Update on the Replacement of the HCV RNA International Standard Sally Baylis & Alan Heath, NIBSC SoGAT XX, Warsaw June 2007

Replacement of the 2 nd International Standard for HCV RNA (96/798) Proposal made at SoGAT 2005 to replace the HCV RNA International Standard as requested by WHO 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 Agreement that HCV 1a genotype would be sourced & would be anti-HCV negative and diluted in plasma rather than cryosupernatant

Candidate Standards for Evaluation 3 anti-HCV negative window period genotype 1a donations have been obtained 3 anti-HCV negative window period genotype 1a donations have been obtained The genotype of each has been confirmed by LiPA & DNA sequencing The genotype of each has been confirmed by LiPA & DNA sequencing Absence of other viral markers confirmed in these stocks Absence of other viral markers confirmed in these stocks Material has been freeze-dried in two batches Material has been freeze-dried in two batches Batch 1 (NIBSC Code 06/100), 2085 vials, fill CV = 0.62% Batch 1 (NIBSC Code 06/100), 2085 vials, fill CV = 0.62% Batch 2 (NIBSC code 06/102), 2100 vials, fill CV = 1.82% Batch 2 (NIBSC code 06/102), 2100 vials, fill CV = 1.82%

Panel for Collaborative study Sample 1 – 2 nd International Standard for HCV RNA (96/798) Sample 1 – 2 nd International Standard for HCV RNA (96/798) Sample 2 - new freeze-dried preparation 06/100 Sample 2 - new freeze-dried preparation 06/100 Sample 3 - new freeze-dried preparation 06/102 Sample 3 - new freeze-dried preparation 06/102 Sample 4 – liquid/frozen bulk material (NIBSC code 06/118) that was used to prepare Samples 2 & 3 Sample 4 – liquid/frozen bulk material (NIBSC code 06/118) that was used to prepare Samples 2 & 3

Collaborative Study Collaborative study commenced in November 2006 Collaborative study commenced in November 2006 Participants requested to test samples in four independent assays Participants requested to test samples in four independent assays 33 laboratories from 14 countries participated in the study 33 laboratories from 14 countries participated in the study 40 data sets received; 25 from quantitative assays & 15 from qualitative assays 40 data sets received; 25 from quantitative assays & 15 from qualitative assays NIBSC collated & analysed data NIBSC collated & analysed data

Types of Quantitative Assays Used by Participants Assay Code AssayNumber of Data-sets ABAbbott RealTime HCV7 BABayer Versant HCV RNA 3.0 (bDNA) 4 CTMCOBAS Ampliprep/COBAS TaqMan 6 CTMHHPS/COBAS TaqMan1 IHRTIn-house real-time3 MOCOBAS Amplicor HCV Monitor 4

Types of Qualitative Assays Used by Participants Assay Code AssayNumber of Data-sets AAmplicor HCV 2.02 ACCOBAS Amplicor HCV 2.01 ASCCOBAS Ampliscreen HCV 2.03 IHQIn-house qualitative1 IHRTQIn-house real-time qualitative3 PProcleix1* PUProcleix Ultrio1 PUTProcleix Ultrio (TIGRIS)1 TSCOBAS TaqScreen MPX1

Estimated IU/ml (log 10 ) from Quantitative Assays SampleNMean95% CISD%CV N - Number of laboratory estimates SD - Standard Deviation of log 10 estimates across laboratories

Estimated PCR Detectable Units/ml (log 10 ) from Qualitative Assays SampleNMean95% CISD%CV N - Number of laboratory estimates SD - Standard Deviation of log 10 estimates across laboratories

Overall Mean Potencies (log 10 IU/ml) Relative to Sample 1 SampleNMean95% CISD%CV N - Number of laboratory estimates SD - Standard Deviation of log 10 estimates across laboratories

Accelerated Degradation Studies Sample 2 Relative potencies of accelerated degradation samples with respect to Sample 2 stored at -20ºC (log 10 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 Incubation Time+4°C (10.5 months) +20°C (1 month) Sample

Proposal for 3 rd International Standard for HCV RNA Real-time & accelerated degradation data indicate that Sample 2 (06/100) is stable & suitable for long term use 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 3 rd International Standard for HCV RNA with a unitage of 5.19 log 10 IU/ml (4.89 log 10 IU/vial) Propose that 06/100 be established as the 3 rd International Standard for HCV RNA with a unitage of 5.19 log 10 IU/ml (4.89 log 10 IU/vial) Report will be submitted to ECBS by July Report will be submitted to ECBS by July

Acknowledgements Nita Shah & David Padley NIBSC Nita Shah & David Padley NIBSC Professor Ewa Brojer, Institute of Hematology & Blood Professor Ewa Brojer, Institute of Hematology & Blood Transfusion, Warsaw Transfusion, Warsaw Dr Richard Smith, National Genetics Institute, Dr Richard Smith, National Genetics Institute, Los Angeles Los Angeles Dr Vivienne James, HPA, Colindale Dr Vivienne James, HPA, Colindale Dr Michael Chudy, WHO, Geneva Dr Michael Chudy, WHO, Geneva Collaborative study participants Collaborative study participants

HCV RNA Collaborative Study Participants S Baylis, N ShahSouth Mimms, UK S Baylis, N ShahSouth Mimms, UK T Cuypers M KoppelmanAmsterdam, The Netherlands T Cuypers M KoppelmanAmsterdam, The Netherlands C DeferLille, France C DeferLille, France L FanningCork, Eire L FanningCork, Eire G GesuMilan, Italy G GesuMilan, Italy A GoglioBergamo, Italy A GoglioBergamo, Italy P GrantLondon, UK P GrantLondon, UK P IudiconeRome, Italy P IudiconeRome, Italy L Jarvis, A ClelandEdinburgh, UK L Jarvis, A ClelandEdinburgh, UK C JorkSpringe, Germany C JorkSpringe, Germany S KerbyBethesda, USA S KerbyBethesda, USA A Klotz, M GessnerVienna, Austria A Klotz, M GessnerVienna, Austria R MadejPleasanton, USA R MadejPleasanton, USA B MillerKarlsruhe, Germany B MillerKarlsruhe, Germany S MizusawaTokyo, Japan S MizusawaTokyo, Japan C NegruTimişoara, Romania C NegruTimişoara, Romania M NüblingLangen, Germany M NüblingLangen, Germany

HCV RNA Collaborative Study Participants JM PawlotskyCréteil, France JM PawlotskyCréteil, France M PecorariModena, Italy M PecorariModena, Italy G PisaniRome, Italy G PisaniRome, Italy A Portela MoreiraMadrid, Spain A Portela MoreiraMadrid, Spain S SauledaBarcelona, Spain S SauledaBarcelona, Spain H SchindlVienna, Austria H SchindlVienna, Austria G SchneiderDes Plaines, USA G SchneiderDes Plaines, USA A da SilvaLisbon, Portugal A da SilvaLisbon, Portugal R SmithLos Angeles, USA R SmithLos Angeles, USA S TassenBrentwood, UK S TassenBrentwood, UK P TaylorLondon, UK P TaylorLondon, UK A van der ZandenApeldoorn, The Netherlands A van der ZandenApeldoorn, The Netherlands YC YangTaipei, Taiwan YC YangTaipei, Taiwan S YerlyGeneva, Switzerland S YerlyGeneva, Switzerland M ZaccanelliTreviglio, Italy M ZaccanelliTreviglio, Italy

Update on the Stability of the HAV RNA International Standard Sally Baylis, Nita Shah & Alan Heath, NIBSC SoGAT XX, Warsaw June 2007

Background – Hepatitis A Virus RNA IS The 1 st International Standard for HAV RNA (00/560) and a second batch (00/562) were prepared in Q1, 2001 The 1 st International Standard for HAV RNA (00/560) and a second batch (00/562) were prepared in Q1, /560 was evaluated in an international collaborative study, with data returned from 16 laboratories 00/560 was evaluated in an international collaborative study, with data returned from 16 laboratories The mean log 10 equivalents/ml were 5.29 (00/560) and 5.07 (00/562) The mean log 10 equivalents/ml were 5.29 (00/560) and 5.07 (00/562) 1 st International Standard (00/560) was established in 2003 with 50,000 IU/vial 1 st 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 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 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 log 10 00/560 – a drop on 0.1 log 10 00/562 – a drop on 1.0 log 10 00/562 – a drop on 1.0 log 10 Conclude that 00/560 is highly stable Conclude that 00/560 is highly stable

Proposal to Replace the Parvovirus B19 DNA International Standard Sally Baylis, NIBSC SoGAT XX, Warsaw June 2007

Replacement of the Parvovirus B19 DNA IS 99/800 The 1 st International Standard for B19V DNA was established by the WHO ECBS in October 2000 The 1 st 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 Estimated date of exhaustion of the IS will be 2008/9 at current rate of usage Materials coded AA (99/800) & BB (99/802) showed no significant difference in potency in the collaborative study 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 ECBS noted that BB (made from the same stock as AA) could be reserved for potential future use as a replacement standard

Candidate 2 nd International Standard Materials coded AA (99/800) & BB (99/802) showed no significant difference in potency in the original collaborative study 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 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 Current study designed to demonstrate the equivalence of the candidate replacement (BB) to AA Real-time data on samples AA & BB Real-time data on samples AA & BB Accelerated degradation data for samples AA & BB Accelerated degradation data for samples AA & BB