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Update on the P. falciparum Standard & Replacement of the HBV & HCV International Standards

Update on the P. falciparum Standard & Replacement of the HBV & HCV International Standards. Sally Baylis, NIBSC SoGAT XIX. NIBSC Proposal for Production of Standards for P. falciparum. For standardisation of NAT assays (qualitative and quantitative)

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Update on the P. falciparum Standard & Replacement of the HBV & HCV International Standards

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  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) 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 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 BB Liquid preparation of P. falciparum cultured in vitro to ~10% ring forms • Sample CC Liquid preparation of blood from patient (~7% 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 • 14 laboratories participated in collaborative study • Participants requested to test samples in four independent assays • Initially at 10-fold dilutions • Subsequently at half log dilutions around the end point • 20 data sets received, 2 from quantitative assays & the rest from qualitative assays • NIBSC collated & analysed data

  5. PCR Detectable Units/ml (log10)

  6. Overall Mean PCR Detectable Units/ml (log10) from End-Point Assays Overall Potencies Relative to AA (log10) from End-Point Assays N - Number of laboratory estimates SD - Standard Deviation of log10 estimates across laboratories

  7. Accelerated Degradation Studies of Sample AA Relative potencies of accelerated degradation samples with respect to sample AA stored at -20ºC (log10 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). • 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 1st International Standard for P. falciparum DNA NAT assays, NIBSC code 04/176. • Proposed potency is 109 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 108 IU/ml. Submission to ECBS by July

  9. Replacement of the 1st International Standard for HBV DNA NIBSC Code 97/746 • The 1st International Standard for HBV DNA was established by the WHO ECBS in October 1999 • Assigned a concentration of 106 IU/ml • Standard made from a dilution of the Eurohep reference 1, genotype A, HBsAg subtype adw • Stocks of 97/746 are extremely low • Study proposed to evaluate replacement at SoGAT 2005

  10. Candidate 2nd International Standard • 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 • Current study designed to demonstrate the equivalence of the candidate replacement (BB) to AA • Real-time data on samples AA & BB • Accelerated degradation data for samples AA & BB

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

  12. Estimated IU/ml (log10) From Quantitative Assays *combining results of lab 2 to give a single lab mean prior to calculating overall mean of laboratories

  13. Estimated PCR-detectable units/ml (log10)

  14. Accelerated Degradation Studies of 97/746 and 97/750 Samples incubated at -20ºC, +4 ºC and +20 ºC for ~4.5 years Estimated IU/ml (log10)

  15. Proposal for 2nd International Standard for HBV DNA • 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 • Propose that 97/750 be established as the 2nd International Standard for HBV DNA with a unitage of 106 IU/ml – submission to ECBS by July

  16. Replacement of the 2nd International Standard for HCV RNA (96/798) • 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

  17. Progress in Replacing 96/798 • 3 anti-HCV negative window period genotype 1a donations have been obtained • The genotype of each has been confirmed by LiPA & DNA sequencing • Absence of other vial markers confirmed in these stocks • 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 • Batch 1, 2085 vials, fill CV = 0.77% • Batch 2, 2100 vials, fill CV = 1.02% • Titre of new freeze-dried preparations is at least double that of the 2nd HCV RNA • After freeze-drying, the activity of the preparations is ~70% that of the original bulk • Call for collaborative study participants

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

  20. Collaborative Study to Establish an International Standard for P.falciparumParticipants • A Calderaro Università degli Studi di Parma, Italy • P Chiodini Hospital for Tropical Diseases, London, UK • A da Silva CDC, USA • C Defer EFS Nord de France • I Felger Swiss Tropical Institute • K Kain Centre for Travel & Tropical Medicine, Canada • S Krishna St. George’s Hospital, London, UK • R Lee Institute of Clinical Pathology & Medical Research, Australia • D Padley NIBSC, UK • F Perandin University of Brescia, Italy • G Pisani ISS, Italy • T Ruckes Qiagen GmbH • R Sauerwein UMC St Radboud, Netherlands • S SauledaLaboratori de Seguretat Transfusional, Spain

  21. Collaborative Study to Establish 2nd International Standard for HBV DNAParticipants • S Baylis/ D Padley NIBSC, UK • M Chudy PEI, Germany • W Gerlich University of Geissen, Germany • S Kerby CBER, USA • A Klotz/M Gessner Baxter, Austria • G Pisani ISS, Italy

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