Post-transplant Donor-specific HLA Antibodies Research Articles

Interrogating post-transplant donor HLA-specific antibody characteristics and effector functions using clinical bead assays

Single antigen bead (SAB) assays are the most common and sensitive method used to detect and monitor post-transplant donor specific HLA antibodies (DSA). However, a direct comparison across traditional and modified SAB assays to improve routine DSA monitoring using pre-treated IgG sera to eliminate interference has not been performed. We performed a technical comparison of 251 post-transplant DSA from n = 91 serum samples tested neat (pre-treated, undiluted), at a single 1:16 dilution, in the C1q bead assay, and for IgG subclasses (IgG1, IgG2, IgG3, IgG4) with IgG-enriched sera. We found that DSAs that are detectable by 1:16 dilution and/or C1q are associated with higher IgG MFI values and results could be predicted by testing neat sera. DSA detected at 1:16 dilution correlated with >7000 IgG MFI in neat sera and identified DSA that exceeded the SAB linear range for semiquantitative measurements. C1q positive DSA correlated with >15,000 IgG MFI in neat sera. C1q binding correlated most strongly with total IgG MFI (Spearman r = 0.82, p = 0.002) and not specific subclasses, demonstrating that DSA C1q binding capacity in this cohort is driven by HLA-specific IgG concentration. Evaluation of engineered pan-HLA class I-specific human IgG1 and IgG2 subclass monoclonal antibodies by SAB C1q and C3d assays revealed that IgG2 antibodies can bind complement at higher concentrations. The strengths and limitations of modified SAB assays must be considered to optimize efficient testing and accurate clinical interpretation.

Immunology of simultaneous liver and kidney transplants with identification and prevention of rejection.

Simultaneous liver and kidney (SLK) transplantation is considered the best treatment modality among selected patients with both chronic kidney disease (CKD) and end-stage liver disease (ESLD). Since the first SLK transplant in 1983, the number of SLK transplants has increased worldwide, and particularly in the United States since the implementation of the MELD system in 2002. SLK transplants are considered a relatively low immunological risk procedure evidenced by multiple studies displaying the immunomodulatory properties of the liver on the immune system of SLK recipients. SLK recipients demonstrate lower rates of both cellular and antibody-mediated rejection on the kidney allograft when compared to kidney transplant-alone recipients. Therefore, SLK transplants in the setting of preformed donor-specific HLA antibodies (DSA) are a common practice, at many centers. Acceptance and transplantation of SLKs are based solely on ABO compatibility without much consideration of crossmatch results or DSA levels. However, some studies suggest an increased risk for rejection for SLK recipients transplanted across high levels of pre-formed HLA DSA. Despite this, there is no consensus regarding acceptable levels of pre-formed DSA, the role of pre-transplant desensitization, splenectomy, or immunosuppressive management in this unique population. Also, the impact of post-transplant DSA monitoring on long-term outcomes is not well-studied in SLK recipients. In this article, we review recent and relevant past articles in this field with a focus on the immunological risk factors among SLK recipients, and strategies to mitigate the negative outcomes among them.

Effects of re-augmenting maintenance immunosuppression on post-transplant donor-specific HLA antibodies in liver transplantation

BackgroundDonor-specific antibodies (DSAs) have various negative short- and long-term effects after organ transplantation. DSAs are prevalent in patients with insufficient immunosuppression; thus, even patients with stable conditions after liver transplantation should be under optimized immunosuppression. However, the effect of re-augmenting immunosuppression therapy for patients with insufficient immunosuppression remains unclear. In this study, we investigated the long-term changes and the effects of immunosuppression (IS) re-augmentation on the DSA status. MethodsTwo DSA screenings were performed in 67 patients during long-term follow-up after liver transplantation. After the first screening, IS re-augmentation was performed in patients with consent. The effects of IS re-augmentation on the DSA status were analyzed using data of the serial DSA screenings. Negative conversion was defined as DSA positivity with MFI > 1000 converted to MFI < 1000. Improvement of DSA status was defined as either a 50% reduction of MFI or negative conversion. ResultsThe median interval between the first and second DSA screening was 50 months. Among 67 patients, 43 were positive for DSAs on the first screening. Among these 43 patients, 30 had minimal to no IS therapy at the time of the first screening. Among the 30 patients, IS re-augmentation was conducted in 19. A comparison between the patients with a re-augmented IS and those with a sustained minimized IS showed that the DSA levels significantly decreased in the former (63% (12/19) vs. 18% (2/11), p = 0.02). ConclusionsThe results of this study indicate that post-liver transplant IS re-augmentation had suppressive effects on the DSA status. However, the clinical significance of DSA-negative conversion and/or mean fluorescence intensity reduction needs to be further investigated through histological evaluation and/or graft survival during longer follow-up periods.

Relevance of donor-specific antibody monitoring after kidney transplantation: Findings from the Collaborative Transplant Study and the Heidelberg Transplant Center.

Monitoring of donor-specific HLA antibodies (DSA) has become part of the clinical routine in kidney transplantation. This paper gives a brief overview on data from the Collaborative Transplant Study (CTS) and the Heidelberg Transplant Center on the clinical relevance of post-transplant DSA monitoring in patients undergoing renal transplantation. The obtained findings underline the importance of DSA monitoring in the post-operative course in immunologically high-risk patients and patients with deterioration of graft function. Especially in patients with a pre-activated immune system, a gap in the immunosuppressive therapy appear to lead to persistence, reappearance or de novo occurrence of strong, complement-activating DSA, resulting in severe antibody-mediated rejection (AMR) and, without timely intervention, in AMR-related graft loss.

Association of posttransplant donor-specific HLA antibody with liver fibrosis after pediatric liver transplantation

Introduction: Although the short-term outcomes of pediatric liver transplantation (LT) have improved, the long-term outcomes, which are especially important in pediatric LT, have not improved to the same extent. This is partly caused by immunological progression of liver graft fibrosis. However, liver function test results are commonly normal even among patients with liver fibrosis; therefore, the indication and timing of liver graft biopsy have not been established. The aim of this study was to evaluate the significance of posttransplant donor-specific antibody (DSA) as a predictor of liver fibrosis during long-term follow-up after pediatric LT. Method: A retrospective analysis of liver graft fibrosis was performed in 34 LT recipients. Patients were divided into two groups based on histological findings, and clinical characteristics among patients with immunological graft fibrosis were assessed. The fibrosis group included patients with fibrosis of F2 or more. Result: Of the 34 patients included in this study, 11 (32%) were included in the fibrosis group and 23 in the non-fibrosis group. No significant between-group differences were found regarding pretransplant characteristics, including age, sex, ABO incompatibility, and immunosuppressive regimen. Episodes of biopsy-proven acute rejection and non-adherence to immunosuppressive drugs were comparable between the two groups. The mean fluorescence intensity (MFI) for anti-DR donor-specific antibodies was significantly higher among the patients with immunological fibrosis than among those without (1655 vs. 389; p = 0.009), and significantly more patients tested positive in the fibrosis group than in the non-fibrosis group (73% vs. 30%; p = 0.03). Conclusion: Posttransplant development of anti-DR DSA is a risk factor of liver fibrosis during long-term follow-up. This finding provides useful information for the implementation of valid histological examinations of liver grafts for patients with higher MFI, especially for anti-DR DSA, after pediatric LT.

Analysis of the Association of Posttransplant Donor-Specific HLA Antibody with Liver Fibrosis After Pediatric Liver Transplantation

Objectives Although the short-term outcomes of pediatric liver transplantation (LT) have improved, the long-term outcomes, which are especially important in pediatric LT, have not improved to the same extent. This is partly caused by immunological progression of liver graft fibrosis. However, liver function test results are commonly normal even among patients with liver fibrosis; therefore, the indication and timing of liver graft biopsy have not been established. The aim of this study was to identify predictive factors of immunological graft fibrosis during long-term follow-up after pediatric LT. Methods We retrospectively identified 90 patients who underwent pediatric living donor LT (LDLT) at our institution between July 1991 and October 2013. Out of 90 LDLT patients, donor-specific antibody (DSA) screening was performed for 71 patients. After the DSA screening, liver biopsies were subsequently performed and graft fibrosis plus findings of chronic rejection were evaluated for 18 patients. Patients were divided into two groups based on histological findings, and clinical characteristics among patients with immunological graft fibrosis were assessed. The degree of fibrosis was diagnosed based on the Metavir fibrosis staging, and the fibrosis group included patients with fibrosis of F2 or more and patients with F1 fibrosis plus findings of chronic rejection, especially including C4d deposition. The cut-off for a positive reaction of Single Antigen Beads assay was set at a mean fluorescence intensity (MFI) of 1,000 or more. Results Of 18 patients, seven were included in the fibrosis group. No significant between-group differences were found regarding pretransplant characteristics, including age, sex, primary disease, ABO incompatibility, and immunosuppressive regimen. Episodes of biopsy-proven acute rejection and non-adherence to immunosuppressive drugs were comparable between the two groups. The mean fluorescence intensity (MFI) for anti-DR DSAs was significantly higher in the fibrosis group than the non-fibrosis group (1655 vs. 216; p = 0.019). There were significant between-group differences regarding the positive rate of anti-DR DSAs (86% vs. 28%; p = 0.012). The MFI for anti-DQ DSA was higher among patients with liver fibrosis, although this between-group difference did not reach statistical significance. The immunosuppression minimization rate was more prevalent among patients with liver fibrosis, although this between-group difference did not reach statistical significance (71% vs. 45%; p = 0.37). Conclusion Posttransplant development of anti-DR DSA is a risk factor of liver fibrosis during long-term follow-up. This finding provides useful information for the implementation of valid histological examinations of liver grafts for patients with higher MFI, especially for anti-DR DSA, after pediatric LT. JSPS KAKENHI Grant Numbers JP17K16502. JSPS KAKENHI Grant Numbers JP26861036.

P025 HLA antibody targets: Pre-transplant, post-transplant, post-graft loss

Aim It has often been noticed that the most frequent target of donor-specific HLA antibodies (DSA) in post-transplant monitoring are DQ antigens, at a rate significantly higher than that observed in patients awaiting transplant. This study is an attempt to quantify that observation. Methods From 2012 to 2017 sera from patients who had received or were waiting to receive a 1st or 2nd second kidney transplant were tested using the LABScreen Single Antigen kit. To avoid counting patients twice within a stage (pre-1st transplant, post-transplant, relisted for 2nd transplant) patients were evaluated for this study only on their most recent serum within each stage. 5896 patients pre-1st transplant (3458 males, 2438 females), 2626 patients who received post-transplant testing for DSA, and 754 patients who were relisted for a 2nd transplant were evaluated. As reliable transfusion and pregnancy information was unavailable for most pre-transplant patients, the difference between male and female sensitization rates was used as a surrogate for the different sensitizing effects of transfusion versus pregnancy in this stage. Results Sensitization to any HLA was 14% in pre-transplant males, 38% in pre-transplant females, 15% in post-transplant monitoring (but only evaluated for DSA), and 71% in patients relisted for a second transplant. Males and females pre-first transplant show significant differences in antibodies against class II, and also against HLA-B and to a lesser extent HLA-C. Post-transplant DSA shows a very high rate of antibodies against HLA-DQ, and relatively little against class I. Patients relisted for a second transplant show extensive antibody production against antigens of all loci, especially HLA-A, B, DRB1 and DQ. Conclusions The three major sources of sensitization to HLA, transfusion, pregnancy and transplantation, show different rates of sensitization to different HLA loci. Among HLA antibody producers, antibodies to DQ are 2–3 times more frequent as DSA than they are as pre-transplant antibody targets. Download : Download high-res image (215KB) Download : Download full-size image

Impact of the Trough Level of Calcineurin Inhibitor on the Prevalence of Donor-Specific Human Leukocyte Antigen Antibodies During Long-Term Follow-Up After Pediatric Liver Transplantation: Antibody Strength and Complement-Binding Ability.

BackgroundIn pediatric patients, long-term immunosuppression after liver transplantation (LT) is typically minimal. However, posttransplant donor-specific HLA antibodies (DSAs) may be prevalent under these conditions. Here, we evaluated the effects of minimized calcineurin inhibitor (CNI) on DSA development to assess the validity of minimized/withdrawn immunosuppression.MethodsWe retrospectively examined 66 patients who underwent pediatric LT at our institution between July 1991 and October 2013. Patients were divided into 2 groups based on the CNI trough level. The cutoff trough levels were 3 and 30 ng/mL for tacrolimus and cyclosporine, respectively. Luminex single-antigen bead assays were performed, and the cutoff for a positive reaction was set at a mean fluorescence intensity (MFI) of at least 1000.ResultsThe mean recipient ages at the time of LT were 29.1 and 77.2 months for the low and regular CNI groups, respectively (P = 0.0007). Univariate logistic regression analysis revealed that recipient age at LT younger than 3 years (P = 0.0099) and low CNI (P < 0.0001) were significantly associated with DSA development. In multivariate analysis, low CNI was an independent risk factor of DSA development (P = 0.0011). Of 15 high-MFI DSAs, 3 were anti-DR, and 12 were anti-DQ. Two of 3 anti-DR DSAs and 11 of 12 anti-DQ DSAs had complement-binding ability and high MFIs.ConclusionsCNI minimization was an independent risk factor for posttransplant DSA during long-term follow-up after pediatric LT. Adjusting CNI to appropriate levels is a safe first step to prevent the immunological effects of DSA.

Effect of Recipient Age at Liver Transplantation on Prevalence of Post-Transplant Donor-Specific HLA Antibody.

BACKGROUND Post-transplant donor-specific HLA antibodies (DSA) may have a detrimental effect on long-term outcomes of organ transplantation. The aim of this study was to specifically evaluate the effect of recipient age on the prevalence of DSA over a long-term follow-up after living donor liver transplantation (LDLT). MATERIAL AND METHODS A retrospective analysis of DSA evaluations was performed in 50 pediatric patients with HLA data available. Patients were divided into 2 groups based on their age at the time of LDLT: younger (Y) group, age <3 years; older (O) group, age ≥3 years. DSA evaluation was performed using Luminex single-antigen bead assays, with a mean fluorescence intensity ≥1000 used as a cut-off for positive results. RESULTS There were no between-group differences in terms of sex, ABO incompatibility or acute rejection. Only one of our 50 patients tested positive for class I DSA. Significantly more patients tested positive for HLA-DR DSA in group Y (40.6%) than in group O (11.1%; p=0.02). Recipients <3 years of age at the time of LDLT may be at a higher risk of testing positive for class II DSA. CONCLUSIONS These findings can inform the implementation of cost-effective screening of post-transplant DSA in pediatric LDLT recipients.

P072 De novo anti-HLA-class II donor specific antibodies are associated with patient mortality after lung transplant failure

Aim The development of donor-specific HLA antibodies (DSA) after lung transplantation has been implicated in acute and chronic rejection. The diagnosis of lung antibody mediated rejection (AMR) is based upon histology, DSA testing and clinical assessment of graft function. Post-transplant DSA monitoring has become more and more common as a guide toward diagnosis and sometimes institution of AMR treatment. In this study, we aimed to assess DSA with biopsy surveillance and associate with patient mortality after lung transplant failure (MLTF). Methods We retrospectively selected 127 patients, transplanted between Jan, 2004 and July, 2015 whose DSA were tested for clinical indications post-transplantation. Protocol biopsies were performed for these patients at 1, 3, 6, 9, and 12-months after transplant for clinical suspicions. DSA were monitored using LabScreen single antigen bead assays. Biopsy HE however, no association was found for Class I DSA. No significant relationship between DSA and histological changes of AMR in lung transplant biopsies was found in this cohort. Conclusion HLA Class II DSA is associated with graft dysfunction and mortality in pulmonary transplant patients although we did not find the correlation with biopsy findings. Our study suggests that post-transplant protocol DSA surveillance may add value to assess allograft function and to treat graft rejection at the early stage.

P002 The consistent inconsistency in HLA antibody testing practices: a collaboration with the Banff workgroup

Detection of donor specific HLA antibodies (DSA) is currently an important component of the Banff criteria for diagnosis of antibody mediated rejection. There is a perception of significant variations in anti-HLA antibody (HLA-Ab) testing practices and interpretation algorithms among different laboratories. In this study, we aimed to survey large transplant centers worldwide to evaluate similarities and differences in HLA-Ab testing practices . METHOD An online survey was sent electronically to 120 histocompatibility laboratories and we received data from 72 laboratories (60% response rate). The survey focused on defining sensitization status, method of reporting HLA-Abs, and post-transplant DSA monitoring practices. Responses from laboratories that support more than 25 transplants per year were analyzed ( n = 66). RESULTS: More than 80% of surveyed laboratories use Luminex based single antigen bead assay to test for HLA-Abs. In non-sensitized patients, absence of HLA-Abs was defined by screening beads (11% of labs), phenotype beads (11%) by single antigen beads (30%) and 2 different assays (48%). For sensitized patients 54% of the laboratories used a combination of assays. Definition of highly sensitized patients varied widely among labs with thresholds ranging from 30% to 80% CPRA. Fifty-five percent of the labs reported MFI values associated with HLA-Abs while 30% reported relative strength (weak, moderate, strong) of the antibody. The definition of relative strength varied among labs with MFI ranges of 500–5000 and 3000–10,000 defining weak and strong antibodies, respectively. Only 39% of the laboratories routinely evaluated sera for the presence of interfering substances/prozone effect. Only 44% of the labs routinely screened for DSA post-transplant; 24% screened only at the time of for-cause and surveillance biopsies and 9% do not monitor for DSA at all. CONCLUSION There is great variability in testing, interpretation and reporting practices among laboratories. Similarly, the definition of a highly sensitize patient varies from center to center. Lack of consistency in practices and definitions hinder our ability to compare patient outcomes from one center to another and highlights the need for standardization and evaluation of clinical relevance of different practice patterns.

DSA and C1q assay in kidney post-transplant monitoring

Aim Predicting the risk of graft loss using C1q assay in the post-transplant DSA monitoring. Methods Retrospective review of post-kidney transplant patients who were tested for DSA after ABO compatible kidney transplantation with negative T and B lymphocyte crossmatch. Patients in post-transplant follow-up since 2007 were tested for donor specific HLA antibody (DSA) IgG class according to schedule by Labscreen phenotype beads/Single Antigen beads (SAB, One Lambda), and if detected, reflexed to C1q assay (One Lambda) since 2011. Results Of the 437 post-kidney transplant patients (range: 1–27 years post-transplant, mean 9.1 years), de novo DSA was detected in 74 (16.9%). C1q assay was performed in 50/74 DSA positive patients: Positive at least once N = 35 (70%) and Negative N = 15. DSA in 26 patients were tested for > 1 year; persistently positive C1q, N = 17, eventually became C1q negative N = 6, or became DSA negative N = 3. Overall, C1q positive DSA presented higher maximum MFI detected by both phenotype beads and SAB, and resulted in higher graft loss. C1q MFI was similar between graft lost and functional (defined by not on dialysis). Regardless of C1q status, graft loss was observed after month-to-years with DSA, indicating chronic antibody mediated rejection. Conclusions Patients with C1q positive DSA had higher MFI (IgG) levels and resulted in higher graft loss. Download : Download full-size image

Endothelial cell antibodies associated with novel targets and increased rejection.

The initial contact point between a recipient's immune system and a transplanted graft is the vascular endothelium. Clinical studies suggest a pathogenic role for non-HLA antiendothelial cell antibodies (AECAs) in allograft rejection; however, evidence linking AECAs of known specificity to in vivo vascular injury is lacking. Here, we used high-density protein arrays to identify target antigens for AECAs isolated from the sera of recipients of kidney transplants experiencing antibody-mediated rejection in the absence of donor-specific HLA antibodies. Four antigenic targets expressed on endothelial cells were identified: endoglin, Fms-like tyrosine kinase-3 ligand, EGF-like repeats and discoidin I-like domains 3, and intercellular adhesion molecule 4; the first three have been implicated in endothelial cell activation and leukocyte extravasation. To validate these findings, ELISAs were constructed, and sera from an additional 150 renal recipients were tested. All four AECAs were detected in 24% of pretransplant sera, and they were associated with post-transplant donor-specific HLA antibodies, antibody-mediated rejection, and early transplant glomerulopathy. AECA stimulation of endothelial cell cultures increased adhesion molecule expression and production of inflammatory cytokines: regulated on activation, normal T cell expressed and secreted PDGF and RESISTIN. These correlations between in vitro experiments and in vivo histopathology suggest that AECAs activate the vascular endothelium, amplifying the alloimmune response and increasing microvascular damage. Given the growing number of transplant candidates, a better understanding of the antigenic targets, beyond HLA, and mechanisms of immune injury will be essential for improving long-term allograft survival.

Looking Behind the Curtain: Pre-Transplant Mismatched Eplet Load May Better Predict Increases in Post-Transplant DSA.

Purpose: Patients receiving renal transplants in the presence of donor specific HLA antibodies (DSA) are at increased risk for antibody mediated rejection (AMR) as well as decreased graft survival. For highly sensitized patients, transplanting in the presence of low/moderate DSA may be the only viable option. We looked at which characteristics of pre-existing DSA are associated with poor post-transplant(tx) outcomes. Methods: We identified 11 patients (mean cPRA=87+7%) with DSAs from a cohort of 165 consecutive renal txs performed between 10/2012 and 12/2013. Patients were desensitized using plasmapheresis(PP) and IVIg and received standard induction and maintenance therapy post-tx. DSA titers and MFI were determined by solid phase assays (pre-Tx, day 3, weekly 1-4, months 2, 3, 6, 9, 12). Donor-recipient eplet mismatches (MM) in the DSA-associated HLA antigens were determined using HLA Matchmaker. Results: Post-tx, 5 of the 11 (45.5%) desensitized patients no longer had clinically significant DSA. Increased DSA were found in the remaining 6 (55.5%) within 2 weeks (MFI=3469-15000, titers 1:16->1:1024) and 5 developed de novo DSA. No association between pre-tx MFI values, positive xmatch (T or B), or previous tx and increased post-tx DSA was found. In contrast, patients with negative DSA post-tx averaged 6.4+3.6 eplet MM in pertinent HLA antigen, whereas those with increasing DSA post-tx averaged 16.8+5.6 MM eplets.Expression of Cytosolic DoubleFigure: No Caption available.Increased DSA were successfully reversed in 2 patients with PP and IVIg and in 1 with increased basal immunosuppression. The remaining 3, after treatment, have DSA levels that are stable or slightly lower with surviving but sub-optimal graft function. Conclusions: Renal transplants may be performed successfully in presence of DSA, but remain at increased risk for graft failure or poor function. Our data suggest that mismatched eplet load is predictive of clinically significant post-tx DSA and may identify patients in need of frequent monitoring and aggressive early intervention to minimize DSA load and improve outcomes.

Mismatched HLA Eplets: Potential Predictor of Donor Specific Antibody Development Risk in Heart Transplantation.

Objectives: Donor Specific HLA antibody (DSA) is associated with poor graft survival. Recent studies suggest that mismatched (MM) HLA eplets may be associated with higher incidents of post-transplant (tx) DSA. Our aim was to investigate whether greater numbers of MM eplets expose heart tx recipients to an increased risk of DSA development and to evaluate whether assessment of MM eplets may provide clinicians with additional tools for more effective monitoring and treatment of DSA. Methods: We evaluated 35 heart recipients transplanted from 2010 to 2013 using solid phase antibody assays. The HLAMatchmaker program was used to calculate the number of donor-recipient HLA MM eplets. Sera were collected at 1, 3, 6, and 12 months or when clinically indicated. DSA were characterized using solid phase assays. Results: The cohort consisted of 71% males and 29% females with a mean age of 48yr. Out of 35 patients (pts), 7 developed DSA (20%) within 18 months post-tx. Among the 7 pts, 5 developed de novo DSA (dnDSA) and 2 had increased levels of historic DSA that were negative at the time of tx. No pts developed DSA against Cw, DR51, or DR52. One pt developed DSA against B and 1 against DR53. For HLA antigens including A, DR, and DQ, the number of MM donor eplets was higher in pts who developed either dnDSA or increased levels of historic DSA. Our data suggest that the number of MM eplets contributing to DSA development may be locus specific. For example, having 8 MM A eplets may be sufficient to induce DSA; in comparison, more than 14 MM DQ eplets appear to be needed. Conclusions: Our results suggest that a higher number of MM eplets may be associated with increased DSA against A, DR, and DQ antigens. Therefore, pts transplanted with a high MM eplet load may require close monitoring for post-tx DSA and early intervention. Further study on a larger population of heart tx pts is required to confirm this finding.Figure: No Caption available.

Significance of C1q-fixing Donor-Specific Antibodies After Kidney Transplantation

ObjectivesDe novo donor-specific HLA antibodies (DSA) are associated with allograft rejection and allograft loss. However, not all DSA are equally detrimental to allograft function. The ability to activate complement may be an important factor differentiating clinically relevant DSA from nonrelevant DSA. The C1q assay detects a subset of HLA antibodies that can fix complement. This study aimed to investigate the correlation between C1q-fixing de novo DSA (dnDSA) and clinical outcomes posttransplant. MethodsThis retrospective study included 193 sera from kidney transplant recipients who underwent posttransplant DSA testing and/or kidney biopsy for clinical causes. Thirty-five of the 193 (18.1%) had immunoglobulin G DSA. Seventeen of the 35 patients were excluded owing to the presence of pretransplant HLA antibodies. We then analyzed C1q DSA at the time of biopsy in 18 recipients who developed dnDSA. The clinical outcomes of patients with C1q-positive DSA and C1q-negative DSA were compared. ResultsC1q-positive DSA were detected in 10 of 18 patients (55.6%). The incidences of transplant glomerulopathy were significantly higher among patients with C1q-positive DSA than patients with C1q-negative DSA (80% vs 0%; P = .001). Although patients with C1q-positive DSA experienced more chronic antibody-mediated rejection and graft loss (80% vs 37.5% [P = .145]; 60% vs 25% [P = .188]), the differences were not significant. The receiver operating characteristic curve analysis showed that the C1q assay was an excellent predictor of transplant glomerulopathy with area under the curve of 0.9 (95% CI, 0.769–1.000). ConclusionThe presence of C1q-positive dnDSA was associated with an increased risk of transplant glomerulopathy. The C1q assay is potentially a powerful method for identifying patients at risk for transplant glomerulopathy.

35-OR

Aim Sensitized heart transplant candidates are evaluated for donor specific HLA antibody (DSA) by Luminex single antigen bead (SAB) testing to assess donor suitability and predict a positive complement dependent cytotoxicity (CDC) crossmatch (XM) by virtual XM (VXM). However, SAB testing imperfectly correlates with VXM, and antibody thresholds for XM prediction are center-specific. A novel Luminex SAB-based assay detecting C1q-binding HLA antibodies adds functional data to SAB testing, but the relationship between SAB strength and C1q positivity is unclear. In this retrospective study, we examine the relationship between HLA antibody titer and complement-binding ability in cardiac allograft recipients and correlate C1q DSA to antibody-mediated rejection (AMR). Methods We identified 15 sensitized pediatric and adult heart recipients with a calculated panel reactive antibody >50%. Sera were tested by conventional SAB antibody testing using undiluted or 1:16 diluted sera and were compared to C1q results. Pre- and post-transplant DSA by both methods were correlated with XM and early AMR. Results In 9 samples, individual Class I antibodies measured by SAB testing in undiluted sera correlated poorly with C1q reactivity (R 2 = 0.07). In contrast, using sera diluted 1:16, conventional SAB results correlated better with C1q testing ( R 2 = 0.73). Receiver operator characteristic curve analysis also showed superior area under the curve (AUC) for predicting C1q positive results in sera diluted 1:16 than in undiluted sera (AUC = 0.99 vs 0.82). Low MFI by conventional SAB testing in alleles with high C1q MFI increased upon dilution or heat-treating sera and correlated with C1q results. Clinically, in 13 recipients C1q DSA predicted CDC-XM positivity and early AMR (positive predictive value (PV) 87.5%; negative PV 100%). Conclusions In conclusion, SAB titer correlated with and predicted C1q reactivity. The risk for positive crossmatch and early AMR in sensitized heart recipients strongly correlated with C1q binding DSA.

75-P

Aim To determine the frequency of donor specific HLA antibody (DSA) to DQ alpha in renal post-transplant recipients. Donor DQA1 typing is not a routine procedure under UNOS. Frequency of post-transplant DQ alpha DSA and possible importance in post-transplant monitoring is not well-studied. Methods Retrospective review of post-transplant HLA antibody test results. Reviewed are all HLA antibody test results between January 2010 and April 2012 for the patients who received a deceased or living donor kidney at our center. HLA typing of donors and recipients was tested by serology or SSO/SSP. HLA antibody was tested by solid phase methods (LABScreen PRA and LABScreen Single Antigen beads, One Lambda). Current antibody monitoring schedule for low risk patients (no pre-formed or de novo DSA and stable creatinine) is 9 times in the first year, every 6 months until the 3rd year, then annually. Additional tests are performed if higher risk or suspected rejections. If DQ alpha antibody is detected, donor DQA1 was typed retrospectively using frozen cells. Results Post-transplant HLA antibody was tested on 158 recipients: 27 pediatrics and 131 adults (average 46.3 years old). Results represent data points from two months to 26 years post-transplant (average 5.0 years). In all patients, pre-transplant crossmatch was either compatible by CDC method, or compatible/weakly incompatible by flow cytometry method. Of the 158 patients, DSA (either de novo or preformed) was detected in 29 patients (18.4%). Detected were class I DSA only in 3 patients, class II DSA only in 20 patients, and both class I/II in 6 patients. Specificities of class II DSAs were DQB1 in 22 patients (85%), DQA1 in 10 patients (39%), DRB1/3/4/5 in 7 patients (27%), and DPB1 in 1 patient (4%). Conclusions HLA-DQ alpha DSA was detected in a substantial percent of DSA positive renal transplant patients, thus DQA1 donor typing is important in renal post-transplant monitoring.