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Our results demonstrate (i) the presence of preformed HLA DSA, both Class I and Class II, in sera of patients transplanted with a negative CDCXM is clinically relevant because it is associated with a lower deathcensored allograft survival, (ii) the presence of anti-HLA antibodies with no DSA has no influence in the outcome of the transplant because this patient group has a similar graft survival as that without anti-HLA antibodies and (iii) MFI values >1500 has no demonstrable effects on graft survival. This study was retrospectively performed and, therefore, the decision to transplant and the immunosuppressive treatment was not conditioned by the results. Several papers analysing the influence of positive virtual XMs in the outcome of patients transplanted with negative CDCXMs have been published. These studies analysing incidence of the acute rejection have reported controversial results, but most of them have important limitations related to the statistical power because of the small number of patients included [13–23]. Our study includes the largest cohort of patients to date and our results are similar to those obtained in the only other two studies including a relatively high number of patients that have been published [24,25]. In our cohort, graft survival was ~75% at 5 years in the group of patients with HLA DSA when compared with the ~90% in both, no DSA and anti-HLA-negative groups. Differences in graft survivals found among our patient groups are similar to those obtained in the cohorts reported by Lefaucheur et al. [24] in patients with DSA pre-existing antibodies and by Amico et al. [25] in patients with DSA and antibody-mediated rejections. In our study, graft survival declines continuously in patients with HLA DSA reaching 48% at the end point (12.5 years) when compared with the ~80% in the no DSA HLA. Regarding the influence of DSA anti-Class I and anti-Class II antibodies, results displayed in Figure 3C and the RRs of graft loss in the different groups suggest influences of both Class I and Class II antibodies. Nevertheless, pre-existing DSA anti-Class I seems to be more important in graft survival, because DSA HLA Class I1II1and HLA Class I1IIÿhad the worst Fig. 4. Kaplan–Meier graft survival curves, stratifying patients in four groups depending on the mean fluorescence intensity (MFI) values: patients with MFI <1500, from 1500 to 3000, from 3000 to 5000 and >5000. Clinical relevance of HLA donor-specific antibodies 1237 at UNIVERSIDAD DE SEVILLA on March 13, 2012http://ndt.oxfordjournals.org/Downloaded from graft survival (66.2 and 68.8% at 4.2 years, respectively) while DSA HLA Class IÿClass II1had an intermediate graft survival (84.9% at the same time) and HLA Class Iÿ Class IIÿhad the best graft survival (96.4%). The only study analysing graft survival taking into account whether DSA antibodies are directed against HLA Class I or Class II donor-specific molecules, reports a similar graft survival for both DSA HLA Class I and Class II but the influence of antibodies directed only against HLA Class I and only against Class II was not analysed separately because they did not stratify in the four possible groups [24]. According to our results, patients with a positive virtual XM should be considered as patients with high immunological risk. Although this condition is not an absolute contraindication to transplantation, it would be desirable to transplant HLA-sensitized patients when they have a negative virtual XM because this is beneficial to patients (better outcome and graft survival) as well as to the transplantation system in both economic terms (a graft is more profitable the longer it is functional) and kidney availability (less re-transplantation candidates on the waiting list). The request of a negative virtual XM would not delay the transplant of HLA highly sensitized patients (PRA >75%); on the contrary, it would increase the likelihood of finding an appropriate donor because in the majority of the allocation schemas only a perfect matched donor is considered for these patients. In the case of less sensitized patients (PRA <75%), algorithms for kidney allocation should get additional points if they have a negative virtual XM proportional to the number of relevant anti-HLA antibodies listed (calculated PRA) [26]. In conclusion, virtual XM is clinically relevant in allograft survival and it could be used for kidney allocation. Acknowledgements. We thank Juan Manuel Praena for the contribution to the statistical analysis. The study was performed according to The Declaration of Helsinki. The Ethical Committee of the Vı´rgen del Rocı´o University Hospital approved this study protocol, and informed written consent was obtained from all patients. This work was supported by grants from Proyecto de Excelencia, P07- CTS-02886, Junta de Andalucı´a, Fondo de Investigaciones Sanitarias, Ministerio de Sanidad y Consumo, FIS PI10/02332 (Spain) and European Community FEDER funds. Conflict of interest statement. None declared. References 1. Gebel HM, Bray RA, Nickerson P. 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Presensitized kidney graft recipients with HLA class I and II antibodies are at increased risk for graft failure: a collaborative transplant study report. Hum Immunol 2009; 70: 569–573 22. Su ¨sal C, Ovens J, Mahmoud K 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 2011; 15: 883–887 23. Willicombe M, Brookes P, Santos-Nunez E et al. Outcome of patients with preformed donor-specific antibodies following alemtuzumab induction and tacrolimus monotherapy. Am J Transplant 2011; 11: 470–477 24. Lefaucheur C, Loupy A, Hill GS et al. Preexisting donor-specific HLA antibodies predict outcome in kidney transplantation. J Am Soc Nephrol 2010; 21: 1398–1406 25. Amico P, Ho¨nger G, Mayr M et al. Clinical relevance of pretransplant donor-specific HLA antibodies detected by single antigen flow-beads. Transplantation 2009; 87: 1681–1688 26. Cecka JM, Kucheryavaya AY, Reinsmoen NL et al. Calculated PRA: initial results show benefits for sensitized patients and a reduction in positive crossmatches. Am J Transplant 2010; 10: 1–6 Received for publication: 4.5.11; Accepted in revised form: 22.6.11 1238 J.L. 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-RVH/XLV&DUR2OHDV      2 75$6 3 8%/,&$&,21(6   ´'RQRUVSHFLILFDQWLERG\GHWHFWLRQFRPSDULVRQRIVLQJOHDQWLJHQDVVD\ DQG/XPLQH[FURVVPDWFKHVµ -/&DUR2OHDV0)*RQ]iOH](VFULEDQR67RUR/ODPDV0-$FHYHGRR0-0DUWLQH]%UDYR,$JXLOHUD DQG$1~xH]5ROGiQ    7LVVXH$QWLJHQ²       Tissue Antigens ISSN 0001-2815 Donor-specific antibody detection: comparison of single antigen assay and Luminex crossmatches J. L. Caro-Oleas, M. F. Gonz´alez-Escribano, S. Toro-Llamas, M. J. Acevedo, M. J. Martinez-Bravo, I. Aguilera & A. N ´u ˜nez-Rold´an Servicio de Inmunolog´ ıa, Instituto de Biomedicina (IBIS), Hospital Universitario Virgen del Roc´ ıo, Universidad de Sevilla, Spain Key words donor-specific antibodies; HLA antibodies; Luminex crossmatches; sensitivity; single antigen assay Correspondence Jos´ e Luis Caro-Oleas Servicio de Inmunolog´ ıa Edificio de Laboratorios Hospital Universitario Virgen del Roc´ ıo Avenida Manuel Siurot s/n, 41013 Seville Spain Tel: +34 95 501 3230 Fax: +34 95 501 3221 e-mail: [email protected] Received 9 December 2009; revised 20 April 2010; accepted 31 May 2010 doi: 10.1111/j.1399-0039.2010.01531.x Abstract Luminex bead-based assays are routinely used in the study of anti-human leukocyte antigen (HLA) donor-specific antibodies (DSA). Single antigen (SA) assays use beads coated with recombinant antigens whereas Luminex crossmatch (Xm-DSA) tests consist of beads coated with isolated donor-specific HLA molecules. The aim of this study was to compare these techniques used to detect DSA. A total of 24 sera recognizing different HLA class I (seven anti-HLA-A and seven anti-HLA-B) as well as class II (seven anti-HLA-DR and three anti-HLA DQ) specificities by complement dependent cytotoxicity were included in the study. These sera were used undiluted and in serial dilutions to perform both class I and II SA and Xm-DSA assays. In the case of Xm-DSA the same serum was checked with different lysates. A total of 42 lysates were used to perform a total of 61 crossmatches: 42 to detect anti-class I and 19 to detect anti-class II antibodies. The maximum positive dilution was higher for SA in 76% of the class I and in 90% of the class II crossmatches. Those cases with a higher sensitivity of the Xm-DSA could not be explained by a larger number of antigen targets. Introduction Screening of sera from patients on the waiting list for transplantation using complement dependent cytotoxicity (CDC) assays has been traditionally used to characterize specificity of anti-human leukocyte antigen (HLA) alloantibodies allowing the prediction of a positive crossmatch (1, 2). The widespread use of anti-T lymphocyte CDC crossmatches to detect donor-specific antibodies (DSA) in the recipient sera prior to transplant has resulted in almost a complete elimination of hyperacute rejection (3, 4). In addition to issues related to sensitivity and specificity (5–7), CDC and other techniques such as anti-human globulin augmented complement dependent cytotoxicity (AHG-CDC) and flow cytometry require the availability of living cells which is an inconvenience for its use in the monitoring of patients transplanted with a cadaveric donor (8–12). Recently, the histocompatibility laboratories have incorporated Luminex bead-based assays among their procedures to detect the presence of anti-HLA antibodies because these techniques are more sensitive and specific and do not require the availability of living cells (13). There are different kinds of available Luminex tests depending on the source of the molecule coated to the color-coded microspheres. In the screening and panel reactive antibody (PRA) assays, beads are coated with HLA molecules isolated from human cell lines whereas in the single antigen (SA) assays, beads are coated with recombinant antigens. Additionally, Luminex crossmatch (Xm-DSA) tests consisting of beads coated with anti-human class I and class II antibodies are available to isolate HLA molecules from a specific donor to be used in donor-specific crossmatches. Xm-DSA permits us to perform a real crossmatch using donor lysates which are easier to keep than living cells for post-transplantation studies. On the other hand, SA assays allow us to take a virtual crossmatch by predicting reactions against HLA specificities. Therefore, both, SA and Xm-DSA could be used to detect the presence of anti-HLA DSA in patient sera. Each technique has particular advantages and disadvantages, for instance, to perform SA it is not necessary to keep donor lysates and it provides additional data about what kind of HLA class I or class II molecule is the target of the response. However, additional donor typing which are not routinely performed in the donation procedures, such as Cw, DQ and DP, are some times necessary to use the SA assay as a virtual crossmatch. Nevertheless, Xm-DSA is a real crossmatch and therefore the existence of DSA against any HLA locus could be theoretically detected without additional donor typing. Meanwhile, Xm-DSA employs 398 2010 John Wiley & Sons A/S · Tissue Antigens 76, 398–403 J. L. Caro-Oleas et al .Comparison of two DSA Luminex-based assays natural human antigen and it is cheaper than the SA test. Thus, regardless of other considerations, it is important to compare both techniques in order to better know any differences related to sensitivity and specificity and to explain discrepant results obtained with both. In the present work, we compare sensitivity of the SA and the Xm-DSA, two assays used to detect the presence of DSA in patient sera. Materials and methods Twenty four sera obtained from pregnant women recognizing different HLA specificities (seven HLA-A, seven HLA-B, seven HLA-DR and three HLA-DQ) by CDC were included in this study. The HLA specificities assigned by CDC in these sera are shown in Table 1. These sera were used in both SA and Xm-DSA Luminex-based assays undiluted and in serial dilutions from 1/2 to 1/256. Table 1 HLA specificities detected by CDC of the sera included in the study. Additional specificities detected in undiluted sera using SA technique are also displayed CDC specificity Additional specificities detected by SA in undiluted sera A1 A36, A29, B35, B53 A2 A68, A69, A24, B78, B40, B35, B18, B42, B51, B53 A3 A11, A34, A66, Cw1, Cw14 A11 A43, A66, A25, A26, B27, B37, B47, B40 A29 A32, A31, A34, A43, A33, A30, A23, A24, A3, A25, B82, B37, B45, B53, B27, B63, B58, B44, B51, B76, B47, B57, B49 A30 A31, B42, B7, B55 A68 A2, A24, A66, A23, A34, A25, B35, B75, B62, B72, B60, B76 B7 A66, A3, B81, B40, B27, B42, B47, B82, B55, B54, B56, B13, B78, B48 B8 A29, B42, B40, Cw8 B13 B75, B62, B76, B27, B61 B35 B53, B75, B56, B62, B72, B63, B78, B58 B40 A31, A66, A29, A33, A30, A68, A34, A74, A25, A2, A26, A43, A32, A69, B27, B7, B81, B47, B73, B42, B41, B48, Cw3, Cw7, Cw17 B41 B40, B42, B45, B47, B37, B54, B7, B13, B44, B50, B8, B49, B81, B55, B39, B18 B44 B47, B27, B13, B76, B45, B37, B82, B49, B53, B77, B41, Cw8, Cw5 DR1 DR4, DR9, DR10, DR51, DR53 DR15+16 DR9, DR1, DR10, DR11, DQ6, DR51 DR3 DR14, DR13, DR8, DR11, DR12, DR52, Poli-DP DR7 DR9, DR12, DQ2, Poli-DP DR10 DR1, DR14, DR9, DR4, DR53, DR51 DR12 DR4, DR13, DR11, DR1, DR14, DR3, DR8, DR52 DR14 DR3, DR11, DR8, DR13, DR15, DR16, DR52, Poli-DP DQ5+6 (a)aDR1, DR9, DQ4, DQ2, DQ8, DQ9, DR51 DQ5+6 (b)aDR13, DR11, DR3, DR14, DR8, DR4, DR16, DR12, DR15, DQ2, DQ4, DR51, DR52 DQ5+6 (c)aDR1, DQ4, DQ2, DR51 HLA, human leukocyte antigen; CDC, complement dependent cytotoxicity; SA, single antigen. aThe three entries DQ5+6 correspond to three different sera. Single antigen assays SA assays were performed using Lifecodes LSAClass I (LSA1) and Class II (LSA2) for the detection of anticlass I and anti-class II antibodies respectively according to the manufacturer’s instructions (Tepnel Lifecode Luminex system, Stanford, CT). Phycoerythrin-conjugated goat antihuman immunoglobulin (Ig) G antibody was used as a second antibody. Samples were analyzed on a Life Match fluoroanalyzer using LUMINEX 100 IS version 2.3 as the software for data acquisition and QUICKTYPE FOR LIFEMATCH as analysis software. Manufacturer’s positive and negative controls were included in each test. To validate one test, the negative control serum had to show mean fluorescence intensity (MFI) <2000 with all the beads and the positive control serum had to recognize a determinate set of microspheres with a batch specific pattern. The mix also included three negative beads whose accepted MFI values are batch specifics and one positive control bead whose MFI value has to be above 2000 with all the sera. To determine if an individual bead is recognized by a serum, three different adjusted (Adj) ratios are calculated by the software. Briefly, the Adj 1 value for each specific bead is the MFI value for this bead minus its background value (batch specific); the Adj 2 value is calculated by dividing the Adj 1 value by the control of the locus (batch specific); and the Adj 3 value is equal to the Adj 2 value divided by the relative quantity of antigen of this specific bead (batch specific). According to the manufacturer’s instructions for the batches used in the present work, a specific bead was considered positive when the MFI raw data was above 3000 and the three Adj ratios were positive (Adj value 1 >3000, and Adj values 2 and 3 >6). Xm-donor specific antibodies assays Lifecodes Donor-Specific Antibody (Tepnel Lifecode Luminex system) was used to obtain 42 lysates from HLA genotyped donors (Table 2). Donors were selected by the presence of one of the HLA specificities detected by CDC in sera included in the study. These lysates were prepared using lymphocytes isolated from the spleen of cadaveric donors with a density gradient (Ficoll). Lymphocytes were treated with a nonionic lysis buffer at a final concentration of 0.275 × 106/µl. HLA molecules were captured from each lysate using 5µl of capture beads for HLA class I or II with 8 µl of lysate per well (2.2 ×106cells). In parallel with our lysates, capture beads were incubated with a control lysate. This control lysate is provided by the manufacturer as a control of the capture step. A total of 61 Xm-DSA assays were performed using these 42 lysates and the 24 sera included in the study (see Tables 3–5 for details). Phycoerythrin-conjugated goat anti-human IgG was used as second antibody. Samples were analyzed on a Life Match fluoroanalyzer using LUMINEX 100 IS version 2.3 as software for data acquisition and QUICKTYPE FOR LIFEMATCH as analysis software. There are three negative and two positive control beads included in 2010 John Wiley & Sons A/S · Tissue Antigens 76, 398–403 399 Comparison of two DSA Luminex-based assays J. L. Caro-Oleas et al . Table 2 HLA typing of the lysates used in the Xm-DSA assaysa Lysate code HLA-A HLA-B HLA-DRB1 HLA-DQB1 005 03/32 35/53 08/14 04/05 08/012 02/26 14/38 01/14 08/014 02/ – 44/ – 03/13 08/027 01/02 44/07 07/ – 08/045 02/30 07/35 01/15 08/059 68/31 51/40 04/10 08/061 02/30 39/44 04/11 08/062 29/34 08/44 03/07 08/067 26/29 18/44 07/11 08/070 02/26 08/41 07/13 08/072 02/24 08/13 11/15 08/073 01/02 13/51 07/11 08/075 02/ – 15/44 03/07 09/002 01/31 15/38 04/11 09/004 24/68 37/57 04/10 03/05 09/006 02/11 07/38 04/ – 09/007 02/29 44/58 07/13 09/008 31/11 44/49 04/07 09/012 02/23 40/44 07/15 02/06 09/013 01/32 50/35 07/13 09/019 31/11 35/38 13/17 09/020 02/29 13/44 07/ – 09/021 02/24 44/40 04/15 03/06 09/023 02/ – 44/41 13/01 09/027 02/32 41/50 03/07 09/031 30/69 07/57 0103/13 09/035 26/29 15/44 07/11 09/039 01/68 08/50 03/13 09/042 01/02 08/44 03/11 09/044 02/ – 07/44 0103/12 09/045 24/29 07/51 07/15 09/050 03/02 51/14 01/04 09/052 01/25 08/44 03/04 09/056 24/68 15/14 04/09 09/059 01/30 44/51 07/09 09/061 02/ – 40/41 11/13 09/065 03/29 35/44 11/15 10/002 01/24 08/44 01/03 10/003 03/32 18/40 03/13 10/004 02/ – 07/18 14/16 10/008 03/23 07/44 07/11 10/009 02/30 35/50 01/14 HLA, human leukocyte antigen; Xm-DSA, Luminex crossmatch. aHLA-DQ was only typed in selected lysates studied with anti-DQ antisera. each test. The negative control beads are used to measure the background and to normalize the signal of the capture beads. Regarding the positive control beads, one of them is coated with human IgG to check the binding of the second antibody, and the other one is coated with a biotinilated protein to check the binding of the streptoavidin-phycoerythrin. To determine if a serum recognizes HLA molecules from a determinate lysate, three different Adj ratios are calculated by the software. Briefly, for each serum, three background values are calculated from the three negative control beads with a batch specific equation for both class I and class II. Each Adj value (for class I and class II) is calculated by subtracting each of these three background values from the MFI value of the serum. To validate a test, the control lysate had to be negative (Adj values 1, 2 and 3 ≤0) with the serum included in the kit as a negative control and positive (Adj values 1, 2 and 3>0) with the positive control serum provided by the manufacturer. Moreover, in order to assure homogeneity in the capture of HLA molecules among different lysates, mean and standard deviation (SD) of the MFI class I and class II values for control lysates were calculated. Those tests with MFI value control out of range of mean ±2SD would be eliminated from analysis. To consider a serum as having antibodies against the class I or class II specificities of one lysate, the three Adj values must be positive (Adj values 1, 2 and 3 >0). As was expected, sera recognized further HLA specificities when they were tested using SA than in the CDC assay (Table 1). Nevertheless, to compare the sensitivity of SA and Xm-DSA, we look at the results of the bead coated with the recombinant antigen corresponding to the HLA specificity detected by CDC because lysates were chosen with this criterion. Analysis of antigen target doses In those cases where differences in sensitivity between both techniques were observed, we analyzed the influence of quantity of antigen target in the results. In this way, we consider whether each lysate was homozygous or heterozygous for the specificity recognized by each serum and also we took into account the number of HLA specificities present in a determinate lysate recognized by the serum. Thus, we considered that there was one antigen target if the following conditions were met: (a) the lysate was heterozygous for the specificity recognized by the serum and (b) only antibodies against one of the specificities present in the lysate were detected by SA. We considered that there were more than one antigen target, in the case of (a) homozygous lysate or (b) antibodies recognizing more than one specificity present in the lysate were detected by SA. Those cases in which anti-Cw or anti-DP antibodies were detected by SA were eliminated from the analysis because we did not perform typing of the lysates for these loci. Results Variability The inter-assay variability, for both SA and Xm-DSA techniques, was checked using one of the sera (anti-DR7) at different dilutions on different days. No differences in the assignation of positive and negative results according to the established criteria were observed (data not shown). In Xm-DSA, none of the tests had MFI values out of the range of mean ±2SD for their control lysates and therefore all were included in analysis. 400 2010 John Wiley & Sons A/S · Tissue Antigens 76, 398–403 J. L. Caro-Oleas et al .Comparison of two DSA Luminex-based assays Table 3 Study of the HLA-A antisera by SA and Xm-DSA assays. Maximum positive dilution obtained with both techniques, code of the lysates used and number of target doses for each Xm-DSA are displayed Serum Dilution SA Dilution Xm-DSA Lysate Number of target dosesa A1 1/8 1/2 09/002 1 1/2 09/013 >1 1/4 09/042 1 A2 1/32 1/64 08/014 >1 1/32 08/045 >1 1/128 08/075 >1 A3 1/2 1/2 09/050 ND 1/4 10/003 ND neat 10/008 ND A11 1/16 1/8 09/006 1 1/4 09/008 1 1/2 09/019 1 A29 1/32 1/16 08/067 >1 1/16 09/007 >1 1/16 09/035 >1 A30 1/16 1/2 08/061 1 1/2 09/031 1 1/2 09/059 >1 A68 1/16 1/16 09/004 >1 1/16 09/039 1 1/8 09/056 >1 SA, single antigen; Xm-DSA, Luminex crossmatch. a1: lysate heterozygous for the specificity recognized by the serum and antibodies against only one specificities present in the lysate. >1: homozygous lysate or antibodies recognizing more than one specificity present in the lysate. ND were cases undetermined because anti-Cw were detected by SA. These cases were eliminated from the analysis because no typing of the lysates in this locus was available. Sensitivity Xm-DSA vs SA HLA-A Seven sera recognizing HLA-A1, A2, A3, A11, A29, A30 and A68 by CDC were tested. These sera were positive by SA up to 1/8, 1/32, 1/2, 1/16, 1/32, 1/16 and 1/16 respectively. Each serum was investigated with three different lysates (total number of Xm-DSA =21). When sera were tested neat in both assays, concordance was 100%. In 14 cases (66.7%) the last positive dilution of the sera was lower in the Xm-DSA than in the SA assay, whereas in four cases (19.0%) the dilution was the same and in three (14.3%) it was higher in the Xm-DSA assay (Table 3). HLA-B Seven sera recognizing HLA-B7, B8, B13, B35, B40, B41 and B44 by CDC were tested. These sera were positive by SA up to 1/32, 1/8, 1/2, 1/8, 1/32, 1/32 and 1/16 respectively. Each serum was investigated with three different lysates (total number of Xm-DSA =21). When sera were tested neat concordance was 86% (18/21) because the serum anti-B8 did not recognize any of the three B8 lysates studied. In 12 cases (57.0%), the last positive dilution of the sera was lower in Xm-DSA than in the SA assay, whereas in 1 case (5.0%) the dilution was the same and in 8 (38.0%) it was higher in the Xm-DSA assay (Table 4). HLA-DR Seven sera recognizing HLA-DR1, DR15+16, DR3, DR7, DR10, DR12 and DR14 by CDC were tested. These sera were positive by SA up to 1/32, 1/32, 1/256, 1/8, 1/64, 1/8 and 1/64 respectively. Each serum was investigated with different lysates (total number of Xm-DSA =15). When sera were investigated neat concordance was 100%. In 13 cases (87.0%) the last positive dilution of the sera was lower in Xm-DSA than in the SA assay, whereas in 2 (13.0%) it was higher in the Xm-DSA assay (Table 5). HLA-DQ Three sera recognizing HLA-DQ5+6 by CDC were tested. These sera were positive by SA from 1/32 up to 1/128. Each serum was investigated with different lysates (total number of Xm-DSA =4). Concordance between both techniques with Table 4 Study of the HLA-B antisera by SA and Xm-DSA assays. Maximum positive dilution obtained with both techniques, code of the lysates used and number of target doses for each Xm-DSA are displayed Serum Dilution SA Dilution Xm-DSA Lysate Number of target dosesa B7 1/32 1/2 09/006 1 1/16 09/031 1 1/8 10/008 >1 B8 1/8 negative 09/039 ND negative 09/042 ND negative 10/002 ND B13 1/2 1/4 08/072 1 1/4 08/073 1 1/4 09/020 1 B35 1/8 1/16 09/013 1 1/2 09/019 1 1/16 10/009 1 B40 1/32 1/16 09/012 ND 1/32 09/061 ND 1/16 10/003 ND B41 1/32 1/8 08/070 >1 1/4 09/023 >1 1/4 09/027 >1 B44 1/16 1/128 08/014 >1 1/32 08/027 ND 1/64 08/075 ND SA, single antigen; Xm-DSA, Luminex crossmatch. a1: lysate heterozygous for the specificity recognized by the serum and antibodies against only one specificities present in the lysate. >1: homozygous lysate or antibodies recognizing more than one specificity present in the lysate. ND were cases undetermined because anti-Cw were detected by SA. These cases were eliminated from the analysis because no typing of the lysates in this locus was available. 2010 John Wiley & Sons A/S · Tissue Antigens 76, 398–403 401 Comparison of two DSA Luminex-based assays J. L. Caro-Oleas et al . Table 5 Study of the HLA-DR and DQ antisera by SA and Xm-DSA assays. Maximum positive dilution obtained with both techniques, code of the lysates used and number of target doses for each Xm-DSA are displayed Serum Dilution SA Dilution Xm-DSA Lysate Number of target dosesa DR1 1/32 1/16 09/023 1 1/16 10/009 1 DR15+16 1/32 1/16 09/045 >1 1/16 09/065 >1 1/4 10/004 >1 DR3 1/256 1/32 09/039 ND 1/32 09/052 ND 1/16 10/003 ND DR7 1/8 1/64 08/075 ND DR10 1/64 1/32 08/059 >1 1/16 09/004 >1 DR12 1/8 1/32 09/044 1 DR14 1/64 1/32 08/012 ND 1/16 10/004 ND 1/32 10/009 ND DQ5+6 (a) 1/128 negative 005 >1 DQ5+6 (b) 1/32 negative 09/004 >1 DQ5+6 (c) 1/64 1/2 09/012 >1 neat 09/021 >1 SA, single antigen; Xm-DSA, Luminex crossmatch. a1: lysate heterozygous for the specificity recognized by the serum and antibodies against only one specificities present in the lysate. >1: homozygous lysate or antibodies recognizing more than one specificity present in the lysate. ND were cases undetermined because anti-Cw were detected by SA. These cases were eliminated from the analysis because no typing of the lysates in this locus was available. neat sera was 50% because two sera did not react neat in Xm-DSA with two different lysates. In the other two cases maximum dilution was higher for SA (Table 5). Antigen target doses We could include a total of 40 cases meeting the criteria detailed above. From the total of 61 assays, we discarded 7 sera (18 crossmatches) in which anti-Cw or anti-DP antibodies were detected and sera another 3 cases were removed because the maximum positive dilution was the same for both assays. One antigen target was observed in 18 and more than one antigen target in 22 of the 40 cases included. The maximum positive dilution was higher for Xm-DSA in 6/18 (33%) of the crossmatches with only one antigen target and in 3/22 (13.6%) of the cases with more than one antigen target. Discussion Currently, both SA and Xm-DSA assays based on Luminex technology are available to detect the presence of DSA and, therefore, it is important to compare both assays in order to clarify possible discrepancies between them. At present, only one study comparing flow cytometry with live cells with Xm-DSA using SA as gold standard has been published (13). This study reported a good concordance between Xm-DSA and SA for class I antibodies and also for HLA-DR. In the present study, all the anti-class I antibodies assigned by CDC were detected using both techniques, except in the case of an anti-HLA-B8 antiserum which was positive by SA up to a 1/8 dilution but negative in the Xm-DSA assay using three different lysates. Billen et al. suggested a deficient capture of HLA-DQ molecules in Xm-DSA, our study supports the same conclusion because two sera anti-DQ5+6 recognizing these specificities by SA up to 1/32 and 1/128 dilutions were negative in the Xm-DSA using undiluted sera. A priori, we could expect a higher variability in the results of Xm-DSA depending on the capture efficiency of the HLA molecules. Nevertheless, the inter-assay variability observed was similar for Xm-DSA and SA. Differences in the mean of MFI for the control lysates among different assays were not found, in fact none of the tests had values of MFI outside the range mean ±2 SD. Additionally, the same serum with different lysates reached a similar maximum positive dilution (±one dilution). All these data suggest a similar capture efficiency inter-assay. In general, sensitivity was higher using SA than Xm-DSA assay for both anti-class I and class II antibodies because positive results were detected up to a higher dilution using recombinant antigens. The smallest differences in sensitivity were found for HLA-B and the greatest for HLA-DQ, although the number of samples in this case was lower. Differences in sensitivity between SA and Xm-DSA could have several explanations. Intuitively, we could expect a higher sensitivity in SA, because in this case each bead is coated with only one antigen, and therefore, the antigen concentration would be higher in the bead surface than in other solid-phase assays, such as Xm-DSA, in which several HLA molecules are attached to one bead. If the sensitivity depended only on the number of molecules bound to the microspheres, we could expect both, a higher sensitivity for SA in all the cases and an increased in Xm-DSA sensitivity when more than one antigen target is captured from the lysate. Nevertheless, according to our results, sensitivity for SA is mostly but not always higher and our results analyzing antigen target doses suggest no differences in sensitivity according to the number of targets, because the ratio of maximum positive dilution for Xm-DSA was even higher when only one antigen target was present. Additionally, if differences in sensitivity were attributed only to the quantity of molecules coated to the beads, we could expect a correspondence between maximum positive dilutions with both techniques, that is to say, a 1/N maximum positive dilution in SA would correspond with a 1/N-X maximum positive dilution in Xm-DSA. Nevertheless, we could not find any correspondence; therefore, our results do not support that the differences in sensitivity be based only in quantitative factors. In this sense, it has been established that the antigen 402 2010 John Wiley & Sons A/S · Tissue Antigens 76, 398–403 J. L. Caro-Oleas et al .Comparison of two DSA Luminex-based assays density is quite variable, both among different antigens and different lots of SA (14), in fact, one of the criteria used to normalize this assay is the relative quantity of antigen in each bead. 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