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Solid phase assays for HLA antibody detection in clinical transplantation
Available online at www.sciencedirect.com
Solid phase assays for HLA antibody detection in clinical transplantation Brian D Tait The complement dependent microlymphocytotoxicity assay has been used for over 40 years for detecting HLA antibodies in transplant patients. This method has been replaced recently by more sensitive solid phase assays such as ELISA and bead based technology including the Luminex method. The introduction of these techniques into clinical practice has revealed previously undetected sensitisation in some patients and allowed the accurate assignment of antibody specificities directed at HLA-DQ and HLA-DP which was not previously possible. However it is emerging that despite the advantage of sensitivity some HLA antibodies defined by these assays are not associated with hyperacute or acute rejection. The role in allograft rejection of antibody titre and non-complement fixing antibodies, which are also detected by these methods, are areas currently under consideration. Address Victorian Transplantation and Immunogenetic Service, Australian Red Cross Blood Service, 2nd Floor Rotary Bone Marrow Research Building, c/o Royal Melbourne Hospital, Grattan St, Parkville 3050, Australia Corresponding author: Tait, Brian D ([email protected])
Current Opinion in Immunology 2009, 21:573–577 This review comes from a themed issue on Immunogenetics and Transplantation Edited by Andrea Velardi and Frank Christiansen
serum can be a confounding factor and CDC does not readily distinguish between IgG and IgM antibodies. Thirdly the relative low sensitivity of the CDC assay and its failure to detect low level antibody has come under question with the introduction of solid phase assays. Solid phase antibody detection assays comprise three types. First introduced was an enzyme-linked immunosorbent assay (ELISA) modified for the detection of HLA antibodies [2–4]. The second major development was the introduction of dye-impregnated beads bound with HLA molecules extracted from cell lines. Two fluorescent dyes are mixed in differing proportions that gives each type of bead a unique fluorescent signal. Test serum containing HLA antibody binds to the relevant bead(s) that is then detected using a fluorescent-labelled anti-human IgG antibody. Two methods for the detection of bound antibody are used. In the first instance the beads are analysed in a flow cytometer and a channel shift indicates the binding of HLA antibody [5]. The second method [6] involves the use of a Luminex (Luminex Corporation, TX, USA) fluorocytometer which has two lasers. One laser excites the dye in the bead and the other excites the phycoerythrin bound to the second antibody. The combination of the two signals indicates firstly the binding of HLA antibody, and secondly the bead carrying a specific HLA molecule to which the antibody is bound.
Available online 16th September 2009 0952-7915/$ – see front matter Crown Copyright # 2009 Published by Elsevier Ltd. All rights reserved. DOI 10.1016/j.coi.2009.07.017
Introduction The complement dependent cytotoxicity assay (CDC) developed over 40 years ago [1] has been the cornerstone technique for the detection of HLA antibodies since its introduction. Although this assay has served us well its limitations have been well documented. Firstly because it measures any antibody capable of binding to a cell surface and fixing complement it cannot discriminate between HLA and non-HLA antibodies. The assignment of HLA specificity is made by comparing the reactivity of the test serum with the HLA typing of the target cells. Since cells display co-dominant expression (i.e. two expressed products from each locus) and some alleles at neighbouring loci display linkage disequilibrium (LD) antibody specificity is assigned based on a probability calculation which is prone to error. Secondly the presence of autoantibodies in the test www.sciencedirect.com
There are three levels of testing for both HLA class 1 and class 2 with the beads at each level varying in the number of specific HLA molecules bound. The third level is the single antigen bead (SAG) in which just one HLA molecule produced by recombinant technology is bound to a specific bead type. SAG testing enables sera with complex mixtures of antibodies to be accurately dissected. Solid phase and, in particular, the bead assays are able to detect pre-sensitisation not previously possible with CDC [7–12,13,14,15,16,17]. This sensitivity has raised important issues concerning the clinical relevance of antibodies detected by these techniques. Since the bead assays are the most common assays now in use the emphasis of the review will be on that technique paying particular attention to the Luminex platform.
Clinical relevance of HLA antibodies detected by bead based assays in organ transplantation Since the first demonstration by Patel and Terasaki [18] that pre-formed HLA class 1 antibodies in renal Current Opinion in Immunology 2009, 21:573–577
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transplant recipients were associated with early rejection it has been an unchallenged rule that a pre-transplant crossmatch is performed to detect donor-specific HLA antibodies (DSHA) in the recipient and that a positive crossmatch with donor T lymphocytes (which detect class 1 but not class 2 antibodies) is a contraindication to transplantation. With the advent of more sensitive bead based assays sensitisation has undergone a re-definition. Gibney et al. [19] reported on a cohort of 136 renal transplant patients of whom 16 had pre-transplant antibodies as defined by CDC. By contrast an additional 39 patients (total 55) were shown to have antibodies to either HLA class 1 or class 2 by the Luminex assay. Twenty of these patients had DSHA and they had a higher rate of primary or delayed graft function, biopsy proven acute rejection (BPAR) and a lower rate of graft survival at six months post-transplant than the non-DSHA group. In this study, therefore, Luminex detected additional antibodies that proved to be clinically relevant. In a retrospective study of renal transplant patients with a negative CDC crossmatch Gupta et al. [20] tested the sera from the day of transplant for HLA antibodies using the Luminex assay. Sixteen out of 121 (13%) patients were shown to have DSHA by the Luminex technique which were undetectable at the time of transplantation by CDC. A further 22 patients were shown to have nonDSHA antibodies present. No hyperacute rejections were observed in the total patient cohort and there was no significant difference in the frequency of BPAR, delayed graft function, median creatinine levels at one year or graft survival at one year between the three groups that is antibody positive (DSHA and non-DHSA) and antibody negative. Multivariate analysis however did show an association of DSHA with long-term graft failure although the power of the study was limited. By contrast, van den Berg-Loonen et al. [21] reported on 34 highly sensitised patients (>85%PRA) transplanted as part of the Eurotransplant acceptable mismatch programme and were able to show a trend towards earlier rejection and graft loss in the Luminex detected DSHA group compared with the non-DSHA group. There was no difference in the long-term graft survival between the groups, however the numbers in the respective groups were small. In an elegant retrospective study of 565 heart transplant patients Smith et al. [22] showed a clear association of Luminex + CDC-DSHA with reduced graft survival. Fourteen patients had either class 1 or class 2 HLA antibodies by CDC but a further 53 patients were identified with HLA antibodies detectable only by the Luminex bead assay 14 of whom had DSHA. The actuarial one year graft survival for patients with CDC + Luminex + DSHA was 40% and 42% in the CDC Luminex + DSHA Current Opinion in Immunology 2009, 21:573–577
group. The graft survival in the group with Luminex + non-DSHA was 77% and 75% in those with no detectable antibodies. A detrimental effect of both HLA class 1 and class 2 antibodies detected only by the Luminex bead assay in a total cohort of 896 liver transplant patients was demonstrated by Castillo-Rama et al. [14]. CDC negativity was defined as a negative crossmatch with donor T and B lymphocytes. When the Luminex + CDC-group were subgrouped into Luminex class 1 + T cell-group, Luminex class 1 + B cell-group, Luminex class 2 + B cellgroup, and Luminex class 1 and 2 + T and B cell-group and compared with the comparable Luminex CDCgroups there was a significant decrease in graft survival in all four subgroups. There are several conclusions that can be drawn from the published data. The first is that transplanting across class 1 or 2 DSHA detectable by Luminex but not by CDC does not appear to cause hyperacute rejection in renal transplantation, and many of the patients in this category have uneventful post-transplant courses. The second observation is that this group of patients have an increased incidence of acute and chronic rejection and possibly a lower graft survival. Increased rejection rates seen in Luminex + CDC-renal patients with DSHA also appears to apply to heart and liver transplant patients. Pre-formed HLA class 1 DSHA detectable by CDC are associated with hyperacute rejection in renal transplantation. The solid phase assays and in particular the most sensitive Luminex based bead assay detect antibodies below the CDC threshold but by definition patients with these antibodies are sensitised. Why is there a difference in the clinical outcome of patients with antibodies detected by these two techniques? One explanation might simply relate to the titre of antibody present. Patients whose antibodies are only detected by the Luminex technique have lower titres than CDC positive patients due to the higher sensitivity of the former assay. The titre of HLA antibodies was shown by Mizutani et al. [23] to be directly related to maximum fluorescence values and the molecules of equivalent soluble fluorochrome (MESF) values as determined by Luminex. The average DSHA MESF values were correlated with graft rejection and the overall average MESF values in the rejection group were approximately 241 000 compared with 31 000 for the graft functioning group. Vaidya et al. have shown that antibodies detected by CDC have an MESF threshold of approximately 250 000 below which they are negative [24]. These findings indicate that the range of values obtained by Luminex will include antibodies that are not detected by CDC but that are able to cause graft rejection, but in addition some of the antibodies detected at the lower end of the MESF range by Luminex appear not to be overtly graft damaging at least www.sciencedirect.com
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in the short term. The long-term effect of these low titre antibodies remains to be seen. The application of Luminex technology therefore represents a paradigm shift and demonstrates that transplantation in a percentage of patients can be successful in the presence of low titres of DSHA. The other important consideration is the fact that the solid phase assays do not rely on complement binding for antibody detection and therefore a proportion of antibodies detected will be noncomplement binding. Non-complement binding antibodies and their detection by solid phase assays
Solid phase assays do not distinguish between complement binding (CB) and non-complement binding (NCB) antibodies. NCB antibodies to non-HLA antigens have been implicated in graft rejection [25] but there has been no systematic study on the clinical effects of NCB HLA antibodies. Their presence however in the eluates of over a quarter of rejected kidneys has been demonstrated [26]. Current practices in the use of solid phase assays exclude patients with NCB antibodies against a prospective donor though there is little evidence that NCB DSHA are harmful. If NCB are not damaging to grafts then compatible transplants are being denied to some patients. There is evidence to conclude that NCB antibodies do occur in a substantial percentage of renal patients awaiting a transplant. Using anti-IgG2 and IgG4 antibodies in modified ELISA and Luminex bead based assays Arnold et al. [27] have demonstrated that 40% of potential re-transplant patients on the waiting list had either class 1 and/or class 2 antibodies of NCB subclasses. Similar results were obtained by Wahrmann et al. [28] modifying the Flow based bead assay (FlowPRA, One Lambda Inc.) by adding normal serum as a source of complement and using anti-C4d as a second antibody. A striking example of the clinical relevance of HLA NCB antibodies was shown by Smith et al. [22] who modified the Luminex bead assay in a similar fashion to Wahrmann et al. [28]. They demonstrated a 20% graft survival in heart transplant patients with CB DSHA compared with 54% for patients with NCB DSA and 91% for patients with non-DSHA. These NCB antibodies may be playing a graft damaging role through a mechanism other than complement binding such as antibody dependent cell mediated cytotoxicity [29,30]. Further studies are required to fully elucidate the role of NCB in graft rejection.
Additional advantages of bead technology for HLA antibody detection Solid phase HLA antibody detection methods and, in particular, the SAG assay have improved our understanding of the role of HLA antibodies in renal graft rejection. Firstly it has allowed for the first time the evaluation of www.sciencedirect.com
HLA-C, HLA-DRB3, HLA-DRB4, HLA-DRB5, HLADQA1, HLA-DQB1, HLA-DPA1, HLA-DPB1 and MICA antibodies in renal graft rejection. The introduction of beads binding just a single molecule has overcome the problem of LD and has revealed antibodies to these other loci products are present in sensitised transplant patients and may play a role in rejection. Secondly solid phase assays have permitted accurate definition of antibodies directed at shared epitopes revealing that many socalled multispecific responses can be shown to be a single antibody to a shared epitope. Using single antigen beads in the Luminex platform and analysing the data by Matchmaker [31] Duquesnoy et al. [32] showed in a cohort of 75 transplant patients that HLA antibodies could be detected to products of HLADRB1, HLA-DRB3, HLA-DRB4, HLA-DPA1, HLADPB1 and to both chains of the DQ heterodimer (DQa, DQb). Interestingly approximately a third of patients had antibodies to either HLA-DPA or HLADPB. This is in agreement with an earlier observation by Piazza et al. [33]. When reactivity was correlated with amino acid sequences it was revealed that the DEAV sequence at positions 84–89 of the DPB1 chain was implicated in 51% of cases suggesting this is a particularly immunogenic epitope. We have previously described a female renal patient who was immunised to this DPB1 epitope from a first transplant and underwent reversible C4d positive rejection of a second transplant mismatched at only one DP allele which carried the DEAV epitope [34]. Other reports have also associated the presence of HLA-DP antibodies with allograft rejection [35,36]. The analysis by Duquesnoy et al. [32] demonstrated 87% of anti-class 2 responses include antibodies to DQB1 coded antigens. The MatchMaker eplet analysis shows there are significantly more potential eplet mismatches for DQB1 than DRB1 which probably explains this observation. Likewise the incidence and damaging role of antibodies to MICA in renal transplant patients undergoing rejection has been convincingly demonstrated by the use of Luminex [37,38,39]. In renal transplantation donor/recipient matching is generally restricted to HLA A, B and DRB1. With the realisation that some patients are immunised to products of other loci such as DQ (A and B), DP (A and B) and MICA more emphasis will be placed in future on avoiding transplanting across these incompatibilities, or at least transplanting in the knowledge that particular care in the form of early rejection prophylaxis may be required. An additional finding which has emerged from the use of single antigen beads has been the discovery that antibodies can be raised to alleles within the same serological group as the antibody producer. For example an A*2403 renal transplant recipient produced a DSHA to A*2402 Current Opinion in Immunology 2009, 21:573–577
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which was associated with rejection [40]. There are other examples appearing in the literature such as an A*0302 individual producing an antibody to A*0301 [41]. The implications of these findings are that some recipient/ donor combinations will require allele typing in order to establish compatibility.
4.
Monien S, Salama A, Schonemann C: ELISA methods detect HLA antibodies with variable sensitivity. Int J Immungenet 2006, 33(3):163-166.
5.
Rebibou JM, Chabod J, Bittencourt MC, Thevenin C, Chalopin JM, Herve P, Tiberghien P: Flow PRA evaluation for antibody screening in patients awaiting kidney transplantation. Transpl Immunol 2000, 8(2):125-128.
6.
Colombo MB, Haworth SE, Poli F, Nocco A, Puglisi G, Innocente A, Serafini M, Messa P, Scalamogne M: Luminex technology for anti-HLA antibody screening: evaluation of performance and of impact on laboratory routine. Cytometry B Clin Cytom 2007, 72(6):4645-4671.
7.
Parissiadis A, Marbach N, Leisenbach R, Wolf P, Cazenave JP, Tongio MM: Identification of HLA class 1 and class 2 antibodies: A comparison of LAT1240 ELISA and cytotoxicity. Eur J Immunogenet 2001, 28(2):360.
8.
Uboldi de Capei M, Pratico L, Curtoni ES: Comparison of different techniques for detection of anti-HLA antibodies in sera from patients awaiting kidney transplantation. Eur J Immunogenet 2002, 29(5):379-382.
9.
Levin MD, de Veld JC, van der Holt B, van’t Veer MB: Screening for alloantibodies in the serum of patients receiving platelet transfusions: a comparison of the ELISA, lymphocytotoxicity and the indirect immunoflourescence method. Transfusion 2003, 43(1):72-77.
Conclusions For the first time solid phase assays have provided tools for exposing low levels of immunisation which was not possible using conventional CDC. The issue, however, is how to use this information to achieve optimal success rates. Some renal transplant patients with antibodies detectable by Luminex but not CDC have uneventful post-transplant courses whereas others have a detrimental outcome. The reason for this is not clear although the lower titre of antibody and the fact that Luminex also detects non-complement binding antibodies are two possibilities. The use of single antigen beads in the Luminex technique has clarified the role of antibodies directed at products of the HLA-C, HLA-DRB3, HLA-DRB4, HLA-DRB5, HLA-DQA1, HLA-DQB1, HLA-DPB1 and MICA loci on renal allograft rejection which was not possible using the CDC technique. The use of single antigen beads also allows accurate designation of shared epitopes between positively reacting HLA molecules which enables avoidance of these epitopes on future transplants. The introduction of solid phase assays and, in particular, the Luminex system has ushered in a new era in the definition of HLA pre-sensitisation. The challenge now is to understand more fully the humoral alloresponse and its association with graft rejection and to use this technique for the optimal management of the transplant patient.
Acknowledgements The author wishes to acknowledge the professional expertise of Linda Cantwell, Fiona Hudson and Gemma Brewin who established the Luminex technique in the VTIS laboratories and who provide ongoing support in the interpretation of results.
References and recommended reading Papers of particular interest, published within the period of review, have been highlighted as: of special interest of outstanding interest 1.
Terasaki PI, McClelland JD: Microdroplet assay of human serum cytotoxins. Nature 1964, 204:998-1000.
2.
Kao KJ, Scornik JC, Small JC: Enzyme linked immunoassay for anti-HLA antibodies — an alternative to panel studies by lymphocytotoxicity. Transplantation 1993, 55(1):192-196.
3.
Worthington JE, Robson AJ, Sheldon S, Langton A, Martin S: A comparison of enzyme-linked immunoabsorbent assays and flow cytometry techniques for the detection of HLA specific antibodies. Hum Immunol 2001, 62(10):1178-1184.
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10. Altemann WW, Seliger B, Sel S, Wendt D, Schlaf G: Comparison of the established standard complement-dependent cytotoxicity and flow cytometric crossmatch assays with a novel ELISA based HLA crossmatch procedure. Histol Histopathol 2006, 21(10–12):1115-1124. 11. Muro M, Llorente S, Gonzales-Soriano MJ, Minguela A, Gimeno L, Alvarez-Lopez MR: In Pre-formed Donor-specific Alloantibodies (DSA) Detected only by Luminex Technology using HLA-coated Microspheres and Causing Acute Humoral Rejection and Kidney Graft Dysfunction. Edited by Terasaki PI. Clinical Transplants Pub. Terasaki Foundation Laboratory; 2006:379-383. 12. Lee PC, Ozawa M: In Reappraisal of HLA Antibody Analysis and Crossmatching in Kidney Transplantation. Edited by Cecka MJ, Terasaki PI. Clinical Transplants Pub Terasaki Foundation Laboratory; 2007:219-226. 13. Fuggle SV, Martin S: Tools for human leukocyte antigen antibody detection and their application to transplanting sensitized patients. Transplantation 2008, 86(3):384-390. Describes various techniques for HLA antibody detection and their application in defining pre-sensitisation in potential transplant patients. 14. Castillo-Rama M, Castro MJ, Bernardo I, Meneu-Diaz JC, Elola Olaso AM, Calleja-Antolin SM, Romo E, Morales P, Moreno E, PazArtal E: Preformed antibodies detected by cytotoxic assay or multibead array decrease liver allograft survival: role of human leukocyte antigen compatibility. Liver Transplant 2008, 14(4):554-562. Demonstrates the importance of the Luminex technique in detecting HLA antibodies which are shown to be associated with rejection in liver transplant patients. 15. Saidman SL: Histocompatibility testing for highly sensitised transplant candidates. Trans Proc 2007, 39(3):673-675. 16. Leffell MS, Montgomery RA, Zachary AA: The changing role of antibody testing in transplantation. In Clin. Transpl.. Edited by Cecka MJ, Terasaki PI. 2005:259-271. 17. Zeevi A, Girnita A, Duquesnoy R: HLA antibody analysis: sensitivity, specificity and clinical significance in solid organ transplantation. Immunol Res 2006, 36(1–3):255-264. HLA antibody detection by solid phase assays particularly the bead based methods have allowed a more accurate analysis of shared epitope structures and their relevance to transplant outcome. 18. Patel R, Terasaki PI: Significance of the positive crossmatch test in kidney transplantation. New Eng J Med 1969, 280(14):735-739. 19. Gibney EM, Cagle LR, Freed B, Warnell SE, Chan L, Wiseman AC: Detection of donor specific antibodies using HLA coated www.sciencedirect.com
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microspheres: another tool for kidney transplant risk stratification. Nephrol Dial Transplant 2006, 21(9):2625-2629. Renal patients transplanted with a negative AHG-CDC donor crossmatch were retrospectively tested by Luminex for the presence of HLA antibodies. Patients with donor-specific antibodies (DSA) had higher rates of biopsy proven rejection and lower graft survival than patients without DSA. 20. Gupta A, Iveson V, Varagunam M, Bodger S, Sinnott P, Thuraisingham RC: Pretransplant donor-specific antibodies in cytotoxic negative crossmatch kidney transplants: are they relevant? Transplantation 2008, 85(8):1200-1204. Renal patients with negative CDC donor crossmatches were tested for the presence of HLA antibodies by solid phase assays. While no shortterm differences in rejection incidence were noted between patients with donor-specific antibodies (DSA), those with non-DSA, and antibody negative patients, there was increased long-term graft loss in the DSA group. 21. van den Berg-Loonen EM, Billen EVA, Voorter CVM, van Heurn LWE, Claas FHJ, van Hooff JP, Christiaans MHL: Clinical relevance of pre transplant donor-directed antibodies detected by single antigen beads in highly sensitised renal transplant patients. Transplantation 2008, 85(8):1086-1090. 22. Smith JD, Hamour IM, Banner NR, Rose ML: C4d fixing Luminex antibodies — a new tool for prediction of graft failure after heart transplantation. Am J Transplant 2007, 7(12):2809-2815. An elegant study describing modifications to the Luminex technique to detect both complement binding (CB) and non-complement binding (NCB) HLA antibodies in heart transplant patients. While a significant negative effect of CB antibodies on graft survival was demonstrated a lesser negative effect of NCB antibodies was also shown. 23. Mizutani K, Terasaki P, Hamdani E, Esquenazi V, Rosen A, Miller J, Ozawa M: The importance of anti-HLA-specific antibody strength in monitoring kidney transplant patients. Am J Transplant 2007, 7:1027-1031. First reported correlation of antibody titre as defined by the Luminex technique and expressed as molecules of equivalent soluble fluorochrome (MESF), with graft outcome. Patients with graft failure had significantly higher MESF values than non-rejection patients. 24. Vaidya S, Partlow DA, Suskind B, Gugliuzzi K: Prediction of crossmatch outcome of highly sensitised patients based on identification of serum HLA class 1 and 2 antibodies by single and/or multiple antigen bead Luminex assay. Abstract no. 670. The First Joint International Transplant Meeting; Boston, MA, July: 2006. 25. Dragun D: Humoral responses directed against non-human leukocyte antigens in solid organ transplantation. Transplantation 2008, 86(8):1019-1025. 26. Heinemann FM, Roth I, Rebmann V, Arnold ML, Witzke O, Wilde B, Spriewald BM, Grosse-Wilde H: Immunoglobulin isotypespecific characterization of anti-human leukocyte antigen antibodies eluted from explanted renal allografts. Hum Immunol 2007, 68(6):500-506. The presence of both complement binding (CB) and non-complement binding (NCB) HLA antibodies were demonstrated in acid eluates of rejected renal allografts. 28% of IgG positive eluates contained NCB antibodies. 27. Arnold M-L, Dechant M, Doxiadis IIN, Spriewald BM: Prevalence and specificity of immunoglobulin G and immunoglobulin A non-complement binding anti-HLA alloantibodies in renal transplant candidates. Tissue Antigens 2008, 72(1):60-66. The Luminex assay was modified in order to detect the four isotypes of IgG. 40% of previously transplanted renal patients’ sera contained HLA antibodies of the non-complement binding isotypes IgG2 and IgG4, the majority of which were donor specific. 28. Wahrmann M, Exner M, Regele H, Derfler K, Kormoczi GF, Lhotta K, Zlabinger GJ, Bohmig GA: Flow cytometry based detection of HLA alloantibody mediated classical complement activation. J Immunol Methods 2003, 275(1–2):149-160.
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A Flow based method is described for detecting the C4 component of complement generated as a result of complement binding and activation by HLA antibodies. 29. Vongwiwatana A, Tasanarong A, Hidalgo LG, Halloran PF: The role of B cells and alloantibody in the host response to human organ allografts. Immunol Rev 2003, 196:197-218. 30. Wehner J, Morrell CN, Reynolds T, Rodriguez ER, Baldwin WM: Antibody and complement in transplant vasculopathy. Circ Res 2007, 100(2):191-203. 31. Duquesnoy RJ, Askar M: HLAMatchmaker: a molecularly based algorithm for histocompatibility determination. V. Eplet matching for HLA-DR, HLA-DQ and HLA-DP. Hum Immunol 2007, 68(1):12-25. This paper describes the design of the HLAMatchmaker programme which explains reactivity of HLA class 2 antibodies in terms of eplet (three amino acid) recognition sequences. This programme has permitted sera which react with multiple HLA molecules to be analysed in terms of shared epitopes. 32. Duquesnoy R, Awadalla Y, Lomago J, Jelinek L, Howe J, Zern D, Hunter B, Martell J, Girnita A, Zeevi A: Retransplant candidates have donor-specific antibodies that react with structurally defined HLA-DR, DQ, DP epitopes. Transplant Immunol 2008, 18(4):352-360. Using the single antigen Luminex assay and the HLAMatchmaker programme (Ref. [31]) the fine specificity of HLA class 2 antibodies are described in terms of eplets and shared epitopes. 33. Piazza A, Poggi E, Ozella G, Borreli L, Scornajenghi KA, Monaco PI, Tisone G, Adorno D: Anti-DP antibodies and graft failure in kidney transplantation. Abstract no. 520. The First Joint International Transplant Meeting; Boston, MA, July: 2006. 34. Blair S, Tait B, Hudson F, Kannellis J: Acute humoral rejection associated with HLA-DPB1 antibody in an HLA identical (A,B,DR,DQ) recipient. Abstract. Transplantation Society of Australia and New Zealand Annual Scientific Meeting; Canberra, Australia, April: 2007. 35. Qui J, Cai J, Terasaki PI, El-Awar N, Lee JH: Detection of antibodies to HLA-DP in renal transplant recipients using single antigen beads. Transplantation 2005, 80(10):1511-1513. 36. Arnold ML, Pei R, Spriewald B, Wassmuth R: Anti-HLA class 2 antibodies in kidney re-transplant patients. Tissue Antigens 2005, 65:370-378. 37. Panigrahi A, Gupta N, Siddiqui JA, Margoob A, Bhowmik D, Guleria S, Mehra NK: Post transplant development of MICA and anti-HLA antibodies is associated with acute rejection episodes and renal allograft loss. Hum Immunol 2007, 68(5):362-367. 38. Terasaki PI, Ozawa M, Castro R: Four year follow-up of a prospective trial of HLA and MICA antibodies on kidney graft survival. Am J Transplant 2007, 7(2):408-415. A four-year analysis of a large cohort of patients demonstrating the role of both HLA and MICA antibodies in solid organ graft rejection. 39. Zou Y, Stastny P, Susal C, Dohler B, Opelz G: Antibodies against MICA antigens and kidney transplant rejection. N Engl J Med 2007, 357(13):1293-1300. 40. Pancoska C, Breed R, Harris P, Sonuga B, Venzon I: Emergent PRA technologies may detect unexpected antibodies in the sera of solid organ transplant recipients. One Lambda Advanced LABMAS Workshop: August 14th–17th; Los Angeles; 2007. 41. Rosen-Bronsen S: A*0301 specific HLA antibody detected in the serum of an A*0302 positive renal transplant recipient. One Lambda Advanced LABMAS Workshop: August 14th–17th; Los Angeles; 2007.
Current Opinion in Immunology 2009, 21:573–577
Solid phase assays for HLA antibody detection in clinical transplantation Brian D Tait The complement dependent microlymphocytotoxicity assay has been used for over 40 years for detecting HLA antibodies in transplant patients. This method has been replaced recently by more sensitive solid phase assays such as ELISA and bead based technology including the Luminex method. The introduction of these techniques into clinical practice has revealed previously undetected sensitisation in some patients and allowed the accurate assignment of antibody specificities directed at HLA-DQ and HLA-DP which was not previously possible. However it is emerging that despite the advantage of sensitivity some HLA antibodies defined by these assays are not associated with hyperacute or acute rejection. The role in allograft rejection of antibody titre and non-complement fixing antibodies, which are also detected by these methods, are areas currently under consideration. Address Victorian Transplantation and Immunogenetic Service, Australian Red Cross Blood Service, 2nd Floor Rotary Bone Marrow Research Building, c/o Royal Melbourne Hospital, Grattan St, Parkville 3050, Australia Corresponding author: Tait, Brian D ([email protected])
Current Opinion in Immunology 2009, 21:573–577 This review comes from a themed issue on Immunogenetics and Transplantation Edited by Andrea Velardi and Frank Christiansen
serum can be a confounding factor and CDC does not readily distinguish between IgG and IgM antibodies. Thirdly the relative low sensitivity of the CDC assay and its failure to detect low level antibody has come under question with the introduction of solid phase assays. Solid phase antibody detection assays comprise three types. First introduced was an enzyme-linked immunosorbent assay (ELISA) modified for the detection of HLA antibodies [2–4]. The second major development was the introduction of dye-impregnated beads bound with HLA molecules extracted from cell lines. Two fluorescent dyes are mixed in differing proportions that gives each type of bead a unique fluorescent signal. Test serum containing HLA antibody binds to the relevant bead(s) that is then detected using a fluorescent-labelled anti-human IgG antibody. Two methods for the detection of bound antibody are used. In the first instance the beads are analysed in a flow cytometer and a channel shift indicates the binding of HLA antibody [5]. The second method [6] involves the use of a Luminex (Luminex Corporation, TX, USA) fluorocytometer which has two lasers. One laser excites the dye in the bead and the other excites the phycoerythrin bound to the second antibody. The combination of the two signals indicates firstly the binding of HLA antibody, and secondly the bead carrying a specific HLA molecule to which the antibody is bound.
Available online 16th September 2009 0952-7915/$ – see front matter Crown Copyright # 2009 Published by Elsevier Ltd. All rights reserved. DOI 10.1016/j.coi.2009.07.017
Introduction The complement dependent cytotoxicity assay (CDC) developed over 40 years ago [1] has been the cornerstone technique for the detection of HLA antibodies since its introduction. Although this assay has served us well its limitations have been well documented. Firstly because it measures any antibody capable of binding to a cell surface and fixing complement it cannot discriminate between HLA and non-HLA antibodies. The assignment of HLA specificity is made by comparing the reactivity of the test serum with the HLA typing of the target cells. Since cells display co-dominant expression (i.e. two expressed products from each locus) and some alleles at neighbouring loci display linkage disequilibrium (LD) antibody specificity is assigned based on a probability calculation which is prone to error. Secondly the presence of autoantibodies in the test www.sciencedirect.com
There are three levels of testing for both HLA class 1 and class 2 with the beads at each level varying in the number of specific HLA molecules bound. The third level is the single antigen bead (SAG) in which just one HLA molecule produced by recombinant technology is bound to a specific bead type. SAG testing enables sera with complex mixtures of antibodies to be accurately dissected. Solid phase and, in particular, the bead assays are able to detect pre-sensitisation not previously possible with CDC [7–12,13,14,15,16,17]. This sensitivity has raised important issues concerning the clinical relevance of antibodies detected by these techniques. Since the bead assays are the most common assays now in use the emphasis of the review will be on that technique paying particular attention to the Luminex platform.
Clinical relevance of HLA antibodies detected by bead based assays in organ transplantation Since the first demonstration by Patel and Terasaki [18] that pre-formed HLA class 1 antibodies in renal Current Opinion in Immunology 2009, 21:573–577
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transplant recipients were associated with early rejection it has been an unchallenged rule that a pre-transplant crossmatch is performed to detect donor-specific HLA antibodies (DSHA) in the recipient and that a positive crossmatch with donor T lymphocytes (which detect class 1 but not class 2 antibodies) is a contraindication to transplantation. With the advent of more sensitive bead based assays sensitisation has undergone a re-definition. Gibney et al. [19] reported on a cohort of 136 renal transplant patients of whom 16 had pre-transplant antibodies as defined by CDC. By contrast an additional 39 patients (total 55) were shown to have antibodies to either HLA class 1 or class 2 by the Luminex assay. Twenty of these patients had DSHA and they had a higher rate of primary or delayed graft function, biopsy proven acute rejection (BPAR) and a lower rate of graft survival at six months post-transplant than the non-DSHA group. In this study, therefore, Luminex detected additional antibodies that proved to be clinically relevant. In a retrospective study of renal transplant patients with a negative CDC crossmatch Gupta et al. [20] tested the sera from the day of transplant for HLA antibodies using the Luminex assay. Sixteen out of 121 (13%) patients were shown to have DSHA by the Luminex technique which were undetectable at the time of transplantation by CDC. A further 22 patients were shown to have nonDSHA antibodies present. No hyperacute rejections were observed in the total patient cohort and there was no significant difference in the frequency of BPAR, delayed graft function, median creatinine levels at one year or graft survival at one year between the three groups that is antibody positive (DSHA and non-DHSA) and antibody negative. Multivariate analysis however did show an association of DSHA with long-term graft failure although the power of the study was limited. By contrast, van den Berg-Loonen et al. [21] reported on 34 highly sensitised patients (>85%PRA) transplanted as part of the Eurotransplant acceptable mismatch programme and were able to show a trend towards earlier rejection and graft loss in the Luminex detected DSHA group compared with the non-DSHA group. There was no difference in the long-term graft survival between the groups, however the numbers in the respective groups were small. In an elegant retrospective study of 565 heart transplant patients Smith et al. [22] showed a clear association of Luminex + CDC-DSHA with reduced graft survival. Fourteen patients had either class 1 or class 2 HLA antibodies by CDC but a further 53 patients were identified with HLA antibodies detectable only by the Luminex bead assay 14 of whom had DSHA. The actuarial one year graft survival for patients with CDC + Luminex + DSHA was 40% and 42% in the CDC Luminex + DSHA Current Opinion in Immunology 2009, 21:573–577
group. The graft survival in the group with Luminex + non-DSHA was 77% and 75% in those with no detectable antibodies. A detrimental effect of both HLA class 1 and class 2 antibodies detected only by the Luminex bead assay in a total cohort of 896 liver transplant patients was demonstrated by Castillo-Rama et al. [14]. CDC negativity was defined as a negative crossmatch with donor T and B lymphocytes. When the Luminex + CDC-group were subgrouped into Luminex class 1 + T cell-group, Luminex class 1 + B cell-group, Luminex class 2 + B cellgroup, and Luminex class 1 and 2 + T and B cell-group and compared with the comparable Luminex CDCgroups there was a significant decrease in graft survival in all four subgroups. There are several conclusions that can be drawn from the published data. The first is that transplanting across class 1 or 2 DSHA detectable by Luminex but not by CDC does not appear to cause hyperacute rejection in renal transplantation, and many of the patients in this category have uneventful post-transplant courses. The second observation is that this group of patients have an increased incidence of acute and chronic rejection and possibly a lower graft survival. Increased rejection rates seen in Luminex + CDC-renal patients with DSHA also appears to apply to heart and liver transplant patients. Pre-formed HLA class 1 DSHA detectable by CDC are associated with hyperacute rejection in renal transplantation. The solid phase assays and in particular the most sensitive Luminex based bead assay detect antibodies below the CDC threshold but by definition patients with these antibodies are sensitised. Why is there a difference in the clinical outcome of patients with antibodies detected by these two techniques? One explanation might simply relate to the titre of antibody present. Patients whose antibodies are only detected by the Luminex technique have lower titres than CDC positive patients due to the higher sensitivity of the former assay. The titre of HLA antibodies was shown by Mizutani et al. [23] to be directly related to maximum fluorescence values and the molecules of equivalent soluble fluorochrome (MESF) values as determined by Luminex. The average DSHA MESF values were correlated with graft rejection and the overall average MESF values in the rejection group were approximately 241 000 compared with 31 000 for the graft functioning group. Vaidya et al. have shown that antibodies detected by CDC have an MESF threshold of approximately 250 000 below which they are negative [24]. These findings indicate that the range of values obtained by Luminex will include antibodies that are not detected by CDC but that are able to cause graft rejection, but in addition some of the antibodies detected at the lower end of the MESF range by Luminex appear not to be overtly graft damaging at least www.sciencedirect.com
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in the short term. The long-term effect of these low titre antibodies remains to be seen. The application of Luminex technology therefore represents a paradigm shift and demonstrates that transplantation in a percentage of patients can be successful in the presence of low titres of DSHA. The other important consideration is the fact that the solid phase assays do not rely on complement binding for antibody detection and therefore a proportion of antibodies detected will be noncomplement binding. Non-complement binding antibodies and their detection by solid phase assays
Solid phase assays do not distinguish between complement binding (CB) and non-complement binding (NCB) antibodies. NCB antibodies to non-HLA antigens have been implicated in graft rejection [25] but there has been no systematic study on the clinical effects of NCB HLA antibodies. Their presence however in the eluates of over a quarter of rejected kidneys has been demonstrated [26]. Current practices in the use of solid phase assays exclude patients with NCB antibodies against a prospective donor though there is little evidence that NCB DSHA are harmful. If NCB are not damaging to grafts then compatible transplants are being denied to some patients. There is evidence to conclude that NCB antibodies do occur in a substantial percentage of renal patients awaiting a transplant. Using anti-IgG2 and IgG4 antibodies in modified ELISA and Luminex bead based assays Arnold et al. [27] have demonstrated that 40% of potential re-transplant patients on the waiting list had either class 1 and/or class 2 antibodies of NCB subclasses. Similar results were obtained by Wahrmann et al. [28] modifying the Flow based bead assay (FlowPRA, One Lambda Inc.) by adding normal serum as a source of complement and using anti-C4d as a second antibody. A striking example of the clinical relevance of HLA NCB antibodies was shown by Smith et al. [22] who modified the Luminex bead assay in a similar fashion to Wahrmann et al. [28]. They demonstrated a 20% graft survival in heart transplant patients with CB DSHA compared with 54% for patients with NCB DSA and 91% for patients with non-DSHA. These NCB antibodies may be playing a graft damaging role through a mechanism other than complement binding such as antibody dependent cell mediated cytotoxicity [29,30]. Further studies are required to fully elucidate the role of NCB in graft rejection.
Additional advantages of bead technology for HLA antibody detection Solid phase HLA antibody detection methods and, in particular, the SAG assay have improved our understanding of the role of HLA antibodies in renal graft rejection. Firstly it has allowed for the first time the evaluation of www.sciencedirect.com
HLA-C, HLA-DRB3, HLA-DRB4, HLA-DRB5, HLADQA1, HLA-DQB1, HLA-DPA1, HLA-DPB1 and MICA antibodies in renal graft rejection. The introduction of beads binding just a single molecule has overcome the problem of LD and has revealed antibodies to these other loci products are present in sensitised transplant patients and may play a role in rejection. Secondly solid phase assays have permitted accurate definition of antibodies directed at shared epitopes revealing that many socalled multispecific responses can be shown to be a single antibody to a shared epitope. Using single antigen beads in the Luminex platform and analysing the data by Matchmaker [31] Duquesnoy et al. [32] showed in a cohort of 75 transplant patients that HLA antibodies could be detected to products of HLADRB1, HLA-DRB3, HLA-DRB4, HLA-DPA1, HLADPB1 and to both chains of the DQ heterodimer (DQa, DQb). Interestingly approximately a third of patients had antibodies to either HLA-DPA or HLADPB. This is in agreement with an earlier observation by Piazza et al. [33]. When reactivity was correlated with amino acid sequences it was revealed that the DEAV sequence at positions 84–89 of the DPB1 chain was implicated in 51% of cases suggesting this is a particularly immunogenic epitope. We have previously described a female renal patient who was immunised to this DPB1 epitope from a first transplant and underwent reversible C4d positive rejection of a second transplant mismatched at only one DP allele which carried the DEAV epitope [34]. Other reports have also associated the presence of HLA-DP antibodies with allograft rejection [35,36]. The analysis by Duquesnoy et al. [32] demonstrated 87% of anti-class 2 responses include antibodies to DQB1 coded antigens. The MatchMaker eplet analysis shows there are significantly more potential eplet mismatches for DQB1 than DRB1 which probably explains this observation. Likewise the incidence and damaging role of antibodies to MICA in renal transplant patients undergoing rejection has been convincingly demonstrated by the use of Luminex [37,38,39]. In renal transplantation donor/recipient matching is generally restricted to HLA A, B and DRB1. With the realisation that some patients are immunised to products of other loci such as DQ (A and B), DP (A and B) and MICA more emphasis will be placed in future on avoiding transplanting across these incompatibilities, or at least transplanting in the knowledge that particular care in the form of early rejection prophylaxis may be required. An additional finding which has emerged from the use of single antigen beads has been the discovery that antibodies can be raised to alleles within the same serological group as the antibody producer. For example an A*2403 renal transplant recipient produced a DSHA to A*2402 Current Opinion in Immunology 2009, 21:573–577
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which was associated with rejection [40]. There are other examples appearing in the literature such as an A*0302 individual producing an antibody to A*0301 [41]. The implications of these findings are that some recipient/ donor combinations will require allele typing in order to establish compatibility.
4.
Monien S, Salama A, Schonemann C: ELISA methods detect HLA antibodies with variable sensitivity. Int J Immungenet 2006, 33(3):163-166.
5.
Rebibou JM, Chabod J, Bittencourt MC, Thevenin C, Chalopin JM, Herve P, Tiberghien P: Flow PRA evaluation for antibody screening in patients awaiting kidney transplantation. Transpl Immunol 2000, 8(2):125-128.
6.
Colombo MB, Haworth SE, Poli F, Nocco A, Puglisi G, Innocente A, Serafini M, Messa P, Scalamogne M: Luminex technology for anti-HLA antibody screening: evaluation of performance and of impact on laboratory routine. Cytometry B Clin Cytom 2007, 72(6):4645-4671.
7.
Parissiadis A, Marbach N, Leisenbach R, Wolf P, Cazenave JP, Tongio MM: Identification of HLA class 1 and class 2 antibodies: A comparison of LAT1240 ELISA and cytotoxicity. Eur J Immunogenet 2001, 28(2):360.
8.
Uboldi de Capei M, Pratico L, Curtoni ES: Comparison of different techniques for detection of anti-HLA antibodies in sera from patients awaiting kidney transplantation. Eur J Immunogenet 2002, 29(5):379-382.
9.
Levin MD, de Veld JC, van der Holt B, van’t Veer MB: Screening for alloantibodies in the serum of patients receiving platelet transfusions: a comparison of the ELISA, lymphocytotoxicity and the indirect immunoflourescence method. Transfusion 2003, 43(1):72-77.
Conclusions For the first time solid phase assays have provided tools for exposing low levels of immunisation which was not possible using conventional CDC. The issue, however, is how to use this information to achieve optimal success rates. Some renal transplant patients with antibodies detectable by Luminex but not CDC have uneventful post-transplant courses whereas others have a detrimental outcome. The reason for this is not clear although the lower titre of antibody and the fact that Luminex also detects non-complement binding antibodies are two possibilities. The use of single antigen beads in the Luminex technique has clarified the role of antibodies directed at products of the HLA-C, HLA-DRB3, HLA-DRB4, HLA-DRB5, HLA-DQA1, HLA-DQB1, HLA-DPB1 and MICA loci on renal allograft rejection which was not possible using the CDC technique. The use of single antigen beads also allows accurate designation of shared epitopes between positively reacting HLA molecules which enables avoidance of these epitopes on future transplants. The introduction of solid phase assays and, in particular, the Luminex system has ushered in a new era in the definition of HLA pre-sensitisation. The challenge now is to understand more fully the humoral alloresponse and its association with graft rejection and to use this technique for the optimal management of the transplant patient.
Acknowledgements The author wishes to acknowledge the professional expertise of Linda Cantwell, Fiona Hudson and Gemma Brewin who established the Luminex technique in the VTIS laboratories and who provide ongoing support in the interpretation of results.
References and recommended reading Papers of particular interest, published within the period of review, have been highlighted as: of special interest of outstanding interest 1.
Terasaki PI, McClelland JD: Microdroplet assay of human serum cytotoxins. Nature 1964, 204:998-1000.
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Worthington JE, Robson AJ, Sheldon S, Langton A, Martin S: A comparison of enzyme-linked immunoabsorbent assays and flow cytometry techniques for the detection of HLA specific antibodies. Hum Immunol 2001, 62(10):1178-1184.
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10. Altemann WW, Seliger B, Sel S, Wendt D, Schlaf G: Comparison of the established standard complement-dependent cytotoxicity and flow cytometric crossmatch assays with a novel ELISA based HLA crossmatch procedure. Histol Histopathol 2006, 21(10–12):1115-1124. 11. Muro M, Llorente S, Gonzales-Soriano MJ, Minguela A, Gimeno L, Alvarez-Lopez MR: In Pre-formed Donor-specific Alloantibodies (DSA) Detected only by Luminex Technology using HLA-coated Microspheres and Causing Acute Humoral Rejection and Kidney Graft Dysfunction. Edited by Terasaki PI. Clinical Transplants Pub. Terasaki Foundation Laboratory; 2006:379-383. 12. Lee PC, Ozawa M: In Reappraisal of HLA Antibody Analysis and Crossmatching in Kidney Transplantation. Edited by Cecka MJ, Terasaki PI. Clinical Transplants Pub Terasaki Foundation Laboratory; 2007:219-226. 13. Fuggle SV, Martin S: Tools for human leukocyte antigen antibody detection and their application to transplanting sensitized patients. Transplantation 2008, 86(3):384-390. Describes various techniques for HLA antibody detection and their application in defining pre-sensitisation in potential transplant patients. 14. Castillo-Rama M, Castro MJ, Bernardo I, Meneu-Diaz JC, Elola Olaso AM, Calleja-Antolin SM, Romo E, Morales P, Moreno E, PazArtal E: Preformed antibodies detected by cytotoxic assay or multibead array decrease liver allograft survival: role of human leukocyte antigen compatibility. Liver Transplant 2008, 14(4):554-562. Demonstrates the importance of the Luminex technique in detecting HLA antibodies which are shown to be associated with rejection in liver transplant patients. 15. Saidman SL: Histocompatibility testing for highly sensitised transplant candidates. Trans Proc 2007, 39(3):673-675. 16. Leffell MS, Montgomery RA, Zachary AA: The changing role of antibody testing in transplantation. In Clin. Transpl.. Edited by Cecka MJ, Terasaki PI. 2005:259-271. 17. Zeevi A, Girnita A, Duquesnoy R: HLA antibody analysis: sensitivity, specificity and clinical significance in solid organ transplantation. Immunol Res 2006, 36(1–3):255-264. HLA antibody detection by solid phase assays particularly the bead based methods have allowed a more accurate analysis of shared epitope structures and their relevance to transplant outcome. 18. Patel R, Terasaki PI: Significance of the positive crossmatch test in kidney transplantation. New Eng J Med 1969, 280(14):735-739. 19. Gibney EM, Cagle LR, Freed B, Warnell SE, Chan L, Wiseman AC: Detection of donor specific antibodies using HLA coated www.sciencedirect.com
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microspheres: another tool for kidney transplant risk stratification. Nephrol Dial Transplant 2006, 21(9):2625-2629. Renal patients transplanted with a negative AHG-CDC donor crossmatch were retrospectively tested by Luminex for the presence of HLA antibodies. Patients with donor-specific antibodies (DSA) had higher rates of biopsy proven rejection and lower graft survival than patients without DSA. 20. Gupta A, Iveson V, Varagunam M, Bodger S, Sinnott P, Thuraisingham RC: Pretransplant donor-specific antibodies in cytotoxic negative crossmatch kidney transplants: are they relevant? Transplantation 2008, 85(8):1200-1204. Renal patients with negative CDC donor crossmatches were tested for the presence of HLA antibodies by solid phase assays. While no shortterm differences in rejection incidence were noted between patients with donor-specific antibodies (DSA), those with non-DSA, and antibody negative patients, there was increased long-term graft loss in the DSA group. 21. van den Berg-Loonen EM, Billen EVA, Voorter CVM, van Heurn LWE, Claas FHJ, van Hooff JP, Christiaans MHL: Clinical relevance of pre transplant donor-directed antibodies detected by single antigen beads in highly sensitised renal transplant patients. Transplantation 2008, 85(8):1086-1090. 22. Smith JD, Hamour IM, Banner NR, Rose ML: C4d fixing Luminex antibodies — a new tool for prediction of graft failure after heart transplantation. Am J Transplant 2007, 7(12):2809-2815. An elegant study describing modifications to the Luminex technique to detect both complement binding (CB) and non-complement binding (NCB) HLA antibodies in heart transplant patients. While a significant negative effect of CB antibodies on graft survival was demonstrated a lesser negative effect of NCB antibodies was also shown. 23. Mizutani K, Terasaki P, Hamdani E, Esquenazi V, Rosen A, Miller J, Ozawa M: The importance of anti-HLA-specific antibody strength in monitoring kidney transplant patients. Am J Transplant 2007, 7:1027-1031. First reported correlation of antibody titre as defined by the Luminex technique and expressed as molecules of equivalent soluble fluorochrome (MESF), with graft outcome. Patients with graft failure had significantly higher MESF values than non-rejection patients. 24. Vaidya S, Partlow DA, Suskind B, Gugliuzzi K: Prediction of crossmatch outcome of highly sensitised patients based on identification of serum HLA class 1 and 2 antibodies by single and/or multiple antigen bead Luminex assay. Abstract no. 670. The First Joint International Transplant Meeting; Boston, MA, July: 2006. 25. Dragun D: Humoral responses directed against non-human leukocyte antigens in solid organ transplantation. Transplantation 2008, 86(8):1019-1025. 26. Heinemann FM, Roth I, Rebmann V, Arnold ML, Witzke O, Wilde B, Spriewald BM, Grosse-Wilde H: Immunoglobulin isotypespecific characterization of anti-human leukocyte antigen antibodies eluted from explanted renal allografts. Hum Immunol 2007, 68(6):500-506. The presence of both complement binding (CB) and non-complement binding (NCB) HLA antibodies were demonstrated in acid eluates of rejected renal allografts. 28% of IgG positive eluates contained NCB antibodies. 27. Arnold M-L, Dechant M, Doxiadis IIN, Spriewald BM: Prevalence and specificity of immunoglobulin G and immunoglobulin A non-complement binding anti-HLA alloantibodies in renal transplant candidates. Tissue Antigens 2008, 72(1):60-66. The Luminex assay was modified in order to detect the four isotypes of IgG. 40% of previously transplanted renal patients’ sera contained HLA antibodies of the non-complement binding isotypes IgG2 and IgG4, the majority of which were donor specific. 28. Wahrmann M, Exner M, Regele H, Derfler K, Kormoczi GF, Lhotta K, Zlabinger GJ, Bohmig GA: Flow cytometry based detection of HLA alloantibody mediated classical complement activation. J Immunol Methods 2003, 275(1–2):149-160.
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A Flow based method is described for detecting the C4 component of complement generated as a result of complement binding and activation by HLA antibodies. 29. Vongwiwatana A, Tasanarong A, Hidalgo LG, Halloran PF: The role of B cells and alloantibody in the host response to human organ allografts. Immunol Rev 2003, 196:197-218. 30. Wehner J, Morrell CN, Reynolds T, Rodriguez ER, Baldwin WM: Antibody and complement in transplant vasculopathy. Circ Res 2007, 100(2):191-203. 31. Duquesnoy RJ, Askar M: HLAMatchmaker: a molecularly based algorithm for histocompatibility determination. V. Eplet matching for HLA-DR, HLA-DQ and HLA-DP. Hum Immunol 2007, 68(1):12-25. This paper describes the design of the HLAMatchmaker programme which explains reactivity of HLA class 2 antibodies in terms of eplet (three amino acid) recognition sequences. This programme has permitted sera which react with multiple HLA molecules to be analysed in terms of shared epitopes. 32. Duquesnoy R, Awadalla Y, Lomago J, Jelinek L, Howe J, Zern D, Hunter B, Martell J, Girnita A, Zeevi A: Retransplant candidates have donor-specific antibodies that react with structurally defined HLA-DR, DQ, DP epitopes. Transplant Immunol 2008, 18(4):352-360. Using the single antigen Luminex assay and the HLAMatchmaker programme (Ref. [31]) the fine specificity of HLA class 2 antibodies are described in terms of eplets and shared epitopes. 33. Piazza A, Poggi E, Ozella G, Borreli L, Scornajenghi KA, Monaco PI, Tisone G, Adorno D: Anti-DP antibodies and graft failure in kidney transplantation. Abstract no. 520. The First Joint International Transplant Meeting; Boston, MA, July: 2006. 34. Blair S, Tait B, Hudson F, Kannellis J: Acute humoral rejection associated with HLA-DPB1 antibody in an HLA identical (A,B,DR,DQ) recipient. Abstract. Transplantation Society of Australia and New Zealand Annual Scientific Meeting; Canberra, Australia, April: 2007. 35. Qui J, Cai J, Terasaki PI, El-Awar N, Lee JH: Detection of antibodies to HLA-DP in renal transplant recipients using single antigen beads. Transplantation 2005, 80(10):1511-1513. 36. Arnold ML, Pei R, Spriewald B, Wassmuth R: Anti-HLA class 2 antibodies in kidney re-transplant patients. Tissue Antigens 2005, 65:370-378. 37. Panigrahi A, Gupta N, Siddiqui JA, Margoob A, Bhowmik D, Guleria S, Mehra NK: Post transplant development of MICA and anti-HLA antibodies is associated with acute rejection episodes and renal allograft loss. Hum Immunol 2007, 68(5):362-367. 38. Terasaki PI, Ozawa M, Castro R: Four year follow-up of a prospective trial of HLA and MICA antibodies on kidney graft survival. Am J Transplant 2007, 7(2):408-415. A four-year analysis of a large cohort of patients demonstrating the role of both HLA and MICA antibodies in solid organ graft rejection. 39. Zou Y, Stastny P, Susal C, Dohler B, Opelz G: Antibodies against MICA antigens and kidney transplant rejection. N Engl J Med 2007, 357(13):1293-1300. 40. Pancoska C, Breed R, Harris P, Sonuga B, Venzon I: Emergent PRA technologies may detect unexpected antibodies in the sera of solid organ transplant recipients. One Lambda Advanced LABMAS Workshop: August 14th–17th; Los Angeles; 2007. 41. Rosen-Bronsen S: A*0301 specific HLA antibody detected in the serum of an A*0302 positive renal transplant recipient. One Lambda Advanced LABMAS Workshop: August 14th–17th; Los Angeles; 2007.
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