QUALITY CONTROL for MOLECULAR DIAGNOSTICS The Altum Building, Todd Campus, West of Scotland Science Park, Glasgow, G20 0XA Scotland Tel: +44 (0) 141 945 6474 Fax: +44 (0) 141 945 5795 www.qcmd.org info@qcmd.org BEIPH Final Report QCMD 2010 Hepatitis B Virus DNA (HBVDNA10A) EQA Programme William G MacKay on behalf of QCMD and its Scientific Council July 2010 Not to be reproduced or quoted without permission of QCMD. Any queries about this report should be addressed to the QCMD Neutral Office. The QCMD programme is organised in collaboration with the European Society for Clinical Virology and the European Society for Clinical Microbiology & Infectious Diseases. Registered in Scotland Reg No: SC219746 Registered Office: 7 Castle Street, Edinburgh EH2 3AH
1. Programme aims The primary aim of this External Quality Assessment Programme was to assess the proficiency of laboratories in the detection and quantitation of Hepatitis B virus (HBV) DNA. 2. Programme details Table 1: Programme details HBVDNA10A Date of panel distribution 12/04/2010 Number of respondents 20 (87%) Number of participants 23 Number of datasets submitted 20 Number of countries 1 Number of qualitative datasets submitted 1 (5%) Number of qualitative and quantitative datasets submitted 19 (95%) Three participants did not return results. One of these withdrew officially, citing 'assay not offered' as the reason. 3. Panel composition This EQA panel for the detection of HBV consisted of eight samples containing various concentrations of HBV and one sample negative for HBV. These samples were obtained through a South African blood bank as a result of a regular screening programme. Negative plasma was obtained from the Groningen blood bank (The Netherlands) and was also used to dilute the samples. The QCMD EQA panels contain a range of samples, designed to look at different aspects of assay performance. Panel members are designated core proficiency samples on the basis of scientific information, clinical relevance and clinical experience (published literature and professional clinical guidelines) and, where available and appropriate, established target performance limits taken from previous QCMD EQA distributions. Laboratories are expected to correctly analyse and report the core proficiency samples in order to show acceptable proficiency. Table 2: Panel composition * conc. content matrix Copies/ml status type HBV10-03 HBV Type A Plasma 5,012 Frequently detected Core HBV10-07 HBV Type A Plasma 505 Frequently detected Core HBV10-05 HBV Type A Plasma 472 Frequently detected Core HBV10-01 HBV Type A Plasma 61 Detected HBV10-08 HBV Type D Plasma 19,055 Frequently detected Core HBV10-06 HBV Type D Plasma 1,950 Frequently detected Core HBV10-02 HBV Type D Plasma 195 Detected HBV10-04 HBV Neg Plasma Plasma Negative Core Key to Table 2 : QCMD panel sample codes for the samples distributed to participants. content: viral content of the panel samples. matrix: material used as a matrix in preparation of the panel samples. conc.: consensus values calculated from all of the data returned by participants in the full EQA programme, once outliers had been removed. The values are not technology specific and should not be used by participants for method comparison or as a target for individual laboratory assessment. status: the sample status assigned to each panel sample consisting of 'Frequently detected', 'Detected', 'Infrequently detected' or 'Negative'. Please see Appendix A for more information. type: panel samples classified as core proficiency samples. Panel samples HBV10-07 and -05 were duplicate samples. * Human plasma negative for HBV DNA. PAGE 2 of 7
4. Programme results 4a. Qualitative analysis of the EQA data The number (percentage) of correct qualitative results are presented in Table 3. Qualitative data were returned by participants as 'positive', 'negative' or 'not determined'. Not determined results were counted as incorrect for all panel samples (positive or negative). QCMD organises datasets according to commercial and in-house technology groups, which are Conventional PCR, Real time PCR, NASBA, SDA, TMA and bdna. Where datasets were reported as other for a technology or kit method this was reviewed by the QCMD Neutral Office and assigned to an appropriate group where possible. Table 3: Number of correct qualitative results per panel member and technology type conc. Total Real time content Copies/ml datasets Commercial c In-house d n=20 n=18 n=2 n % n % n % HBV10-03 HBV Type A 5,012 20 100.0 18 100.0 2 100.0 HBV10-07 HBV Type A 505 20 100.0 18 100.0 2 100.0 HBV10-05 HBV Type A 472 20 100.0 18 100.0 2 100.0 HBV10-01 HBV Type A 61 15 75.0 14 77.8 1 50.0 HBV10-08 HBV Type D 19,055 20 100.0 18 100.0 2 100.0 HBV10-06 HBV Type D 1,950 20 100.0 18 100.0 2 100.0 HBV10-02 HBV Type D 195 19 95.0 18 100.0 1 50.0 HBV10-04 HBV Neg Plasma 20 100.0 18 100.0 2 100.0 Key to Table 3 : QCMD panel sample codes for the samples distributed to participants. content: viral content of the panel samples. conc.: consensus values calculated from all of the data returned by participants in the full EQA programme, once outliers had been removed. The values are not technology specific and should not be used by participants for method comparison or as a target for individual laboratory assessment. Total datasets: number and percentage of datasets reporting the correct qualitative result for each panel sample. A breakdown of the results for all datasets is also provided based on technology type. c: Abbott RealTime HBV test (n=8), QIAGEN artus HBV PCR Kit (RG) (n=3), Roche COBAS AmpliPrep/COBAS TaqMan HBV Test (n=3), Roche COBAS TaqMan HBV Test (n=2), Roche COBAS TaqMan HBV Test For Use With The High Pure System (n=2). d: Details not presented. PAGE 3 of 7
4b. Qualitative performance scores Table 4: Qualitative performance scores per technology type Key to Table 4 : QCMD panel sample codes for the samples distributed to participants. status: the sample status assigned to each panel sample. Please see Appendix A for more information. Total. All technologies: number of datasets awarded each score (0 to 3). A breakdown of the results for all datasets is also provided based on technology type. These data are presented graphically in Figure 1. c: Abbott RealTime HBV test (n=8), QIAGEN artus HBV PCR Kit (RG) (n=3), Roche COBAS AmpliPrep/COBAS TaqMan HBV Test (n=3), Roche COBAS TaqMan HBV Test (n=2), Roche COBAS TaqMan HBV Test For Use With The High Pure System (n=2). d: Details not presented. Status Total All technologies n=20 Real time Commercial c In-house d n=18 n=2 0 1 2 3 0 1 2 3 0 1 2 3 HBV10-03 Frequently detected 20 0 0 0 18 0 0 0 2 0 0 0 HBV10-07 Frequently detected 20 0 0 0 18 0 0 0 2 0 0 0 HBV10-05 Frequently detected 20 0 0 0 18 0 0 0 2 0 0 0 HBV10-01 Detected 15 0 5 0 14 0 4 0 1 0 1 0 HBV10-08 Frequently detected 20 0 0 0 18 0 0 0 2 0 0 0 HBV10-06 Frequently detected 20 0 0 0 18 0 0 0 2 0 0 0 HBV10-02 Detected 19 0 1 0 18 0 0 0 1 0 1 0 HBV10-04 Negative 20 0 0 0 18 0 0 0 2 0 0 0 Figure 1: Percentage of qualitative performance scores per technology type 100 90 80 70 60 3 % 50 2 1 40 0 30 20 10 0 c d c d c d c d c d c d c d c d HBV10-03 HBV10-07 HBV10-05 HBV10-01 HBV10-08 HBV10-06 HBV10-02 HBV10-04 Technology group per panel sample c: Real time commercial PCR, d: Real time in-house PCR. PAGE 4 of 7
4c. Analysis of the EQA data by assay target gene Participants were asked to specify the target gene of their assay when submitting their results. Out of the 20 datasets received by QCMD 12 (60%) contained information on the target gene. These data are summarised in Table 5. Table 5: Analysis of the qualitative data by assay target gene conc. content Copies/ml Total datasets Gene C Gene S n=20 n=5 n=5 Other* n % n % n % n % n % HBV10-03 HBV Type A 5,012 20 100.0 5 100.0 5 100.0 2 100.0 8 100.0 HBV10-07 HBV Type A 505 20 100.0 5 100.0 5 100.0 2 100.0 8 100.0 HBV10-05 HBV Type A 473 20 100.0 5 100.0 5 100.0 2 100.0 8 100.0 HBV10-01 HBV Type A 61 15 75.0 4 80.0 4 80.0 2 100.0 5 62.5 HBV10-08 HBV Type D 19,055 20 100.0 5 100.0 5 100.0 2 100.0 8 100.0 HBV10-06 HBV Type D 1,950 20 100.0 5 100.0 5 100.0 2 100.0 8 100.0 HBV10-02 HBV Type D 195 19 95.0 4 80.0 5 100.0 2 100.0 8 100.0 HBV10-04 HBV Neg Plasma 20 100.0 5 100.0 5 100.0 2 100.0 8 100.0 Key to Table 5 : QCMD panel sample codes for the samples distributed to participants. content: viral content of the panel samples. conc.: consensus values calculated from all of the data returned by participants in the full EQA programme, once outliers had been removed. The values are not technology specific and should not be used by participants for method comparison or as a target for individual laboratory assessment. Total datasets: number and percentage of datasets reporting the correct qualitative result for each panel sample. A breakdown of the results for all datasets is also provided based on target gene. n=2 Not reported n=8 * 5'UTR (n=1), X gene & pol (n=1). PAGE 5 of 7
4d. Quantitative analysis of the EQA data Table 6: Consensus concentration values for the BEIPH participants Consensus Log 10 virus concentration All participants BEIPH n=200 n=19 Mean SD Mean SD HBV10-03 3.700 0.352 3.525 0.506 HBV10-07 2.703 0.336 2.693 0.279 HBV10-05 2.675 0.348 2.582 0.328 HBV10-01 1.784 0.568 1.619 0.538 HBV10-08 4.280 0.383 4.051 0.523 HBV10-06 3.290 0.346 3.051 0.423 HBV10-02 2.291 0.355 2.173 0.218 Key to Table 6 : QCMD panel sample codes for the samples distributed to participants. Consensus Log10 virus concentration: the mean quantitative value and standard deviation value for each panel sample expressed in log10 units and calculated once outlying values had been removed. SD refers to the Standard Deviation. All participants: values calculated from results submitted by all participants in the QCMD 2010 Hepatitis B Virus DNA EQA Programme. BEIPH: values calculated from results submitted by BEIPH participants only. Panel samples HBV10-07 and -05 were duplicate samples. Technology consensus concentration Table 7: Technology consensus concentration values for the BEIPH participants Real time Commercial Tech Consensus Log 10 virus concentration All participants BEIPH n=164 17 (c) Mean SD Mean SD HBV10-03 3.697 0.338 3.454 0.506 HBV10-07 2.713 0.324 2.633 0.279 HBV10-05 2.683 0.327 2.538 0.328 HBV10-01 1.752 0.590 1.619 0.538 HBV10-08 4.306 0.385 3.987 0.523 HBV10-06 3.317 0.339 3.001 0.423 HBV10-02 2.310 0.338 2.173 0.218 Key to Table 7 : QCMD panel sample codes for the samples distributed to participants. Tech. Consensus Log10 virus concentration: the mean quantitative value and standard deviation value for each panel sample expressed in log10 units and calculated once outlying values had been removed. SD refers to the Standard Deviation. All participants: values calculated from results submitted by all participants in the QCMD 2010 Hepatitis B Virus DNA EQA Programme. BEIPH: values calculated from results submitted by BEIPH participants only. c: Abbott RealTime HBV test (n=8), QIAGEN artus HBV PCR Kit (RG) (n=3), Roche COBAS AmpliPrep/COBAS TaqMan HBV Test (n=3), Roche COBAS TaqMan HBV Test (n=1), Roche COBAS TaqMan HBV Test For Use With The High Pure System (n=2). Acknowledgements Data analysis and report generation were performed by Stuart West and William MacKay of the QCMD Neutral Office. QCMD 2010. The QCMD EQA programme samples, associated reports and data generated during this programme are intended for External Quality Assessment (EQA) and Proficiency Testing (PT) purposes only. QCMD operates according to a strict Code of Practice which is in line with ISO/IEC 17043 and associated standards. Data reported in QCMD programmes is representative of a laboratory s standard diagnostic testing protocols irrespective of the technology they use. The data provided in the reports are based on technical information provided by the individual laboratories as part of the assessment process, as such it does not constitute a formal technology method comparison. All text and images produced by QCMD are the property of QCMD unless otherwise stated. The reproduction and use of these materials is not permitted without the express written consent of QCMD. The use of the information provided in QCMD reports for commercial purposes is strictly prohibited. PAGE 6 of 7
Appendix A Assigning the sample status status is assigned by peer-group consensus, based on the qualitative results returned by all participants in the full EQA programme. It is not a measure of the 'strength' of a positive sample nor is it technology-dependent, and is used solely for the scoring of the EQA data. The rationale for the sample status is: Frequently detected: More than 95% of datasets recorded the correct positive result. Detected: Between 65% and 95% of datasets recorded the correct positive result. Infrequently detected: Less than 65% of datasets recorded the correct positive result. Negative: A panel sample that does not contain the target and produces an unequivocal negative result. Scoring system for qualitative EQA data The scores awarded for qualitative EQA data were based on the sample status. The scoring system is represented in the following table, where 0 is 'highly satisfactory' and 3 is 'highly unsatisfactory'. Colour has been included as an extra visual aid. Scoring system based on the assigned sample status status Participant's result Negative Not determined Positive Frequently detected 3 3 0 Detected 2 2 0 Infrequently detected 1 1 0 Negative 0 3 3 PAGE 7 of 7