1. NUCLEIC ACID AMPLIFICATION TECHNOLOGY HCV-RNA / HBV-DNA / HIV-RNA testing blood and blood components for transfusion Italian External Quality Assessment Program, 2008 IT NAT EQA 2008 CENTER FOR IMMUNOBIOLOGICALS RESEARCH AND EVALUATION Biologicals Unit ITALIAN NATIONAL BLOOD CENTRE Transfusion Safety Area

2. This study was organized and promoted by the Italian National Blood Centre in cooperation with the Centre for Immunobiologicals Research and Evaluation, Istituto Superiore di Sanità (ISS) IT NAT EQA 2008 Study F.Luciani - SOGAT – Brussels 28-29 May 2009 IT NAT EQA 2008 - ISS

3. The IT NAT EQA 2008 study was organized for assessing the analytical performance of the qualitative NAT assays/systems currently used in Italian blood centers for HCV RNA, HIV RNA and HBV DNA screening. The study was extended, on a voluntary basis, also to other european and international testing labs and blood products manufacturers. Scope and Participants F.Luciani - SOGAT – Brussels 28-29 May 2009 IT NAT EQA 2008 - ISS

4. Participants A total of 122 laboratories participated in the IT NAT EQA 2008 study Italy98 Austria1 Germany3 United Kingdom1 Greece1 Spain9 Switzerland1 Lithuania1 Thailand7 F.Luciani - SOGAT 28-29 May 2009 IT NAT EQA 2008 - ISS

5. 1 st phase June 2008 to July 2008 2 nd phase Nov 2008 to Dec 2008 IT NAT EQA 2008 Study Timeline F.Luciani - SOGAT – Brussels 28-29 May 2009 IT NAT EQA 2008 - ISS

6. Design and rationale of the study (1) It would not be possible to draw definitive conclusions using either: -low viral load (i.e. close to 95% DL): participants could miss the target due to its random distribution in the plasma matrix, or -high viral load: it would produce 100% of correct results hiding any occurrence of procedural mistakes. Thus, panels were prepared taking into account the 95% detection limit (DL) of the methods most commonly used by laboratories involved in blood screening by NAT. (Pisani et al. Vox Sanguinis 2008, 95:8–12). F.Luciani - SOGAT – Brussels 28-29 May 2009 IT NAT EQA 2008 - ISS

7. A load of 3x 95% DL should be found positive by all participant in 100% of the assays and any error in the procedure, even a minor one, would be detected. This concentration is also currently recommended to evaluate the robustness of qualitative NAT methods in the context of validation studies [Guidelines for validation of NAT for the detection of HCV RNA in plasma pools (PA/PH/OMCL (98) 22, DEF)] Panel sample F.Luciani - SOGAT – Brussels 28-29 May 2009 IT NAT EQA 2008 - ISS

8. Assays F.Luciani - SOGAT – Brussels 28-29 May 2009 IT NAT EQA 2008 - ISS

9. International Standard The majority of participating laboratories use commercial kits for NAT blood screening for which the 95% DL was calculated by the manufacturers using WHO standards. Thus, WHO International Standards were selected for the preparation of the panels. F.Luciani - SOGAT – Brussels 28-29 May 2009 IT NAT EQA 2008 - ISS

10. Negative samples: they were prepared using a plasma pool made up of 25 donations tested negative for HCV, HIV and HBV by serological and NAT tests. Positive samples: they were obtained spiking negative plasma pool with the relevant WHO International Standard to obtain the following concentrations : 10, 33 and 85 IU/ml of the HCV RNA WHO IS 96/798, genotype 1 60, 150 and 235 IU/mL of the HIV RNA WHO IS 97/650, genotype B 12, 15 and 30 IU/ml of the HBV DNA WHO IS 97/746, genotype A A total of 3000 vials, including negative and positive samples, were prepared and stored at –80°C, numbered from 0001 to 3000. Materials and Methods F.Luciani - SOGAT – Brussels 28-29 May 2009 IT NAT EQA 2008 - ISS

11. HCV HIV HBV neg TMA-panel AMP-panel CTM-panel Panels F.Luciani - SOGAT – Brussels 28-29 May 2009 IT NAT EQA 2008 - ISS 10 30 60 10 30 60 10 30 60 85 15 235 85 15 235 85 15 235 33 12 150 33 12 150 33 12 150

12. Design and rationale of the study (2) The panel samples were tested in multiple runs To better simulate a routine testing, participants were invited to test four samples (one sub-panel) per day in a timeframe of 2–3 weeks, by different operators, where possible. Two identical panels were sent to the participants, to be tested separately in the 1 st and 2 nd phase of the study Three samples with the same viral concentration were included in the panel, to verify the consistency of the results obtained in separate runs on different days. F.Luciani - SOGAT – Brussels 28-29 May 2009 IT NAT EQA 2008 - ISS

13. To confirm the negative and positive status of the samples (homogeneity), the selection of the vials to be tested was carried out randomly during the filling. For each dilution, 5 samples were tested before the study (t=0), 5 samples were tested at the end of the study (t= 6 months) Quality control F.Luciani - SOGAT – Brussels 28-29 May 2009 IT NAT EQA 2008 - ISS Shipment Panels were shipped in dry ice (48 hours delivery). Participants were asked to check the integrity of the parcel, the presence of dry ice and the status of the samples and to fax this information to the ISS using the acknowledgement of receipt sheet. Each participant subscribed a responsibility sheet to acknowledge that the samples received were potentially infectious

14. Assays ASSAY CODE 1 st phase2 nd phase TMA Ultrio AssayTMA 42 COBAS Ampliprep/TaqmanCTM 4154 COBAS AmpliscreenAMP 4029 In-HouseIH 32 F.Luciani - SOGAT – Brussels 28-29 May 2009 IT NAT EQA 2008 - ISS

15. A total of 2848 samples were tested and the results returned to ISS by fax or e-mail: HCV RNA (716 data) HIV RNA (713 data) HBV DNA (699 data) Negative samples (720 data) RESULTS Samples F.Luciani - SOGAT – Brussels 28-29 May 2009 IT NAT EQA 2008 - ISS

17. 36 deviations were observed (21 in EQA-1 and 15 in EQA-2), classified as follows: 1)Time schedule not observed 2)One or more samples not tested 3)One sub-panel not tested 4)Dedicated panel (e.g. for TMA) tested with a different assay RESULTS Protocol deviations F.Luciani - SOGAT – Brussels 28-29 May 2009 IT NAT EQA 2008 - ISS

18. A total of 247 different operators participated in EQA study testing one or more sub-panels: 32 laboratories - 1 operator 55 laboratories - 2 operators 35 laboratories - 3 operators RESULTS Operators F.Luciani - SOGAT – Brussels 28-29 May 2009 IT NAT EQA 2008 - ISS

19. A total of 28 errors (20 laboratories) occurred. RESULTS EQA Ph-1EQA Ph-2 N° of laboratories who reported at least 1 error119 Total errors observed1216 unspecified analitytcal error43 analytical errors classified as contamination26 pre/post analytical errors67 Errors F.Luciani - SOGAT – Brussels 28-29 May 2009 IT NAT EQA 2008 - ISS

20. Participant Coordinator Results sheet Feedback 24-48 h Laboratories failing to correctly identify the positive or negative samples were invited to critically evaluate each single step in order to identify the cause of the mistake.

21. CONCLUSIONS The study allowed participants to verify their performance. Under these conditions, it was demonstrated that, despite the high level of automation reached by NAT assays, human errors can still occur. Laboratories reporting a failure are encouraged to better comply with the good laboratory practice in terms of operator training and control of contamination. F.Luciani - SOGAT – Brussels 28-29 May 2009 IT NAT EQA 2008 - ISS

22. Acknowledgments CRIVIB Biologicals Unit CNS Transfusion Safety Area Giulio Pisani Karen Cristiano Francesco Marino Claudio Mele Guillermo Bisso Andrea Gaggioli Daniela Adriani Maria Wirz Simonetta Pupella Vanessa Piccinini Silvia Vitali Claudio Mele Maria Wirz Secretarial Assistance Katia Colombo Cristina Marra F.Luciani - SOGAT – Brussels 28-29 May 2009 IT NAT EQA 2008 - ISS

23. Back-up slides

24. 95% DL 3x 95% DL 40 IU/mL 100 IU/mL 20 IU/mL HCV-RNAHIV-RNAHBV-DNA

25. 95% DL 3x 95% DL 10 IU/mL HCV-RNAHIV-RNAHBV-DNA 33 IU/mL 85 IU/mL 60 IU/mL 150 IU/mL 235 IU/mL 30 IU/mL 12 IU/mL 15 IU/mL 10 33 85 60 150 235 30 12 15