1. Paris, May 2004SoGAT XVII SoGAT and the Development of Standards Harvey Holmes Division of Retrovirology NIBSC, UK

2. Paris, May 2004SoGAT XVII SoGAT and the Development of Standards NIBSC/EPFA NAT workshop held in Helsinki in 1994 led to the setting up of the SoGAT Working Group SoGAT (Standardisation of Gene Amplification Techniques) first met in April 1995 at NIBSC SoGATs terms of reference included: – Standardisation of methodologies – New developments in technology – Development, evaluation and provision of standards SoGAT provided forum in which the views of all groups (control labs, fractionators, kit manufacturers, reference labs etc) could be voiced and could influence development of standards Co-sponsored by WHO Over the last 9 years great strides made in providing standards for main blood markers

3. Paris, May 2004SoGAT XVII SoGAT and the Development of Standards 1997: 1 st international Standard for HCV (96/790) – 50,000 IU per vial, genotype 1a 1999: 1 st international Standard for HIV-1 (97/656) – 100,000 IU per vial, genotype B 1999: 1 st international Standard for HBV (97/746) – 500,000 IU per vial, genotype A 2000: 1 st international Standard for B19 (99/800) – 500,000 IU per vial 2002: 1 st international Standard for HAV (00/560) – 50,000 IU per vial 2002: 1 st international Reference panel for HIV-1 Genotypes (01/466) – genotypes A-H – no unitage assigned

4. Paris, May 2004SoGAT XVII SoGAT and the Development of Standards Standards may be International (Primary) or Working (Secondary): International Standard – WHO ECBS establishes International Standards and Reference Reagents – It is a preparation by means of which the WHO defines an International Unit after an international study has been completed – An International Unitage is assigned to the 1 st International Standard – Replacement standards are calibrated against the previous standard – Usually freeze-dried – International Units are arbitrary units, not SI units – There is no primary method – a range of methodologies is preferred Working standards – National or laboratory reference materials – Working (secondary) reference materials for day-to-day use – Calibrated against International Standard (in IU) – May be freeze-dried or frozen liquid – ready for use

5. Paris, May 2004SoGAT XVII Stages in the Development of Standards Planning the study Selecting suitable candidate materials Source material and suitable diluent Aliquoting and freeze-drying Labelling Characterisation International collaborative study Study protocol Data analysis Report and recommendation to WHO ECBS Establishment of standard

6. Paris, May 2004SoGAT XVII Development of Standards Will describe the process of developing standards with some examples Starting / source material – Should closely resemble samples that will be assayed against it – Stable – Appropriate specific activity – Homogenous – Sufficient to provide several years supply (~10y) Bulk material / diluent – Diluent should closely resemble samples that will be assayed against it – Diluent important – may influence performance of standard – Preservative should be chosen with care – should not affect preparation or interfere with assays likely to be used – Free from other blood viruses – Prepare standard from homogeneous bulk

7. Paris, May 2004SoGAT XVII Development of 1 st International Standard for HIV-1 RNA Candidate:XXYYZZ Subtype:BBB Virus:Primary isolate Plasmapheresis donation T-cell line virus Propagated:PBMCsNoT cell line Copy number:HighMediumLow Freeze-dried:Yes No Diluent:Plasma Volume:1ml 0.5ml

8. Paris, May 2004SoGAT XVII Development of Standards: Evaluation of Plasma Diluents To determine if different plasma diluents influence assay of RNA concentration Diluents evaluated: – Citrated Human plasma – EDTA human plasma – Cryosupernatant Each diluent spiked with subtype B virus used for PWS-1/2 Batch frozen at -70 o C and coded sample A, B or C Small collaborative study organised Participants asked to assay samples in duplicate 3 assays Results to date based on three different quantitative assays

9. Paris, May 2004SoGAT XVII Development of Standards: Evaluation of Plasma Diluents (Preliminary analysis of data received to date) AssayEDTA-PlasmaACD-PlasmaCryospnt 13.523.42 3.50 23.493.53 3.48 33.283.53 3.45 Mean3.433.49 3.48

10. Paris, May 2004SoGAT XVII Development of Standards Freeze-drying – Provides greater long-term stability (standards stored at -20 o C) – Provides greater ease of distribution – avoids need for dry ice – Precise fill volume – low coefficient of variance (<0.25%) – Ampoules preferred, but vials safer for biohazardous materials – Suitability of glass and stoppers must be established Study at NIBSC showed that stoppered vials gain moisture and oxygen when stored at > 0 o C for several weeks) – Freeze-drying cycle important – pilot study may be required Characterisation – Potency – confirm that this is satisfactory and no undue loss of potency during processing – Moisture content and residual oxygen – Stability – degradation studies to predict loss of potency

11. Paris, May 2004SoGAT XVII Development of Standards International Collaborative Study: – Needed before standard can be established by WHO ECBS – Usually compare two or more candidate standards – To determine which candidate material is suitable to serve as standard for assay of preparations from different sources – May use duplicates to assess within-lab reproducibility – To assign a potency to the standard on the basis of valid assays that have been statistically analysed – To determine if different assay methods measure same or different properties of proposed reference material – 4-10 labs or more for complex studies of new test materials – Storage temperature and shipment important – Freeze-dried samples can be shipped at ambient – Each lab carries out a minimum number of independent assays – Results coded to ensure anonymity

12. Paris, May 2004SoGAT XVII Development of Standards Data collation and analysis – Each lab provides information on assay method used – Details of dilutions etc on assay results sheet – Each assay is analysed for assay validity, relative potency and precision (95% confidence limits) – For each candidate, results combined and potencies and confidence limits calculated Report to WHO ECBS – Draft sent to participants to check – Final report includes recommendation on which candidate to use and suggested potency

13. Paris, May 2004SoGAT XVII Development of 1 st International Standard for HIV-1 RNA Candidate XX Candidate YY Candidate ZZ --- bDN --- Monitor--Nuclisens --- Abbott --- in-house

14. Paris, May 2004SoGAT XVII Development of Standards 1 st International Standard for HIV-1 RNA (code 97/656) – Candidate YY established by ECBS – Assigned a potency of 100,000 IU per vial Progress report on replacement HIV-1 standard to be given tomorrow by Clare Davis

15. Paris, May 2004SoGAT XVII Development of Standards – Concluding Remarks SoGAT has overseen establishment of International Standards to all the main blood-transmitted viruses WHO International Standard has proven track record: – Closely resemble materials being assayed complex macromolecules / microorganisms that are not chemically defined – Should be suitable for use with wide range of assays – There is no single approved reference method often there is no agreement on which methods are best – Are assigned arbitrary International Units, not SI units Can accommodate changes is assay precision – Traceability - replacement candidates calibrated against previous standard We need to proceed cautiously in any move towards the adoption of SI units for NAT standards