1. Karen Cristiano Biologicals Unit, CRIVIB Calibration against the WHO Standards of National Reference Preparations for detection of blood viruses by NAT: the Italian experience SOGAT XXI Brussels, 28-29 May 2009

2. Karen Cristiano Biologicals Unit, CRIVIB European Pharmacopoeia European Pharmacopoeia (I) 2.6.21 Nucleic Acid Amplification Techniques 7.3.1 External Controls

3. Karen Cristiano Biologicals Unit, CRIVIB European Pharmacopoeia European Pharmacopoeia (II) External Controls Extraction Amplification Detection Negative control: a sample of the same matrix already proven to be free of the target sequences Positive control: this contains a defined number of target-sequence copies, the number being determined individually for each assay system and indicated as a multiple of the positive cut- off value of the test system External Controls

4. Karen Cristiano Biologicals Unit, CRIVIB External Controls Commercial NAT test kits include positive and negative external controls for the assay validation. However, for the positive controls there are some drawbacks:  viral load unknown (in some cases too high compared to the detection limit)  integrity of the virus (?)  in some cases (e.g. Ampliscreen) the control does not cover the whole NAT method Additional external controls: in-house positive run controls

5. Karen Cristiano Biologicals Unit, CRIVIB ISS Reference Preparations for NAT assays A total of 7500 vials of ISS Reference Preparations have been distributed since 1998:  3500 for HCV (3 batches)  1500 for HIV (2 batches)  2500 for HBV (3 batches)

6. Karen Cristiano Biologicals Unit, CRIVIB ISS Ref. Prep. for NAT assays: the Italian approach (I)  Selection and characterisation of an appropriate positive plasma donation with respect to the viral load and the genotype  Small scale production in order to evaluate which dilution of the positive sample is adequate (viral load: 3000-5000 IU/ml)  Large scale preparation: dilution of the positive plasma, filling into vials…  Homogeneity test on the positive preparation (at least 10 assays in duplicate). Result of this test also used for stability studies (T=0)  Stability studies (RT 24 h, 4°C 1 week, -80°C 6 month- intervals)

7. Karen Cristiano Biologicals Unit, CRIVIB ISS Ref. Prep. for NAT assays: the Italian approach (II) CALIBRATION through a mini collaborative study (10-14 participants):  50% laboratories using NAT assay A (e.g. TMA § )  50% laboratories using NAT assay B (e.g. PCR * ) § now automated on Tigris platform * now automated on COBAS S201 system (Real Time PCR)

8. Karen Cristiano Biologicals Unit, CRIVIB HCV WHO International Standard Example of ISS Collaborative study (HCV) (I) Samples Dil. 10 -3* 10 -3.5 # 10 - 4 # 10 -4.5 # 10 -5 # IU/mL 100 32 10 3 1 HCV ISS Reference Preparation^ Dil. § 10 -1.6 10 -2.1 10 -2.6 10 -3.1 10 -3.6 *Provided to participants (pre-diluted by the ISS) # Dilutions to be carried out by participants ^ Provided undiluted to participants § Dilutions to be carried out by participants (based on the preliminary titer)

9. Karen Cristiano Biologicals Unit, CRIVIB Example of ISS Collaborative study (HCV) (II) Testing scheme  Four indipendent dilutions of both the WHO International Standard (IS) and the ISS reference preparation are tested in four separate runs  Results are sent to the ISS for statistical analysis

10. Karen Cristiano Biologicals Unit, CRIVIB Example of ISS Collaborative study (HCV) (III) Statistical analysis by the ISS (Probit) 0.63

11. Karen Cristiano Biologicals Unit, CRIVIB HCV RNA ISS 1005 ISS Last Collab. Study (2007) (I) PARTICIPANTS NAT assays Cobas Ampliscreen (6 laboratories) TMA Ultrio (7 laboratories) 1  11 Italian BTCs  1 Spanish BTC  ISS

12. Karen Cristiano Biologicals Unit, CRIVIB HCV RNA ISS 1005 - ISS Last Collab. Study (2007) (II) Approx. 5700 IU/mL

13. Karen Cristiano Biologicals Unit, CRIVIB HCV RNA ISS 1005 - ISS Last Collab. Study (2007) (III) Deviation of each estimated value with respect to the mean titre (—) and the interval of confidence (mean ± geometric coeff. of variation ( --- ))

14. Karen Cristiano Biologicals Unit, CRIVIB HCV WHO IS HCV ISS/1005 HCV RNA ISS 1005 - ISS Last Collab. Study (2007) (IV) Distribution of results

15. Karen Cristiano Biologicals Unit, CRIVIB When a failure confirms the validity of your approach HBV ISS/0905 HBV ISS/0905: not suitable as a reference preparation! HBV WHO IS

16. Karen Cristiano Biologicals Unit, CRIVIB What is the right concentration for a positive run control? Probability % 0 20 40 60 80 100 IU/ mL (log) 01101001000 95% cut-off 3-4 x 95% cut-off Positive samples/total number tested

17. Karen Cristiano Biologicals Unit, CRIVIB What is the right concentration for an ISS reference preparation? On-going pilot study to determine the correct use of the curent ISS reference preparations (HCV, HIV, HBV):  Each laboratory receives a standard protocol to dilute the ISS reference preparations to obtain a concentration 4 x the 95% cut-off of the NAT assay (TMA or Real Time PCR)

18. Karen Cristiano Biologicals Unit, CRIVIB Ultrio Tigris (TMA-Novartis) HCV RNAHIV RNAHBV DNA ~ 3 UI/mL ~ 20 UI/mL ~ 10 UI/mL ~ 12 UI/mL ~ 80 UI/mL ~ 40 UI/mL 95% DL as stated by the manufacturer RUN CONTROL 4 X 95% DL as suggested by the ISS

19. Karen Cristiano Biologicals Unit, CRIVIB 95% DL as stated by the manufacturer RUN CONTROL 4 X 95% DL as suggested by the ISS Real Time PCR (S201-Roche) HCV RNAHIV RNAHBV DNA ~ 11UI/mL ~ 49 UI/mL ~ 4 UI/mL ~ 45 UI/mL ~ 200 UI/mL ~ 16 UI/mL

20. Karen Cristiano Biologicals Unit, CRIVIB What is the right concentration for an ISS reference preparation? On-going pilot study to determine the correct use of the curent ISS reference preparations (HCV, HIV, HBV):  Run controls are to be used with each run on a routine basis  All the results, recorded in an Excel file, are sent to the ISS on a monthly basis  Based on the results, collected up to June 2009, we will decide whether to use or not the 4 x the 95% cut-off

21. Karen Cristiano Biologicals Unit, CRIVIB What’s next? ISS Collaborative Study 2009:  HCV RNA ISS 1008 (Genotype 1)  HIV RNA ISS 0109 (Subtype F)  10 Italian transfusion centres will participate  Same approach as just described

22. Karen Cristiano Biologicals Unit, CRIVIB Thank you for your attention!