Complement-fixing HLA Research Articles

Getting on target: Development of the novel, prozone-resistant, dual antibody rapid test (DART) for the LABScreen single antigen bead (SAB) assay

A major limitation of the single antigen bead (SAB) assay is the so called prozone effect, whereby the detection of high titer complement fixing HLA antibodies is compromised due to complement split product (from C3 and C4 components) deposition and interference with the reporter anti-IgG-PE antibody binding. Strategies to minimize prozone include serum titration or treatment with heat, dithiotreitol (DTT), or ethylenediaminetetraacetic acid (EDTA). While effective, these treatments may compromise HLA antibody binding and detection. Here we describe the Dual Antibody Rapid Test (DART), a modified version of the rapid optimized SAB (ROB) protocol, in which we use an IgG-PE/C3d-PE antibody cocktail to simultaneously detect bead bound IgG and C3d, which allows for detection of HLA antibodies independent of the prozone effect. Twenty prozone positive sera (10 class I and 10 class II), identified by titration, were tested by the ROB protocol, with or without EDTA pre-treatment, using three reporter antibody cocktails: (1) IgG-PE, (2) C3d-PE, or (3) IgG-PE/C3d-PE (DART). Mean fluorescence intensity (MFI) values were then compared. IgG negative (n = 735) vs IgG positive (n = 1185) reactions were identified using a 1000 MFI IgG EDTA cutoff. IgG positive reactions were classified based on ΔMFI (IgG EDTA – IgG) as follows: (1) prozone negative (ΔMFI < 3000; n = 737), (2) slight prozone (ΔMFI 3001–5000; n = 49), (3) moderate prozone (ΔMFI 5001–10,000; n = 93), and (4) marked prozone (ΔMFI > 10,001; n = 306). No C3d deposition was present on IgG negative beads, and the majority of prozone positive specificities (438/448; 98%) fixed complement and were detected with the C3d-PE reporter. Interestingly, C3d-PE MFI was directly proportional to the degree of prozone (mean C3d-PE MFI = 4419.5 ± 1606.3 for slight, 5991.0 ± 2302.7 for moderate, and 12,417.4 ± 2969.9 for marked prozone specificities). Interestingly, EDTA treatment was found to have a negative impact on MFI of up to 15% of prozone negative specificities. Importantly, the DART protocol detected all prozone positive specificities while MFI for prozone negative specificities correlated well with those seen with the IgG-PE reporter alone (R2 = 0.97). In conclusion, the DART protocol accurately detects HLA antibodies independent of the prozone effect. Implementation of DART is an easy way to overcome the prozone effect without compromising HLA antibody detection.

(334) - Complement-Fixing Donor-Specific HLA Antibodies Detected By Novel C3d Assay Are Associated With Antibody Mediated Rejection in Heart Transplant Recipients

The Luminex single antigen bead (SAB) technology is ultra-sensitive and detects all IgG donor-specific antibodies (DSA) non-selectively. In this study, we used a novel C3d-SAB assay to identify clinically relevant complement-fixing antibodies and examined the association of C3d+DSA with antibody mediated rejection (AMR) in heart transplant recipients.

OR09: KEEP IT COOL. A NOVEL INHIBITOR COMPLEX EXCLUSION (ICE) PROTOCOL FOR LABSCREEN THAT PREVENTS THE “PROZONE” EFFECT

Background An important limitation of the single antigen bead (SAB) assay is its sensitivity to the prozone effect, where high titer complement fixing HLA antibodies are undetected due to complement mediated interference with IgG-PE detection antibody. Strategies used to minimize the prozone effect include serum treatment with heat, DTT or EDTA, all inhibiting complement fixation. However, these treatments may compromise HLA antibody integrity and/or binding. Given that complement activation is markedly reduced at low temperatures we hypothesized that SAB testing performed at Methods 5 prozone positive and 5 prozone negative sera were tested in parallel using three protocols: (1) standard LABscreen (SL), (2) EDTA pre-treatment (ESL) and (3) inhibitor complex exclusion (ICE) protocol in which sera are incubated with SABs on ice instead of room temperature. Reactions were classified into inhibitor positive (IP), questionable (IQ) or negative (IN) based on SL to ESL MFI increase >10 K, 4 K-10 K and Results Parallel testing of 5 prozone positive sera with SL and ESL protocols identified 79 IP, 24 IQ and 857 IN rxns. 32/79 IP rxn had SL MFI Conclusion We have developed a novel ICE protocol, which prevents the prozone effect while maintaining excellent HLA antibody detection sensitivity. Importantly, ICE protocol avoids any potential interference with HLA antibody testing that may be caused by pre-treatment of sera.

1041-LBP: Establishment of a consistent single antigen bead (SAB) IgG subtype alloantibody assay. Correlation with C1q complement fixing alloantibody

Aim Clinically relevant assays that help assessing the risk of rejection in transplanted patients remain a priority in the transplantation field. In this study we determined the frequency of different complement and non-complement fixing HLA antibody (Ab) subtypes in transplanted patients. We also examined the correlation of these assays with C1q-binding alloantibodies. Methods Sera from patients with high titers of HLA antibodies were tested for standard total IgG SAB and C1q-alloantibody binding according to the manufacturer’s protocol (OneLambda). The frequency of each Ab subtype was determined using the same SAB Luminex technology, but replacing the secondary Ab conjugated to PE for human IgG1, IgG2, IgG3 and IgG4 subclasses from Southern Biotech. A titration analysis was performed using 1:5 and 1:100 dilutions of the standard concentration. Saturating secondary titers were chosen for evaluation. Samples were pre-heat inactivated for all the assays. Results The distribution of IgG1, IgG2, IgG3 and IgG4 in the population analyzed was 59%, 26%, 9% and 6% respectively. Complement-fixing IgG subtypes (IgG1 and IgG3) accounted for 68% of the anti-HLA Ab present in the analyzed patients, while non-fixing complement IgG subtypes (IgG2 and IgG4) accounted for 32%. Correlation between the total IgG SAB and the IgG subtypes assays were 78% for class I and 92% for class II Ab, and 37% for class I and 67% for class II Ab between IgG subtypes and C1q assays. Conclusions This study reveals the potential clinical application of IgG subtype analysis for the risk rejection assessment of transplanted patients.

60-OR

Aim The C1qScreen™ assay detects complement binding HLA antibodies. Recent studies suggest that the presence of C1q positive donor specific antibodies (DSA) is associated with poor post-transplant outcomes. However, in some patients with documented episodes of antibody mediated rejection or graft failure, C1q binding DSA are not detected by the C1qScreen™ suggesting poor assay sensitivity. The goal of this study was to develop an enhanced C1q assay to improve the detection of complement binding HLA DSA. Methods Nine patient sera with well characterized (using IgG single antigen bead (SAB) assay) post-transplant DSA were tested for C1q binding using the standard C1qScreen™ and three modified protocols: 1) extended incubation (EI), 2) wash modified (WM) and 3) anti-human globulin (AHG) C1q enhanced (ACE) protocol. The number of class I/II DSA detected and DSA MFI values obtained with each protocol were compared. Results The C1qScreen™ was positive for only 30% of Class I (9/30) and 52% of Class II (15/29) IgG DSA specificities. The average MFI DSA values were markedly reduced compared with the IgG-SAB assay. The EI and WM protocols exhibited higher average DSA MFI values (2.25 and 3.4 fold higher respectively) compared to the standard protocol but only 2 additional DSA specificities were detected. In contrast, the ACE protocol identified 10 additional Class I (19/30; 63%) and 4 additional Class II (19/29; 66%) DSA specificities with an average increase in DSA MFI of 4.7 fold when compared with the standard C1qScreen™. Conclusions The C1qScreen™ exhibits poor assay sensitivity and fails to adequately identify many post-transplant complement fixing HLA DSA. The sensitivity of the C1qScreen™ to detect DSA can be improved with various protocol modifications. The ACE protocol was the most sensitive of the assays tested and detected the majority of post transplant HLA DSA. Future studies will investigate the clinical significance of HLA DSA detected by the ACE protocol.

Structural aspects of HLA class I epitopes reacting with human monoclonal antibodies in Ig-binding, C1q-binding and lymphocytotoxicity assays

This study addresses the reactivity patterns of human cytotoxic HLA class I epitope-specific monoclonal antibodies in Ig-binding and complement component C1q-binding Luminex assays in comparison with complement-dependent lymphocytotoxicity data reported at the 13th International HLA Workshop. Some monoclonal antibodies reacted similarly with epitope-carrying alleles in all three assays but others showed different reactivity patterns. These reactivity differences were analyzed with HLAMatchmaker and we incorporated the concept that eplets are essential parts of structural epitopes which can contact the six Complementarity Determining Regions (CDRs) of antibody. The data show that technique-dependent reactivity patterns are associated with distinct differences between polymorphic amino acid configurations on eplet-defined structural epitopes. The findings have been viewed in context of antigen-antibody complex formation that results in the release of free energy necessary to stabilize binding and to induce conformational changes in the antibody molecule to expose the C1q binding site, the first step of complement activation. Moreover the amount of free energy should be sufficient to induce a conformational change of C1q thereby initiating the first stages of the classical complement cascade leading to lymphocytotoxicity. The complement-fixing properties of HLA antibodies require not only specific recognition of eplets but also depend on interactions of other CDRs with critical amino acid configurations within the structural epitope. Eplet-carrying alleles that lack such configurations may only bind with antibody. This concept is important to our understanding whether or not complement-fixing donor-specific HLA antibodies can initiate antibody-mediated rejection.

C1q-Fixing Human Leukocyte Antigen Assay in Immunized Renal Patients: Correlation Between Luminex SAB-C1q and SAB-IgG

BackgroundThere is no consensus about the impact of thresholds of complement-fixing antibody assays. Recently, a C1q-SAB assay has been developed to identify complement-fixing HLA antibodies with high sensitivity and specificity. Our aim was to determine the correlation between IgG single antigens beads (SAB) and C1q-SAB assay results among patients on the renal waiting list. Patients and MethodsSerum samples from immunized renal waiting list patients as well as negative and positive controls were valided by Luminex (LMX). These sera, which were positive for 166 antibody specificities, were tested for HLA class I in parallel by LMX-IgG and LMX-C1q. ResultsComparison of antibody detection revealed no correlation based on median fluorescent intensity (MFI), levels between the IgG SAB and the C1qSAB assay (P > .05). IgG-positive sera with MFIs as low as 700 were able to fix C1q, whereas other sera with MFIs as high 14,500 did not. Furthermore, there appeared to be disparities in the profiles of class I antigens able to fix C1q-SAB. In our series, only 34% class I IgG SAB antibodies were also C1qSAB+. In several patients, we detected C1qSAB+ against IgGSAB- that was surely due to IgM antibodies. So, the C1qSAB assay detected IgM antibodies that fix complement. ConclusionThese data suggested that the C1q-SAB assay could be an important method to evaluate pretransplant virtual crossmatch and to define nonpermitted specificities (C1q-fixing) in kidney transplantation.

Clinical Relevance of C1q-Fixing HLA Antibodies in Kidney Transplantation

Introduction: De novo production of donor-specific HLA antibodies (DSA) represents the major risk factor of graft failure in kidney transplantation. However, some patients show persistent presence of circulating DSA without occurrence of graft dysfunction/loss. The complement-dependent citotoxicity (CDC) assay has demonstrated low sensitivity in detecting DSA. Newer solid phase assays, such as Luminex Single Antigen (LSA) beads (microbeads coated with single HLA class I or class II molecules), are highly sensitive but less predictive of transplant outcome because of detection of both complement-fixing and less clinically relevant no complement-fixing HLA antibodies. Methods: Using a modified LSA assay that identifies antibodies able to fix C1q, we investigate the clinical relevance of de novo HLA-DSA in relation to their capability to fix complement. In serum samples from 40 kidney transplanted patients who had developed IgG-LSA DSA after transplantation, we measured C1q-fixing ability of detected DSA by Class I and Class II LSA beads (One Lambda, CA). As for transplant outcome, 22 (55%) patients suffered graft failure (within 10±1 months from DSA appearance) and 18 (45%) had good graft function (GF) during all the follow up (mean follow up from DSA appearance: 54±34 months). Results: Twenty-three of the 40 patients showed production of C1q-positive DSA; the remaining 17 patients produced C1q-negative DSA. Correlating graft outcome and capability of DSA to fix C1q, we evidenced that graft failure due to antibody-mediated rejection occurred in 20 of the 23 patients showing C1q-positive DSA; only 2 of the 17 patients showing C1q-negative DSA suffered graft failure (87% vs. 12% respectively, P< 0.0001; RR = 7.39; Sensitivity=0.91; Specificity=0.83; PPV=0.87; NPV=0.88). It is to underlay that both C1q-positive and C1q-negative DSA were mainly specific for DQ mismatched molecules of the donor (74% and 53% respectively). Analyzing the target molecules of DQ-specific DSA in relation to the ability to fix complement, we evidenced that 53% (9/17) of anti-DQ C1q-positive DSA were specific for DQ1 molecules while 75% (6/8) of anti-DQ C1q-negative DSA were specific for DQ2 molecules. Conclusion: Our findings demonstrate that C1q-LSA assay shows the capability to identify the subset of IgG-LSA DSA strongly associated to antibody-mediated rejection and graft loss in kidney transplantation. Moreover the ability of C1q assay in distinguishing less harmful no complement-fixing DSA from clinically relevant C1q-fixing DSA represent a non-invasive tool for identifying patients that need specific immunosuppressive-therapy strategy to prolong graft survival.

15-OR

Aim Complement-fixing (CF) HLA antibodies (Ab) play an important role in transplantation because they can initiate inflammatory processes within the graft that lead to rejection. The Luminex Ig-binding assay (LUM-Ig) is now the principal method for HLA Ab testing. The C1q-binding assasy (LUM-C1q) can distinguish between CF and non-CF Abs. We show here evidence that the differential reactivity of CF HLA Ab depends on the structural composition of epitope (Ep)-carrying alleles (Al). Methods Human cytotoxic class I Ep-specific monoclonal antibodies (mAb) were tested in LUM-Ig and LUM-C1q (OneLambda). Results were compared to CDC reactivity with informative cells from the 13th International Workshop. Results Two reactivity patterns were observed. For some mAb, Ep-carrying Al reacted similarly in all assays. But certain mAb reacted with more Al in LUM-Ig than in LUM-C1q and CDC. Reactivity was analyzed with HLAMatchmaker, incorporating the concept that eplets are essential parts of structural Eps which can contact the 6 Complementarity Determining Regions (CDRs) of Ab. The data show that LUM-Ig mAb are specific for eplets but that LUM-C1q and CDC reactivity require an additional polymorphic amino acid configuration on eplet-carrying Al. Conclusions The findings must be viewed in context of formation of an antigen-Ab complex resulting in release of free energy necessary to stabilize binding and to induce conformational change in the Fc part of the Ab structure to expose the C1q receptor, the first step of Complement activation. The CF properties of HLA Ab require not only specific recognition of an eplet but also depend on interactions of other CDRs with critical amino acid configurations within the structural Ep. Eplet-carrying Al that lack such critical configurations will bind with Ab but the amount of free energy is insufficient to expose the C1q-binding site and there is no CF. This concept is important to our understanding whether or not CF donor-specific HLA Ab can initiate Ab-mediated rejection.

45-OR

Aim De novo production of donor-specific HLA antibodies (DSA) represents the major risk factor of graft failure in kidney transplantation. However, some patients show persistent presence of circulating DSA without occurrence of graft dysfunction/loss. Newer solid phase assay Luminex Single Antigen (LSA) beads, is highly sensitive respect to complement-dependent citotoxicity assay but less predictive of transplant outcome because of detection of both complement-fixing and less clinically relevant no complement-fixing HLA antibodies. Methods Using Class I and II C1q-LSA assay (One Lambda,CA), that identifies antibodies able to fix C1q, we investigate the clinical relevance of de novo HLA-DSA in 40 kidney transplanted patients. As for transplant outcome, 22 patients suffered graft failure (within 10 ± 1 months from DSA appearance) and 18 had good graft function during all the follow up (mean 54 ± 34 months from DSA appearance). Results Twenty-three patients showed production of C1q-positive DSA while 17 produced C1q-negative DSA. Correlating graft outcome and capability of DSA to fix C1q, graft failure occurred in 20/23 C1q-positive DSA patients; only 2/17 C1q-negative DSA patients suffered graft failure (87% vs.12%, P Conclusions C1q-LSA assay showed the capability to identify the subset of IgG-LSA DSA strongly associated to antibody-mediated rejection and graft loss in kidney transplantation; moreover, its ability in distinguishing less harmful no complement-fixing DSA from clinically relevant C1q-fixing DSA, allows to identify patients that need specific immunosuppressive strategy to prolong graft survival.

In Vitro Detection of C4d-Fixing HLA Alloantibodies: Associations With Capillary C4d Deposition in Kidney Allografts

Capillary C4d deposition is a valuable marker of antibody-mediated rejection (AMR). In this analysis, flow cytometric detection of alloantibody-triggered C4d deposition to HLA antigen-coated microparticles ([C4d]FlowPRA) was evaluated for its value as a marker for C4d deposition in renal allografts. For comparative analysis, 105 first renal biopsies performed for graft dysfunction and an equal number of concurrent sera were subjected to immunohistochemistry and [C4d] plus standard [IgG]FlowPRA, respectively. C4d deposition/fixation was detected in 17 biopsies and, applying [C4d]FlowPRA HLA class I and II screening, also in a small number of corresponding sera (N = 20). IgG reactivity detected by standard [IgG]FlowPRA was more frequent (49% of sera). Comparing [C4d]FlowPRA screening with capillary C4d staining, we found a high level of specificity (0.92 [95% confidence interval: 0.86-0.98]), which far exceeded that calculated for [IgG]FlowPRA (0.60 [0.50-0.70]). [IgG]FlowPRA screening, however, turned out to be superior in terms of sensitivity (0.94 [0.83-1.05] vs. 0.76 [0.56-0.97] calculated for C4d-fixing panel reactivity). Remarkably, posttransplant single antigen testing for identification of complement-fixing donor-specific alloreactivities failed to improve the predictive value of FlowPRA-based serology. In conclusion, our results suggest that detection of complement-fixing HLA panel reactivity could provide a specific tool for monitoring of C4d-positive AMR.

Pivotal Role of Complement-Fixing HLA Alloantibodies in Presensitized Kidney Allograft Recipients

Recipient presensitization represents a major risk factor for kidney allograft loss. Complement fixation may be a critical attribute of deleterious alloantibodies. We investigated clinical impact of complement-fixing HLA presensitization employing [C4d]FlowPRA, a novel assay permitting selective detection of HLA panel reactive antibody (PRA)-triggered C4 complement split product deposition. A cohort of 338 kidney transplants was evaluated for presensitization applying [C4d]FlowPRA together with [IgG]FlowPRA and complement-dependent cytotoxicity (CDC)-PRA. Analysis of HLA class I alloreactivities revealed a high incidence of C4d-positive graft dysfunction in [IgG]FlowPRA(+)/[C4d]FlowPRA(+) and [IgG]FlowPRA(+)/[C4d]FlowPRA(-) recipients (23% and 22% vs. 3% in [IgG]FlowPRA(-) patients). Only patients with complement-fixing HLA class I immunization had inferior graft survival [75% (3 years) vs. 91% and 89%, respectively (p=0.036)]. Despite frequent finding of capillary C4d deposition (28%), complement-fixing HLA class II immunization was not associated with inferior survival rates. This may have been due to reduction of clinical effects by intense immunosuppression in presensitized patients. Evaluating CDC, 29% of CDC-PRA(+)/[C4d]FlowPRA(+) recipients had C4d-positive graft dysfunction. For these patients 3-year graft survival was worst, followed by CDC-PRA(+)/[C4d]FlowPRA(-) and CDC-PRA(-) patients (76% vs. 81% vs. 90%, p=0.014). Results highlight a strong impact of complement-fixing HLA presensitization. Discerning complement-activating abilities of HLA alloantibodies, [C4d]FlowPRA may help identify recipients at particularly high risk for graft rejection and loss.

Elution of HL-A-Specific Antibodies from Platelets

The preconditions and kinetics of absorption and elution of HL-A specific antibodies from platelets, their antibody binding capacity (‘activity’) and the stability of HL-A receptors on platelets were studied. It was found that the acid elution technique is the only useful method as compared to the ether or heat elution. The optimal ratio of absorbed serum to eluate volume, platelet number, incubation temperature and time were evaluated. At pH 3.0-3.5 (range tested between pH 1.5 and 5.0) the strongest eluate activity was obtained. The fixation of HL-A specific antibodies to platelet receptors was not reduced by repeated washings with buffered saline, hypo- and hyperosmolaric salt solutions (range 0.077 to 2.464 mol NaCl). The capacity of antigenic receptors of platelets to bind complement-fixing HL-A antibodies remained unaltered after treatment with distilled water, hypo- and hyperosmolaric salt solutions (range as above) and incubation between pH 5 and 9. Freezing (-20 °C), heating (56 °C) and short incubation at pH 10 moderately altered HL-A receptors on platelets, which clearly deteriorated at pH 3 or after incubation at 100 °C. A standardized elution procedure is recommended.