SoGAT and the Development of Standards

1. SoGAT and theDevelopment of Standards Harvey Holmes Division of Retrovirology NIBSC, UK SoGAT XVII

2. 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 SoGAT XVII

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

4. 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 1st 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 SoGAT XVII

5. 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 SoGAT XVII

6. 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 SoGAT XVII

7. Development of 1st International Standard for HIV-1 RNA SoGAT XVII

8. 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 -70oC 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 SoGAT XVII

9. Development of Standards: Evaluation of Plasma Diluents(Preliminary analysis of data received to date) AssayEDTA-PlasmaACD-PlasmaCryospnt 1 3.52 3.42 3.50 2 3.49 3.53 3.48 3 3.28 3.53 3.45 Mean 3.43 3.49 3.48 SoGAT XVII

10. Development of Standards • Freeze-drying • Provides greater long-term stability (standards stored at -20oC) • 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 > 0oC 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 SoGAT XVII

11. 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 SoGAT XVII

12. 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 SoGAT XVII

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

14. Development of Standards • 1st 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 SoGAT XVII

15. 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 SoGAT XVII