SOGAT XXI Brussels, 28-29 May 2009

1. 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. European Pharmacopoeia(I) 2.6.21 Nucleic Acid Amplification Techniques 7.3.1 External Controls

3. European Pharmacopoeia(II) External Controls Negative control: a sample of the same matrix already proven to be free of the target sequences Extraction 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 Amplification Detection External Controls

4. 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 External Controls Additional external controls: in-house positive run controls

5. 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) ISS Reference Preparations for NAT assays

6. 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) ISS Ref. Prep. for NAT assays: the Italian approach (I)

7. 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) ISS Ref. Prep. for NAT assays: the Italian approach (II)

8. Example of ISS Collaborative study (HCV) (I) Samples HCV WHO International Standard *Provided to participants (pre-diluted by the ISS) #Dilutions to be carried out by participants Dil. 10-3* 10-3.5 # 10- 4 #10-4.5 #10-5# IU/mL 100 32 10 31 HCV ISS Reference Preparation^ ^Provided undiluted to participants §Dilutions to be carried out by participants (based on the preliminary titer) Dil.§ 10-1.6 10-2.1 10-2.6 10-3.1 10-3.6

9. 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 Example of ISS Collaborative study (HCV) (II)

10. Example of ISS Collaborative study (HCV) (III)Statistical analysis by the ISS (Probit) 0.63

11. HCV RNA ISS 1005 ISS Last Collab. Study (2007) (I) PARTICIPANTS 1 • 11 Italian BTCs • 1 Spanish BTC • ISS • NAT assays • Cobas Ampliscreen (6 laboratories) • TMA Ultrio (7 laboratories)

12. HCV RNA ISS 1005 - ISS Last Collab. Study (2007) (II) Approx. 5700 IU/mL

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

14. HCV RNA ISS 1005 - ISS Last Collab. Study (2007) (IV)Distribution of results HCV WHO IS HCV ISS/1005

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

16. What is the right concentration for a positive run control? 100 95% cut-off 80 60 40 Probability % 20 0 0 1 10 100 1000 IU/ mL (log) Positive samples/total number tested 3-4 x 95% cut-off

17. 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) What is the right concentration for an ISS reference preparation?

18. Ultrio Tigris (TMA-Novartis) 95% DL as stated by the manufacturer RUN CONTROL 4 X 95% DL as suggested by the ISS

19. Real Time PCR (S201-Roche) 95% DL as stated by the manufacturer RUN CONTROL 4 X 95% DL as suggested by the ISS

20. 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 What is the right concentration for an ISS reference preparation?

21. 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 What’s next?

22. Thank you for your attention!