1. The Sense of Sensitivity I Dr. Matthias Gessner Plasma Control Europe/Plasma Analytics Baxter AG, Vienna

2. Matthias Gessner/Gerold Zerlauth Regulatory Sensitivity WHO NAT Standards for HAV HBV HCV HIV Parvo B19 Guidelines validation of NAT ICH Guidelines on the validation of analytical assays. (CPMP/ICH/381/95 (Q2A) und CPMP/ICH/281/95 (Q2B)) Anforderungen an Validierung bzw. Routinebetrieb der HCV-NAT im Blutspendewesen (Paul Ehrlich Institut, 1998) Guidelines for validation of nucleic acid amplification technology (NAT) for the detection of hepatitis C virus (HCV) RNA in plasma pools (Council of Europe, PA/PH/OMCL(98)22, DEF, 1999). "European Pharmacopoeia, 4th edition"; published by the „Directorate for the Quality of Medicines of the Council of Europe (EDQM)“; January 1, 2002

3. Matthias Gessner/Gerold Zerlauth Regulatory Sensitivity Conversion „copies“ zu „IU“ –HCV: 1 IU = 2 cp (Collaborative Studies for WHO Standard) 1 IU = 4 cp (VQC) –HIV 1 IU = 0.4 cp (Collaborative Studies for WHO Standard) 1 IU = 1 cp (VQC) Factor of 4-5 between HCV and HIV International Units expressed in copies

4. Matthias Gessner/Gerold Zerlauth Regulatory Sensitivity Requirements for HCV, HIV NAT –Original:5000 copies/ml –Converted:5000 IU/ml

5. Matthias Gessner/Gerold Zerlauth Conversion „copies“ zu „IU“ The original requirement Thus it was implemented That‘s how it should have been implemented

6. Matthias Gessner/Gerold Zerlauth Conclusion If there was a basis for the original requirement in copies/ml – did it change? If not - apparently the outer packing seemed more important than the acutal content Scientists must be aware of this fact when creating standards – public perception may not coincide with scientific knowledge

7. The Sense of Sensitivity II Dr. Gerold Zerlauth Plasma Control Europe Baxter AG, Vienna

8. Matthias Gessner/Gerold Zerlauth Poolsize Virus: RNA / DNA Antibodies Antibody negative window NAT negative window INFECTIONDetection pool of 96SeroconversionDetection Single donation

9. Matthias Gessner/Gerold Zerlauth HCVHIV Infection to Seroconversion [d] 7022 Reduction by PCR [d] 41-6010-15 Busch et al., Transfusion 2000; 40; 143-59 Diagnostic Window

10. Matthias Gessner/Gerold Zerlauth HCV Doubling time 17h 96-fold amplification –4.7 d HIV Doubling time 22h 96-fold amplification –6 d Busch et al., Transfusion 2000; 40; 143-59 Poolsize

11. Matthias Gessner/Gerold Zerlauth Single donation testing HCV Virus: RNA / DNA Antibodies Antibody negative window NAT negative window 60-41 d4.7 d 0.6/Mio.*0.05/Mio. *Roth et al., Transfus.Clin.Biol. 2001; 8; 282-4

12. Matthias Gessner/Gerold Zerlauth Theoretical gain by reducing poolsize HCVHIV NAT pos./ Million in pools of 96 0.6 Theor. NAT pos. /Million single donations 0.650.9

13. Matthias Gessner/Gerold Zerlauth Pool-size reduction in reality HCV ARC S.L.Stramer et al,,US experience with Nucleic Acid Testing (NAT) for HIV-1 and HCV, 03/2001 Samples per pool 12816 1 NAT pos. in 334.043302.805 Positive per Mil. 3,03,3 Samples tested 2.338.3049.084.162

14. Matthias Gessner/Gerold Zerlauth Pool-size reduction in reality HCV ARC S.L.Stramer et al,,US experience with Nucleic Acid Testing (NAT) for HIV-1 and HCV, 03/2001 JRC Dr. Murokawa, JRC NAT Research Group, Minisymposium of JRC, 2002 Samples per pool 1281650050 1 NAT pos. in 334.043302.805333.785336.155 Positive per Mil. 3,03,33,0 Samples tested 2.338.3049.084.1621.668.92616.483.458

15. Matthias Gessner/Gerold Zerlauth Pool-size reduction in reality HIVHIV ARC S.L.Stramer et al,,US experience with Nucleic Acid Testing (NAT) for HIV-1 and HCV, 03/2001 Samples per pool 12816 1 NAT pos. in > 2.338.3044.542.081 Positive per Mil. ---0,22 Samples tested 2.338.3049.084.162

16. Matthias Gessner/Gerold Zerlauth Pool-size reduction in reality HIVHIV ARC S.L.Stramer et al,,US experience with Nucleic Acid Testing (NAT) for HIV-1 and HCV, 03/2001 JRC Dr. Murokawa, JRC NAT Research Group, Minisymposium of JRC, 2002 Samples per pool 1281650050 1 NAT pos. in > 2.338.3044.542.081>1.668.9263.025.397 Positive per Mil. ---0,22---0,33 Samples tested 2.338.3049.084.1621.668.92616.483.458

17. Matthias Gessner/Gerold Zerlauth Conclusion Increased sensitivity or smaller pool sizes do not necessarely yield higher detection rate of NAT- only positives Recent experience with pool testing have to be taken into consideration prior to establish requirements for sensitivity Optimal sensitivity/pool sizes can then be evaluated for optimal detection of targeted viruses