Detection Of Donor-specific anti-HLA Antibodies Research Articles

An Optimal Multi-Locus HLA-Typing in Potential Donors of Allogeneic Hematopoietic Stem Cells

Background. HLA-typing and matched donor selection as well as the detection of donor-specific anti-HLA antibodies are essential for allogeneic hematopoietic cell transplantation (allo-HSCT). In accordance with the guidelines of the Center for International Blood and Marrow Transplant Research (CIBMTR) optimal HLA-typing is performed on 11 HLA genes (-A, ‐B, ‐C, ‐DRB1, ‐DRB3/4/5, ‐DQA1, ‐DQB1, ‐DPA1, and ‐DPB1) with an adequate coverage aiming to obtain the values at the two-field level.
 Aim. To assess the results of multi-locus HLA-typing in bone marrow/hematopoietic cell donors from the database at the National Research Center for Hematology in terms of their conformance with the CIBMTR guidelines for allo-HSCT and to analyze the frequency and distribution of HLA alleles and multi-locus HLA haplotypes.
 Materials & Methods. The study enrolled 3485 donors who were HLA-typed by next-generation sequencing.
 Results. In all donors, the alleles of HLA class I genes were identified at the fourth-field level (nucleotide sequence). When the results were reduced to the second-field level (amino acid sequence), 61 HLA-A, 92 HLA-B, and 49 HLA-C alleles were detected. The alleles of class II genes were discovered either at the two-field or high-resolution levels. Among the HLA-DRB locus genes, 57 DRB1, 11 DRB3, 6 DRB4, and 5 DRB5 alleles were identified. Also, 23 HLA-DQA1, 30 HLA-DQB1, 14 HLA-DPA1, and 33 HLA-DPB1 alleles were detected. There were reported 3289 different HLA haplotypes of A-B-C-DRB1-DQA1-DQB1-DPA1-DPB1 genes.
 Conclusion. The database created at the National Research Center for Hematology includes potential bone marrow/hematopoietic stem cell donors typed for 11 classical polymorphic genes HLA-A, ‐B, ‐C, ‐DRB1, ‐DRB3/4/5, ‐DQA1, ‐DQB1, ‐DPA1, and -DPB1, which is in line with the guidelines of CIBMTR. The frequency and distribution of HLA alleles and multi-locus HLA haplotypes in our donors correspond to those in populations of European origin. HLA-typing and donor selection with regard to 11 HLA genes will contribute to improving the outcomes of both unrelated and haploidentical HSCTs.

The Role of Major Histocompatibility Complex in Organ Transplantation- Donor Specific Anti-Major Histocompatibility Complex Antibodies Analysis Goes to the Next Stage.

Organ transplantation has progressed with the comprehension of the major histocompatibility complex (MHC). It is true that the outcome of organ transplantation largely relies on how well rejection is managed. It is no exaggeration to say that to be well acquainted with MHC is a shortcut to control rejection. In human beings, MHC is generally recognized as human leukocyte antigens (HLA). Under the current circumstances, the number of alleles is still increasing, but the function is not completely understood. Their roles in organ transplantation are of vital importance, because mismatches of HLA alleles possibly evoke both cellular and antibody-mediated rejection. Even though the control of cellular rejection has improved by recent advances of immunosuppressants, there is no doubt that antibody-mediated rejection (AMR), which is strongly correlated with donor-specific anti-HLA antibodies (DSA), brings a poor outcome. Thus, to diagnose and treat AMR correctly is a clear proposition. In this review, we would like to focus on the detection of intra-graft DSA as a recent trend. Overall, here we will review the current knowledge regarding MHC, especially with intra-graft DSA, and future perspectives: HLA epitope matching; eplet risk stratification; predicted indirectly recognizable HLA epitopes etc. in the context of organ transplantation.

Unmanipulated haploidentical transplantation for adult patients with hematological malignancies.

Unmanipulated haploidentical transplantation for adult patients with hematological malignancies.

Complement-Binding Donor-Specific Anti-HLA Antibodies and Risk of Primary Graft Failure in Hematopoietic Stem Cell Transplantation

Detection of donor-specific anti-HLA antibodies (DSA) has been associated with graft rejection in all forms of transplantation. The mechanism by which DSA increase the risk of graft failure remains unclear. We hypothesized that complement-binding DSA are associated with engraftment failure in hematopoietic stem cell transplantation (HSCT) and analyzed 122 haploidentical transplant recipients tested prospectively for DSA. Retrospective analysis to detect C1q binding DSA (C1q+DSA) was performed on 22 allosensitized recipients. Twenty-two of 122 patients (18%) had DSA, 19 of which were women (86%). Seven patients with DSA (32%) rejected the graft. Median DSA level at transplant for patients who failed to engraft was 10,055 mean fluorescence intensity (MFI) versus 2065 MFI for those who engrafted (P = .007). Nine patients with DSA were C1q positive in the initial samples with median DSA levels of 15,279 MFI (range, 1554 to 28,615), compared with 7 C1q-negative patients with median DSA levels of 2471 MFI (range, 665 to 12,254) (P = .016). Of 9 patients who were C1q positive in the initial samples, 5 patients remained C1q positive at time of transplant (all with high DSA levels [median, 15,279; range, 6487 to 22,944]) and experienced engraftment failure, whereas 4 patients became C1q negative pretransplant and all engrafted the donor cells (P = .008). In conclusion, patients with high DSA levels (>5000 MFI) and complement-binding DSA antibodies (C1q positive) appear to be at much higher risk of primary graft failure. The presence of C1q+DSA should be assessed in allosensitized patients before HSCT. Reduction of C1q+DSA levels might prevent engraftment failure in HSCT.

Circulating NK-Cell Subsets in Renal Allograft Recipients With Anti-HLA Donor-Specific Antibodies

Detection of posttransplant donor-specific anti-HLA antibodies (DSA) constitutes a risk factor for kidney allograft loss. Together with complement activation, NK-cell antibody-dependent cell mediated cytotoxicity (ADCC) has been proposed to contribute to the microvascular damage associated to humoral rejection. In the present observational exploratory study, we have tried to find a relationship of circulating donor-specific and non donor-specific anti-HLA antibodies (DSA and HLA non-DSA) with peripheral blood NK-cell subsets and clinical features in 393 renal allograft recipients. Multivariate analysis indicated that retransplantation and pretransplant sensitization were associated with detection of posttransplant DSA. Recipient female gender, DR mismatch and acute rejection were significantly associated with posttransplant DSA compared to HLA non-DSA. In contrast with patients without detectable anti-HLA antibodies, DSA and HLA non-DSA patients displayed lower proportions of NK-cells, associated with increased CD56(bright) and NKG2A(+) subsets, the latter being more marked in DSA cases. These differences appeared unrelated to retransplantation, previous acute rejection or immunosuppressive therapy. Although preliminary and observational in nature, our results suggest that the assessment of the NK-cell immunophenotype may contribute to define signatures of alloreactive humoral responses in renal allograft recipients.

Solid phase-based cross-matching as solution for kidney allograft recipients pretreated with therapeutic antibodies.

In order to select recipients without donor-specific anti-HLA antibodies, the complement-dependent cytotoxicity crossmatch (CDC-CM) was established as the standard procedure about 40 years ago. However, the interpretability of this functional assay strongly depends on the vitality of isolated donors' lymphocytes. Since the application of therapeutic antibodies for the immunosuppressive regimen falsifies the outcome of the CDC-crossmatch as a result of these antibodies' complement-activating capacity in the recipients' sera, we looked for an alternative methodical approach. We here present 27 examples of AB0 blood group-incompatible living kidney allograft recipients who, due to their treatment with the humanized chimeric monoclonal anti-CD20 antibody Rituximab, did not present valid outcomes of CDC-based pretransplant cross-matching. Additionally, four cases of posttransplant cross-matching after living kidney allografting and consequent treatment with the therapeutic anti-CD25 antibody Basiliximab (Simulect) due to acute biopsy-proven rejection episodes are presented and compared regarding CDC- and ELISA-based crossmatch outcomes. In all cases, it became evident that the classical CDC-based crossmatch was completely unfeasible for the detection of donor-specific anti-HLA antibodies, whereas ELISA-based cross-matching not requiring vital cells was not artificially affected. We conclude that ELISA-based cross-matching is a valuable tool to methodically circumvent false positive CDC-based crossmatch results in the presence of therapeutically applied antibodies.

P036 : C1Q SINGLE ANTIGEN BEAD ASSAY ONLY DETECTS HIGH TITER/AVIDITY CLASS-I ANTI-HLA ANTIBODIES DETECTED BY SINGLE ANTIGEN BEADS

Detection of donor-specific anti-HLA antibodies (DSA) by solid phase assay (SpA) helps donor selection by predicting the actual crossmatch (xM), a process referred as a virtual crossmatch (VxM). At our center we find good correlation ( R 2 = 0.85) between flow xM (FxM) and VxM using single antigen bead (SAB) assay to identify DSA. The SAB assay is considered a sensitive SpA; however, a concern is that clinically insignificant anti-HLA antibodies (HLA-Ab) may be detected and preclude the recipient from receiving a transplant. The complement fixing (C1q) SAB assay has been proposed as a way to minimize the detection of clinically insignificant HLA-Ab. We present a summary of our evaluation of this assay. 20 positive sera, previously used for serological typing, were tested with SAB, at 1:8 dilution with SAB and by C1q SAB method as per the manufacturer’s protocol. Serological HLA-Ab specificities were compared with those that were obtained by SAB and C1q SAB. For the SpA, specificities >5000 MFI were considered as positive. Specificities were grouped into two categories: 5000–10,000 MFI and >10,000 MFI as in our center >5000 MFI correlates with a positive FxM and >10,000 MFI correlates with cytotoxicity xM. In total there were 53 HLA-Ab specificities as defined by serology, 328 as defined by SAB, and 123 as defined by C1q. SAB identified all 53 specificities defined by serology; 49 with MFI >10,000. Of the 4 with MFI 10,000 on 1:8 dilution and other 2 were not detected by C1q assay. In addition, C1q assay did not detect 3 specificities identified by serology (all with MFI >10,000 by SAB). C1q assay identified 123/179 (69%) of the specificities with MFI >10,000 and 0/151 specificities with MFI: 5000–10,000 as detected by SAB (table). For Class I HLA-Ab, C1q assay only detects high titer/avidity antibodies as defined by SAB. As DSA with SAB MFI: 5000–10,000 can result in a positive FxM, additional investigation is warranted to determine the clinical significance of these HLA-Ab. Serology positive N % SAB Total positive 53 100 MFI: >10,000 49 92 MFI: 5000–10,000 4 8 Total negative 0 0 C1q SAB Total positive 47 89 MFI: >10,000 47 89 MFI: 5000–10,000 0 0 Total negative 5 11 M.J. Gandhi: Consultant; Company/Organization; Thermo Fisher-One Lambda.

Comparative Performance Evaluation of a Donor-Specific Bead-Based Crossmatch Technique for the Detection of Donor-Specific Anti-HLA Antibodies in Heart Transplantation

IntroductionRecently new human leukocyte antigen (HLA) antibody screening methods have been shown to significantly improve thoracic transplantation outcomes. The best combination of assays for both allocation and monitoring is still under investigation. Herein, we evaluated the correlation and clinical relevance of three methodologies to detect donor-specific anti-HLA antibodies (DSA). MethodsFor the same donor-recipient combinations we compared the results of the Luminex donor-specific crossmatch (DSA-LX), using donor-isolated HLA antigens coated onto microbeads, with those of a flow-cytometric crossmatch (FCXM) and of the Luminex single-antigen bead methodology (SA-LX), wherein recombinant HLA molecules are coated onto microbeads. We compared 46 pre- (n = 27) and post-transplant (n = 19) serum samples using DSA-LX and SA-LX, while 27 pretransplant samples were additionally analyzed by FCXM. ResultsAmong the pretransplant sera, the 3 methods were in agreement for class I DSA in 22/27 (81.5%) samples and for class II DSA in 17/27 (63.0%) of tested samples. When the results of the 2 bead-based methodologies were compared with the FCXM methodology, the SA-LX results better correlated than the DSA-LX results both for class I and class II antibodies. Furthermore, the SA-LX results showed greater clinical relevance. The reproducibility scores for both the SA-LX and FCXM were 100%, whereas for the DSA-LX it was 85.5%. ConclusionsDSA-LX did not correlate with FCXM and SA-LX to detect DSA, particularly for class II antibodies. Furthermore, in comparison to FCXM and SA-LX, DSA-LX showed lower reproducibility rendering the method not eligible for prediction, allocation, or monitoring purposes.

Donor Specific Anti-HLA Antibody Detection in Heart Transplantation: Comparison of a Donor-Specific Bead-Based Crossmatch Technique with Flow-Crossmatch and Single-Antigen Bead Methodology

Introduction: Progress in assays detecting and characterizing anti-human leukocyte antigen (HLA) antibodies have significantly improved outcomes in thoracic organ transplantation. However, due to the complicated nature of humoral alloresponses practices are still evolving in laboratories to determine the best combination of assays that can be used for graft allocation in prospective heart allograft (HTx) recipients, as well as for post-transplant monitoring of sensitized patients. In the present study we analyzed the efficiency of three different laboratory methodologies for the detection of donor-specific anti-HLA antibodies (DSA), their degree of correlation and their clinical relevance. Methods: The results of a Luminex donor-specific crossmatch (DSA-LX), in which donor-isolated HLA antigens are coated onto capture microbeads (Gen-Probe Transplant Diagnostics, USA), were compared with the results of a flow-cytometric crossmatch (FCM-X) for the same donor-recipient combination and with the results of a Luminex single-antigen bead methodology (SA-LX) in which recombinant HLA class I or class II molecules are coated onto microbeads (One Lambda, USA). A total of 46 sera samples obtained pre- (27 samples) and post-transplant (19 samples) were included in the study. All 46 samples were tested with the DSA-LX and SA-LX methodology, while the 27 pre-transplant samples were also analyzed by the FCM-X methodology. Results: For the pre- transplant sera, the results of the three methods were in agreement for class I DSA in 21/27 (77.8%) samples and for class II DSA in 17/27 (63.0%) samples. When the results of the two bead-based methodologies (DSA-LX and SA-LX) were compared to the results of the FCM-X methodology, the SA-LX results showed better correlation with the flow-cytometric crossmatch results than the DSA-LX results both for class I and for class II antibodies. The results of the two bead-based assays SA-LX and DSA-LX, coincided for 40/46 (87.0%) and for 33/46 (71.7%) of sera tested for class I and class II HLA-DSA respectively. Furthermore, in the group of transplanted patients the SA-LX results had more clinical relevance than the DSA-LX results, particularly when samples were tested positive by SA-LX and negative by DSA-LX. The methodology reproducibility score for both the SA-LX and the FCM-X was 100% whereas for DSA-LX that score was 88.9%. Conclusions: The results of the DSA-LX methodology do not have a good correlation with the results of the more established FCM-X and SA-LX techniques for the detection of DSA, particularly as far as class II antibodies. Furthermore, in comparison to FCM-X and SA-LX, the DSA-LX methodology has a lower reproducibility performance and a lower clinical relevance which renders it unsuitable for reliable graft allocation and patient monitoring.

Clinical Relevance of Non-HLA-Antibodies after Intestinal and Multivisceral Transplantation

Non-HLA-allo- and autoantibodies are involved in allograft rejection in kidney and heart transplantation. Their role in intestinal transplantation (ITX) has not yet been described. We examined the development of anti-Angiotensin II-Type I receptor antibodies (anti-AT1R) and anti-Endothelin-Type A receptor antibodies (anti-ETAR) in association with the clinical course and histopathological findings of 20 ITX-recipients. Between 06/2000 and 08/2011, 30 patients with a median age of 37.6±9.8 years received an isolated intestinal graft (n=18) or a multivisceral transplantation (MVTX, n=12). Since 2005 anti-AT1R and anti-ETAR were screened regularly. Levels of >12 U/l were considered as highly-positive. All non-HLA antibody levels were evaluated retrospectively in regards of simultaneous rejection episodes or other clinical events. Anti-AT1R and anti-ETAR levels were determined in 20 out of 30 ITX- and MVTX-recipients. Fifteen of the 20 patients (75%) developed high levels of either anti-AT1R, or anti-ETAR, or both. Twelve of the 15 patients (80%) had rejection episodes around the time of positive non-HLA antibody sampling. Rejection episodes were either exclusively cellular rejections (n=4) or mixed cellular and antibody-mediated rejections (AMR) with positive detection of donorspecific anti-HLA antibodies (DSA, n=8). The other 3 patients showed non-donorspecific anti-HLA antibodies and viral infections (n=2) during the time of anti-AT1R- or anti-ETAR antibody detection, but did not have any signs of rejection. Our data suggest that antibody-mediated mechanisms targeting antigens beyond HLA- may additionally trigger and accelerate immune responses. Given the possibility of pharmacologic targeting of both receptors, future studies will focus on the explanation of mechanisms how non-HLA antibodies may enhance allograft rejection and deteriorate long-term allograft survival after ITX and MVTX.

HLA Antibodies Detected by Luminex-Single Antigen Beads and Virtual Crossmatch in Renal Transplantation

Introduction: The most important immunologic consideration for kidney transplantation is the presence of preformed donor-specific anti-HLA antibodies (DSA). Solid-phase assays, such as Luminex-Single Antigen Beads (LSA), allow an accurate detection and characterization of pre-existing DSA in transplant candidates. But the ability of LSA in detecting low level of antibodies makes hard to correctly predict crossmatch results in donor selection, that is to perform a virtual-crossmatch (v-XM). In this study was to retrospectively analyze the accuracy of our v-XM protocol in predicting the results of actual-crossmatch (a-XM) in renal transplantation. We also investigated correlation between a-XM negative results and strength/specificity of pre-formed DSA. Methods: We retrospectively analyzed the correlation between v-XMs and a-XMs performed within January 2007 and June 2011 at Regional Transplant Center of Lazio (Italy). In carrying out v-XM, we considered as “unacceptable mismatches” the donor HLA class I and class II molecules against which the patients showed LSA-detected DSA with normalized MFI ≥5000. The LSA-DSA showing MFI lower than the established cutoff were defined as “acceptable DSA” for v-XM. All donors were routinely typed for HLA-A, -B, -DR and -DQB1 molecules by SSP methods. Consequently, in performing v-XM, we didn’t consider anti-HLA-C, -DQA and -DP DSA. On the basis of a negative v-XM, we performed 328 a-XMs between sera from 194 sensitized renal transplant candidates (%PRA class I = 47±39, %PRA class II = 59±32) and T/B lymphocytes from 219 cadaver donors using both cytotoxic-crossmatch (CDCXM) and flow-cytometry-crossmatch (FCXM). Results: The v-XM results predicted by LSA-DSA showed good correlation with both CDC and FC aXMs (96% and 90% respectively; Sensitivity = 100%). The high sensitivity of v-XM was related to the lack of false-negative DSA results. The limited specificity with both the XM techniques (CDCXM = 78%, FCXM = 81%) was due to the presence of “acceptable DSA” and/or anti-DQA/-DP DSA in sera used to perform 86 a-XMs. Ten of these a-XMs gave positive results by both CDC and FC assays, 20 gave positive results by FCXM only and 56 were negative by both the assays. During the study period, 104 (54%) of the 194 sensitized patients received a kidney transplant. Twenty-eight of the 104 (27%) transplanted patients had “acceptable” DSA and/or anti-DQA/-DP DSA. Late antibody mediate rejection due to preformed DSA was observed in 2 patients only; anti-DP DSA at “unacceptable” levels (MFI>5000) were present in these patients. Conclusion: Our v-XM protocol showed high sensitivity in predicting donor-recipient immunologic compatibility. Data presented here demonstrate the importance to evaluate DSA strength for implementing v-XM results in selection of recipient for kidney transplantation. Nevertheless prediction based on LSA assay feels the amount of antigen on beads and may not be representative of the amount found on cells. The findings of anti-DPB/-DQA DSA highlight the importance to define all the donor HLA molecules to perform an accurate v-XM. Finally, the good outcome of transplants carried out in patients with low levels of DSA and negative XM underlines the importance to discard discarding weak and clinically irrelevant DSA in performing v-XM.

HLA Antibodies Detected by Luminex-Single Antigen Beads and Virtual Crossmatch in Renal Transplantation

Introduction: The most important immunologic consideration for kidney transplantation is the presence of preformed donor-specific anti-HLA antibodies (DSA). Solid-phase assays, such as Luminex-Single Antigen Beads (LSA), allow an accurate detection and characterization of preexisting DSA in transplant candidates. But the ability of LSA in detecting low level of antibodies makes hard to correctly predict crossmatch results in donor selection, that is to perform a virtual-crossmatch (v-XM). In this study was to retrospectively analyze the accuracy of our v-XM protocol in predicting the results of actual-crossmatch (a-XM) in renal transplantation. We also investigated correlation between a-XM negative results and strength/specificity of pre-formed DSA. Methods: We retrospectively analyzed the correlation between v-XMs and a-XMs performed within January 2007 and June 2011 at Regional Transplant Center of Lazio (Italy). In carrying out v-XM, we considered as “unacceptable mismatches” the donor HLA class I and class II molecules against which the patients showed LSA-detected DSA with normalized MFI ≥5000. The LSA-DSA showing MFI lower than the established cutoff were defined as “acceptable DSA” for v-XM. All donors were routinely typed for HLA-A, -B, -DR and -DQB1 molecules by SSP methods. Consequently, in performing v-XM, we didn't consider anti-HLA-C, -DQA and -DP DSA. On the basis of a negative v-XM, we performed 328 a-XMs between sera from 194 sensitized renal transplant candidates (%PRA class I = 47±39, %PRA class II = 59±32) and T/B lymphocytes from 219 cadaver donors using both cytotoxic-crossmatch (CDCXM) and flow-cytometry-crossmatch (FCXM). Results: The v-XM results predicted by LSA-DSA showed good correlation with both CDC and FC aXMs (96% and 90% respectively; Sensitivity = 100%). The high sensitivity of v-XM was related to the lack of false-negative DSA results. The limited specificity with both the XM techniques (CDCXM = 78%, FCXM = 81%) was due to the presence of “acceptable DSA” and/or anti-DQA/-DP DSA in sera used to perform 86 a-XMs. Ten of these a-XMs gave positive results by both CDC and FC assays, 20 gave positive results by FCXM only and 56 were negative by both the assays. During the study period, 104 (54%) of the 194 sensitized patients received a kidney transplant. Twenty-eight of the 104 (27%) transplanted patients had “acceptable” DSA and/or anti-DQA/-DP DSA. Late antibody mediate rejection due to preformed DSA was observed in 2 patients only; anti-DP DSA at “unacceptable” levels (MFI>5000) were present in these patients. Conclusion: Our v-XM protocol showed high sensitivity in predicting donor-recipient immunologic compatibility. Data presented here demonstrate the importance to evaluate DSA strength for implementing v-XM results in selection of recipient for kidney transplantation. Nevertheless prediction based on LSA assay feels the amount of antigen on beads and may not be representative of the amount found on cells. The findings of anti-DPB/-DQA DSA highlight the importance to define all the donor HLA molecules to perform an accurate v-XM. Finally, the good outcome of transplants carried out in patients with low levels of DSA and negative XM underlines the importance to discard discarding weak and clinically irrelevant DSA in performing v-XM.

A novel ELISA-based crossmatch procedure to detect donor-specific anti-HLA antibodies responsible for corneal allograft rejections

Previous studies had shown that donor-specific anti-HLA antibodies may highly influence the survival rate of corneal allografts, although the anterior chamber generally represents an immune‐privileged compartment of the eye. We postulated that the introduction of a novel crossmatch procedure for the detection of donor-specific anti-HLA antibodies in recipients awaiting a corneal graft would be adequate to investigate their influence on the outcome of the graft survival. The Antibody Monitoring System (AMS) HLA class I & II crossmatch ELISA was adapted for the use of material from the outer scleral rim instead of blood lymphocytes to isolate the donors' HLA molecules. In case of detectable donor-specific anti-HLA class I and/or class II antibodies (DSA) this result was confirmed using an identification ELISA to specify the detectable recipient's anti-HLA antibodies. PCR-based genetic tissue typing of the donors was performed also using their outer scleral rims. 45 recipients of corneal grafts were analyzed for DSA prior to or after grafting, respectively. 75% of the recipients with preformed DSA exhibited immunological complications up to the complete graft loss in four cases during the first two months. In contrast 77% of the recipients without DSA did not show any complications during the follow up period of averagely 18months. Only two cases of graft loss were observed in this group after 17 and 23months, respectively. The results demonstrate the impact of preventing donor-specific anti-HLA antibodies which are for the first time reliably detectable in any laboratory's daily work using the adapted AMS-ELISA.

Comparison of the established standard complement-dependent cytotoxicity and flow cytometric crossmatch assays with a novel ELISA-based HLA crossmatch procedure.

The detection of donor-specific anti-HLA antibodies by standard procedures such as complement-dependent cytotoxicity assay (CDC) or flow cytometric (FACS) analysis is limited by its low sensitivity and the quality of the donor cells. Therefore, an ELISA-based technique was employed using solid phase-immobilized monoclonal antibodies to capture HLA class I or class II molecules of the donor, respectively. In this HLA class I and class II antibody monitoring system (AMS) the donor-specific anti-HLA antibodies from the sera of recipients bind to the HLA molecules of the donor which have been immobilized by monoclonal antibodies (mAb) recognizing non-polymorphic epitopes. Upon binding of donor-specific anti-HLA antibodies they are recognized by secondary enzyme-conjugated anti-human immunoglobulin (Ig) antibodies. A newly established modification of the standard protocol allows the differentiation between bound antibodies of the IgG and IgM isotype. Furthermore, this assay was adapted for investigating small amounts of solid tissue of donors from whom no other cells (e.g. from blood) were available. We here provide an overview of the classical crossmatch methods with their advantages and limits. In addition, the design of the novel AMS-ELISA is described in terms of quality and sensitivity of the approach using exemplary cases of different application. The selected cases show that the AMS-ELISA represents a valuable tool for the post-transplantation monitoring of donor-specific anti-HLA antibodies during reaction crisis, after transfusion reactions and in particular cases of tissue transplantations lacking single cells.

Detection of donor-specific anti-HLA antibodies with flow cytometry in eluates and sera from renal transplant recipients with chronic allograft nephropathy.

Chronic allograft nephropathy (CAN), which remains the main cause of graft loss after kidney transplantation, is still poorly understood. Because anti-HLA antibodies may be involved in the pathogenesis of CAN, this study was performed to look for donor-specific antibodies (DSA) fixed onto renal transplants with CAN. DSA were identified after elution with flow cytometric assay and/or flow cytometric crossmatches in 20 transplants removed after irreversible graft failure caused by CAN and in control samples from 2 transplants with relapsing glomerulopathy, 2 transplants lost after vascular thrombosis, and 4 normal kidneys. The results were compared with those obtained in the serum samples 1 year after grafting, at the time of transplantectomy, and within 2 months after transplantectomy. IgG anti-class I, anti-class II, or both DSA were identified in 70.6% of eluates versus 73.6% of posttransplantectomy serum samples (NS), 42.1% of 1-year postgrafting serum samples (P<0.05), and 31.6% of serum samples at the time of transplantectomy (P<0.05). Our data show a good correlation between the target of anti-HLA antibodies found in both eluates and posttransplantectomy serum samples, but the precise specificity of anti-HLA antibodies is more often assigned in posttransplantectomy serum samples than in eluates. This problem needs further evaluation. This study shows that testing for anti-HLA DSA in eluates from removed kidney transplants using flow cytometry can be achieved and is highly efficient. It already suggests that both anti-class I and anti-class II HLA antibodies can be involved in CAN. Further studies are now needed to evaluate the possibility of identifying such antibodies in the eluates of transplant biopsy specimens from recipients experiencing CAN.