FINAL REPORT HIV Serology External Quality Assessment Scheme Programme codes: HIVC4310, HIVC435 Panel ID: 2008/Mar/11
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The NRL is a: • NATA-accredited proficiency testing provider, complying with ILAC-G13:2000 • WHO Collaborating Centre on HIV/AIDS
Report prepared by Kathy Smeh and distributed in the week beginning 20008/Mar/31
National Serology Reference Laboratory, Australia (NRL) th
4 Floor, Healy Building, 41 Victoria Parade, Fitzroy, Victoria 3065 Australia
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1 INTRODUCTION The National Serology Reference Laboratory, Australia (NRL) distributes three Human Immunodeficiency Virus (HIV) Serology External Quality Assessment Scheme (EQAS) panels each year to participants that conduct HIV 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 participants, NRL, HMX and AcroMetrix. This report presents and discusses the results reported for HIV Serology EQAS Panel ID 2008/Mar/11. The aims of this panel were to: • investigate instances of sample carryover, particularly on automated analysers; • examine testing strategies used by participants; • examine the integrity of result reporting.
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 HIV Serology EQAS panel for programme code HIVC4310 consisted of ten plasma samples. The HIV Serology EQAS panel for programme code HIVC435 consisted of five samples which were the same as the first five samples included in the panel for programme code HIVC4310. The HIV reactivity of each sample is detailed in Tables A1 and A2. The samples were obtained from blood donors from Africa, Australia and Southeast Asia. Each pooled sample was prepared by mixing equal 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 A2). 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. HIV Serology EQAS Panel ID 2008/Mar/11 was distributed to 206 participants in 38 countries.
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2.2 Evaluation of results 2.2.1 Qualitative evaluation Results reported by participants for assay interpretations and status were compared with the relevant reference results (Tables A1 and A2). 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. An aberrant status is one that did not agree with the relevant reference result or consensus status (if applicable). 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 Results from 187 participants were received for HIV Serology EQAS Panel ID 2008/Mar/11. Results were not received by the closing date for the acceptance of results from Participants NRL0014, NRL0016, NRL0100, NRL0146, NRL0183, NRL0276, NRL0346, NRL0347, NRL0348, NRL0351, NRL0352, NRL0353, NRL0354, NRL0355, NRL0356, NRL0439, NRL0485, NRL0501 and NRL0525. Participant NRL0276 contacted the NRL after panels had been distributed and indicated that it was not able to test the panel because it had temporarily ceased testing for HIV. Participants NRL0076, NRL0091, NRL0092, NRL0095, NRL0101, NRL0117, NRL0128 and NRL0321 used kits 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. 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.
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Table 1. Participants that reported outlying test results, aberrant assay interpretations or status, or results that warranted comment Laboratory ID*
Sample ID
Reference Result
Assay
Type of Result Identified Outlying Test Results**
Aberrant Assay Interpretations***
Aberrant Status****
NRL0010
E
Negative
Abbott PRISM HIV O Plus ChLIA
●
NRL0012
F
Negative
Abbott ARCHITECT HIV Ag/Ab Combo CMIA
●
NRL0018
B, C, D, H, I
Positive
♦
NRL0026
B, C, D, H, I
Positive
♦
NRL0029
B, C, D, H, I
Positive
NRL0030
J
Negative
NRL0049
B, C, D, H, I
Positive
NRL0060
J
NRL0062
Negative
■ Abbott ARCHITECT HIV Ag/Ab Combo CMIA
● ♦
Abbott IMx HIV-1/HIV-2 III Plus MEIA
♦
Negative
♦
B, C, D, H, I
Positive
♦
NRL0066
B, C, D, H, I
Positive
♦
NRL0075
A, E, F, G, J
Negative
NRL0084
A
Negative
NRL0090
A, E, F, G, J
Negative
J. Mitra HIV WB
NRL0099
G
Negative
Bio-Rad Genscreen Plus HIV Ag-Ab EIA
B, C, D, H, I
p24 Ag Negative
bioMerieux VIDAS HIV DUO Ultra ELFA
J
Negative
NRL0113
A, E, F, G, J
Negative
Bio-Rad NEW LAV BLOT 1 WB
NRL0115
A
Negative
Abbott ARCHITECT HIV Ag/Ab Combo CMIA
●
F
Negative
Abbott ARCHITECT HIV Ag/Ab Combo CMIA
●
NRL0109
NRL0119
MP Diagnostics HIV BLOT 2.2 WB
■ ♦ ■ ●
♦ ♦ ♦ ■
B, C, D, H, I
Positive
NRL0126
E, J
Negative
Abbott ARCHITECT HIV Ag/Ab Combo CMIA
●
NRL0138
A
Negative
Abbott PRISM HIV O Plus ChLIA
●
♦
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Table 1. Participants that reported outlying test results, aberrant assay interpretations or status, or results that warranted comment (continued) Laboratory ID*
Sample ID
Reference Result
Assay
Type of Result Identified Outlying Test Results**
Aberrant Assay Interpretations***
Aberrant Status****
NRL0144
B, C, D, H, I
Positive
♦
NRL0150
B, C, D, H, I
Positive
♦
NRL0161
G
Negative
Abbott AxSYM HIV 1/2 gO MEIA
●
NRL0162
J
Negative
Bayer ADVIA Centaur HIV1/O/2 Enhanced (EHIV) Assay
NRL0174
G
Negative
Abbott AxSYM HIV Ag/Ab Combo MEIA
● ●
NRL0180
B, C, D, H, I
Positive
NRL0235
J
Negative
♦ Abbott AxSYM HIV 1/2 gO MEIA
●
♦
NRL0248
J
Negative
Abbott Murex HIV-1.2.O EIA
●
NRL0249
B, D
Reactive
bioMerieux Vironostika HIV Uni-Form II Plus O ELISA
●
NRL0250
B, C, D, H, I
Positive
NRL0282
G
Negative
G
♦ Ortho Vitros Anti-HIV 1+2 Assay
●
Abbott Determine HIV-1/2 Immunoassay
♦
Fujirebio SFD HIV-1/2
♦
Negative
NRL0292 I
Reactive
♦ Abbott Determine HIV-1/2 Immunoassay
♦
Fujirebio SFD HIV-1/2
♦
Positive
♦
NRL0305
E, J
Negative
Abbott ARCHITECT HIV Ag/Ab Combo CMIA
NRL0373
A, E, F, G, J
Negative
Bio-Rad NEW LAV BLOT 1 WB
NRL0421
B, C, D, H, I
Positive
NRL0427
A, E, F, G, J
Negative
MP Diagnostics HIV BLOT 2.2 WB
B, C, D, H, I
Reactive
Bio-Rad Genscreen HIV-1/2 Version 2 EIA
A, E, F, G, J
Negative
Innogenetics INNO-LIA HIV I/II Score
NRL0441
♦
Negative
Negative
● ■ ♦
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Table 1. Participants that reported outlying test results, aberrant assay interpretations or status, or results that warranted comment (continued) Laboratory ID*
Sample ID
Reference Result
Assay
Type of Result Identified Outlying Test Results**
NRL0460
Aberrant Assay Interpretations***
Aberrant Status****
A, E
Negative
Chiron RIBA HIV-1/HIV-2 SIA
A
Negative
bioMerieux Vironostika HIV Uni-Form II Plus O ELISA
B, D
Reactive
bioMerieux Vironostika HIV Uni-Form II Plus O ELISA
C, H, I
Reactive
bioMerieux Vironostika HIV Uni-Form II Plus O ELISA
J
Negative
bioMerieux Vironostika HIV Uni-Form II Plus O ELISA
B, C, D, H, I
Reactive
Li Zhu Diagnostic Kit for Antibody to HIV (Sandwich ELISA)
NRL0487
B, C, D, H, I
Positive
♦
NRL0500
B, C, D, H, I
Positive
♦
NRL0481
Note:
Miscellaneous Comments ■
♦ ●
■ ■
● ■
* **
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. **** An aberrant status is one that did not agree with the relevant reference result or consensus status (if applicable).
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4 DISCUSSION Six participants each reported at least one assay interpretation that was considered to be aberrant. Nineteen participants each reported at least one status that was considered to be aberrant. Participants NRL0018, NRL0026, NRL0049, NRL0062, NRL0066, NRL0119, NRL0144, NRL0150, NRL0180, NRL0250, NRL0441, NRL0487 and NRL0500 each assigned an aberrant HIV serology status to the HIV positive samples B, C, D, H and I. The participants assigned an inconclusive/indeterminate status based on testing all samples in a single assay (except for Participant NRL0500 that used two assays). In addition, all participants that assigned a serology status to the HIV positive samples B, C, D, H and I after testing in only one assay should note the following: When performing serological testing, participants use a variety of testing strategies that may involve testing with a combination of screening, supplemental and/or confirmatory assays. A final serology status (final interpretation) of positive, negative or indeterminate should only be assigned to a sample after consideration of all test results (screening, supplemental and confirmatory). Participants that test samples using only one assay should refer any reactive samples for further testing in order to distinguish true from false reactivity. When reporting a final interpretation in DigitalPT, a participant that would not report a status/final interpretation but would refer a sample for further testing should submit their results as: Serology Status Refer for Further Testing Select problem code
Do not select any option Do not select any option Would not report a status based on these results
Otherwise, participants that would report a status/final interpretation should assign an appropriate status and select “No” for “Refer for Further Testing”. Participant NRL0029 tested all of the panel samples once in the Abbott AxSYM HIV Ag/Ab Combo MEIA. The participant obtained reactive results for the HIV positive samples B, C, D, H and I and indicated that these samples would be referred for further testing. The participant also indicated that it would assign a positive status to each of these samples but that it also ‘Would not report a status based on’ the results obtained. Participant NRL0060 tested all of the panel samples once in the Abbott IMx HIV-1/HIV-2 III Plus MEIA. The participant obtained a reactive test result that was over the cut-off for the assay (S:Co 6.34) for the HIV negative sample J. The test result obtained for sample J may be due to sample carry over because the two samples preceding sample J in the panel were HIV positive samples. The result does not appear to be due to testing a positive sample twice because the S/Co values obtained by the participant for the HIV positive panel samples were substantially higher than the result obtained for sample J. No other participant that tested the sample in the same assay reported a reactive assay interpretation for the sample. Participant NRL0060 assigned a status to all of the panel samples even though the participant indicated that it would refer all of the samples for further testing. Participant NRL0084 assigned a positive HIV status to the HIV negative sample A. Following the release of the preliminary report, Participant NRL0084 contacted the NRL and indicated that difficulty had been experienced with data entry into DigitalPT, especially for sample A. Participant NRL0084 indicated that it was necessary to enter the results for sample A twice and that an error had occurred. This participant was inexperienced in the use of DigitlPT. Participants that experience difficulty with DigitalPT should refer to the DigitalPT User Guide and/or contact the NRL. The DigitalPT user Guide is available from the ‘DOCUMENTS’ section of the DigitalPT website.
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Participant NRL0099 tested all of the panel samples in the Abbott Determine HIV-1/2 Immunoassay and reported results that agreed with the reference results. The participant then proceeded to test all of the samples once in each of two test runs in the Bio-Rad Genscreen Plus HIV Ag-Ab EIA. The participant reported reactive results that were just above the cut-off for the assay (S:Co 1.99; 4.77) for the HIV negative sample G. The participant interpreted sample G to be reactive in the Bio-Rad Genscreen Plus HIV Ag-Ab EIA. All of the other participants that had tested sample G in the same assay had reported results that agreed with the reference results. The participant then proceeded to test the sample in an immunoblot and obtained a negative immunoblot result for the sample. The participant assigned a negative HIV status to the sample. Participant NRL0109 tested all of the panel samples in the bioMerieux VIDAS HIV DUO Ultra ELFA. The participant reported reactive p24 antigen test results and assay interpretations for samples B, C, D, H and I. The assay interpretations for these samples were considered to be aberrant. All of the panel samples were negative for p24 antigen. Two other participants had tested samples B, C, D, H and I for p24 antigen in the same assay and reported negative results. Three participants had tested all of the panel samples in the same assay and reported reactive anti-HIV-1/2 test results and assay interpretations for samples B, C, D, H and I. It appears that Participant NRL0109 had tested the panel samples in the bioMerieux VIDAS HIV DUO Ultra ELFA only for anti-HIV-1/2 and had reported these results as p24 antigen results. Participant NRL0109 also assigned a positive HIV status to the HIV negative sample J. This aberrant status may be due to a clerical error (based on the results that were reported) because the participant had reported negative anti-HIV-1/2 (Innovacon HIV 1/2/O Tri-line HIV Rapid Test Device) and p24 antigen (bioMerieux VIDAS HIV DUO Ultra ELFA) assay interpretations for the sample, which agreed with the reference results for the sample. Participant NRL0235 tested all of the panel samples once in the Abbott AxSYM HIV 1/2 gO MEIA. The participant had reported a reactive test result that was interpreted as reactive for the HIV negative sample J. The test result obtained for sample J may be due to sample carry over because the two samples preceding sample J in the panel were HIV positive samples. The result does not appear to be due to testing an HIV sample twice because the S:Co values obtained by the participant for the HIV positive panel samples were higher than the result obtained for sample J. No other participant that tested sample J in the same assay reported a reactive assay interpretation for the sample. Participant NRL0235 assigned a positive HIV status to sample J. The participant however also indicated that it would refer the sample for further testing. The assay manufacturer for the assay concerned recommends, in the package insert that is available in Australia, that samples that are reactive on initial testing be retested in duplicate. Had the participant retested sample J in duplicate, the participant may have obtained negative test results and been able to interpret the sample as negative. Participant NRL0292 had tested all of the samples once in the Bio-Rad Genscreen Plus HIV Ag-Ab EIA and had reported test results and assay interpretations for all of the samples that agreed with the reference results. The participant then tested all of the samples once in each of two supplemental assays: Fujirebio SFD HIV-1/2 and Abbott Determine HIV-1/2 Immunoassay. The participant reported reactive results for the HIV negative sample G and negative results for the HIV positive sample I in each of the supplemental assays. All of the other participants that tested samples G and I in the same assays reported results that agreed with the reference results. The participant assigned a positive status to sample G and a negative status to sample I. The aberrant results may be due to a sample mix-up that occurred after the testing in the Bio-Rad Genscreen Plus HIV Ag-Ab EIA had taken place. The aberrant results may also be due to human error and /or the individual interpretation of the results obtained for the supplemental assays. Both of the supplemental assays used are read subjectively. Subjectively read assays are prone to human error and/or individual interpretation. The NRL recommends that two operators score subjectively read assays independently of each other, and that a third opinion is sought if they disagree. It is also recommended that staff are periodically assessed for competence following initial training. HIV Serology EQAS Panel ID 2008/Mar/11 Template SerolEqTmpl-0001
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Participant NRL0481 tested all of the panels samples twice, once in each of two assays: bioMerieux Vironostika HIV Uni-Form II Plus O ELISA and Li Zhu Diagnostic Kit for Antibody to HIV (Sandwich ELISA). The participant reported a reactive assay interpretation for the HIV negative sample A in the bioMerieux Vironostika HIV Uni-Form II Plus O ELISA, which appears to be due to a clerical error. The participant reported obtaining a S:Co value of 0.05 for sample A which was interpreted as reactive. However, the participant had also reported the same S:Co value for two other samples that had been tested in the same assay which were interpreted as negative. The participant reported a negative assay interpretation for sample A in the Li Zhu Diagnostic Kit for Antibody to HIV (Sandwich ELISA). The participant did not indicate whether or not any of the panel samples would be referred for further testing or whether a status would be assigned to any of the panel samples. Participant NRL0481 reported results for samples found to be reactive as S:Co 3.00 rather than as > 3.00 in all of the assays used. Consequently, these test results were included in statistical analyses (where a peer group was able to be formed). The reactive test results for samples B, D and J in the bioMerieux Vironostika HIV Uni-Form II Plus O ELISA were identified to be outlying. Participants NRL0075, NRL0090, NRL0113, NRL0373, NRL0427, NRL0441 and NRL0460 tested all of the panel samples in an immunoblot irrespective of the screening results obtained. A screening assay is a first line assay used to test all samples. Supplemental and confirmatory assays are used to test samples repeatedly reactive in the screening assay to differentiate true from false reactivity. Samples that are not reactive in a screening assay should not be tested in immunoblots because the samples may display non-specific reactivity and then be reported unnecessarily as indeterminate. On this occasion, the participants did not report any aberrant immunoblot interpretations.
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 HIV serology testing, will be welcomed by Kathy Smeh on +61 3 9418 1110 or via email on
[email protected].
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5 APPENDIX Table A1. Composition of HIV Serology EQAS Panel ID 2008/Mar/11 Sample Identification by Programme
Sample Origin
Sample Type
HIV Status
HIVC4310
HIVC435
A
A
Australian blood donors
Pooled plasma
Negative
B
B
African blood donors
Pooled plasma
Positive
C
C
Southeast Asian blood donors
Pooled plasma
Positive
D
D
Southeast Asian blood donors
Pooled plasma
Positive
E
E
Australian blood donors
Pooled plasma
Negative
F
Not applicable
Australian blood donors
Pooled plasma
Negative
G
Australian blood donors
Pooled plasma
Negative
H
African blood donors
Pooled plasma
Positive
I
Southeast Asian blood donors
Pooled plasma
Positive
J
Australian blood donors
Pooled plasma
Negative
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Table A2. Characterisation of the samples that comprised HIV Serology EQAS Panel ID 2008/Mar/11 Sample Identification by Programme
Abbott Murex HIV-1.2.O
Bio-Rad Genetic Systems HIV-1 Ag EIA
HIVC4310
HIVC435
Test Result (S/Co*)
Test Result (S/Co)
A
A
0.38
Negative
B
B
20.08
C
C
D
NRL In-house HIV-1 WB
Assay Interpretation
Test Result
Assay Interpretation
0.09
Negative
Not tested
Not applicable
Reactive
0.12
Negative
Not tested
Not applicable
++++
+++
++
+++
+++
+++
20.08
Reactive
0.11
Negative
Not tested
Not applicable
++
++++
+
++
+++
D
20.08
Reactive
0.12
Negative
Not tested
Not applicable
+++
++++
+++
+++
+++
E
E
0.29
Negative
0.12
Negative
Not tested
Not applicable
F
Not applicable
0.34
Negative
0.12
Negative
Not tested
0.29
Negative
0.11
Negative
H
20.08
Reactive
0.12
I
20.08
Reactive
J
0.29
Negative
G
Assay Interpretation
Bio-Rad Genetic Systems HIV-1 Ag Confirmatory p18
p24
p31
p34
gp41
p53
HIV Status p68
Not tested
Assay Interpretation Not applicable
Negative
++++
Positive
Positive
+++
+++
Positive
Positive
+++
++++
Positive
Positive
Not tested
Not applicable
Negative
Not applicable
Not tested
Not applicable
Negative
Not tested
Not applicable
Not tested
Not applicable
Negative
Negative
Not tested
Not applicable
+++
++++
+
++
+++
++
++++
Positive
Positive
0.10
Negative
Not tested
Not applicable
+++
++++
+++
+++
++++
+++
++++
Positive
Positive
0.11
Negative
Not tested
Not applicable
Not applicable
Negative
Not tested
Note: * Sample to cut-off ratio, where ≥1 is reactive.
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