TRIM-00946; No of Pages 8 Transplant Immunology xxx (2014) xxx–xxx
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Transplant Immunology journal homepage: www.elsevier.com/locate/trim
Concordance and discordance in anti-HLA antibody testing Moshe Israeli a,b,⁎, Marilyn S. Pollack c, Carley A.E. Shaut d, Anne Halpin e, Nicholas R. DiPaola f, Danny Youngs g, Susan L. Saidman h, On behalf of the ASHI Proficiency Testing Program a
Tissue Typing Laboratory, Rabin Medical Center, Beilinson Campus, Zabotinski Road, Petach-Tikva 49100, Israel Department of Immunohematology and Blood Transfusion, Leiden University Medical Center, Leiden, The Netherlands Department of Pathology, University of Texas Health Science Center, 7703 Floyd Curl Drive, San Antonio, TX 78229, USA d School of Medicine, Oregon Health & Science University, 2611 SW 3rd Ave Suite 360, Portland, OR 97239, USA e Histocompatibility Laboratory, University of Alberta Hospital, 8220-112 St, Edmonton, AB T6G 2B7, Canada f Clinical Histocompatibility Lab, Wexner Medical Center, The Ohio State University, N943 Doan Hall, 410 W 10th Avenue, Columbus, OH 43210, USA g Puget Sound Blood Center, 921 Terry Ave., Seattle, WA 98104, USA h Histocompatibility Laboratory, Massachusetts General Hospital, 55 Fruit St, Room GRJ 220, Boston, MA 02114, USA b c
a r t i c l e
i n f o
Article history: Received 17 September 2014 Received in revised form 10 October 2014 Accepted 10 October 2014 Available online xxxx Keywords: Antibodies ASHI Consensus HLA Proficiency testing Luminex
a b s t r a c t Background: Correct identification of the specificity of antibodies directed against HLA using single antigen Luminex beads (SALB) is essential in current HLA laboratory practice for transplantation. The aim of this study was to investigate the magnitude of concordance and discordance among laboratories in testing for anti-HLA antibodies using SALB. Method: 35 sera were distributed by the ASHI Proficiency Testing Program to HLA laboratories worldwide. We analyzed 4335 test results submitted between April 2010 and April 2013 by participating laboratories. Results: SALB was used by approximately 94% of the participating laboratories, yet concordant assignment of antibody specificity was imperfect. For each serum, the assignment of an average of 10 antibody specificities was discordant. Disagreement was observed for antibodies directed against common as well as uncommon antigens. The assignment of an average of 15 antibody specificities in each “positive” serum appeared to be influenced by vendor-dependent causes. Inter-vendor concordance was lower than intra-vendor concordance, indicating that vendor dependent factors may be a central cause for disagreement. Conclusions: Our study illustrates the prevalence of concordance and discordance, also affected by unpremeditated causes, in reporting SALB antibody results. Insufficient concordance and standardization in antibody testing may have practical implications for organ allocation and organ sharing programs. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Decades have passed since the crucial importance of anti-HLA antibodies in organ transplantation was revealed [1], but the discussion about the optimal method for their detection and specificity determination has only recently been triggered. The wide variety of assays currently available for the detection of anti-HLA antibodies offers laboratories diverse options for screening patients for antibody presence and for identification of their antigen specificities [2–4]. Considerable discussion concerns the Luminex® analyzer and its accompanying diagnostic reagent kits [5–7]. Single antigen Luminex beads (SALB) can provide superior detection of low levels of antibodies and better determination of antibody Abbreviations: ASHI, American Society for Histocompatibility and Immunogenetics; HLA, Human leukocyte antigens; MFI, Mean fluorescence intensity; PT, Proficiency testing; SALB, Single antigen Luminex beads. ⁎ Corresponding author at: Department of Immunohematology and Blood Transfusion, Leiden University Medical Center, Leiden, The Netherlands. Tel.: +31 6 13644948, +31 20 8220360, +31 71 5263804; fax: +31 71 5265267. E-mail addresses: [email protected], [email protected] (M. Israeli).
specificities [8–10], but standardization of the technique and clinical relevance of antibodies detected only by SALB are still an unsettled issue. Recently, cooperative efforts have been dedicated to the establishment of guidelines and standardization schemes for optimal utilization of SALBbased antibody detection, in order to promote comparable diagnostic and analytical practices among HLA laboratories [11,12]. The mission of the American Society for Histocompatibility and Immunogenetics (ASHI) Proficiency Testing (PT) Program is to promote quality practice in clinical HLA laboratories through objective and consistent evaluation of their performance. The ASHI PT Program supplies specimens that are as similar as possible to actual clinical samples and that challenge laboratory personnel, procedures and facilities to survey their competence by evaluation of the laboratory's agreement with consensus results. One of the proficiency surveys facilitated by the PT Program evaluates laboratory performance in serum screening for the presence of HLA antibodies, HLA antibody specificity identification and crossmatching. The cumulative data collected by the PT Program from hundreds of laboratory submissions over years of activity provide an invaluable opportunity to study antibody testing in a broad group of
http://dx.doi.org/10.1016/j.trim.2014.10.003 0966-3274/© 2014 Elsevier B.V. All rights reserved.
Please cite this article as: Israeli M, et al, Concordance and discordance in anti-HLA antibody testing, Transpl Immunol (2014), http://dx.doi.org/ 10.1016/j.trim.2014.10.003
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HLA laboratories. The aim of the current investigation was to analyze the accumulated data of this survey from recent years and to describe the results with respect to concordance and discordance in SALB antibody testing. 2. Materials and methods 2.1. Samples This investigation encompassed the ASHI PT Program AC survey results for 35 sera or recalcified plasma samples distributed to participating laboratories between April 2010 and April 2013 in seven shipments. Samples were collected from non-randomly selected anonymous apparently healthy blood donors and are not necessarily representative of the general population. All samples were provided by the PT contracted vendor, Immucor (Norcross, GA, USA; formerly GenProbe/GTI, Wisconsin, USA).
study was carried out using the web-based statistical calculator at http://StatPages.info/ctab2x2.html, part of the StatPages web site (created by Prof. John C. Pezzullo, Department of Medicine, Georgetown University, Washington DC, USA). Statistical significance was declared when p b 0.05. Fisher's exact test output and the binomial confidence intervals were validated using the “R” statistical software package (R Foundation for Statistical Computing, Vienna, Austria). 3. Results 3.1. Utilization of SALB for antibody identification is almost ubiquitous As shown in Fig. 1, the percentage of laboratories using the SALB method to report Class I antibody results grew steadily during the study time period from 78.2% in 2010 to 94.6% in 2013 (p = 0.0002, Fisher's exact test). An identical trend was found for Class II antibody identification by SALB, growing from 77.4% in 2010 to 93.2% in 2013 (p = 0.0003, Fisher's exact test). 3.2. Positive and negative samples
2.2. Participating laboratories An average of 124 laboratories reported results derived from SALB assays in each of the 7 surveys described (range: 116–132). Laboratories were free to choose their test method(s) and standard operating procedure(s) for antibody testing. They were asked to report the following: (1) methods used and the identity of the commercial vendor providing the test kit(s); (2) antibody screening/detection results (positive or negative) for each serum/method; and (3) antibody identification results indicating a positive or a negative assignment for each possible antibody specificity for each “positive” serum. Reagent lot numbers, protocols for sera treatment, mean fluorescence intensity (MFI) values and cutoff threshold levels were not reported. The current study focused on laboratory reports on antibodies directed against HLA-A, -B, -DR and -DQB1 antigens. Since the beginning of 2011 (5 of 7 surveys), laboratories were penalized for overcalling extraneous antibody specificities. An average of 86% of the participating laboratories were located in North America, 7% in Asia and Oceania, 4% in South America, 2% in Europe and 1% in Africa and the Middle East. 2.3. Definitions Laboratory results submitted to the PT survey were graded using the criteria described in the survey instructions that accompanied each send-out. The current analysis is based on the following definitions: 2.3.1. Antibody assignment ‘in agreement’ An antibody specificity that was formally assigned as ‘positive by consensus’ according to the survey grading criteria. For an antibody specificity to be declared consensus positive at least 10 participants must have submitted results for that method, and that specificity had to be reported by ≥80% (for surveys in 2010–2011) or ≥90% (for surveys in 2012–2013) of the participating laboratories. 2.3.2. ‘Discordant’ antibody assignment An antibody specificity that was reported as positive by at least 20% of the participating laboratories yet did not reach the required threshold to be declared as ‘consensus positive’ according to PT committee rules. 2.3.3. ‘Vendor dependent’ antibody assignment An antibody specificity for which a statistically significant difference (Fisher's exact test, p b 0.05) was observed between the users of the two available commercial reagent vendors (referred to as Vendor A and Vendor B) for assignment of positivity or negativity. 2.4. Statistical analysis Fisher's exact test for comparing rates of positive results from the two commercial vendors and for additional calculations in the present
Of the 35 sera distributed to participating laboratories, 21 reached consensus for the presence of Class I antibodies with an average of 15 consensus positive antibody specificities in each positive sample (SD: ±8; range: 2–27). Within these same 35 samples, a different subset of 21 sera reached consensus for the presence of Class II antibodies with an average of 6 consensus positive antibody specificities in each positive serum (SD: ±6; range: 1– 12). Table 1 lists all the antibody specificities that were called positive by at least 20% of the laboratories that tested the sample (n = 86) and shows how often the assignment was ‘in agreement’. 14 of the 35 sera were reported as negative for Class I antibodies by at least 80% of the laboratories. Another subset of 14 sera was negative for Class II antibodies. In the consensus negative samples, an average of 6 antibody specificities were assigned as positive in each negative serum by b10% of the laboratories. A list of the outlying antibody specificities that were reported in consensus negative samples is provided in Table 2. Only one sample was unanimously agreed to be completely negative for the presence of antibodies by all participating laboratories. 3.3. ‘Discordant’ antibody assignments In each positive serum an average of 8 (SD: ±5; range: 2–19) Class I and an average of 2 (SD: ±1; range: 1–4) Class II antibody specificities were ‘discordant’, which means that the participating laboratories did not concur for a negative or a positive consensus assignment for those antibodies. Table 1 also lists the instances of ‘discordant’ assignments for each of the antibody specificities called positive by at least 20% of the laboratories. Interestingly, 18 of the 86 (21%) antibody specificities assigned were ‘discordant’ between laboratories in more than 50% of the incidents of their assignment. This means that the majority of their assignments were ‘discordant’. It should be noted that the specificities with the poorest concordance were often those which appeared in only a few sera. Nevertheless, some specificities showed high percentages of discordance even though they were assigned in multiple sera (e.g. A66, B48, B38). In contrast, the assignment of 9 (10%) antibody specificities was never ‘discordant’ between the reporting laboratories. 3.4. ‘Vendor dependent’ antibody assignments In each positive serum an average of 12 (SD: ±7; range: 2–28) Class I and an average of 3 (SD: ±2; range: 1–7) Class II antibody specificity assignments were identified as ‘vendor dependent’, where a statistically significant difference was detected in the positive or the negative assignment between users of the two SALB vendors (Table 3). Vendor A users reported a positive antibody assignment in 93% of the Class I ‘vendor dependent’ incidents (245 of a total 263, p b 0.0001), while vendor B users reported positivity in 59% of the Class II incidents (41 of a total 70, p = 0.06). 3.5. Intra-vendor versus inter-vendor concordance The assignments of 28 of the 86 (33%) antibody specificities were more frequently ‘vendor dependent’ than ‘in agreement’ between laboratories. Furthermore, the majority of the total 333 ‘vendor dependent’ assignments were in agreement among the laboratories using the same vendor. Vendor A and vendor B users reported agreement within their own group in 58% (n = 192) and 73% (n = 243), respectively, of the cases that were in inter-vendor disagreement. This indicates that overall inter-vendor concordance is lower than intra-vendor concordance. 3.6. Discordant assignments in common antigen specificities Disagreement in antibody assignments was not only restricted to antibodies against rare antigens (e.g., B59, B67, and B76 which are among the 10 rarest HLA Class I antigens in the North American population) but also included antibodies against antigens that are common in North American populations [13]. Several antibody specificities directed against common HLA Class I antigens were consistently found to be ‘discordant’ (e.g., A26, A29, B18 and B27 which are among the 20 most common HLA Class I antigens
Please cite this article as: Israeli M, et al, Concordance and discordance in anti-HLA antibody testing, Transpl Immunol (2014), http://dx.doi.org/ 10.1016/j.trim.2014.10.003
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Fig. 1. Portion of participating laboratories that reported results using the SALB kits in the seven studied surveys for testing antibodies directed against HLA Class I (a) and Class II (b). in the North American population). Furthermore, common HLA Class I antigens were also affected by the ‘vendor dependent’ phenomenon (e.g., A32, B13, B44, B51, B57 as well as B53 which is common in the African-American population and B39 which is common in the Hispanic population). Similarly, the assignment of antibodies against common DR antigens (DR15, DR1, and DR17) was also identified as ‘vendor dependent’.
4. Discussion The proper clinical utilization of solid-phase assays for anti-HLA antibody detection in general [14,15] and for SALB in particular [16,17] is a point of debate. The usage of SALB assay results for clinical decision making varies greatly among different transplant centers [18], with differences not only in the interpretation of the assay results as positive or negative for particular specificities [19–21] but also in the practical utilization of the assay results for clinical decision making before and after transplantation [22–24]. Some studies have promoted normalization and agreement among HLA laboratories both in standardization of practical procedures for SALB assays and in clinical utilization of their results [11,12,25]. This study provides a unique overview of HLA antibody testing by SALB among a large group of HLA laboratories. The summary and comparison of SALB assay results submitted to the ASHI PT Program by more
than a hundred laboratories for 35 sera over a three year period provide a singular opportunity to compare the results reported by these laboratories. It is clear that the use of SALB for anti-HLA antibody analysis is a routine procedure in almost all participating laboratories, as 94% of the HLA laboratories reported SALB-based antibody results to the PT Program in 2013. Although consensus in the assignment of many antibody specificities was found, for others consensus was less common than disagreement between laboratories. This confirms earlier studies on inter- and intra-laboratory variation in the results of SALB tests [26,27]. Most recently, Reed et al. [12] reported that standardization among laboratories is achievable if strict adherence to identical operating procedures and similar reagents is enforced. Nonetheless our study demonstrates the current reality of SALB result reporting. The disagreement between laboratories may be attributed to several factors such as differences in testing procedures or in assay output interpretation. A drawback of our study is the absence of information about the assay protocols (for example, whether sera were diluted or pretreated with Dithiothreitol or EDTA, which MFI threshold(s) were used to assign positive reactions, or which reagent lot numbers were used for testing the sera) as this information was not collected for the
Please cite this article as: Israeli M, et al, Concordance and discordance in anti-HLA antibody testing, Transpl Immunol (2014), http://dx.doi.org/ 10.1016/j.trim.2014.10.003
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Table 1 Antibody specificity assignments ‘in agreement’ or ‘discordant’ between laboratories. Antibody specificity
Total # of sera where specificity was assigneda
# of ‘in agreement’ assignments
# of ‘discordant’ assignments
% of ‘discordant’ assignmentsb
Class I A3 A31 A30 A74 A29 A66 A26 B39 B65 B64 A80 A33 B48 A36 B18 B38 B76 B55 A34 B46 B67 A43 B77 B71 B75 B73 A1 B42 B54 B27 A24 A32 A68 B37 B53 A2 A23 A25 B45 B72 B44 B52 B35 A11 B50 B78 B59 B62 B51 B49 B47 B8 A69 B81 B82 B57 B41 B58 B63 B13 B7 B60 B61 B56
1 2 2 1 5 10 6 6 3 3 6 5 10 5 7 9 9 6 6 4 6 4 11 7 7 7 5 5 5 13 8 8 8 11 11 6 6 9 9 6 10 11 8 4 8 8 12 5 12 12 12 7 7 7 7 8 8 8 9 10 6 10 10 6
0 0 0 0 1 3 2 2 1 1 2 2 4 2 3 4 4 3 3 2 3 2 6 4 4 4 3 3 3 8 5 5 5 7 7 4 4 6 6 4 7 8 6 3 6 6 9 4 10 10 10 6 6 6 6 7 7 7 8 9 6 10 10 6
1 2 2 1 4 7 4 4 2 2 4 3 6 3 4 5 5 3 3 2 3 2 5 3 3 3 2 2 2 5 3 3 3 4 4 2 2 3 3 2 3 3 2 1 2 2 3 1 2 2 2 1 1 1 1 1 1 1 1 1 0 0 0 0
100.0% 100.0% 100.0% 100.0% 80.0% 70.0% 66.7% 66.7% 66.7% 66.7% 66.7% 60.0% 60.0% 60.0% 57.1% 55.6% 55.6% 50.0% 50.0% 50.0% 50.0% 50.0% 45.5% 42.9% 42.9% 42.9% 40.0% 40.0% 40.0% 38.5% 37.5% 37.5% 37.5% 36.4% 36.4% 33.3% 33.3% 33.3% 33.3% 33.3% 30.0% 27.3% 25.0% 25.0% 25.0% 25.0% 25.0% 20.0% 16.7% 16.7% 16.7% 14.3% 14.3% 14.3% 14.3% 12.5% 12.5% 12.5% 11.1% 10.0% 0.0% 0.0% 0.0% 0.0%
Class II DQ4 DQ7 DQ8 DQ9 DR10 DR4 DR1 DQ6 DR9
4 7 8 8 3 4 4 5 10
1 4 5 5 2 3 3 4 8
3 3 3 3 1 1 1 1 2
75.0% 42.9% 37.5% 37.5% 33.3% 25.0% 25.0% 20.0% 20.0%
Please cite this article as: Israeli M, et al, Concordance and discordance in anti-HLA antibody testing, Transpl Immunol (2014), http://dx.doi.org/ 10.1016/j.trim.2014.10.003
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Table 1 (continued) Antibody specificity
Total # of sera where specificity was assigneda
# of ‘in agreement’ assignments
# of ‘discordant’ assignments
Class II DR7 DR17 DR14 DR16 DR103 DR18 DR8 DR12 DQ2 DR15 DR13 DQ5 DR11
11 7 7 7 7 7 10 11 3 5 9 2 10
9 6 6 6 6 6 9 10 3 5 9 2 10
2 1 1 1 1 1 1 1 0 0 0 0 0
% of ‘discordant’ assignmentsb 18.2% 14.3% 14.3% 14.3% 14.3% 14.3% 10.0% 9.1% 0.0% 0.0% 0.0% 0.0% 0.0%
a
Number of times the specificity was called positive by at least 20% of the participating laboratories. Values N50% indicate specificities of antibodies for which the majority of their assignments were ‘discordant’. Values =0% indicate specificities of antibody assignments that were never ‘discordant’. b
Table 2 Outlying antibody assignments in consensus negative sera. Antibody specificity
# of negative sera where specificity was assigned by b10% of laboratoriesa
Class I A43 A66 B76 B82 A24 A34 B13 B44 B45 B67 A11 A2 A25 A26 B37 B46 B47 B53 B58 B7 B73 B81
3 3 3 3 2 2 2 2 2 2 1 1 1 1 1 1 1 1 1 1 1 1
Class II DR11 DQ7 DQ6 DR13 DR14 DR15 DR17 DR18 DQ2 DQ4 DQ8 DR8 DR1 DR103 DR4 DR9 DR12 DR16 DR7 DQ5 DQ9 DR10
7 6 5 5 5 5 5 5 4 4 4 4 3 3 3 3 2 2 2 1 1 1
a Consensus negative serum is defined as a sample in which N80% of the participating laboratories did not report the presence of anti-HLA antibodies.
PT survey. Thus, besides pure technical factors, center-specific parameters such as the MFI cutoff used will influence the agreement in HLAantibody testing. Laboratories use their own criteria to interpret positive reactions from MFI values and one can expect considerable variations. Interestingly, even under the controlled circumstances of the study by Reed et al., there was still a 25% variability in antibody specificity assignment [12]. A fundamental conclusion of our investigation is that beyond factors that are related to the laboratories or personnel performing the assay, the identity of the commercial vendor that manufactured the SALB reagent kit is a paramount reason for discordant results. ‘Vendor dependent’ specificities were highly prevalent. In each positive serum sample, an average of 15 antibody assignments was significantly different between users of the two available SALB kits. For Class I specificities, most ‘vendor dependent’ assignments occurred when vendor A users reported positive antibody identification and vendor B users reported the specificity as negative. An opposite trend was identified in Class II specificities where most ‘vendor dependent’ assignments were due to vendor B users reporting the specificities as positive, in contrast to vendor A users who reported them as negative. Moreover, we found that intra-vendor concordance is clearly higher than intervendor concordance, both in regard to laboratories using vendor A or vendor B. This emphasizes that vendor dependent factors are a central cause for discordance between laboratories' results. Some of the ‘vendor dependent’ assignments are clearly due to differences in the antigen panel represented on the SALB provided by the vendors. For example, B59 and B67 were not present in SALB panels of vendor B throughout the studied time period, hence their anticipated ‘vendor dependent’ assignment. Many of the other recurrent ‘vendor dependent’ assignments are antibody specificities that have been previously reported to be against denatured antigen molecules on the surface of the beads. For example, A25, A32, A66, B27, B44, B45, B48, B55, B57, B65 and B76 were also reported by El-Awar et al. [28] in their study on ‘natural’ HLA antibodies in non-sensitized healthy males. Some ‘vendor dependent’ specificities (A25, A66, B13, B18, B38, B44 and B51) were described by Otten et al. as prone to be identified by SALB due to denatured antigen [29]. Therefore, it is possible that the vendors' different manufacturing procedures are a reason for the difference in the reactivity patterns of the SALB kits. In addition to the publication by Morales-Buenrostro et al. [30], which implied that antibodies directed against rare HLA antigens are more likely to be influenced by the imprecise identification by SALB due to the cross-reactivity with natural antibodies, our findings show that ‘vendor dependent’ and ‘discordant’ assignments were also prevalent among antibodies against common HLA antigens. This is consistent with recent clinical case reports of antibodies that were shown to be directed against denatured common antigens on the surface of SALB, such as HLA-A2 and -B44 [31,32].
Please cite this article as: Israeli M, et al, Concordance and discordance in anti-HLA antibody testing, Transpl Immunol (2014), http://dx.doi.org/ 10.1016/j.trim.2014.10.003
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Table 3 ‘Vendor dependent’ antibody specificity assignments. Antibody specificity
Total # of sera where specificity was assigneda
# of ‘vendor dependent’ assignments
% of ‘vendor dependent’ assignmentsb
Class I A31 A74 B65 B64 B38 B46 B73 B59c B39 B55 B67c B52 A1 B13 B76 B49 B77 B18 A66 B44 A26 A80 A25 B45 A32 B50 B57 A33 B48 B54 B51 B75 A30 A34 A43 A24 A11 B41 B27 B63 B71 B81 A29 A36 B42 B61 B35 B58 B53 A2 A23 B72 B47 B7 B60 B8 B37 B62 B56 A69 B82 B78 A3 A68
2 1 3 3 9 4 7 12 6 6 6 11 5 10 9 12 11 7 10 10 6 6 9 9 8 8 8 5 10 5 12 7 2 6 4 8 4 8 13 9 7 7 5 5 5 10 8 8 11 6 6 6 12 6 10 7 11 5 6 7 7 8 1 8
2 1 3 3 9 4 7 12 5 5 5 9 4 8 7 9 8 5 7 7 4 4 6 6 5 5 5 3 6 3 7 4 1 3 2 4 2 4 6 4 3 3 2 2 2 4 3 3 4 2 2 2 4 2 3 2 3 1 1 1 1 1 0 0
100.0% 100.0% 100.0% 100.0% 100.0% 100.0% 100.0% 100.0% 83.3% 83.3% 83.3% 81.8% 80.0% 80.0% 77.8% 75.0% 72.7% 71.4% 70.0% 70.0% 66.7% 66.7% 66.7% 66.7% 62.5% 62.5% 62.5% 60.0% 60.0% 60.0% 58.3% 57.1% 50.0% 50.0% 50.0% 50.0% 50.0% 50.0% 46.2% 44.4% 42.9% 42.9% 40.0% 40.0% 40.0% 40.0% 37.5% 37.5% 36.4% 33.3% 33.3% 33.3% 33.3% 33.3% 30.0% 28.6% 27.3% 20.0% 16.7% 14.3% 14.3% 12.5% 0.0% 0.0%
Class II DQ5 DR1 DR15 DQ2 DQ6 DR103 DR17 DQ4 DQ9
2 4 5 3 5 7 7 4 8
2 4 5 3 5 6 6 3 4
100.0% 100.0% 100.0% 100.0% 100.0% 85.7% 85.7% 75.0% 50.0%
Please cite this article as: Israeli M, et al, Concordance and discordance in anti-HLA antibody testing, Transpl Immunol (2014), http://dx.doi.org/ 10.1016/j.trim.2014.10.003
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Table 3 (continued) Antibody specificity
Total # of sera where specificity was assigneda
Class II DR4 DR14 DR18 DQ8 DR10 DQ7 DR16 DR12 DR13 DR11 DR8 DR7 DR9
4 7 7 8 3 7 7 11 9 10 10 11 10
# of ‘vendor dependent’ assignments 2 3 3 3 1 2 2 3 2 2 1 1 0
% of ‘vendor dependent’ assignmentsb 50.0% 42.9% 42.9% 37.5% 33.3% 28.6% 28.6% 27.3% 22.2% 20.0% 10.0% 9.1% 0.0%
a
Number of times the specificity was called positive by at least 20% of the participating laboratories. Values N50% indicate specificities of antibodies for which the majority of their assignments were ‘vendor dependent’. Values =0% indicate specificities of antibody assignments that were never ‘vendor dependent’. c B59 and B67 were not present in SALB panels of vendor B throughout the studied time period. b
In agreement with our findings, Gombos et al. [33] have recently reported substantially discrepant results obtained by testing 10 sera with the SALB kits from the two major commercial vendors. Our current investigation summarizing the results of hundreds of laboratories with numerous serum samples highlights the inconsistency between laboratories performing anti-HLA antibody testing. The novelty of the current study is in its scope, providing an exceptional large-scale overview of the current practice among HLA laboratories. Another limitation of our study is the fact that, in the PT surveys analyzed in this study, laboratories were not able to report when antibodies reacted with different alleles of the same antigen (for example B*44:02 or B*44:03) [34]. Hence our investigation could not address the influence of allelic differences in antibody assignments. Furthermore, although laboratories are instructed to do the testing in the same manner as they test clinical samples, it is possible that some took extreme quality measures for analysis and reporting of PT survey results in a manner which does not reflect daily clinical routine. Hence it is possible that the discordance in real clinical practice may be even more abundant. Alternatively, some laboratories may call more specificities in the PT surveys than they might when testing patients, especially in the 2010 surveys where there was no penalty for overcalling antibody specificities. Our study is unique in the extent of its analysis, displaying the concordance and discordance in HLA antibody testing across multiple laboratory results during a period of more than three years. We have shown that several antibody specificity assignments, some of them for very common HLA antigens, are frequently in disagreement between laboratories that tested the same serum to the extent that disagreement may be more prevalent than agreement. Whether or not there is a need for standardization in anti-HLA antibody testing is open for debate alongside the many other differences in clinical practice between transplant centers. However, it may be advisable to examine the practical implications of the widespread discordance on organ allocation and organ sharing programs. Conflict of interest All authors declare no conflict of interest. Acknowledgments The authors express gratitude to Prof. Frans Claas of the Leiden University Medical Center in The Netherlands for his critical review of the paper and to Prof. John C. Pezzullo, Department of Medicine, Georgetown University, Washington DC, USA for his support in the statistical analysis. The authors thank Cecilia Blair of the ASHI Executive Office,
the volunteer members of the ASHI PT Program committee and all laboratories that participated in the ASHI PT surveys.
References [1] Jeannet M, Pinn VW, Flax MH, Winn HJ, Russell PS. Humoral antibodies in renal allotransplantation in man. N Engl J Med Jan 15 1970;282(3):111–7. [2] Fuggle SV, Martin S. Tools for human leukocyte antigen antibody detection and their application to transplanting sensitized patients. Transplantation Aug 15 2008;86(3): 384–90. [3] Lachmann N, Todorova K, Schulze H, Schönemann C. Luminex(®) and its applications for solid organ transplantation, hematopoietic stem cell transplantation, and transfusion. Transfus Med Hemother Jun 2013;40(3):182–9. [4] Eckels DD, Stehlik J, Kfoury AG. The detection and role of circulating antibodies in rejection. Curr Opin Organ Transplant 2013;18:589–94. [5] Tait BD. Solid phase assays for HLA antibody detection in clinical transplantation. Curr Opin Immunol Oct 2009;21(5):573–7. [6] Gupta A, Sinnott P. Clinical relevance of pretransplant human leukocyte antigen donor-specific antibodies in renal patients waiting for a transplant: a risk factor. Hum Immunol Aug 2009;70(8):618–22. [7] Zoet YM, Brand-Schaaf SH, Roelen DL, Mulder A, Claas FH, Doxiadis II. Challenging the golden standard in defining donor-specific antibodies: does the solid phase assay meet the expectations? Tissue Antigens Mar 2011;77(3):225–8. [8] Vlad G, Ho EK, Vasilescu ER, Colovai AI, Stokes MB, Markowitz GS, et al. Relevance of different antibody detection methods for the prediction of antibody-mediated rejection and deceased-donor kidney allograft survival. Hum Immunol Aug 2009;70(8): 589–94. [9] Süsal C, Opelz G, Morath C. Role and value of Luminex(®)-detected HLA antibodies before and after kidney transplantation. Transfus Med Hemother Jun 2013;40(3): 190–5. [10] Roelen DL, Doxiadis II, Claas FH. Detection and clinical relevance of donor specific HLA antibodies: a matter of debate. Transpl Int Jun 2012;25(6):604–10. [11] Tait BD, Süsal C, Gebel HM, Nickerson PW, Zachary AA, Claas FH, et al. Consensus guidelines on the testing and clinical management issues associated with HLA and non-HLA antibodies in transplantation. Transplantation Jan 15 2013;95(1):19–47. [12] Reed EF, Rao P, Zhang Z, Gebel H, Bray RA, Guleria I, et al. Comprehensive assessment and standardization of solid phase multiplex-bead arrays for the detection of antibodies to HLA. Am J Transplant Jul 2013;13(7):1859–70. [13] Leffell MS, Cherikh WS, Land G, Zachary AA. Improved definition of human leukocyte antigen frequencies among minorities and applicability to estimates of transplant compatibility. Transplantation Apr 15 2007;83(7):964–72. [14] Morris GP, Phelan DL, Jendrisak MD, Mohanakumar T. Virtual crossmatch by identification of donor-specific anti-human leukocyte antigen antibodies by solid-phase immunoassay: a 30-month analysis in living donor kidney transplantation. Hum Immunol Mar 2010;71(3):268–73. [15] Gebel HM, Liwski RS, Bray RA. Technical aspects of HLA antibody testing. Curr Opin Organ Transplant Aug 2013;18(4):455–62. [16] Süsal C, Ovens J, Mahmoud K, Döhler B, Scherer S, Ruhenstroth A, et al. No association of kidney graft loss with human leukocyte antigen antibodies detected exclusively by sensitive Luminex single-antigen testing: a Collaborative Transplant Study report. Transplantation Apr 27 2011;91(8):883–7. [17] Caro-Oleas JL, González-Escribano MF, González-Roncero FM, Acevedo-Calado MJ, Cabello-Chaves V, Gentil-Govantes MÁ, et al. Clinical relevance of HLA donorspecific antibodies detected by single antigen assay in kidney transplantation. Nephrol Dial Transplant Mar 2012;27(3):1231–8. [18] Süsal C, Roelen DL, Fischer G, Campos EF, Gerbase-DeLima M, Hönger G, et al. Algorithms for the determination of unacceptable HLA antigen mismatches in kidney transplant recipients. Tissue Antigens Aug 2013;82(2):83–92.
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[19] Reinsmoen NL, Lai CH, Vo A, Cao K, Ong G, Naim M, et al. Acceptable donor-specific antibody levels allowing for successful deceased and living donor kidney transplantation after desensitization therapy. Transplantation Sep 27 2008;86(6):820–5. [20] Dunn TB, Noreen H, Gillingham K, Maurer D, Ozturk OG, Pruett TL, et al. Revisiting traditional risk factors for rejection and graft loss after kidney transplantation. Am J Transplant Oct 2011;11(10):2132–43. [21] van den Berg-Loonen EM, Billen EV, Voorter CE, van Heurn LW, Claas FH, van Hooff JP, et al. Clinical relevance of pretransplant donor-directed antibodies detected by single antigen beads in highly sensitized renal transplant patients. Transplantation Apr 27 2008;85(8):1086–90. [22] Zachary AA, Lucas DP, Detrick B, Leffell MS. Naturally occurring interference in Luminex assays for HLA-specific antibodies: characteristics and resolution. Hum Immunol Jul 2009;70(7):496–501. [23] Waterboer T, Sehr P, Pawlita M. Suppression of non-specific binding in serological Luminex assays. J Immunol Methods Feb 20 2006;309(1–2):200–4. [24] Schnaidt M, Weinstock C, Jurisic M, Schmid-Horch B, Ender A, Wernet D. HLA antibody specification using single-antigen beads — a technical solution for the prozone effect. Transplantation Sep 15 2011;92(5):510–5. [25] Kobashigawa J, Crespo-Leiro MG, Ensminger SM, Reichenspurner H, Angelini A, Berry G, et al. Report from a consensus conference on antibody-mediated rejection in heart transplantation. J Heart Lung Transplant Mar 2011;30(3):252–69. [26] Gandhi MJ, Degoey S, Falbo D, Jenkins S, Stubbs JR, Noreen H, et al. Inter and intra laboratory concordance of HLA antibody results obtained by single antigen bead based assay. Hum Immunol Mar 2013;74(3):310–7.
[27] Friedlander R, Putheti P, Diaz E, Menon A, Ponce B, Muthukumar T, et al. On the detection of anti-HLA antibodies using single antigen bead Luminex assay: lot-to-lot variations in MFI. Transplantation Aug 27 2013;96(4):e24–6. [28] El-Awar N, Terasaki PI, Nguyen A, Sasaki N, Morales-Buenrostro LE, Saji H, et al. Epitopes of human leukocyte antigen class I antibodies found in sera of normal healthy males and cord blood. Hum Immunol Oct 2009;70(10):844–53. [29] Otten HG, Verhaar MC, Borst HP, van Eck M, van Ginkel WG, Hené RJ, et al. The significance of pretransplant donor-specific antibodies reactive with intact or denatured human leucocyte antigen in kidney transplantation. Clin Exp Immunol Sep 2013;173(3):536–43. [30] Morales-Buenrostro LE, Terasaki PI, Marino-Vázquez LA, Lee JH, El-Awar N, Alberú J. “Natural” human leukocyte antigen antibodies found in nonalloimmunized healthy males. Transplantation Oct 27 2008;86(8):1111–5. [31] Poli F, Poli F, Benazzi E, Innocente A, Nocco A, Cagni N, Gianatti A, et al. Heart transplantation with donor-specific antibodies directed toward denatured HLA-A*02:01: a case report. Hum Immunol Nov 2011;72(11):1045–8. [32] Jacob EK, De Goey SR, Gandhi MJ. Positive virtual crossmatch with negative flow crossmatch results in two cases. Transpl Immunol Jul 2011;25(1):77–81. [33] Gombos P, Opelz G, Scherer S, Morath C, Zeier M, Schemmer P, et al. Influence of test technique on sensitization status of patients on the kidney transplant waiting list. Am J Transplant Aug 2013;13(8):2075–82. [34] Lomago J, Jelenik L, Zern D, Howe J, Martell J, Zeevi A, et al. How did a patient who types for HLA-B*4403 develop antibodies that react with HLA-B*4402? Hum Immunol Feb 2010;71(2):176–8.
Please cite this article as: Israeli M, et al, Concordance and discordance in anti-HLA antibody testing, Transpl Immunol (2014), http://dx.doi.org/ 10.1016/j.trim.2014.10.003
Contents lists available at ScienceDirect
Transplant Immunology journal homepage: www.elsevier.com/locate/trim
Concordance and discordance in anti-HLA antibody testing Moshe Israeli a,b,⁎, Marilyn S. Pollack c, Carley A.E. Shaut d, Anne Halpin e, Nicholas R. DiPaola f, Danny Youngs g, Susan L. Saidman h, On behalf of the ASHI Proficiency Testing Program a
Tissue Typing Laboratory, Rabin Medical Center, Beilinson Campus, Zabotinski Road, Petach-Tikva 49100, Israel Department of Immunohematology and Blood Transfusion, Leiden University Medical Center, Leiden, The Netherlands Department of Pathology, University of Texas Health Science Center, 7703 Floyd Curl Drive, San Antonio, TX 78229, USA d School of Medicine, Oregon Health & Science University, 2611 SW 3rd Ave Suite 360, Portland, OR 97239, USA e Histocompatibility Laboratory, University of Alberta Hospital, 8220-112 St, Edmonton, AB T6G 2B7, Canada f Clinical Histocompatibility Lab, Wexner Medical Center, The Ohio State University, N943 Doan Hall, 410 W 10th Avenue, Columbus, OH 43210, USA g Puget Sound Blood Center, 921 Terry Ave., Seattle, WA 98104, USA h Histocompatibility Laboratory, Massachusetts General Hospital, 55 Fruit St, Room GRJ 220, Boston, MA 02114, USA b c
a r t i c l e
i n f o
Article history: Received 17 September 2014 Received in revised form 10 October 2014 Accepted 10 October 2014 Available online xxxx Keywords: Antibodies ASHI Consensus HLA Proficiency testing Luminex
a b s t r a c t Background: Correct identification of the specificity of antibodies directed against HLA using single antigen Luminex beads (SALB) is essential in current HLA laboratory practice for transplantation. The aim of this study was to investigate the magnitude of concordance and discordance among laboratories in testing for anti-HLA antibodies using SALB. Method: 35 sera were distributed by the ASHI Proficiency Testing Program to HLA laboratories worldwide. We analyzed 4335 test results submitted between April 2010 and April 2013 by participating laboratories. Results: SALB was used by approximately 94% of the participating laboratories, yet concordant assignment of antibody specificity was imperfect. For each serum, the assignment of an average of 10 antibody specificities was discordant. Disagreement was observed for antibodies directed against common as well as uncommon antigens. The assignment of an average of 15 antibody specificities in each “positive” serum appeared to be influenced by vendor-dependent causes. Inter-vendor concordance was lower than intra-vendor concordance, indicating that vendor dependent factors may be a central cause for disagreement. Conclusions: Our study illustrates the prevalence of concordance and discordance, also affected by unpremeditated causes, in reporting SALB antibody results. Insufficient concordance and standardization in antibody testing may have practical implications for organ allocation and organ sharing programs. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Decades have passed since the crucial importance of anti-HLA antibodies in organ transplantation was revealed [1], but the discussion about the optimal method for their detection and specificity determination has only recently been triggered. The wide variety of assays currently available for the detection of anti-HLA antibodies offers laboratories diverse options for screening patients for antibody presence and for identification of their antigen specificities [2–4]. Considerable discussion concerns the Luminex® analyzer and its accompanying diagnostic reagent kits [5–7]. Single antigen Luminex beads (SALB) can provide superior detection of low levels of antibodies and better determination of antibody Abbreviations: ASHI, American Society for Histocompatibility and Immunogenetics; HLA, Human leukocyte antigens; MFI, Mean fluorescence intensity; PT, Proficiency testing; SALB, Single antigen Luminex beads. ⁎ Corresponding author at: Department of Immunohematology and Blood Transfusion, Leiden University Medical Center, Leiden, The Netherlands. Tel.: +31 6 13644948, +31 20 8220360, +31 71 5263804; fax: +31 71 5265267. E-mail addresses: [email protected], [email protected] (M. Israeli).
specificities [8–10], but standardization of the technique and clinical relevance of antibodies detected only by SALB are still an unsettled issue. Recently, cooperative efforts have been dedicated to the establishment of guidelines and standardization schemes for optimal utilization of SALBbased antibody detection, in order to promote comparable diagnostic and analytical practices among HLA laboratories [11,12]. The mission of the American Society for Histocompatibility and Immunogenetics (ASHI) Proficiency Testing (PT) Program is to promote quality practice in clinical HLA laboratories through objective and consistent evaluation of their performance. The ASHI PT Program supplies specimens that are as similar as possible to actual clinical samples and that challenge laboratory personnel, procedures and facilities to survey their competence by evaluation of the laboratory's agreement with consensus results. One of the proficiency surveys facilitated by the PT Program evaluates laboratory performance in serum screening for the presence of HLA antibodies, HLA antibody specificity identification and crossmatching. The cumulative data collected by the PT Program from hundreds of laboratory submissions over years of activity provide an invaluable opportunity to study antibody testing in a broad group of
http://dx.doi.org/10.1016/j.trim.2014.10.003 0966-3274/© 2014 Elsevier B.V. All rights reserved.
Please cite this article as: Israeli M, et al, Concordance and discordance in anti-HLA antibody testing, Transpl Immunol (2014), http://dx.doi.org/ 10.1016/j.trim.2014.10.003
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HLA laboratories. The aim of the current investigation was to analyze the accumulated data of this survey from recent years and to describe the results with respect to concordance and discordance in SALB antibody testing. 2. Materials and methods 2.1. Samples This investigation encompassed the ASHI PT Program AC survey results for 35 sera or recalcified plasma samples distributed to participating laboratories between April 2010 and April 2013 in seven shipments. Samples were collected from non-randomly selected anonymous apparently healthy blood donors and are not necessarily representative of the general population. All samples were provided by the PT contracted vendor, Immucor (Norcross, GA, USA; formerly GenProbe/GTI, Wisconsin, USA).
study was carried out using the web-based statistical calculator at http://StatPages.info/ctab2x2.html, part of the StatPages web site (created by Prof. John C. Pezzullo, Department of Medicine, Georgetown University, Washington DC, USA). Statistical significance was declared when p b 0.05. Fisher's exact test output and the binomial confidence intervals were validated using the “R” statistical software package (R Foundation for Statistical Computing, Vienna, Austria). 3. Results 3.1. Utilization of SALB for antibody identification is almost ubiquitous As shown in Fig. 1, the percentage of laboratories using the SALB method to report Class I antibody results grew steadily during the study time period from 78.2% in 2010 to 94.6% in 2013 (p = 0.0002, Fisher's exact test). An identical trend was found for Class II antibody identification by SALB, growing from 77.4% in 2010 to 93.2% in 2013 (p = 0.0003, Fisher's exact test). 3.2. Positive and negative samples
2.2. Participating laboratories An average of 124 laboratories reported results derived from SALB assays in each of the 7 surveys described (range: 116–132). Laboratories were free to choose their test method(s) and standard operating procedure(s) for antibody testing. They were asked to report the following: (1) methods used and the identity of the commercial vendor providing the test kit(s); (2) antibody screening/detection results (positive or negative) for each serum/method; and (3) antibody identification results indicating a positive or a negative assignment for each possible antibody specificity for each “positive” serum. Reagent lot numbers, protocols for sera treatment, mean fluorescence intensity (MFI) values and cutoff threshold levels were not reported. The current study focused on laboratory reports on antibodies directed against HLA-A, -B, -DR and -DQB1 antigens. Since the beginning of 2011 (5 of 7 surveys), laboratories were penalized for overcalling extraneous antibody specificities. An average of 86% of the participating laboratories were located in North America, 7% in Asia and Oceania, 4% in South America, 2% in Europe and 1% in Africa and the Middle East. 2.3. Definitions Laboratory results submitted to the PT survey were graded using the criteria described in the survey instructions that accompanied each send-out. The current analysis is based on the following definitions: 2.3.1. Antibody assignment ‘in agreement’ An antibody specificity that was formally assigned as ‘positive by consensus’ according to the survey grading criteria. For an antibody specificity to be declared consensus positive at least 10 participants must have submitted results for that method, and that specificity had to be reported by ≥80% (for surveys in 2010–2011) or ≥90% (for surveys in 2012–2013) of the participating laboratories. 2.3.2. ‘Discordant’ antibody assignment An antibody specificity that was reported as positive by at least 20% of the participating laboratories yet did not reach the required threshold to be declared as ‘consensus positive’ according to PT committee rules. 2.3.3. ‘Vendor dependent’ antibody assignment An antibody specificity for which a statistically significant difference (Fisher's exact test, p b 0.05) was observed between the users of the two available commercial reagent vendors (referred to as Vendor A and Vendor B) for assignment of positivity or negativity. 2.4. Statistical analysis Fisher's exact test for comparing rates of positive results from the two commercial vendors and for additional calculations in the present
Of the 35 sera distributed to participating laboratories, 21 reached consensus for the presence of Class I antibodies with an average of 15 consensus positive antibody specificities in each positive sample (SD: ±8; range: 2–27). Within these same 35 samples, a different subset of 21 sera reached consensus for the presence of Class II antibodies with an average of 6 consensus positive antibody specificities in each positive serum (SD: ±6; range: 1– 12). Table 1 lists all the antibody specificities that were called positive by at least 20% of the laboratories that tested the sample (n = 86) and shows how often the assignment was ‘in agreement’. 14 of the 35 sera were reported as negative for Class I antibodies by at least 80% of the laboratories. Another subset of 14 sera was negative for Class II antibodies. In the consensus negative samples, an average of 6 antibody specificities were assigned as positive in each negative serum by b10% of the laboratories. A list of the outlying antibody specificities that were reported in consensus negative samples is provided in Table 2. Only one sample was unanimously agreed to be completely negative for the presence of antibodies by all participating laboratories. 3.3. ‘Discordant’ antibody assignments In each positive serum an average of 8 (SD: ±5; range: 2–19) Class I and an average of 2 (SD: ±1; range: 1–4) Class II antibody specificities were ‘discordant’, which means that the participating laboratories did not concur for a negative or a positive consensus assignment for those antibodies. Table 1 also lists the instances of ‘discordant’ assignments for each of the antibody specificities called positive by at least 20% of the laboratories. Interestingly, 18 of the 86 (21%) antibody specificities assigned were ‘discordant’ between laboratories in more than 50% of the incidents of their assignment. This means that the majority of their assignments were ‘discordant’. It should be noted that the specificities with the poorest concordance were often those which appeared in only a few sera. Nevertheless, some specificities showed high percentages of discordance even though they were assigned in multiple sera (e.g. A66, B48, B38). In contrast, the assignment of 9 (10%) antibody specificities was never ‘discordant’ between the reporting laboratories. 3.4. ‘Vendor dependent’ antibody assignments In each positive serum an average of 12 (SD: ±7; range: 2–28) Class I and an average of 3 (SD: ±2; range: 1–7) Class II antibody specificity assignments were identified as ‘vendor dependent’, where a statistically significant difference was detected in the positive or the negative assignment between users of the two SALB vendors (Table 3). Vendor A users reported a positive antibody assignment in 93% of the Class I ‘vendor dependent’ incidents (245 of a total 263, p b 0.0001), while vendor B users reported positivity in 59% of the Class II incidents (41 of a total 70, p = 0.06). 3.5. Intra-vendor versus inter-vendor concordance The assignments of 28 of the 86 (33%) antibody specificities were more frequently ‘vendor dependent’ than ‘in agreement’ between laboratories. Furthermore, the majority of the total 333 ‘vendor dependent’ assignments were in agreement among the laboratories using the same vendor. Vendor A and vendor B users reported agreement within their own group in 58% (n = 192) and 73% (n = 243), respectively, of the cases that were in inter-vendor disagreement. This indicates that overall inter-vendor concordance is lower than intra-vendor concordance. 3.6. Discordant assignments in common antigen specificities Disagreement in antibody assignments was not only restricted to antibodies against rare antigens (e.g., B59, B67, and B76 which are among the 10 rarest HLA Class I antigens in the North American population) but also included antibodies against antigens that are common in North American populations [13]. Several antibody specificities directed against common HLA Class I antigens were consistently found to be ‘discordant’ (e.g., A26, A29, B18 and B27 which are among the 20 most common HLA Class I antigens
Please cite this article as: Israeli M, et al, Concordance and discordance in anti-HLA antibody testing, Transpl Immunol (2014), http://dx.doi.org/ 10.1016/j.trim.2014.10.003
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Fig. 1. Portion of participating laboratories that reported results using the SALB kits in the seven studied surveys for testing antibodies directed against HLA Class I (a) and Class II (b). in the North American population). Furthermore, common HLA Class I antigens were also affected by the ‘vendor dependent’ phenomenon (e.g., A32, B13, B44, B51, B57 as well as B53 which is common in the African-American population and B39 which is common in the Hispanic population). Similarly, the assignment of antibodies against common DR antigens (DR15, DR1, and DR17) was also identified as ‘vendor dependent’.
4. Discussion The proper clinical utilization of solid-phase assays for anti-HLA antibody detection in general [14,15] and for SALB in particular [16,17] is a point of debate. The usage of SALB assay results for clinical decision making varies greatly among different transplant centers [18], with differences not only in the interpretation of the assay results as positive or negative for particular specificities [19–21] but also in the practical utilization of the assay results for clinical decision making before and after transplantation [22–24]. Some studies have promoted normalization and agreement among HLA laboratories both in standardization of practical procedures for SALB assays and in clinical utilization of their results [11,12,25]. This study provides a unique overview of HLA antibody testing by SALB among a large group of HLA laboratories. The summary and comparison of SALB assay results submitted to the ASHI PT Program by more
than a hundred laboratories for 35 sera over a three year period provide a singular opportunity to compare the results reported by these laboratories. It is clear that the use of SALB for anti-HLA antibody analysis is a routine procedure in almost all participating laboratories, as 94% of the HLA laboratories reported SALB-based antibody results to the PT Program in 2013. Although consensus in the assignment of many antibody specificities was found, for others consensus was less common than disagreement between laboratories. This confirms earlier studies on inter- and intra-laboratory variation in the results of SALB tests [26,27]. Most recently, Reed et al. [12] reported that standardization among laboratories is achievable if strict adherence to identical operating procedures and similar reagents is enforced. Nonetheless our study demonstrates the current reality of SALB result reporting. The disagreement between laboratories may be attributed to several factors such as differences in testing procedures or in assay output interpretation. A drawback of our study is the absence of information about the assay protocols (for example, whether sera were diluted or pretreated with Dithiothreitol or EDTA, which MFI threshold(s) were used to assign positive reactions, or which reagent lot numbers were used for testing the sera) as this information was not collected for the
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Table 1 Antibody specificity assignments ‘in agreement’ or ‘discordant’ between laboratories. Antibody specificity
Total # of sera where specificity was assigneda
# of ‘in agreement’ assignments
# of ‘discordant’ assignments
% of ‘discordant’ assignmentsb
Class I A3 A31 A30 A74 A29 A66 A26 B39 B65 B64 A80 A33 B48 A36 B18 B38 B76 B55 A34 B46 B67 A43 B77 B71 B75 B73 A1 B42 B54 B27 A24 A32 A68 B37 B53 A2 A23 A25 B45 B72 B44 B52 B35 A11 B50 B78 B59 B62 B51 B49 B47 B8 A69 B81 B82 B57 B41 B58 B63 B13 B7 B60 B61 B56
1 2 2 1 5 10 6 6 3 3 6 5 10 5 7 9 9 6 6 4 6 4 11 7 7 7 5 5 5 13 8 8 8 11 11 6 6 9 9 6 10 11 8 4 8 8 12 5 12 12 12 7 7 7 7 8 8 8 9 10 6 10 10 6
0 0 0 0 1 3 2 2 1 1 2 2 4 2 3 4 4 3 3 2 3 2 6 4 4 4 3 3 3 8 5 5 5 7 7 4 4 6 6 4 7 8 6 3 6 6 9 4 10 10 10 6 6 6 6 7 7 7 8 9 6 10 10 6
1 2 2 1 4 7 4 4 2 2 4 3 6 3 4 5 5 3 3 2 3 2 5 3 3 3 2 2 2 5 3 3 3 4 4 2 2 3 3 2 3 3 2 1 2 2 3 1 2 2 2 1 1 1 1 1 1 1 1 1 0 0 0 0
100.0% 100.0% 100.0% 100.0% 80.0% 70.0% 66.7% 66.7% 66.7% 66.7% 66.7% 60.0% 60.0% 60.0% 57.1% 55.6% 55.6% 50.0% 50.0% 50.0% 50.0% 50.0% 45.5% 42.9% 42.9% 42.9% 40.0% 40.0% 40.0% 38.5% 37.5% 37.5% 37.5% 36.4% 36.4% 33.3% 33.3% 33.3% 33.3% 33.3% 30.0% 27.3% 25.0% 25.0% 25.0% 25.0% 25.0% 20.0% 16.7% 16.7% 16.7% 14.3% 14.3% 14.3% 14.3% 12.5% 12.5% 12.5% 11.1% 10.0% 0.0% 0.0% 0.0% 0.0%
Class II DQ4 DQ7 DQ8 DQ9 DR10 DR4 DR1 DQ6 DR9
4 7 8 8 3 4 4 5 10
1 4 5 5 2 3 3 4 8
3 3 3 3 1 1 1 1 2
75.0% 42.9% 37.5% 37.5% 33.3% 25.0% 25.0% 20.0% 20.0%
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Table 1 (continued) Antibody specificity
Total # of sera where specificity was assigneda
# of ‘in agreement’ assignments
# of ‘discordant’ assignments
Class II DR7 DR17 DR14 DR16 DR103 DR18 DR8 DR12 DQ2 DR15 DR13 DQ5 DR11
11 7 7 7 7 7 10 11 3 5 9 2 10
9 6 6 6 6 6 9 10 3 5 9 2 10
2 1 1 1 1 1 1 1 0 0 0 0 0
% of ‘discordant’ assignmentsb 18.2% 14.3% 14.3% 14.3% 14.3% 14.3% 10.0% 9.1% 0.0% 0.0% 0.0% 0.0% 0.0%
a
Number of times the specificity was called positive by at least 20% of the participating laboratories. Values N50% indicate specificities of antibodies for which the majority of their assignments were ‘discordant’. Values =0% indicate specificities of antibody assignments that were never ‘discordant’. b
Table 2 Outlying antibody assignments in consensus negative sera. Antibody specificity
# of negative sera where specificity was assigned by b10% of laboratoriesa
Class I A43 A66 B76 B82 A24 A34 B13 B44 B45 B67 A11 A2 A25 A26 B37 B46 B47 B53 B58 B7 B73 B81
3 3 3 3 2 2 2 2 2 2 1 1 1 1 1 1 1 1 1 1 1 1
Class II DR11 DQ7 DQ6 DR13 DR14 DR15 DR17 DR18 DQ2 DQ4 DQ8 DR8 DR1 DR103 DR4 DR9 DR12 DR16 DR7 DQ5 DQ9 DR10
7 6 5 5 5 5 5 5 4 4 4 4 3 3 3 3 2 2 2 1 1 1
a Consensus negative serum is defined as a sample in which N80% of the participating laboratories did not report the presence of anti-HLA antibodies.
PT survey. Thus, besides pure technical factors, center-specific parameters such as the MFI cutoff used will influence the agreement in HLAantibody testing. Laboratories use their own criteria to interpret positive reactions from MFI values and one can expect considerable variations. Interestingly, even under the controlled circumstances of the study by Reed et al., there was still a 25% variability in antibody specificity assignment [12]. A fundamental conclusion of our investigation is that beyond factors that are related to the laboratories or personnel performing the assay, the identity of the commercial vendor that manufactured the SALB reagent kit is a paramount reason for discordant results. ‘Vendor dependent’ specificities were highly prevalent. In each positive serum sample, an average of 15 antibody assignments was significantly different between users of the two available SALB kits. For Class I specificities, most ‘vendor dependent’ assignments occurred when vendor A users reported positive antibody identification and vendor B users reported the specificity as negative. An opposite trend was identified in Class II specificities where most ‘vendor dependent’ assignments were due to vendor B users reporting the specificities as positive, in contrast to vendor A users who reported them as negative. Moreover, we found that intra-vendor concordance is clearly higher than intervendor concordance, both in regard to laboratories using vendor A or vendor B. This emphasizes that vendor dependent factors are a central cause for discordance between laboratories' results. Some of the ‘vendor dependent’ assignments are clearly due to differences in the antigen panel represented on the SALB provided by the vendors. For example, B59 and B67 were not present in SALB panels of vendor B throughout the studied time period, hence their anticipated ‘vendor dependent’ assignment. Many of the other recurrent ‘vendor dependent’ assignments are antibody specificities that have been previously reported to be against denatured antigen molecules on the surface of the beads. For example, A25, A32, A66, B27, B44, B45, B48, B55, B57, B65 and B76 were also reported by El-Awar et al. [28] in their study on ‘natural’ HLA antibodies in non-sensitized healthy males. Some ‘vendor dependent’ specificities (A25, A66, B13, B18, B38, B44 and B51) were described by Otten et al. as prone to be identified by SALB due to denatured antigen [29]. Therefore, it is possible that the vendors' different manufacturing procedures are a reason for the difference in the reactivity patterns of the SALB kits. In addition to the publication by Morales-Buenrostro et al. [30], which implied that antibodies directed against rare HLA antigens are more likely to be influenced by the imprecise identification by SALB due to the cross-reactivity with natural antibodies, our findings show that ‘vendor dependent’ and ‘discordant’ assignments were also prevalent among antibodies against common HLA antigens. This is consistent with recent clinical case reports of antibodies that were shown to be directed against denatured common antigens on the surface of SALB, such as HLA-A2 and -B44 [31,32].
Please cite this article as: Israeli M, et al, Concordance and discordance in anti-HLA antibody testing, Transpl Immunol (2014), http://dx.doi.org/ 10.1016/j.trim.2014.10.003
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Table 3 ‘Vendor dependent’ antibody specificity assignments. Antibody specificity
Total # of sera where specificity was assigneda
# of ‘vendor dependent’ assignments
% of ‘vendor dependent’ assignmentsb
Class I A31 A74 B65 B64 B38 B46 B73 B59c B39 B55 B67c B52 A1 B13 B76 B49 B77 B18 A66 B44 A26 A80 A25 B45 A32 B50 B57 A33 B48 B54 B51 B75 A30 A34 A43 A24 A11 B41 B27 B63 B71 B81 A29 A36 B42 B61 B35 B58 B53 A2 A23 B72 B47 B7 B60 B8 B37 B62 B56 A69 B82 B78 A3 A68
2 1 3 3 9 4 7 12 6 6 6 11 5 10 9 12 11 7 10 10 6 6 9 9 8 8 8 5 10 5 12 7 2 6 4 8 4 8 13 9 7 7 5 5 5 10 8 8 11 6 6 6 12 6 10 7 11 5 6 7 7 8 1 8
2 1 3 3 9 4 7 12 5 5 5 9 4 8 7 9 8 5 7 7 4 4 6 6 5 5 5 3 6 3 7 4 1 3 2 4 2 4 6 4 3 3 2 2 2 4 3 3 4 2 2 2 4 2 3 2 3 1 1 1 1 1 0 0
100.0% 100.0% 100.0% 100.0% 100.0% 100.0% 100.0% 100.0% 83.3% 83.3% 83.3% 81.8% 80.0% 80.0% 77.8% 75.0% 72.7% 71.4% 70.0% 70.0% 66.7% 66.7% 66.7% 66.7% 62.5% 62.5% 62.5% 60.0% 60.0% 60.0% 58.3% 57.1% 50.0% 50.0% 50.0% 50.0% 50.0% 50.0% 46.2% 44.4% 42.9% 42.9% 40.0% 40.0% 40.0% 40.0% 37.5% 37.5% 36.4% 33.3% 33.3% 33.3% 33.3% 33.3% 30.0% 28.6% 27.3% 20.0% 16.7% 14.3% 14.3% 12.5% 0.0% 0.0%
Class II DQ5 DR1 DR15 DQ2 DQ6 DR103 DR17 DQ4 DQ9
2 4 5 3 5 7 7 4 8
2 4 5 3 5 6 6 3 4
100.0% 100.0% 100.0% 100.0% 100.0% 85.7% 85.7% 75.0% 50.0%
Please cite this article as: Israeli M, et al, Concordance and discordance in anti-HLA antibody testing, Transpl Immunol (2014), http://dx.doi.org/ 10.1016/j.trim.2014.10.003
M. Israeli et al. / Transplant Immunology xxx (2014) xxx–xxx
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Table 3 (continued) Antibody specificity
Total # of sera where specificity was assigneda
Class II DR4 DR14 DR18 DQ8 DR10 DQ7 DR16 DR12 DR13 DR11 DR8 DR7 DR9
4 7 7 8 3 7 7 11 9 10 10 11 10
# of ‘vendor dependent’ assignments 2 3 3 3 1 2 2 3 2 2 1 1 0
% of ‘vendor dependent’ assignmentsb 50.0% 42.9% 42.9% 37.5% 33.3% 28.6% 28.6% 27.3% 22.2% 20.0% 10.0% 9.1% 0.0%
a
Number of times the specificity was called positive by at least 20% of the participating laboratories. Values N50% indicate specificities of antibodies for which the majority of their assignments were ‘vendor dependent’. Values =0% indicate specificities of antibody assignments that were never ‘vendor dependent’. c B59 and B67 were not present in SALB panels of vendor B throughout the studied time period. b
In agreement with our findings, Gombos et al. [33] have recently reported substantially discrepant results obtained by testing 10 sera with the SALB kits from the two major commercial vendors. Our current investigation summarizing the results of hundreds of laboratories with numerous serum samples highlights the inconsistency between laboratories performing anti-HLA antibody testing. The novelty of the current study is in its scope, providing an exceptional large-scale overview of the current practice among HLA laboratories. Another limitation of our study is the fact that, in the PT surveys analyzed in this study, laboratories were not able to report when antibodies reacted with different alleles of the same antigen (for example B*44:02 or B*44:03) [34]. Hence our investigation could not address the influence of allelic differences in antibody assignments. Furthermore, although laboratories are instructed to do the testing in the same manner as they test clinical samples, it is possible that some took extreme quality measures for analysis and reporting of PT survey results in a manner which does not reflect daily clinical routine. Hence it is possible that the discordance in real clinical practice may be even more abundant. Alternatively, some laboratories may call more specificities in the PT surveys than they might when testing patients, especially in the 2010 surveys where there was no penalty for overcalling antibody specificities. Our study is unique in the extent of its analysis, displaying the concordance and discordance in HLA antibody testing across multiple laboratory results during a period of more than three years. We have shown that several antibody specificity assignments, some of them for very common HLA antigens, are frequently in disagreement between laboratories that tested the same serum to the extent that disagreement may be more prevalent than agreement. Whether or not there is a need for standardization in anti-HLA antibody testing is open for debate alongside the many other differences in clinical practice between transplant centers. However, it may be advisable to examine the practical implications of the widespread discordance on organ allocation and organ sharing programs. Conflict of interest All authors declare no conflict of interest. Acknowledgments The authors express gratitude to Prof. Frans Claas of the Leiden University Medical Center in The Netherlands for his critical review of the paper and to Prof. John C. Pezzullo, Department of Medicine, Georgetown University, Washington DC, USA for his support in the statistical analysis. The authors thank Cecilia Blair of the ASHI Executive Office,
the volunteer members of the ASHI PT Program committee and all laboratories that participated in the ASHI PT surveys.
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