doi: 10.1111/iji.12184
Meeting report: 3rd international transplant conference: how much risk can you take? D. Lowe*, S. Daga†,‡, D. Briggs§, N. Khovanova¶, D. Mitchell†, R. Higgins‡ & N. Krishnan‡
Summary The 3rd International Transplant Conference took place on 31st October and 1st November 2014 at the University of Warwick, Coventry, UK. Key focal points of the meeting were the exploration of the molecular basis of antibody–antigen interactions and their relation to clinical practice and to share experiences and knowledge regarding strategies to transplant the ‘high-risk’ patient. In addition, lively debate sessions were hosted where controversial clinical and immunological themes were discussed by leading experts in the field.
Introduction The growing disparity between the number of patients presenting with renal failure necessitating transplantation and the availability of donor organs has led to concerted efforts to increase the rates of live donor transplantation. Given that over 30% of patients awaiting transplantation in the UK are now sensitized against HLA antigens, it has increasingly become standard practice to transplant against both HLA and ABO antibody barriers. As outlined in the previous meeting back in 2012, the sharing of knowledge and clinical procedures in these high-risk cases is key to successful management and prolonged graft survival (Lowe et al., 2013). This meeting explored the factors that contribute to HLA immunogenicity and the factors that influence
*Transplant Immunology, Royal Liverpool and Broadgreen University Hospital, Liverpool, UK, †Clinical Sciences Research Laboratories, University of Warwick, Coventry, UK, ‡Renal Department, University Hospital Coventry and Warwickshire, Coventry, UK, §Department of Histocompatibility and Immunogenetics, NHS Blood and Transplant, Birmingham, UK and ¶School of Engineering, University of Warwick, Coventry, UK Received 12 December 2014; revised 9 January 2015; accepted 27 January 2015 Correspondence: Dave Lowe, Transplant Immunology Department, Royal Liverpool and Broadgreen University Hospital, L7 8XP Liverpool, UK. Tel: 0151 706 4366; Fax: 0151 706 5814; E-mail: [email protected]
© 2015 John Wiley & Sons Ltd International Journal of Immunogenetics, 2015, 0, 1–10
the pathogenicity of donor-specific antibody responses. Following on from the previous meeting in 2012 which comprehensively reviewed the prevalence and importance of transplant infectious disease, the meeting this time focussed on clinical risk management in renal transplantation, pinpointing not just the transplant recipient but also on factors which impact on donor assessment and selection.
Antibody-Mediated Rejection (AMR) and aspects of antibody–antigen interactions: 31st October 2014 Professor Rob Higgins set the scene by summarizing key issues around antibody-incompatible kidney transplantation (AiT) from a UK perspective. He highlighted difficulties in diagnosing antibody-mediated damage to the allograft with current technologies. He noted that conventional immunohistochemistry often does not demonstrate antibody in biopsy material, even during acute antibody-mediated rejection. Antibody is presumably present, as indicated by studies eluting donor-specific HLA antibodies from graft biopsies (Martin et al., 2003). Similarly, complement binding and activation may not be universal during acute antibody-mediated rejection and hence the recent down-grading of C4d-positive staining in the latest Banff criteria (Haas et al., 2014). A key marker of AMR is usually high cellular infiltration and staining for CD45 (expressed at high levels on nucleated hematopoietic cells) observed early, combined with CD68 & CD3 staining for mononuclear cells and T cells that are positive during AMR (Higgins et al., 2010). Graft outcome is linked to the level of antibodies, and positive complement-dependant cytotoxicity (CDC) crossmatch prior to transplant was particularly associated with diminished graft function at 10 years (in the Coventry series, 85%. Derek O’Neill from Beaumont Hospital, Dublin, Ireland, routinely tests
© 2015 John Wiley & Sons Ltd International Journal of Immunogenetics, 2015, 0, 1–10
How much risk can you take?
HLA-specific antibodies using two commercially available LUMINEX kits and only considers specificities defined by both as truly positive. The outcomes of grafts with single test antibodies (STAs) were comparable to DSA negative cases. However, it was noted that seven of 12 cases with STAs had a positive flow cytometry (FC) cross-match, and they received augmented immunosuppressive treatment. The next abstract, also from Dublin and presented by Dr Colm Magee, focused on the Irish experience of linking into the paired exchange program in the UK. Of 39 cases listed, twelve were transplanted (four via paired kidney exchange, five direct donations with AiT, the remainder from standard deceased donation). The analysis pointed out that despite multiple runs of the paired exchange scheme there are significant numbers of cases not getting an offer. This was followed by data from Dr Sian Griffin from Cardiff, the UK, on integrating desensitization within the national living donor kidney sharing scheme (NLDKSS) in UK. Selective delisting of antibodies (HLA-specificities with MFI < 5000 in LUMINEX testing using a neat sample that falls with dilution at 1:16) resulted in decreased cRF and allowed five paired donations to proceed. These recipients had good graft function (mean eGFR 69 17 mL min 1) and no episodes of AMR during follow-up of 4–30 months post-transplantation. The final abstract in this session was on the use of machine learning and decision tree analysis from Ms Torgyn Shaikhina, a PhD student in the School of Engineering at the University of Warwick, Coventry, UK. She demonstrated how this sophisticated analysis tool could handle complex clinical and laboratory data with multiple parameters and predict rejection with 86% accuracy. She noted that, based on the AiT programme at Coventry, the top three risk factors for AMR were single highest DSA MFI (the MFI of the highest reactive donor-specific antigen bead), total number of mismatches and interestingly the presence or absence of IgG4 DSA pretransplantation. The afternoon session focused on antibody–antigen interactions and presentations involved techniques used for exploring antigenicity/immunogenicity of antigen (epitopes) and pathogenicity of HLA-specific antibodies. Dr Dave Lowe, from Liverpool, UK, presented data demonstrating the potential underlying mechanisms resulting in positive CDC cross-match results. He demonstrated that absolute values of luminex derived MFI did not predict a positive CDC cross-match and that multiple antibodies specific for multiple epitopes on the same HLA molecule or epitopes presented on different HLA molecules presented on the same cell could explain positive CDC results. This is due to cross-linking of C1q complexes between antibodies binding to different epitopes, a crucial requirement for effective complement fixation and cell lysis. He also demonstrated antibodies recognizing combinations of epitopes situated in very close proximity on the cell surface results in a
© 2015 John Wiley & Sons Ltd International Journal of Immunogenetics, 2015, 0, 1–10
3
blunting of the CDC response and presented further data which may explain the reduced MFI values often observed at neat in samples with high levels of antibodies measured by microbead assays at serial dilution. This was followed by a talk from Dr Dermot Mallon from Cambridge University, UK, who demonstrated a novel way of predicting immunogenicity of antigen/epitope using electrostatic potential measurements and computational HLA-alloantibody interactions (Mallon et al., 2014). He made the key point that alloantigen sequence information alone would only provide limited insight into the key determinants of immunogenicity. The assessment of the electrostatic properties of the surface residues of HLA molecules could be crucial to our understanding of HLA immunogenicity and key to informing clinical practice when transplanting across HLA-specific antibody barriers. Dr Sunil Daga from Warwick University, Coventry, UK, then described his work involving real-time biosensor assays based on surface plasmon resonance to measure the binding kinetics and affinity of HLAspecific antibodies. Using human monoclonal antibodies generously donated from the University of Leiden and HLA proteins from the University of Oklahoma, binding interactions between HLA protein and these antibodies were studied. Interestingly, the binding kinetics and strength were different for same epitope– paratope interactions between different alleles of HLA. The higher affinities were shown to be for alleles that were the same as the primary immunizing antigen. Preliminary work on polyclonal HLA-specific antibodies carefully isolated from patient sera showed heterogeneity of affinity, not previously demonstrated using specific molecular binding assays. Further clinical correlation studies are ongoing to ascertain how detailed knowledge of HLA-specific antibody affinity and avidity can be used to inform clinical practice. The day was concluded by a talk given by Prof Richard Pleass from the Parasitology department at the University of Liverpool, UK who completed the session’s focus on antibody–antigen interactions by describing novel in vitro alternatives to intravenous immunoglobulin (IVIg). Currently, 46 standard blood donations are required in order to isolate enough specific immunoglobulin to constitute a single therapeutic dose of IVIg. He described how current IVIg treatment also involves large infusion volumes as only 5% of injected IVIg is active in vivo, and he suggested that this could be due to fewer terminal sialyic acid residues on the N-linked oligosaccharide located on the Fc domain of human IgG1. He then went on to describe a novel hexameric form of IgG that could be more effective than IVIg. This exciting molecule has been described as naturally occurring and can be manufactured in laboratories with the potential to revolutionize this therapeutic area (Diebolder et al., 2014).
4
D. Lowe et al.
Histocompatibility and immunogenetics workshop: 1st November 2014 The workshop session began with Professor Derek Middleton from the Transplant Immunology laboratory at the Royal Liverpool University Hospital, UK, providing a local perspective on how laboratories should approach the difficult task of assigning HLAspecific antibodies. The advent of solid phase assays, in particular the single antigen bead (SAB) assay on the LUMINEX platform, has led to unheralded levels of HLA-specific antibody characterization. However, the use of SAB data provides a series of difficult challenges and devising robust strategies to accurately define which antibodies could be clinically relevant is challenging (Middleton et al., 2013, 2014). The current understanding of the advantages and disadvantages of SAB assays was discussed and how data from these assays are used to support a Virtual Crossmatching (VXM) programme in Liverpool. The criteria used in Liverpool for determining which specificities detected by SAB assay are to be listed as unacceptable antigens was discussed and showed that this is an area where much further investigation is needed. Assigning positive cut-off thresholds for unacceptable antigens, determining which reactions were due to genuine HLA-reactivity, and identifying when luminex assays may have saturated are all issues to be considered when defining clinical relevance of HLA-specific antibodies. It was also suggested that the increasing understanding of HLA epitopes will lead to enhanced matching in solid organ transplantation. Data were presented which demonstrated how relatively poorly matched grafts assessed by standard antigen levelmatching criteria could in fact turn out to be completely free of disparity when the repertoire of epitopes expressed by both donor and recipient were analysed. Miss Maria Irvine, an undergraduate student from Liverpool John Moore’s University, UK, and on placement at the Transplant Immunology Department in Liverpool, presented data which compared the relative merits of SAB technology and traditional CDC analysis as a means of identifying clinically relevant HLAspecific antibodies. As expected, the mean number of antibody specificities detected by SAB was far greater than CDC; however, there were a small number of cases studied which gave clear antibody specificities on CDC analysis which were initially negative by SAB assays. It was only when serum was further analysed following serial dilution that these specificities were then detectable by SAB assay. This highlights the potential dangers of relying solely on microbeads for antibody analysis and also the need for potential costprohibitive repeat testing of samples under various test conditions to ensure all antibody specificities are detected. Dr Natasha Khovanova from the University of Warwick, Coventry, UK then presented novel data highlighting the impact of pretransplant donor-specific
IgG4 on graft survival following AiT. In a cohort of 77 AiT cases, it was clearly demonstrated that when pretransplant DSA of the IgG4 subclass was positive there was a significant increase in the likelihood of acute antibody-mediated rejection. The use of sophisticated binary regression analysis also showed that the occurrence of acute AMR seen (approaching 50% and confirmed earlier in the meeting in US data by Dr Orandi) was heavily influenced by three factors: total IgG4 MFI levels, highest single DSA MFI level and the total number of mismatches between donor and recipient. For example, for each additional 1000 MFI increase in IgG4 DSA level, there was a 30% increase in the likelihood of rejection. Long-term graft survival was also shown to be significantly reduced in IgG4 DSA positive cases (P = 0.004), highlighting the potential of pretransplant IgG4 to be used as a biomarker for kidney transplant rejection. Mr James Jones from the Transplant Immunology department, Royal Liverpool University Hospital, UK, then presented his recent work on developing computer-based algorithms for defining HLA-specific reactivity patterns in both SAB assays and CDC analysis data. Alternative methods of antibody analysis (HLAMatchmaker and the epitope analysis module within One Lambda’s HLA Fusion software) were explored, and it was shown that particular components of each can be inadequate. It was also shown how epitopes can be incorporated into an automated algorithm to improve antibody analysis. Parameters of 2 9 2 contingency tables were discussed with emphasis on sensitivity and True Positive frequency (Antigen Present/ Test Positive). These are more reliable measures to define antibodies than the R value correlation coefficient used widely, especially for serum with antibodies to many HLA-specificities. The algorithm allowed complex CDC assay reaction patterns to be resolved to the epitope level, where parameters such as epitope frequency and combinations within different analysis panels could investigated. Antibody profiles of SAB assays could also be translated accurately into epitopes, which allowed MFI thresholds to be better placed by assessing grouped reactive beads by the shared epitopes identified and in addition provided a fresh perspective on how to recognize those bead reactions attributable to variable HLA target quality. Completing the morning session was Mr Harry Moyse, another PhD student from Warwick University School of Engineering, Coventry, UK. He presented key work from his thesis exploring the mathematical modelling of antibody–antigen interactions (Evans et al., 2012). This work is carried out on the biosensor assays described on day one by Dr Daga. He described how it is possible to develop highly refined computational modelling techniques in order to obtain accurate estimates of affinities of HLA, and ABO-specific antibodies both in monoclonal antibody preparations but also more impressively in complex polyclonal mixtures of antibodies as would be expected in the serum com-
© 2015 John Wiley & Sons Ltd International Journal of Immunogenetics, 2015, 0, 1–10
How much risk can you take?
partment of highly sensitized transplant patients. It was clear from the data presented that with the development of ever more sophisticated assays to define antibodies comes a clear need for the kind of high level mathematical and computational analysis demonstrated here. The workshop then concluded with a debate in which the relative merits of the long-standing CDC assay and its place in the 21st century tissue typing laboratory testing repertoire were passionately discussed. It is only about 50 years since the microcytotoxicity assay was developed by Terasaki and McClelland (Terasaki & McClelland, 1964). In essence, this assay is still widely used today, mainly for a pretransplant cross-match, because of the 1969 publication showing the risk of early rejection associated with a positive CDC cross-match (Patel & Terasaki, 1969). There are many methodological versions of this assay, but also many more recent alternatives, so Dr Robert Vaughan of Guy’s/Kings/Viapath, London, UK, proposed the assertion ‘50 years of CDC: enough is enough’ and debated this with Professor D Briggs of NHSBT Birmingham, UK, who took a position of ‘not really’. Guy’s HLA laboratory was an early adopter of flow cytometry (FC)-based detection of HLA-specific antibodies and after 30 years has continued its innovation with this technology, including using a FC titre to predict how many plasma exchange treatments are necessary to remove DSA to an acceptable level for AiT [Shaw O, PhD thesis, University of London.]. Dr Vaughan asserted that this is one of many examples where FC is superior to CDC for detection of HLAspecific antibody: it is a more sensitive technique, the read-out is quantitative and it is far better at indicating relative levels. In addition, FC is intrinsically more objective in its measurements. The inferiority of CDC in these assay parameters suggests that 50 years are more than enough of what can seem an archaic assay. Comparing CDC with other HLA antibody detection methods on the basis of sensitivity and quantitation is, however, a profound mistake said Prof Briggs: they measure different things. FC in this context measures how much antibody binds to the cell, while CDC measures activity of the antibodies bound to that cell. Briggs therefore made the point that CDC continues to be a relevant histocompatibility technique because it measures the one thing that none of the others does and such a functional assay has obvious clinical relevance. Using CDC to detect antibody is clearly not sensible – there are far better methods for this, particularly FC. Anti-human globulin (AHG) augmentation of CDC is probably not a useful assay variation in this respect because this becomes primarily a detection assay but with a functional read-out (i.e. detection by AHG, as in FC). Vaughan and Briggs agreed that the use of rabbit serum as the source of complement in the CDC assay is a problem because this does not resemble the physiological state. While conceding low
© 2015 John Wiley & Sons Ltd International Journal of Immunogenetics, 2015, 0, 1–10
5
sensitivity may seem to be a flaw of CDC used for cross-matching, Briggs did suggest that, on the contrary, this assay might be too sensitive and would be improved by replacing the rabbit serum with human serum, perhaps autologous serum. The audience was asked to vote, and in the absence of a formal count, the result was not recorded, but this part of this meeting report was written by the victor – CDC lives to fight another day.
Clinical session: 1st November 2014 End-stage renal disease (ESRD) is increasing worldwide. As potent immunosuppressive medications, intense pretransplant screening and improved surgical techniques are increasingly accessible, more and more high-risk patients are getting transplanted. As a consequence, there is a push to increase the donor numbers, which inadvertently results in acceptance of borderline donors for transplantation. The clinical session on day 2 aimed to discuss this potentially dangerous territory under the theme of how much risk we can take in kidney transplantation. Dr Nithya Krishnan, Coventry, UK, discussed the issue of body mass index in kidney transplantation recipients. Obesity is on the increase worldwide, as is end-stage renal disease (ESRD) (Van Lerberghe, 2008). Kidney transplantation is the treatment of choice for ESRD. However, patients with high BMI are disadvantaged worldwide as obesity is considered a contraindication for transplantation. There have been contradictory reports with regard to transplanting patients with a high BMI (Meier-Kriesche et al., 2002) (Glanton et al., 2003; Ng et al., 2014). Dr Krishnan presented their study, which investigated the effect of BMI on mortality in transplanted and patients remaining on the waiting list in the UK. The group analysed the UK Renal Registry and NHSBT Organ Donation and Transplantation, UK, data for patients listed from 1st January 2004 to 31st December 2010, with follow-up until 31st December 2011. Kaplan–Meier survival plots and Cox proportional hazards models were used to estimate and compare patient and graft survival for transplanted and listed patients with BMI recorded. A total of 17 681 patients were listed during the study period, with BMI recorded for 13 526 (77%). Five-year patient survival was significantly better in all BMI bands (
Meeting report: 3rd international transplant conference: how much risk can you take? D. Lowe*, S. Daga†,‡, D. Briggs§, N. Khovanova¶, D. Mitchell†, R. Higgins‡ & N. Krishnan‡
Summary The 3rd International Transplant Conference took place on 31st October and 1st November 2014 at the University of Warwick, Coventry, UK. Key focal points of the meeting were the exploration of the molecular basis of antibody–antigen interactions and their relation to clinical practice and to share experiences and knowledge regarding strategies to transplant the ‘high-risk’ patient. In addition, lively debate sessions were hosted where controversial clinical and immunological themes were discussed by leading experts in the field.
Introduction The growing disparity between the number of patients presenting with renal failure necessitating transplantation and the availability of donor organs has led to concerted efforts to increase the rates of live donor transplantation. Given that over 30% of patients awaiting transplantation in the UK are now sensitized against HLA antigens, it has increasingly become standard practice to transplant against both HLA and ABO antibody barriers. As outlined in the previous meeting back in 2012, the sharing of knowledge and clinical procedures in these high-risk cases is key to successful management and prolonged graft survival (Lowe et al., 2013). This meeting explored the factors that contribute to HLA immunogenicity and the factors that influence
*Transplant Immunology, Royal Liverpool and Broadgreen University Hospital, Liverpool, UK, †Clinical Sciences Research Laboratories, University of Warwick, Coventry, UK, ‡Renal Department, University Hospital Coventry and Warwickshire, Coventry, UK, §Department of Histocompatibility and Immunogenetics, NHS Blood and Transplant, Birmingham, UK and ¶School of Engineering, University of Warwick, Coventry, UK Received 12 December 2014; revised 9 January 2015; accepted 27 January 2015 Correspondence: Dave Lowe, Transplant Immunology Department, Royal Liverpool and Broadgreen University Hospital, L7 8XP Liverpool, UK. Tel: 0151 706 4366; Fax: 0151 706 5814; E-mail: [email protected]
© 2015 John Wiley & Sons Ltd International Journal of Immunogenetics, 2015, 0, 1–10
the pathogenicity of donor-specific antibody responses. Following on from the previous meeting in 2012 which comprehensively reviewed the prevalence and importance of transplant infectious disease, the meeting this time focussed on clinical risk management in renal transplantation, pinpointing not just the transplant recipient but also on factors which impact on donor assessment and selection.
Antibody-Mediated Rejection (AMR) and aspects of antibody–antigen interactions: 31st October 2014 Professor Rob Higgins set the scene by summarizing key issues around antibody-incompatible kidney transplantation (AiT) from a UK perspective. He highlighted difficulties in diagnosing antibody-mediated damage to the allograft with current technologies. He noted that conventional immunohistochemistry often does not demonstrate antibody in biopsy material, even during acute antibody-mediated rejection. Antibody is presumably present, as indicated by studies eluting donor-specific HLA antibodies from graft biopsies (Martin et al., 2003). Similarly, complement binding and activation may not be universal during acute antibody-mediated rejection and hence the recent down-grading of C4d-positive staining in the latest Banff criteria (Haas et al., 2014). A key marker of AMR is usually high cellular infiltration and staining for CD45 (expressed at high levels on nucleated hematopoietic cells) observed early, combined with CD68 & CD3 staining for mononuclear cells and T cells that are positive during AMR (Higgins et al., 2010). Graft outcome is linked to the level of antibodies, and positive complement-dependant cytotoxicity (CDC) crossmatch prior to transplant was particularly associated with diminished graft function at 10 years (in the Coventry series, 85%. Derek O’Neill from Beaumont Hospital, Dublin, Ireland, routinely tests
© 2015 John Wiley & Sons Ltd International Journal of Immunogenetics, 2015, 0, 1–10
How much risk can you take?
HLA-specific antibodies using two commercially available LUMINEX kits and only considers specificities defined by both as truly positive. The outcomes of grafts with single test antibodies (STAs) were comparable to DSA negative cases. However, it was noted that seven of 12 cases with STAs had a positive flow cytometry (FC) cross-match, and they received augmented immunosuppressive treatment. The next abstract, also from Dublin and presented by Dr Colm Magee, focused on the Irish experience of linking into the paired exchange program in the UK. Of 39 cases listed, twelve were transplanted (four via paired kidney exchange, five direct donations with AiT, the remainder from standard deceased donation). The analysis pointed out that despite multiple runs of the paired exchange scheme there are significant numbers of cases not getting an offer. This was followed by data from Dr Sian Griffin from Cardiff, the UK, on integrating desensitization within the national living donor kidney sharing scheme (NLDKSS) in UK. Selective delisting of antibodies (HLA-specificities with MFI < 5000 in LUMINEX testing using a neat sample that falls with dilution at 1:16) resulted in decreased cRF and allowed five paired donations to proceed. These recipients had good graft function (mean eGFR 69 17 mL min 1) and no episodes of AMR during follow-up of 4–30 months post-transplantation. The final abstract in this session was on the use of machine learning and decision tree analysis from Ms Torgyn Shaikhina, a PhD student in the School of Engineering at the University of Warwick, Coventry, UK. She demonstrated how this sophisticated analysis tool could handle complex clinical and laboratory data with multiple parameters and predict rejection with 86% accuracy. She noted that, based on the AiT programme at Coventry, the top three risk factors for AMR were single highest DSA MFI (the MFI of the highest reactive donor-specific antigen bead), total number of mismatches and interestingly the presence or absence of IgG4 DSA pretransplantation. The afternoon session focused on antibody–antigen interactions and presentations involved techniques used for exploring antigenicity/immunogenicity of antigen (epitopes) and pathogenicity of HLA-specific antibodies. Dr Dave Lowe, from Liverpool, UK, presented data demonstrating the potential underlying mechanisms resulting in positive CDC cross-match results. He demonstrated that absolute values of luminex derived MFI did not predict a positive CDC cross-match and that multiple antibodies specific for multiple epitopes on the same HLA molecule or epitopes presented on different HLA molecules presented on the same cell could explain positive CDC results. This is due to cross-linking of C1q complexes between antibodies binding to different epitopes, a crucial requirement for effective complement fixation and cell lysis. He also demonstrated antibodies recognizing combinations of epitopes situated in very close proximity on the cell surface results in a
© 2015 John Wiley & Sons Ltd International Journal of Immunogenetics, 2015, 0, 1–10
3
blunting of the CDC response and presented further data which may explain the reduced MFI values often observed at neat in samples with high levels of antibodies measured by microbead assays at serial dilution. This was followed by a talk from Dr Dermot Mallon from Cambridge University, UK, who demonstrated a novel way of predicting immunogenicity of antigen/epitope using electrostatic potential measurements and computational HLA-alloantibody interactions (Mallon et al., 2014). He made the key point that alloantigen sequence information alone would only provide limited insight into the key determinants of immunogenicity. The assessment of the electrostatic properties of the surface residues of HLA molecules could be crucial to our understanding of HLA immunogenicity and key to informing clinical practice when transplanting across HLA-specific antibody barriers. Dr Sunil Daga from Warwick University, Coventry, UK, then described his work involving real-time biosensor assays based on surface plasmon resonance to measure the binding kinetics and affinity of HLAspecific antibodies. Using human monoclonal antibodies generously donated from the University of Leiden and HLA proteins from the University of Oklahoma, binding interactions between HLA protein and these antibodies were studied. Interestingly, the binding kinetics and strength were different for same epitope– paratope interactions between different alleles of HLA. The higher affinities were shown to be for alleles that were the same as the primary immunizing antigen. Preliminary work on polyclonal HLA-specific antibodies carefully isolated from patient sera showed heterogeneity of affinity, not previously demonstrated using specific molecular binding assays. Further clinical correlation studies are ongoing to ascertain how detailed knowledge of HLA-specific antibody affinity and avidity can be used to inform clinical practice. The day was concluded by a talk given by Prof Richard Pleass from the Parasitology department at the University of Liverpool, UK who completed the session’s focus on antibody–antigen interactions by describing novel in vitro alternatives to intravenous immunoglobulin (IVIg). Currently, 46 standard blood donations are required in order to isolate enough specific immunoglobulin to constitute a single therapeutic dose of IVIg. He described how current IVIg treatment also involves large infusion volumes as only 5% of injected IVIg is active in vivo, and he suggested that this could be due to fewer terminal sialyic acid residues on the N-linked oligosaccharide located on the Fc domain of human IgG1. He then went on to describe a novel hexameric form of IgG that could be more effective than IVIg. This exciting molecule has been described as naturally occurring and can be manufactured in laboratories with the potential to revolutionize this therapeutic area (Diebolder et al., 2014).
4
D. Lowe et al.
Histocompatibility and immunogenetics workshop: 1st November 2014 The workshop session began with Professor Derek Middleton from the Transplant Immunology laboratory at the Royal Liverpool University Hospital, UK, providing a local perspective on how laboratories should approach the difficult task of assigning HLAspecific antibodies. The advent of solid phase assays, in particular the single antigen bead (SAB) assay on the LUMINEX platform, has led to unheralded levels of HLA-specific antibody characterization. However, the use of SAB data provides a series of difficult challenges and devising robust strategies to accurately define which antibodies could be clinically relevant is challenging (Middleton et al., 2013, 2014). The current understanding of the advantages and disadvantages of SAB assays was discussed and how data from these assays are used to support a Virtual Crossmatching (VXM) programme in Liverpool. The criteria used in Liverpool for determining which specificities detected by SAB assay are to be listed as unacceptable antigens was discussed and showed that this is an area where much further investigation is needed. Assigning positive cut-off thresholds for unacceptable antigens, determining which reactions were due to genuine HLA-reactivity, and identifying when luminex assays may have saturated are all issues to be considered when defining clinical relevance of HLA-specific antibodies. It was also suggested that the increasing understanding of HLA epitopes will lead to enhanced matching in solid organ transplantation. Data were presented which demonstrated how relatively poorly matched grafts assessed by standard antigen levelmatching criteria could in fact turn out to be completely free of disparity when the repertoire of epitopes expressed by both donor and recipient were analysed. Miss Maria Irvine, an undergraduate student from Liverpool John Moore’s University, UK, and on placement at the Transplant Immunology Department in Liverpool, presented data which compared the relative merits of SAB technology and traditional CDC analysis as a means of identifying clinically relevant HLAspecific antibodies. As expected, the mean number of antibody specificities detected by SAB was far greater than CDC; however, there were a small number of cases studied which gave clear antibody specificities on CDC analysis which were initially negative by SAB assays. It was only when serum was further analysed following serial dilution that these specificities were then detectable by SAB assay. This highlights the potential dangers of relying solely on microbeads for antibody analysis and also the need for potential costprohibitive repeat testing of samples under various test conditions to ensure all antibody specificities are detected. Dr Natasha Khovanova from the University of Warwick, Coventry, UK then presented novel data highlighting the impact of pretransplant donor-specific
IgG4 on graft survival following AiT. In a cohort of 77 AiT cases, it was clearly demonstrated that when pretransplant DSA of the IgG4 subclass was positive there was a significant increase in the likelihood of acute antibody-mediated rejection. The use of sophisticated binary regression analysis also showed that the occurrence of acute AMR seen (approaching 50% and confirmed earlier in the meeting in US data by Dr Orandi) was heavily influenced by three factors: total IgG4 MFI levels, highest single DSA MFI level and the total number of mismatches between donor and recipient. For example, for each additional 1000 MFI increase in IgG4 DSA level, there was a 30% increase in the likelihood of rejection. Long-term graft survival was also shown to be significantly reduced in IgG4 DSA positive cases (P = 0.004), highlighting the potential of pretransplant IgG4 to be used as a biomarker for kidney transplant rejection. Mr James Jones from the Transplant Immunology department, Royal Liverpool University Hospital, UK, then presented his recent work on developing computer-based algorithms for defining HLA-specific reactivity patterns in both SAB assays and CDC analysis data. Alternative methods of antibody analysis (HLAMatchmaker and the epitope analysis module within One Lambda’s HLA Fusion software) were explored, and it was shown that particular components of each can be inadequate. It was also shown how epitopes can be incorporated into an automated algorithm to improve antibody analysis. Parameters of 2 9 2 contingency tables were discussed with emphasis on sensitivity and True Positive frequency (Antigen Present/ Test Positive). These are more reliable measures to define antibodies than the R value correlation coefficient used widely, especially for serum with antibodies to many HLA-specificities. The algorithm allowed complex CDC assay reaction patterns to be resolved to the epitope level, where parameters such as epitope frequency and combinations within different analysis panels could investigated. Antibody profiles of SAB assays could also be translated accurately into epitopes, which allowed MFI thresholds to be better placed by assessing grouped reactive beads by the shared epitopes identified and in addition provided a fresh perspective on how to recognize those bead reactions attributable to variable HLA target quality. Completing the morning session was Mr Harry Moyse, another PhD student from Warwick University School of Engineering, Coventry, UK. He presented key work from his thesis exploring the mathematical modelling of antibody–antigen interactions (Evans et al., 2012). This work is carried out on the biosensor assays described on day one by Dr Daga. He described how it is possible to develop highly refined computational modelling techniques in order to obtain accurate estimates of affinities of HLA, and ABO-specific antibodies both in monoclonal antibody preparations but also more impressively in complex polyclonal mixtures of antibodies as would be expected in the serum com-
© 2015 John Wiley & Sons Ltd International Journal of Immunogenetics, 2015, 0, 1–10
How much risk can you take?
partment of highly sensitized transplant patients. It was clear from the data presented that with the development of ever more sophisticated assays to define antibodies comes a clear need for the kind of high level mathematical and computational analysis demonstrated here. The workshop then concluded with a debate in which the relative merits of the long-standing CDC assay and its place in the 21st century tissue typing laboratory testing repertoire were passionately discussed. It is only about 50 years since the microcytotoxicity assay was developed by Terasaki and McClelland (Terasaki & McClelland, 1964). In essence, this assay is still widely used today, mainly for a pretransplant cross-match, because of the 1969 publication showing the risk of early rejection associated with a positive CDC cross-match (Patel & Terasaki, 1969). There are many methodological versions of this assay, but also many more recent alternatives, so Dr Robert Vaughan of Guy’s/Kings/Viapath, London, UK, proposed the assertion ‘50 years of CDC: enough is enough’ and debated this with Professor D Briggs of NHSBT Birmingham, UK, who took a position of ‘not really’. Guy’s HLA laboratory was an early adopter of flow cytometry (FC)-based detection of HLA-specific antibodies and after 30 years has continued its innovation with this technology, including using a FC titre to predict how many plasma exchange treatments are necessary to remove DSA to an acceptable level for AiT [Shaw O, PhD thesis, University of London.]. Dr Vaughan asserted that this is one of many examples where FC is superior to CDC for detection of HLAspecific antibody: it is a more sensitive technique, the read-out is quantitative and it is far better at indicating relative levels. In addition, FC is intrinsically more objective in its measurements. The inferiority of CDC in these assay parameters suggests that 50 years are more than enough of what can seem an archaic assay. Comparing CDC with other HLA antibody detection methods on the basis of sensitivity and quantitation is, however, a profound mistake said Prof Briggs: they measure different things. FC in this context measures how much antibody binds to the cell, while CDC measures activity of the antibodies bound to that cell. Briggs therefore made the point that CDC continues to be a relevant histocompatibility technique because it measures the one thing that none of the others does and such a functional assay has obvious clinical relevance. Using CDC to detect antibody is clearly not sensible – there are far better methods for this, particularly FC. Anti-human globulin (AHG) augmentation of CDC is probably not a useful assay variation in this respect because this becomes primarily a detection assay but with a functional read-out (i.e. detection by AHG, as in FC). Vaughan and Briggs agreed that the use of rabbit serum as the source of complement in the CDC assay is a problem because this does not resemble the physiological state. While conceding low
© 2015 John Wiley & Sons Ltd International Journal of Immunogenetics, 2015, 0, 1–10
5
sensitivity may seem to be a flaw of CDC used for cross-matching, Briggs did suggest that, on the contrary, this assay might be too sensitive and would be improved by replacing the rabbit serum with human serum, perhaps autologous serum. The audience was asked to vote, and in the absence of a formal count, the result was not recorded, but this part of this meeting report was written by the victor – CDC lives to fight another day.
Clinical session: 1st November 2014 End-stage renal disease (ESRD) is increasing worldwide. As potent immunosuppressive medications, intense pretransplant screening and improved surgical techniques are increasingly accessible, more and more high-risk patients are getting transplanted. As a consequence, there is a push to increase the donor numbers, which inadvertently results in acceptance of borderline donors for transplantation. The clinical session on day 2 aimed to discuss this potentially dangerous territory under the theme of how much risk we can take in kidney transplantation. Dr Nithya Krishnan, Coventry, UK, discussed the issue of body mass index in kidney transplantation recipients. Obesity is on the increase worldwide, as is end-stage renal disease (ESRD) (Van Lerberghe, 2008). Kidney transplantation is the treatment of choice for ESRD. However, patients with high BMI are disadvantaged worldwide as obesity is considered a contraindication for transplantation. There have been contradictory reports with regard to transplanting patients with a high BMI (Meier-Kriesche et al., 2002) (Glanton et al., 2003; Ng et al., 2014). Dr Krishnan presented their study, which investigated the effect of BMI on mortality in transplanted and patients remaining on the waiting list in the UK. The group analysed the UK Renal Registry and NHSBT Organ Donation and Transplantation, UK, data for patients listed from 1st January 2004 to 31st December 2010, with follow-up until 31st December 2011. Kaplan–Meier survival plots and Cox proportional hazards models were used to estimate and compare patient and graft survival for transplanted and listed patients with BMI recorded. A total of 17 681 patients were listed during the study period, with BMI recorded for 13 526 (77%). Five-year patient survival was significantly better in all BMI bands (
