John Saldanha Regulatory Requirements: Precision and Accuracy of Quantitative NAT Tests XXII SoGAT Meeting, 14 15 April, 2011 Istituto Superiore di Sanita (ISS), Rome, Italy 2 June 2011 page 1 2011 Roche
Biological Substance Cannot be completely characterised by physico-chemical means alone Requires use of some form of bioassay Bioassay: comparison of response of test substance with that of reference material potency 2 June 2011 page 2 2011 Roche
NAT NAT tests measure amount of nucleic acid Biological substance has defined biological effect that can be measured by in vitro or in vivo assays Nucleic acids? May be inactive Mixture of complete and incomplete genomes 2 June 2011 page 3 2011 Roche
NAT Physically/chemically well-defined compounds traceable to SI unit Nucleic acids - Biological analytes potency defined by IU 2 June 2011 page 4 2011 Roche
NAT Assays Establishment of International Standards have facilitated move to quantitative tests Accuracy and precision of current quantitative tests Regulatory requirements for NAT tests 2 June 2011 page 5 2011 Roche
Regulatory Requirement for B19V Final plasma pool : 1E + 04 IU/mL OMCL Guideline for Validation of Nucleic Acid Amplification Techniques (NAT) for Quantitation of B19 Virus (B19V) DNA in Plasma Pools (EDQM, 2008) Accuracy Precision 2 June 2011 page 6 2011 Roche
95% LOD of B19 Test (cobas TaqScreen DPX Test) B19 95% LOD calculation 8 dilution steps 189 replicates/dilution No. of replicates/dilution (8 dilutions) 95% LOD (IU/mL) 95% lower CI (IU/mL 95% upper CI (IU/mL) 189 replicates 11.48 10.56 12.91 2 June 2011 page 7 2011 Roche
Multi-European DPX Evaluation: B19 Accuracy, Mean Estimates Nominal concentration (log 10 IU/mL) N Mean (log 10 IU/mL) Lower 95% CI Limit (log 10 IU/mL) Upper 95% CI Limit (log 10 IU/mL) 3.0 144 3.12 3.07 3.16 4.0 143 4.04 3.99 4.09 5.0 144 4.86 4.76 4.97 2 June 2011 page 8 2011 Roche
Multi-European DPX Evaluation: B19 Accuracy, Standard Deviation Nominal concentration (log 10 I U/mL) N Total standard deviation of Log 10 concentration 3.0 144 0.053 4.0 143 0.043 5.0 144 0.066 2 June 2011 page 9 2011 Roche
B19V International Standard, 99/800 Log 10 NAT detectable units/ml Mean Standard Deviation Minimum Maximum Range 5.92 0.47 4.84 6.72 1.88 2 June 2011 page 10 2011 Roche
Major Concerns Establishment of replacement International Standards WHO collaborative study to establish standard includes all laboratories bias, inconsistent quantitation due to different mix of laboratories, qualitative and quantitative tests Stringent requirements by regulatory authorities regarding assay performance necessitates accurate calibration of replacement standard with regard to previous standards 2 June 2011 page 11 2011 Roche
Issues with NAT Quantitative Tests
Issues Requirement by testing community for precise quantitation is this possible for biological assay? Accuracy and precision of NAT quantitative assays continue to improve Shift from biological standards to synthetic/physico-chemical standards? Lack of international reference method 2 June 2011 page 13 2011 Roche
Issues Only International Standard has assigned value no uncertainty Secondary standards, calibrators have increasing uncertainty of measurement Not taken into account when expressing quantitation 2 June 2011 page 14 2011 Roche
Conclusions Establishment of replacement International Standards Equitable mix of different assays to remove bias Use quantitative assays calibrated against the International Standard Nucleic acids, present status somewhere between biological material and chemical analyte What are we measuring? Infectious virus Complete genomes (active and inactive) Bits of nucleic acid? Potency (activity biological preparations used in prophylaxis or therapy) X X X 2 June 2011 page 15 2011 Roche
Acknowledgements Multi-European DPX Evaluation Dr. Susanna Wessberg, Finnish Red Cross Dr. Lutz Pichl, DRK-Blutspendedienst West Dr. Marco Koppelman, Sanquin ROCHE, COBAS, TAQSCREEN and LIFE NEEDS ANSWERS are trademarks of Roche. 2 June 2011 page 16 2011 Roche