV-RNA short term stability in liquid phase Temp Time copies/ml in bdna 3.0 assay (hours) liquid. lyoph. inact. lyoph. 20 C C C C C C

39 Impact of lyophilization and pasteurization on HIV-RNA stability in liquid phase Temp Time copies/ml in bdna 3.0 assay (hours) 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%

NASBA Log copies/ml Stability of HIV-RNA run control* at 4 C and room temperature in QT-NASBA Log linear regression analysis estimated 15% degradation at 41 and 54 hours for RT

40 NASBA Log copies/ml Stability of HIV-RNA run control* at 4 C and room temperature in QT-NASBA Log linear regression analysis estimated 15% degradation at 41 and 54 hours for RT and 4 C at -70 C 74% after 120h 63% after 120h Time (hours) *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

41 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 (100%) (97%) (115%) (107%) (100%) (104%) (113%) (107%) (100%) (113%) (111%) (93%) mean quadruplicate Ct (potency) inactivated HBV-DNA hours -70 C 2-8 C 20 C 37 C (100%) (102%) (117%) (121%) (100%) (111%) (125%) (137%) (100%) (117%) (117%) (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)

42 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 (100%) (100%) (92%) (66%) (100%) (102%) (98%) (45%) (100%) (81%) (87%) (37%) mean quadruplicate Ct (potency) native HCV-RNA gt 3a hours -70 C 2-8 C 20 C 37 C (100%) (74%) (81%) (47%) (100%) (78%) (78%) (60%) (100%) (72%) (74%) (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)

43 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 (100%) (74%) (81%) (47%) (100%) (78%) (78%) (60%) (100%) (72%) (74%) (47%) mean quadruplicate Ct (potency) inactivated HCV-RNA gt 3a hours -70 C 2-8 C 20 C 37 C (100%) (87%) (77%) (38%) (100%) (74%) (57%) (35%) (100%) (66%) (77%) (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)

44 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 (100%) (100%) (115%) (102%) (100%) (107%) (111%) (95%) (100%) (71%) (76%) (48%) mean quadruplicate Ct (potency) inactivated HIV-RNA clade B hours -70 C 2-8 C 20 C 37 C (100%) (102%) (121%) (95%) (100%) (95%) (100%) (98%) (100%) (69%) (74%) (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)

45 Safety Assessment of secondary inactivated standards for NAT blood screening

46 Inactivation of viral standards for preparation of NAT blood screening run controls BQC standard HBV- DNA HCV- RNA HIV-1 RNA inactivation method 10 hours pasteurization at 65 C, 1:100 diluted in PBS 0.14% betapropiolactone for 5 hours at 23 C, 18h 4 C 2 hours pasteurization at 65 C, 1:10 diluted in PBS before inactivation cps/ml ID50/ ml 2,15 E+09 4,39 E+07 1,05 E+08 6,80 E+08 1,38E +07 1,66 E+06 in standard after inactivation cps/ml ID50/mL yield 7,23 E+06 4,11 E+07 2,62 E+06 <2,30 E+00 <4,11 E+03 <6,60 E-01 84% <500 94% <500 62% <500 in run controls cps/ml ID50/mL <1,60 E-04 <5,00 E-02 1,27 E-04

47 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

Stability of native, lyophilized and inactivated standards

Stability of native, lyophilized and inactivated standards Harry van Drimmelen Bio Quality Control, Heiloo, Netherlands Satellite Meeting before IPFA-PEI 25 th Workshop Twenty-five Years Standardization