1. Update on the P. falciparum Standard & Replacement of the HBV & HCV International Standards Sally Baylis, NIBSC SoGAT XIX

2. NIBSC Proposal for Production of Standards for P. falciparum For standardisation of NAT assays (qualitative and quantitative) For standardisation of NAT assays (qualitative and quantitative) Screening of blood for transfusion and tissues: exclusion of infected donations (run controls); determination of levels of parasitaemia where TTIs occur; validation of assay sensitivities Screening of blood for transfusion and tissues: exclusion of infected donations (run controls); determination of levels of parasitaemia where TTIs occur; validation of assay sensitivities Diagnosis and clinical management of malaria: standardisation of commercial and in-house tests – harmonisation of results to a single reference material Diagnosis and clinical management of malaria: standardisation of commercial and in-house tests – harmonisation of results to a single reference material Vaccine studies: cross-comparison of studies, parasite loads, efficacy etc. Vaccine studies: cross-comparison of studies, parasite loads, efficacy etc.

3. Candidate P. falciparum Standards Sample AA Freeze-dried blood from patient (~10% parasitaemia) Sample AA Freeze-dried blood from patient (~10% parasitaemia) Sample BB Liquid preparation of P. falciparum cultured in vitro to ~10% ring forms Sample BB Liquid preparation of P. falciparum cultured in vitro to ~10% ring forms Sample CC Liquid preparation of blood from patient (~7% parasitaemia) Sample CC Liquid preparation of blood from patient (~7% parasitaemia) Sample DD Liquid preparation of blood from patient (~0.007% parasitaemia) Sample DD Liquid preparation of blood from patient (~0.007% parasitaemia)

4. Collaborative Study to Establish an International Standard for P. falciparum Commenced April 2005 Commenced April 2005 14 laboratories participated in collaborative study 14 laboratories participated in collaborative study Participants requested to test samples in four independent assays Participants requested to test samples in four independent assays Initially at 10-fold dilutions Initially at 10-fold dilutions Subsequently at half log dilutions around the end point Subsequently at half log dilutions around the end point 20 data sets received, 2 from quantitative assays & the rest from qualitative assays 20 data sets received, 2 from quantitative assays & the rest from qualitative assays NIBSC collated & analysed data NIBSC collated & analysed data

5. PCR Detectable Units/ml (log 10 )

6. Overall Mean PCR Detectable Units/ml (log 10 ) from End- Point Assays N - Number of laboratory estimates SD - Standard Deviation of log 10 estimates across laboratories SampleNMeanMinMaxRangeSD AA178.517.4410.463.010.74 BB188.457.629.852.230.69 CC178.356.359.923.570.83 DD185.514.516.862.360.60 SampleNMeanRangeSD BB17-0.092.310.67 CC16-0.223.060.76 DD17-3.042.350.60 Overall Potencies Relative to AA (log 10 ) from End-Point Assays

7. Accelerated Degradation Studies of Sample AA Incubation Time-20°C+4°C+20°C+37°C+45°C 8 months0.00-0.040.260.410.44 12 months0.000.060.350.62 Relative potencies of accelerated degradation samples with respect to sample AA stored at -20ºC (log 10 drop)

8. Summary & Conclusions The mean log10 “equivalents”/ml were 8.51 for sample AA, 8.45 for sample BB, 8.35 for sample CC, and 5.51 for sample DD (NIBSC code 04/112). The mean log10 “equivalents”/ml were 8.51 for sample AA, 8.45 for sample BB, 8.35 for sample CC, and 5.51 for sample DD (NIBSC code 04/112). Predictions of the stability of the freeze-dried preparation AA indicate that it is extremely stable and suitable for long term use. Predictions of the stability of the freeze-dried preparation AA indicate that it is extremely stable and suitable for long term use. Propose that AA be established as the 1 st International Standard for P. falciparum DNA NAT assays, NIBSC code 04/176. Propose that AA be established as the 1 st International Standard for P. falciparum DNA NAT assays, NIBSC code 04/176. Proposed potency is 10 9 International units per ml. Each vial contains the equivalent of 0.5 ml of material, and the content of each vial would be 5 x 10 8 IU/ml. Submission to ECBS by July Proposed potency is 10 9 International units per ml. Each vial contains the equivalent of 0.5 ml of material, and the content of each vial would be 5 x 10 8 IU/ml. Submission to ECBS by July

9. Replacement of the 1 st International Standard for HBV DNA NIBSC Code 97/746 The 1 st International Standard for HBV DNA was established by the WHO ECBS in October 1999 The 1 st International Standard for HBV DNA was established by the WHO ECBS in October 1999 Assigned a concentration of 10 6 IU/ml Assigned a concentration of 10 6 IU/ml Standard made from a dilution of the Eurohep reference 1, genotype A, HBsAg subtype adw Standard made from a dilution of the Eurohep reference 1, genotype A, HBsAg subtype adw Stocks of 97/746 are extremely low Stocks of 97/746 are extremely low Study proposed to evaluate replacement at SoGAT 2005 Study proposed to evaluate replacement at SoGAT 2005

10. Candidate 2 nd International Standard Materials coded AA (97/746) & BB (97/750) showed no significant difference in potency in the collaborative study Materials coded AA (97/746) & BB (97/750) 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 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

11. Collaborative Study to Establish 2 nd International Standard for HBV DNA 6 laboratories participated in study 6 laboratories participated in study The 1 st International Standard and candidate BB were recoded as samples 1 and 2 respectively The 1 st International Standard and candidate BB were recoded as samples 1 and 2 respectively Participants requested to test samples in four independent assays Participants requested to test samples in four independent assays Initially at 10-fold dilutions Initially at 10-fold dilutions Subsequently at half log dilutions around the end point Subsequently at half log dilutions around the end point 8 sets of data were received, 5 from qualitative assays and 3 from quantitative assays 8 sets of data were received, 5 from qualitative assays and 3 from quantitative assays

12. Estimated IU/ml (log 10 ) From Quantitative Assays Laboratory number Sample 97/74697/750 15.995.97 2A6.085.99 2B6.065.92 45.945.86 Mean*6.005.93 *combining results of lab 2 to give a single lab mean prior to calculating overall mean of laboratories

13. Estimated PCR-detectable units/ml (log 10 ) Laboratory number Sample 97/74697/750 3A6.486.58 3B6.906.68 3C6.566.35 56.496.25 66.516.59

14. Accelerated Degradation Studies of 97/746 and 97/750 Storage temperatureSample 97/74697/750 -20°C6.025.92 +4°C5.925.91 +20°C5.946.03 Samples incubated at -20ºC, +4 ºC and +20 ºC for ~4.5 years Estimated IU/ml (log10)

15. Proposal for 2 nd International Standard for HBV DNA Real-time & accelerated degradation data indicate 97/746 & 97/750 are very stable & suitable for long term use Real-time & accelerated degradation data indicate 97/746 & 97/750 are very stable & suitable for long term use No significant differences found in estimated IU/ml or PCR-detectable units/ml for 97/746 & 97/750 No significant differences found in estimated IU/ml or PCR-detectable units/ml for 97/746 & 97/750 Propose that 97/750 be established as the 2 nd International Standard for HBV DNA with a unitage of 10 6 IU/ml – submission to ECBS by July Propose that 97/750 be established as the 2 nd International Standard for HBV DNA with a unitage of 10 6 IU/ml – submission to ECBS by July

16. 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

17. Progress in Replacing 96/798 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 vial markers confirmed in these stocks Absence of other vial markers confirmed in these stocks Titres determined by qPCR and Roche Monitor assay by comparison to 96/798 Titres determined by qPCR and Roche Monitor assay by comparison to 96/798

18. Progress in Replacing 96/798 contd. Material has been freeze-dried in two batches Material has been freeze-dried in two batches Batch 1, 2085 vials, fill CV = 0.77% Batch 1, 2085 vials, fill CV = 0.77% Batch 2, 2100 vials, fill CV = 1.02% Batch 2, 2100 vials, fill CV = 1.02% Titre of new freeze-dried preparations is at least double that of the 2 nd HCV RNA Titre of new freeze-dried preparations is at least double that of the 2 nd HCV RNA After freeze-drying, the activity of the preparations is ~70% that of the original bulk After freeze-drying, the activity of the preparations is ~70% that of the original bulk Call for collaborative study participants Call for collaborative study participants

19. Acknowledgements David Padley, Alan Heath & Nita Shah, NIBSC David Padley, Alan Heath & Nita Shah, NIBSC Peter Chiodini, Hospital for Tropical Diseases, London Peter Chiodini, Hospital for Tropical Diseases, London Claire Swales, LSHTM, London Claire Swales, LSHTM, London Collaborative study participants Collaborative study participants

20. Collaborative Study to Establish an International Standard for P.falciparum Participants A CalderaroUniversità degli Studi di Parma, Italy A CalderaroUniversità degli Studi di Parma, Italy P ChiodiniHospital for Tropical Diseases, London, UK P ChiodiniHospital for Tropical Diseases, London, UK A da SilvaCDC, USA A da SilvaCDC, USA C DeferEFS Nord de France C DeferEFS Nord de France I FelgerSwiss Tropical Institute I FelgerSwiss Tropical Institute K KainCentre for Travel & Tropical Medicine, Canada K KainCentre for Travel & Tropical Medicine, Canada S KrishnaSt. George’s Hospital, London, UK S KrishnaSt. George’s Hospital, London, UK R LeeInstitute of Clinical Pathology & Medical Research, Australia R LeeInstitute of Clinical Pathology & Medical Research, Australia D PadleyNIBSC, UK D PadleyNIBSC, UK F Perandin University of Brescia, Italy F Perandin University of Brescia, Italy G PisaniISS, Italy G PisaniISS, Italy T RuckesQiagen GmbH T RuckesQiagen GmbH R SauerweinUMC St Radboud, Netherlands R SauerweinUMC St Radboud, Netherlands S Sauleda Laboratori de Seguretat Transfusional, Spain S Sauleda Laboratori de Seguretat Transfusional, Spain

21. Collaborative Study to Establish 2 nd International Standard for HBV DNA Participants S Baylis/ D PadleyNIBSC, UK S Baylis/ D PadleyNIBSC, UK M Chudy PEI, Germany M Chudy PEI, Germany W GerlichUniversity of Geissen, Germany W GerlichUniversity of Geissen, Germany S KerbyCBER, USA S KerbyCBER, USA A Klotz/M GessnerBaxter, Austria A Klotz/M GessnerBaxter, Austria G PisaniISS, Italy G PisaniISS, Italy