FINAL REPORT HBV Serology External Quality Assessment Scheme Programme codes: HBVC4310, HBVC435 Panel ID: 2008/Oct/28 HBV Serology EQAS Panel ID 2008/Oct/28 Page 1 of 11
The NRL is a: NATA-accredited proficiency testing provider, complying with ILAC-G13:2000 World Health Organization (WHO) Collaborating Centre for Diagnostics and Laboratory Support for HIV and AIDS and Other Blood-borne Infections Report prepared by Wayne Dimech and distributed in the week beginning 2008/Nov/24 National Serology Reference Laboratory, Australia (NRL) 4 th Floor, Healy Building, 41 Victoria Parade, Fitzroy, Victoria 3065 Australia HBV Serology EQAS Panel ID 2008/Oct/28 Page 2 of 11
1 INTRODUCTION The National Serology Reference Laboratory, Australia (NRL) distributes three Hepatitis B Virus (HBV) Serology External Quality Assessment Scheme (EQAS) panels each year to participants that conduct HBV serology testing. The NRL manages its EQAS using the DigitalPT informatics system (DigitalPT) developed and operated by HealthMetrx Canada Inc. (HMX). AcroMetrix Corporation and its subsidiaries (AcroMetrix) provide logistical support for participants enrolled through AcroMetrix. All participants in the NRL s EQAS are provided with a unique and confidential identifier. This identifier is known only by the participant, NRL, HMX and AcroMetrix. This report presents and discusses the results reported for HBV Serology EQAS Panel ID 2008/Oct/28. The aim of this panel was to examine the integrity of result reporting and detect instances of sample carry over. 1.1 Copyright The information and data included in this report are the intellectual property of the NRL, a division of St Vincent s Institute. They may not be reproduced, in whole or in part, for any purpose without the written permission of the NRL; nor may they be used in any type of advertising or sales promotion. 2 METHODS 2.1 Panel composition, preparation and distribution The HBV Serology EQAS panel for programme code HBVC4310 consisted of ten samples. The HBV Serology EQAS panel for programme code HBVC435 consisted of five samples which were the same as the first five samples included in the panel for programme code HBVC4310. The reactivity to Hepatitis B surface antigen (HBsAg), Hepatitis B e antigen (HBeAg), antibody to HBeAg (anti-hbe), antibody to HBsAg (anti-hbs) and total antibody to Hepatitis B core antigen (anti-hbct) of each sample is detailed in Table A1. The samples were obtained from blood donors from Australia, Southeast Asia and the Western Pacific region. All 10 panel samples were pooled samples. Each panel sample was prepared by mixing similar volumes of at least two samples that were from the same country of origin and that had similar antibody and antigen profiles. The samples were tested by the NRL in a range of assays to confirm their reactivity (Table A1). The samples were aliquotted and their homogeneity was confirmed by testing conducted at the NRL. Representative samples were tested by the NRL following shipment to participants to confirm their stability. HBV Serology EQAS Panel ID 2008/Oct/28 was distributed to 144 participants in 36 countries. HBV Serology EQAS Panel ID 2008/Oct/28 Page 3 of 11
2.2 Evaluation of results Reference results were not available for IgM antibody to Hepatitis B core antigen (anti-hbcigm). Consequently, assay interpretations reported for anti-hbcigm assays were not evaluated. 2.2.1 Qualitative evaluation s reported by participants for assay interpretations were compared with the relevant reference results (Table A1). In instances where an assay interpretation was not provided for a sample tested in a rapid or agglutination assay, the reactivity for the sample that was reported by the participant was taken to also be the assay interpretation. An aberrant assay interpretation is one that did not agree with the relevant reference result. 2.2.2 Statistical analyses Tukey s Outlier Filter was used to identify outlying results. An outlying test result is a numerical test result that is found to be statistically different from other test results reported by participants that tested the same sample in the same assay. Occasionally the EQAS coordinator may manually flag a result as outlying, which will cause it to be removed from the statistical analysis. This will only occur when inclusion of the result will erroneously bias the statistical analysis e.g. when the result is from the testing of an incorrect sample. 3 RESULTS s from 132 participants were received for HBV Serology EQAS Panel ID 2008/Oct/28. s were not received by the closing date for the acceptance of results from Participants NRL0108, NRL0118, NRL0259, NRL0323, NRL0354, NRL0355, NRL0485, NRL0501, NRL0558, NRL0561 and NRL0591. s in an appropriate format were not received by the closing date for the acceptance of results from Participant NRL0580. Participants NRL0108 and NRL0354 submitted results after the closing date for the acceptance of results. These results were not evaluated. Participants NRL0039, NRL0214, NRL0531, NRL0559 and NRL0565 used at least one kit that had expired. The NRL does not recommend the use of kits once the expiry date has passed and urges participants to check expiry dates prior to testing. Participant NRL0451 reported an incorrect test run date for Abbott AxSYM Anti-HBe 2.0 MEIA and Abbott AxSYM CORE MEIA assays. All participants that reported outlying test results, aberrant assay interpretations or results that warranted comment are listed in Table 1. Each participant is advised to check Table 1 and to review Section 4 (DISCUSSION), which comments on other results that may need review. HBV Serology EQAS Panel ID 2008/Oct/28 Page 4 of 11
Table 1. Participants that reported outlying test results, aberrant assay interpretations or results that warranted comment Participant ID* NRL0001 Sample ID Reference Assay Outlying Test s** Type of Identified Aberrant Assay Interpretations*** C Negative Abbott PRISM HBsAg ChLIA D,F Reactive Abbott AxSYM AUSAB MEIA NRL0003 D,F Reactive Abbott AxSYM AUSAB MEIA NRL0005 I Reactive Abbott AxSYM CORE MEIA D,F Reactive Abbott AxSYM AUSAB MEIA NRL0007 B Reactive Abbott Murex HBsAg Version 3 EIA NRL0008 B Reactive Abbott AxSYM Anti-HBe 2.0 MEIA NRL0010 D,F Reactive Abbott AxSYM AUSAB MEIA NRL0013 B Reactive Abbott Murex HBsAg Version 3 EIA NRL0026 C Negative Abbott ARCHITECT HBsAg CMIA Abbott ARCHITECT HBsAg Confirmatory V.1/Abbott ARCHITECT HBsAg CMIA Abbott ARCHITECT Anti-HBc CMIA E,G Reactive Abbott ARCHITECT Anti-HBe CMIA C Negative NRL0029 G Negative Abbott AxSYM AUSAB MEIA NRL0046 A,C,D,F.H,I,J Negative Ortho Vitros HBsAg Assay/Ortho Vitros HBsAg Confirmatory NRL0064 I Reactive Abbott AxSYM Anti-HBe 2.0 MEIA NRL0071 A,C,D,F.H,I,J Negative Abbott AxSYM HBsAg Confirmatory Assay/Abbott AxSYM HBsAg Version 2 MEIA NRL0095 H Negative ACON HBsAg One Step Test Strip Miscellaneous Comments HBV Serology EQAS Panel ID 2008/Oct/28 Page 5 of 11
Table 1. Participants that reported outlying test results, aberrant assay interpretations or results that warranted comment (continued) Participant ID* Sample ID Reference Assay HBV Serology EQAS Panel ID 2008/Oct/28 Page 6 of 11 Outlying Test s** Type of Identified Aberrant Assay Interpretations*** NRL0237 F Negative Ortho Vitros HBsAg Confirmatory/Ortho Vitros HBsAg Assay NRL0255 A, D, F Negative Bio-Rad MONOLISA HBsAg ULTRA EIA B,E,G Reactive NRL0275 A,J Negative Abbott PRISM HBsAg ChLIA NRL0282 A,C,D,H,I,J Negative Ortho Vitros HBsAg Assay NRL0287 A,C Negative Abbott AxSYM HBsAg Version 2 MEIA NRL0304 G Reactive J Negative NRL0312 H Negative Chemtron Biotech Chemtrue HBsAg Serum/Plasma One-Step Test Strip J. Mitra Hepalisa HBsAg J. Mitra Hepacard HBsAg NRL0325 A Negative Abbott PRISM HBsAg ChLIA NRL0343 A-J Abbott PRISM HBsAg ChLIA NRL0356 B,E,G Negative D,I Reactive NRL0399 H Negative Ortho Vitros Anti-HBs Assay Innovacon Foresight HBsAg EIA Test Kit Atlas Link HBsAg ELISA NRL0417 H Negative Abbott ARCHITECT Anti-HBc CMIA NRL0529 A Negative NRL0533 A,C,D Negative B,E Reactive VECTOR-BEST VectoHep B-HBs-antigen (Complects 1, 3, 5, 7) EIA VECTOR-BEST VectoHep B-HBs-antigen (Complects 1, 3, 5, 7) EIA Miscellaneo us Comments
Table 1. Participants that reported outlying test results, aberrant assay interpretations or results that warranted comment (continued) Participant ID* Sample ID Reference Assay Outlying Test s** NRL0560 A Negative Abbott AxSYM HBsAg Version 2 MEIA Type of Identified Aberrant Assay Interpretations*** NRL0580 A-J NRL0588 F Reactive Abbott PRISM HBcore ChLIA H Negative NRL0596 E Reactive Abbott ARCHITECT Anti-HBe CMIA NRL0600 H Negative MBU HBs-antigen-DS ELISA NRL0604 H Negative NRL0606 F,I H Reactive Negative VECTOR-BEST VectoHep B-HBs-antigen (Complects 1, 3, 5, 7) EIA VEDA. LAB HBcAb-CHECK-1 VEDA. LAB HBeAb-CHECK-1 D,F Reactive VEDA. LAB HBsAb-CHECK-1 H Negative VECTOR-BEST VectoHep B-HBs-antigen (Complects 1, 3, 5, 7) EIA NRL0607 F Negative DiaSorin LIAISON HBsAg CLIA Miscellaneo us Comments Note: * Identification. ** An outlying test result is a numerical test result that is found to be statistically different from the other test results reported by participants that tested the same sample in the same assay. *** An aberrant assay interpretation is one that did not agree with the relevant reference result. HBV Serology EQAS Panel ID 2008/Oct/28 Page 7 of 11
4 DISCUSSION Overall, twenty-one participants each reported at least one assay interpretation that was considered to be aberrant. Of these: Fourteen participants each reported at least one aberrant HBsAg assay interpretation; Three participants each reported at least one aberrant anti-hbe assay interpretation; Seven participants each reported at least one aberrant anti-hbs assay interpretation Three participants each reported at least one aberrant anti-hbct assay interpretation. s for sample F were not evaluated for participants testing in the Abbott AxSYM HBsAg Version 2 MEIA or the Ortho Vitros HBsAg Assay. Participants testing in the Abbott AxSYM HBsAg Version 2 MEIA reported HBsAg S/Co values that ranged from 0.72 to 1.48, while participants testing in the Ortho Vitros HBsAg Assay reported HBsAg S/Co values that ranged from 0.63 to 2.47 for sample F. Of the 15 participants reporting HBsAg assay interpretations for the Abbott AxSYM HBsAg Version 2 MEIA, four reported the result as reactive and one as inconclusive. One of these five participants correctly reported a negative HBsAg neutralisation result for this sample. The other four participants failed to report neutralisation test results for sample F. Only one of five participants testing in the Ortho Vitros HBsAg Assay reported a reactive HBsAg assay interpretation for sample F. This participant correctly reported a negative HBsAg neutralisation test result. Similarly, results for sample H were not evaluated for participants testing in the Abbott PRISM HBsAg ChLIA. Participants testing sample H in the assay reported HBsAg S/Co values that ranged from 0.33 to 1.08. One of the 13 participants testing sample H in the Abbott PRISM HBsAg ChLIA reported a reactive HBsAg assay interpretation but also reported a negative HBsAg neutralisation test result. Another participant, testing in the same assay reported an inconclusive HBsAg assay interpretation but failed to report a HBsAg neutralisation test result. A number of participants, using a range of HBsAg assays, reported aberrant HBsAg assay interpretations for sample H (Table 1). None performed neutralisation testing and would have reported a falsely reactive HBsAg test result if sample H had been a clinical sample. It is recommended that HBsAg neutralisation be performed on all HBsAg reactive samples and results of testing for other hepatitis B virus markers be reviewed to distinguish between true and false reactivity. Participants NRL0001, NRL0003, NRL0005 and NRL0010, testing in the Abbott AxSYM AUSAB MEIA, reported anti-hbs negative interpretations for samples D and F, despite reporting test results ranging from 35.0 to 81.5 miu/ml. All four participants used internal policies as the guide for anti-hbs interpretation, which defined the cut-off for anti-hbs as 100 miu/ml. Participant NRL0029 testing in the Abbott AxSYM AUSAB MEIA reported a reactive assay interpretation for sample G. The result reported for this sample was 1.1 miu/ml. The manufacturer s instructions states Samples whose concentrations are greater than or equal to 10.0 miu/ml are reactive. Participant NRL0001 testing in the Abbott PRISM HBsAg ChLIA, reported an aberrant assay interpretation for HBsAg negative sample C, with a S/Co value of 0.49. Participant NRL0008 testing in the Abbott AxSYM Anti-HBe 2.0 MEIA reported an aberrant negative assay interpretation for anti-hbe reactive sample B, despite the S/Co result being 0.03. Both aberrant results appear to be due to data entry errors. Participants are encouraged to cross check results using the Data Confirmation Reports available on DigitalPT prior to the closing date of each test event. Participant NRL0026, testing in the Abbott ARCHITECT HBsAg CMIA, reported an aberrant assay interpretation for HBsAg negative sample C. The sample was tested in triplicate with results HBV Serology EQAS Panel ID 2008/Oct/28 Page 8 of 11
ranging from 0.2 to 0.8 IU/mL (cut-off for this assay is 0.05 IU/mL). The HBsAg neutralisation assay result for sample C was reported as positive by this participant. None of the other 17 participants using the Abbott ARCHITECT HBsAg CMIA reported a reactive assay interpretation for this sample. Participant NRL0026 also tested the sample for anti-hbct in the Abbott ARCHITECT Anti-HBc CMIA and anti-hbeab in the Abbott ARCHITECT Anti-HBe CMIA. Aberrant reactive assay interpretations were reported for both these tests. The preceding sample B was strongly reactive for HBsAg, HBcT and HBeAb. The possibility of carry-over should be excluded. However, no aberrant test results were reported by this participant for sample H, which also followed the strongly reactive Sample G. Participants NRL0046 and NRL0071, tested HBsAg negative samples A, C, D, F, H, I and J in HBsAg confirmatory assays, despite reporting negative HBsAg screening assay interpretations. Samples screened negative in HBsAg assays should not be tested in confirmatory assays. Participant NRL0237 reported results for all ten panel samples as being tested in the Ortho Vitros HBsAg Confirmatory/Ortho Vitros HBsAg Assay. However, the values reported by the participant appear to be HBsAg S/Co values rather than HBsAg percentage neutralisation. It appears that the participant selected the wrong assay when entering the results. Participant NRL0255 testing in the Bio-Rad MONOLISA HBsAg ULTRA EIA reported aberrant assay interpretations for samples A, B, D, E, F and G. None of the other 16 participant using this assay reported aberrant assay interpretations. The NRL is investigating whether the wrong panel was tested. Participant NRL0275, testing in the Abbott PRISM HBsAg ChLIA, reported aberrant assay interpretations for the HBsAg negative samples A and J. The participant tested the samples in three test runs and reported S/Co values of 0.78, 1.01 and 0.73 for sample A and 0.90, 0.89 and 0.45 for sample J. The participant assigned a reactive HBsAg interpretation to sample A and an inconclusive HBsAg interpretation to sample J. The manufacturer s instructions state that samples with S/Co less than the cut-off should be interpreted as HBsAg negative. The participant did not perform HBsAg confirmatory testing. Participant NRL0282, testing in the Ortho Vitros HBsAg Assay, reported HBsAg S/Co values that were identified as outlying for six of the seven HBsAg negative samples. The HBsAg S/Co values reported by the participant for samples A, C, D, H, I and J were consistently higher than the peer group mean indicating a possible systematic problem with the instrument. The NRL recommends the participant check instrument maintenance and calibration records. Several participants NRL0290, NRL0343, and NRL0588 testing in the Abbott PRISM HBsAg ChLIA reported aspiration errors for one or more samples. Participant NRL0343 could not report any HBsAg test results using the Abbott PRISM HBsAg ChLIA but did report Abbott AxSYM HBsAg Version 2 MEIA results that were concordant with the reference results for all samples whose results were evaluated. Participant NRL0356 testing in the Ortho Vitros Anti-HBs Assay, reported aberrant assay interpretations for five of the ten samples. The results reported by the participant wee consistent with HBsAg results rather than anti-hbs results, indicating the participant may have selected the wrong assay at data entry. s reported by Participant NRL0580 were not received in the specified format. Participants are reminded that results should be submitted via DigitalPT or on the approved result forms. s not reported in the specified format will not be accepted. Participant NRL0606, reported aberrant negative assay interpretations on samples F and I using both VEDA. LAB HBeAb-CHECK-1 and VEDA. LAB HBcAb-CHECK-1 and aberrant reactive assay interpretations on sample H using VEDA. LAB HBeAb-CHECK-1 and VECTOR-BEST VectoHep B- HBV Serology EQAS Panel ID 2008/Oct/28 Page 9 of 11
HBs-antigen (Complects 1, 3, 5, 7) EIA. In addition, aberrant negative assay interpretations were reported for samples D and F when tested in the VEDA. LAB HBsAb-CHECK-1. It is recommended that participant NRL0606 review its processes. Acknowledgements The NRL thanks our colleagues who supply samples for the EQAS. We thank all our participants for their cooperation. Comments or queries about this report, or about any problems you might experience with HBV serology testing, will be welcomed by Kathy Smeh on +61 3 9418 1110 or via email on kathy@nrl.gov.au. HBV Serology EQAS Panel ID 2008/Oct/28 Page 10 of 11
5 APPENDIX Table A1. Characterisation of the samples that comprised HBV Serology EQAS Panel ID 2008/Oct/28 Sample Identification by Programme HBVC4310 HBVC435 Sample Origin Sample Type A A Australian blood donor Pooled B B Australian blood donor Pooled C C Australian blood donor Pooled D D Southeast Asian blood donor Pooled E E Australian blood donor Pooled F G Southeast Asian blood donor Western Pacific blood donor Pooled Pooled H Australian blood donor Pooled I J Not applicable Southeast Asian blood donor Australian blood donor Pooled Pooled Abbott Murex HBsAg Version 3 MEIA Test (S/Co ) AI % Reduction Abbott Murex HBsAg Confirmatory Version 3 AI biomérieux VIDAS HBeAg/Anti-HBe ELFA Test (IV*) HBeAg AI Test (IV ) Anti-HBe AI biomérieux VIDAS anti-hbs Total Quick ELFA Test (miu/mlˆ) AI biomérieux VIDAS Anti-HBc Total II ELFA Test (IV ) 0.69, 0.70 Negative Not tested Not applicable 0.00 Negative 0.91 Negative <5 Negative 1.90 Negative 42.75 Reactive 99.0 Positive 0.00 Negative 0.00 Reactive <5 Negative 0.00 Reactive 0.56, 0.58 Negative Not tested Not applicable 0.00 Negative 0.96 Negative <5 Negative 2.11 Negative 0.57, 0.56 Negative Not tested Not applicable 0.00 Negative 0.84 Negative 92 Reactive 1.97 Negative 34.37 Reactive 99.0 Positive 0.00 Negative 0.00 Reactive <5 Negative 0.00 Reactive 0.86, 0.61 Negative Not tested Not applicable 0.00 Negative 0.02 Reactive 127 Reactive 0.04 Reactive 42.73 Reactive 99.0 Positive 0.00 Negative 0.00 Reactive <5 Negative 0.00 Reactive 0.59, 0.70 Negative Not tested Not applicable 0.00 Negative 0.86 Negative <5 Negative 2.04 Negative 0.77, 0.64 Negative Not tested Not applicable 0.00 Negative 0.01 Reactive >500 Reactive 0.11 Reactive 0.70, 0.78 Negative Not tested Not applicable 0.00 Negative 0.82 Negative <5 Negative 1.90 Negative AI Note: HBsAg Hepatitis B surface antigen. Sample divided by cut-off, where 1 is reactive. HBeAg Hepatitis B e antigen. * Index value, where 0.1 is reactive. Anti-HBe Antibody to Hepatitis B e antigen. Index value, where < 0.4 is reactive. Anti-HBs Antibody to Hepatitis B surface antigen. ˆ miu/ml (milli-international Units per millilitre), where > 12 is reactive. Anti-HBc Total Total antibody to Hepatitis B core antigen. Index value, where < 1 is reactive. AI Assay interpretation. HBV Serology EQAS Panel ID 2008/Oct/28 Page 11 of 11