External Quality Assessment Programmes (EQAPs) for blood-borne viruses: the Italian experience

Transcription

1 External Quality Assessment Programmes (EQAPs) for blood-borne viruses: the Italian experience Giulio Pisani Biologicals Unit CRIVIB, ISS, Rome SOGAT XXII 14 th 15 th April Istituto Superiore di Sanità, Rome, Italy

2 External Quality Assurance Programmes (EQAPs) for blood-borne viruses: the Italian experience 2010 EQAP for HCV RNA, HIV RNA and HBV DNA Inter-laboratory Study for WNV RNA Inter-laboratory Study for HIV-2 RNA

3 Mandatory introduction of NAT in Italian Blood Centers: June 2002 HCV RNA April 2008 HIV RNA and HBV DNA

4 Italian NAT EQAPs for HCV, HIV and HBV Since 2008, the National Blood Centre and the National Centre for Immunobiologics Research and Evaluation have been promoting and organizing on an annual basis NAT EQAPs for HCV, HIV and HBV

5 Scope EQAPs are aimed at assessing the performance of laboratories in carrying out qualitative NAT assays for HCV RNA, HIV RNA and HBV DNA

6 Scope but they also provide participants with a tool to 1. evaluate the effectiveness of its quality system by detecting any possible weakness in the preand post-analytical steps of the process 2. estimate the ability of analytical procedures to generate results that meet the parameters and the technical specifications of the assay

7 Participants Italian NAT EQAPs, though addressed to the Italian blood centres, are also open to foreign laboratories and blood centres involved in NAT testing of plasma for viral markers.

8 Participants Italy Argentina Austria Estonia Germany United Kingdom Spain Slovenia Switzerland Lithuania Thailand

9 2010 EQAP

10 2010 NAT EQAP Phase 1 May - June Phase 2 November December

11 Home Page

12 UserID e Password

13 Registration Form

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19 EQA samples

20 NAT EQA 2010 Starting Material Italian Reference Preparations calibrated in IU/mL 180 IU/mL 330 IU/mL 100 IU/mL 1:3 dilution 1:3 dilution 1:3 dilution 60 IU/mL 110 IU/mL 33 IU/mL HCV RNA Genotype 1 HIV RNA Subtype F HBV DNA Genotype A

21 NAT EQA 2010 Low Level Target (LLT) Samples* HCV HIV HBV :6 dilution 55 1:6 dilution 12 1:6 dilution 10 1:18 dilution 27 1:18 dilution 8 1:18 dilution * Not taken into consideration for the evaluation of results

22 NAT EQA 2010 Negative samples Starting material: human pooled plasma tested negative for molecular and serological markers

23 NAT EQA 2010 RESULTS HCV samples

24 NAT EQA 2010 HCV RNA 180 IU/mL 369 positive / 370 samples tested 1 failing laboratory: AMP user 99.7 % 60 IU/mL 365 positive / 368 samples tested 99.2 % 3 failing laboratories: two AMP users and one CA/CT user

25 NAT EQA 2010 RESULTS HIV samples

26 NAT EQA 2010 HIV RNA 330 IU/mL 369 positive / 1 failing laboratory: AMP user 370 samples tested 99.7 % 110 IU/mL 369 positive / 370 samples tested 99.7 % 1 failing laboratory: CTS user (post-analytical error)

27 NAT EQA 2010 RESULTS HBV samples

28 NAT EQA 2010 HBV DNA 100 IU/mL 370 positive / 370 samples tested 100 % 33 IU/mL 370 positive / 370 samples tested 100 %

29 NAT EQA 2010 RESULTS Negative samples

30 NAT EQA 2010 Negative samples 732 negative / 740 samples tested 98.9 % 8 failures: two post-analytical errors and six false positive results

31 NAT EQA 2010 List of failures (analytical and pre/post-analytical errors) EQA Phases errors 11* participants 5 false negative results 4 laboratories 6 false positive results 6 laboratories 3 post-analytical errors 1 laboratories * One participant reported one error in each EQA phase

32 NAT EQA 2010 EQAPs: a three-years experience N of non proficient laboratories False negative results False positive results Pre/post-analytical errors

33 NAT EQA 2010 RESULTS Samples with a low level of target (LLT samples)

34 NAT EQA 2010 LLT Samples: n react / n tests (%) HCV CTS TMA AMP IN-H ~ 30 IU/mL 69/74 38/39 3/7 4/6 (93.2 %) (97.4 %) (42.8 %) (66.6 %) ~ 10 IU/mL 48/74 26/39 1/6 1/6 (64.8%) (66.6 %) (16.0 %) (16.0 %) 95%DL ~ 10 ~ 3 ~ 28 (IU/mL)

35 NAT EQA 2010 LLT Samples: n react / n tests (%) HIV CTS TMA AMP IN-H ~ 55 IU/mL 74/74 37/39 3/7 5/5 (100 %) (94.9 %) (42.8 %) (100 %) ~ 27 IU/mL 67/74 22/39 2/7 3/5 (90.5%) (56.5 %) (28.5 %) (60.0 %) 95%DL ~ 49 ~ 20 ~ 78 (IU/mL)

36 NAT EQA 2010 LLT Samples: n react / n tests (%) HBV CTS TMA AMP IN-H ~ 16 IU/mL 74/74 36/39 7/7 6/6 (100 %) (92.3 %) (100 %) (100 %) ~ 8 IU/mL 74/74 31/38 7/7 5/6 (100%) (81.5 %) (100 %) (83.3 %) 95%DL ~ 4 ~ 10 ~ 5 (IU/mL)

37 NAT EQA 2010 How can the failure to detect the LLT samples be explained? 95% detection limit of the assays 95% confidence limit of the Italian Ref. Prep. used pre-analitycal steps used by the participant

38 NAT EQA 2010 CONCLUSIONS Design of this EQAP allowed participants to verify their routine activity making them confident regarding their performance especially when the level of molecular target is low (near or below the detection limit of the NAT assay used)

39 External Quality Assurance Programmes (EQAPs) for blood-borne viruses: the Italian experience 2010 EQAP for HCV RNA, HIV RNA and HBV DNA Inter-laboratory Study for WNV RNA Inter-laboratory Study for HIV-2 RNA

40 Participants 11 Blood Centres

41 NAT Methods Assay (code) 95%DL copies/ml No. panel tested Procleix WNV kit (TMA) ~ 10 6 Cobas TaqScreen WNV kit (CTS) ~ 40 6

42 Panel A WNV RNA Ref Prep copies/ml Dilution factor cp/ml Vial Code 1:3.16 ~ :10 ~ :31.6 ~ 31 5 negative negative

43 Panel A WNV RNA Ref Prep copies/ml Panel B WNV RNA Ref Prep copies/ml Dilution factor cp/ml Vial Code Dilution factor cp/ml Vial Code 1:3.16 ~ :10 ~ :31.6 ~ 31 5 negative negative : : : negative negative

44 RESULTS WNV panels

45 Panel A Panel B ID sample Copies/mL negative negative negative negative lab code Method 11 TMA POS POS POS neg neg POS POS neg neg neg 15 TMA POS POS POS neg neg POS POS POS neg neg 27 TMA POS POS POS neg neg POS POS neg neg neg 72 TMA POS POS POS neg neg POS POS POS neg neg 98 TMA POS POS POS POS neg POS neg neg neg neg 130 TMA POS POS POS neg neg POS POS POS neg neg 20 CTS POS POS POS neg neg POS neg neg neg neg 44 CTS POS POS POS neg neg POS neg POS neg neg 46 CTS POS POS POS neg neg POS POS neg neg neg 51 CTS POS POS neg neg neg neg neg neg neg neg 129 CTS POS POS POS neg neg POS POS neg neg neg 130 CTS POS POS POS neg neg POS neg neg neg neg

46 CONCLUSIONS All participants carried out the inter-laboratory study with favorable results, correctly identifying negative and positive samples. The observed differences between the two NAT assays in terms of the probability of detecting low level WNV RNA samples reflect their 95% Detection Limit as stated by the manufacturers

47 External Quality Assurance Programmes (EQAPs) for blood-borne viruses: the Italian experience 2010 EQAP for HCV RNA, HIV RNA and HBV DNA Inter-laboratory Study for WNV RNA Inter-laboratory Study for HIV-2 RNA

48 1 st WHO International Standard HIV-2 RNA 1000 IU/mL HIV-2 RNA Italian Ref. Preparation Batch ISS 0210 ~ 400 IU/mL

49 Participants 27 Italian Blood Centres NAT Assay TaqScreen MPX system on COBAS S201

50 HIV-2 RNA Panel Composition Dilution factor IU/ml Vial Code ~ ~ ~ ~ 1 3 negative

51 RESULTS / CONCLUSIONS HIV-2 panel

52 IU/ml n positive/n tested (mean Crossing Point and SD) ~ / 27 ( ) ~ / 27 ( ) ~ 4 9 / 27 ( ) ~ 1 1 / 27 Not determined / 27 Not determined

53 Acknowledgment CRiVIb Francesco Marino Karen Cristiano Francesca Luciani Guillermo Bisso Andrea Gaggioli Claudio Mele Daniela Adriani Katia Colombo Cristina Marra Maria Wirz Carlo Pini Simonetta Pupella Giuliano Grazzini Blood Centers