Early Antibody-mediated Rejection Research Articles

Preformed Complement-Activating Low-Level Donor-Specific Antibody Predicts Early Antibody-Mediated Rejection in Renal Allografts

BACKGROUND.: Donor-specific anti-HLA antibodies (DSA) are a major cause of alloimmune injury. Transplant recipients with negative complement-dependent cytotoxic crossmatch (CDC-XM) and donor cell-based flow cytometric crossmatch (flow-XM) but low level DSA (i.e., by Luminex) have worse outcomes compared with nonsensitized patients. The aim of this study was to establish whether complement-activating ability in this low-level DSA, present before transplantation, as determined by this technique is important in dictating pathogenicity. METHODS.: We retrospectively studied 52 patients with preformed DSA detected by single-antigen flow cytometric fluorescent beads (SAFBs). Patients were transplanted using a steroid-sparing regimen consisting of alemtuzumab induction, 1 week of corticosteroids and tacrolimus monotherapy.Fifteen (29%) of 52 patients experienced antibody-mediated rejection (AMR), whereas 37 (71%) patients did not. There were no demographic differences between patients with AMR and those without. Pretransplant sera were retested using a modified (SAFB) assay, which detects the presence of the complement fragment C4d as a result of DSA-induced complement activation. RESULTS.: C4d+DSA were detected in 10 (19%) of 52 patients. Biopsy-proven AMR occurred in 7 (70%) of the 10 patients with C4d+DSA and in 8 (19%) of 42 patients with C4d-DSA. AMR-free survival was worse in patients with C4d+DSA (P<0.001). CONCLUSIONS.: The ability of preformed, low-level, DSA to trigger C4d fixation in vitro in patients with negative conventional crossmatch tests is predictive for AMR. C4d SAFB is potentially a powerful tool for risk stratification prior to transplantation and may allow identification of unacceptable donor antigens, or patients who may require enhanced immunosuppression.

The Use of Bortezomib as a Rescue Treatment for Acute Antibody-Mediated Rejection: Report of Three Cases and Review of Literature

Antibody-mediated rejection (AMR) typically occurs early after transplantation in approximately 5%–7% of recipients. The literature reports suggest that 12%–37% of kidney transplant recipients with acute AMR do not respond to treatment and eventually lose their grafts. The proteasome inhibitor bortezomib is currently approved by the Food and Drug Administration for the treatment of multiple myeloma. It has been demonstrated both in vitro and in vivo to possess apoptotic properties against mature plasma cells. Herein we have described a series of 3 patients with positive cross-matches who developed early AMR after kidney transplantation. Bortezomib rescue treatment was administered after the patients failed to respond to plasmapheresis/intravenous immunoglobulin and splenectomy. All 3 patients responded with full, durable recovery of renal function. In conclusion, bortezomib is useful to treat refractory AMR after kidney transplantation.

Outcome of Late Antibody Mediated Rejection in Renal Allografts after Conventional and Novel Therapies

Background: Antibody-mediated rejection (AMR) often develops early (within days or weeks) after kidney transplantation (txp). However, late AMR, manifesting after 6 months, can also occur. Although several strategies for treating early AMR have been investigated, there is little data on treatment of late AMR. In this study, we present single-center data of 23 patients treated for late AMR. Methods: Patients diagnosed with late AMR between Oct' 09 - Dec '11 were included. Late AMR was diagnosed using Banff 2007 criteria along with presence of donor specific antibody (DSA) and acute rise in serum creatinine (SCr). Patients were treated with plasmapheresis (PP) followed by low dose IVIG +/- rituximab +/- bortezomib. Serial DSA levels were followed throughout and after treatment. A response to therapy was defined as return of SCr to baseline or decline by at least 50% of peak SCr. Histologic improvement and decline in DSA strength were evaluated. Results: Patients' characteristics are presented in the table.Table: [Patients’ Characteristics]Eighteen patients (78%) were treated with PP/low-dose IVIG+rituximab; 9 of them (39%) received 1-3 cycles of bortezomib (4 dose-cycle). Four patients (17%) received PP/IVIG, and 1 received high-dose IVIG only. Patient received a median of 13 (6-22) PP sessions. Only 5 patients (22%) had improvement in all three criteria: histologic, DSA and SCr; 4 of whom had Class I in addition to Class II DSA; 3 of these responders had received bortezomib in addition to PP/IVIG+rituximab. Serious infectious complications required hospital admission developed in 7 patients (30%). There was a significant improvement in mean SCr (p=0.02) after therapy (2.5±1.0mg/dL) compared with SCr at the time of AMR diagnosis (2.9±1.92mg/dL), though in most cases (87%) renal function did not return to baseline. Eight patients (35%) had no improvement in histology and SCr, despite a decline in DSA strength in 5 of them. Lack of response among these patients was associated with higher interstitial fibrosis (ci) score ≥2 (p=0.01) and vascular fibrous intimal thickening (cv) score ≥2 (p=0.02) on index biopsy, but not with SCr at presentation. Conclusion: Reduction in maintenance immunosuppression is highly associated with subsequent late AMR. Our data demonstrate an inadequate response of late AMR to current and novel (bortezomib) therapies used successfully for early AMR. Although the combination of PP/IVIG+rituximab+bortezomib resulted in substantial improvement at least in some patients, the infectious complication risk remained high. The benefits of therapy need to be counterweighed with the potential adverse effects especially in patients with higher biopsy chronicity scores.

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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.

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Aim Bortezomib has activity against mature plasma cells, the source of potentially harmful donor-specific antibody (DSA) and antibody-mediated rejection (AMR). We present the use of bortezomib in 2 children with AMR after heart transplant (Tx). Methods Case 1: 3 yo with repaired congenital heart disease (CHD) with VAD placed for ventricular dysfunction. 2 DSAs were present at Tx with X-match positive. Initial therapy with Simulect (induction), plasmapheresis (Px), and IVIG 2 gm/kg cleared DSA. 18 days later a new DSA appeared. C4d on biopsy was negative. IVIG was repeated. DSA levels later increased with positive C4d and hemodynamic change. Bortezomib protocol was started: 1.3 mg/m2 IV every 3-4 days for 4 doses, Solumedrol 5 mg/kg with each dose, Px prior to bortezomib dose then for 3 consecutive days 3 days after the last dose, and rituximab 375 mg/m2 the day before protocol started. Serial HLA antibody testing was performed. Case 2: 3 yo with CHD transplanted at 11 mo developed acute AMR following viral illness. Steroids and IVIG produced no response. Bortezomib protocol was started without rituximab. Results DSA MFIs over time are presented below. Tx = transplant; Px = pheresis; Bor = bortezomib. Case 1 [Fig. 1] Case 2 [Fig. 2]. Download : Download full-size image Download : Download full-size image Conclusions Bortezomib can be used successfully to treat early and late AMR associated with DSA. Neither patient has had recurrence of antibody after 1 year follow-up.

HLA antibody profiling in thoracic transplantation undergoing desensitization therapy

Desensitization therapies in sensitized patients have been implemented to lower the human leukocyte antigen (HLA) antibody burden and facilitate thoracic transplantation. This review will focus on the characterization of HLA antibodies pre- and post-intervention, to assess the changes in antibody titer and function that might promote transplantation with low risk of early antibody-mediated rejection (AMR). A steady increase in the proportion of both adult and pediatric sensitized thoracic patients awaiting transplantation has been observed over the last years. Patients with elevated panel reactive antibodies at the time of listing have a poorer outcome compared to nonsensitized patients. Advances in solid phase assays for HLA antibodies have benefited the detection accuracy and characterization of clinically relevant anti-HLA antibodies. Therefore, monitoring the level and function of HLA antibodies following desensitization protocols can be used to determine the success of achieving acceptable antibody levels for safe transplantation and also the risk of AMR. Allosensitization among candidates considered for thoracic transplantation remains a great challenge. Determining the efficacy of desensitizing protocols by incorporating new tools for monitoring antibody profiles can better stratify a candidate's immunologic risk, thereby increasing the likelihood of transplantation and minimizing AMR.

The impact of terminal complement blockade on the efficacy of induction with polyclonal rabbit antithymocyte globulin in living donor renal allografts

BackgroundEculizumab, a potent inhibitor of terminal complement activation, appears promising in reducing early antibody-mediated rejection in positive crossmatch kidney transplantation. However, its concomitant use with polyclonal rabbit antithymocyte globulin (rATG) might reduce the efficacy of rATG. This study aimed to evaluate the effect of eculizumab on the efficacy of rATG in vivo and determine the role of complement in rATG-induced lymphocyte cell depletion. Patients and methodsThirty-six kidney transplant recipients were classified into 3 groups according to induction regime: anti-IL-2 receptor antibody alone induction group (basiliximab, n=8); rATG induction (n=20), and rATG+eculizumab induction group (n=8). Peripheral blood T-cell subsets and NK cells were measured 3–4days after transplant (after 3 doses of rATG). ResultsCompared to anti-IL-2 receptor antibody induction group, both groups treated with rATG demonstrated significant depletion of all T-cell subsets (CD3-positive cells) (P<0.0001 for rATG vs. anti-IL-2 receptor antibody induction group; P<0.001 for rATG+eculizumab vs. anti-IL-2 receptor antibody group). However, while T-cell counts were low in all rATG-treated patients, eculizumab treatment resulted in higher peripheral blood T-cell counts in rATG treated patients (P=0.005). Before induction, median total lymphocyte counts were normal for the three study groups. By 1, 4months and 1year, median the total lymphocyte count was normal for the anti-IL-2 receptor antibody group but was below normal range or at the lower edge of normality for rATG and rATG+eculizumab groups. ConclusionsThis small-sample size study suggests that peripheral T cells are depleted by rATG in the presence of terminal complement inhibition. However, eculizumab appears to have a mild inhibitory effect on peripheral blood T-cell depletion by rATG in kidney transplant recipients.

Proteasome Inhibition for Antibody-Mediated Allograft Rejection

Antibody-mediated rejection (AMR) is a major risk factor for graft loss following kidney transplantation. Traditional anti-humoral therapies provide suboptimal therapy and they do not deplete plasma cells, which are the source of antibody production. Proteasome inhibitors (PI) have been shown to deplete both transformed and nontransformed plasma cells in human transplant recipients and animal models; and therefore, offer a new paradigm for AMR, ie, plasma cell-targeted therapy. Bortezomib, a first in class proteasome inhibitor approved by the US Food and Drug Administration for treatment of multiple myeloma, has been used to treat AMR in several solid organ transplant recipients. The greatest experience with PI therapy for treating AMR is in kidney transplant recipients. Experiences to date with PI therapy have demonstrated that: (1) early AMR (within the first 6 months post-transplant) responds better than late AMR, and (2) the nature of the plasma cell clonal population influences sensitivity to PI therapy with plasma subsets greater than long-lived bone marrow niche-resident plasma cells. In conclusion, plasma cell-targeted therapy with PIs is a method for targeting plasma cells (the source of antibody production) with a well-elucidated mechanism of action and subsequent points of synergy, thereby providing an exciting new potential means for enhancing anti-humoral therapies.

Terminal Complement Inhibition Decreases Antibody-Mediated Rejection in Sensitized Renal Transplant Recipients

Sensitized renal transplant recipients with high levels of donor-specific alloantibody (DSA) commonly develop antibody-mediated rejection (AMR), which may cause acute graft loss or shorten allograft survival. We examined the efficacy of terminal complement inhibition with the humanized anti-C5 antibody, eculizumab, in the prevention AMR in renal transplant recipients with a positive crossmatch against their living donor. The incidence of biopsy-proven AMR in the first 3 months posttransplant in 26 highly sensitized recipients of living donor renal transplants who received eculizumab posttransplant was compared to a historical control group of 51 sensitized patients treated with a similar plasma exchange (PE)-based protocol without eculizumab. The incidence of AMR was 7.7% (2/26) in the eculizumab group compared to 41.2% (21/51) in the control group (p = 0.0031). Eculizumab also decreased AMR in patients who developed high levels of DSA early after transplantation that caused proximal complement activation. With eculizumab, AMR episodes were easily treated with PE reducing the need for splenectomy. On 1-year protocol biopsy, transplant glomerulopathy was found to be present in 6.7% (1/15) eculizumab-treated recipients and in 35.7% (15/42) of control patients (p = 0.044). Inhibition of terminal complement activation with eculizumab decreases the incidence of early AMR in sensitized renal transplant recipients (ClincalTrials.gov number NCT006707).

Clinical and investigational use of proteasome inhibitors for transplant rejection

Introduction:The presence of donor-specific anti-human leukocyte antigen (HLA) antibodies (DSA) in patients experiencing acute cellular rejection and antibody-mediated rejection (AMR) is associated with poor renal allograft survival in kidney transplant recipients. Traditional therapies for AMR provide variable results, and do not deplete the cellular source of antibody production, that is, the plasma cell.Areas covered:Physiologic effects of proteasome inhibitors (PIs) are reviewed in the context of recent clinical reports of PI therapy in solid organ transplantation for AMR and desensitization.Expert opinion:PI-based therapy is a novel approach for treating AMR that is being employed with increasing frequency in transplantation. Initial reports of PI-based regimens for treating AMR have demonstrated the ability of bortezomib to significantly reduce DSA levels and improve histology and allograft function. Use of PI agents have recently been evaluated in a large multicenter collaborative consisting of over 100 solid organ transplant recipients treated with a common PI-based regimen. Increasing experience with PI-based regimens for AMR have indicated that PI therapy (similar to other AMR therapies) provides excellent results in early AMR, with late AMR demonstrating a greater degree of therapeutic resistance. A substantial number of strategies exist for enhancement of therapeutic results with PI therapy for AMR.

Early and Late Acute Antibody-Mediated Rejection Differ Immunologically and in Response to Proteasome Inhibition

The efficacy of plasma cell targeted therapies for antibody-mediated rejection (AMR) has not been defined in detail. The purpose of this study was to compare early and late acute AMR in terms of immunologic characteristics and responses with proteasome inhibitor (PI) therapy. Renal transplant recipients with acute AMR were treated with PI-based regimens. Early acute AMR was defined as occurring within 6 months posttransplant. Immunodominant donor-specific antibody (iDSA) was defined as the DSA with the highest level. Results are expressed as early or late acute AMR. Thirty AMR episodes (13 early, 17 late) were treated in 12 and 16 patients. Early but not late AMR was associated with presensitization. Late AMR iDSA levels were higher, and specificities were primarily class II (DQ being most frequent). Early AMR patients demonstrated greater reduction in iDSA at 7, 14, and 30 days and at the posttreatment nadir (81.5%+21.2% vs. 51.4%+27.6%; P<0.01). Early AMR patients were more likely to demonstrate histologic resolution/improvement (87.5% vs. 53.8%; P=0.13). Both groups demonstrated significant improvement in renal function. Early and late AMR exhibit distinct immunologic characteristics and respond differently to PI therapy.

Salvage therapy for refractory rejection and persistence of donor-specific antibodies after intestinal transplantation using the proteasome inhibitor bortezomib

Salvage therapy for refractory rejection and persistence of donor-specific antibodies after intestinal transplantation using the proteasome inhibitor bortezomib

The strength of donor-specific antibody is a more reliable predictor of antibody-mediated rejection than flow cytometry crossmatch analysis in desensitized kidney recipients

The aim of this study was to evaluate the utility of donor-specific antibodies (DSA) and flow cytometry crossmatch (FCCM) as tools for predicting antibody-mediated rejection (AMR) in desensitized kidney recipients. Sera from 44 patients with DSA at the time of transplant were reviewed. Strength of DSA was determined by single antigen Luminex bead assay and expressed as mean fluorescence intensity (MFI). T- and B-cell FCCM results were expressed as mean channel shift (MCS). AMR was diagnosed by C4d deposition on biopsy. Incidence of early AMR was 31%. Significant differences in the number of DSAs (p = 0.0002), cumulative median MFI in DSA class I (p = 0.0004), and total (class I + class II) DSA (p < 0.0001) were found in patients with and without AMR. No significant difference was seen in MCS of T and B FCCM (p = 0.095 and p = 0.307, respectively). The three-yr graft survival in desensitized patients with DSA having total MFI < 9500 was 100% compared to 76% with those having total MFI > 9500 (p = 0.022). Desensitized kidney transplant recipients having higher levels of class I and total DSA MFI are at high risk for AMR and poor graft survival. Recipient DSA MFI appears to be a more reliable predictor of AMR than MCS of FCCM.

75-P: Preformed Low Reactive Anti-HLA Antibodies Are Associated With Majority of Early Antibody-Mediated Rejection in Renal Transplantation

75-P: Preformed Low Reactive Anti-HLA Antibodies Are Associated With Majority of Early Antibody-Mediated Rejection in Renal Transplantation

C4d-Fixing Capability of Low-Level Donor-Specific HLA Antibodies Is Not Predictive for Early Antibody-Mediated Rejection

Recent studies indicate that not all low-level donor-specific human leukocyte antigen (HLA) antibodies (HLA-DSA) (i.e., positive by solid-phase assays, negative by complement-dependent cytotoxic-crossmatch) have a detrimental clinical impact. The aim of this study was to investigate whether the pretransplant C4d-fixing capability allows distinguishing harmful from presumably clinically irrelevant HLA-DSA. We retrospectively investigated 64 patients with low-level HLA-DSA detected by single-antigen flow beads (SAFB). Thirty-four of 64 patients (53%) experienced early antibody-mediated rejection (AMR), whereas 30 patients (47%) did not. HLA-DSA characteristics (i.e., number, class, and strength), frequency of retransplants, and immunosuppressive regimens were not different in these two groups. Pretransplant sera were reanalyzed using a modified SAFB assay measuring C4d fixation induced by HLA antibodies. C4d-fixing HLA-DSA were observed in 11 of 64 patients (17%). AMR occurred in 6 of 11 patients (55%) with C4d-fixing HLA-DSA and in 28 of 53 patients (53%) without C4d-fixing HLA-DSA (P=1.0). Positive C4d staining in peritubular capillaries was detected in 6 of 11 patients (55%) with C4d-fixing HLA-DSA and in 21 of 53 patients (40%) without C4d-fixing HLA-DSA (P=0.50). The pretransplant capability of low-level HLA-DSA to trigger C4d fixation in vitro is not predictive for early AMR or C4d deposition in peritubular capillaries in vivo. This argues against pretransplant C4d SAFB testing to define the clinical relevance of low-level HLA-DSA.

Proteasome Inhibitor-Based Primary Therapy for Antibody-Mediated Renal Allograft Rejection

Rapid and complete elimination of donor-specific anti-human leukocyte antigen antibodies (DSA) during antibody-mediated rejection (AMR) is rarely achieved with traditional antihumoral therapies. Proteasome inhibitor-based therapy has been shown to effectively treat refractory AMR, but its use as a primary therapy for AMR has not been presented. Our initial experience with proteasome inhibition as a first-line therapy for AMR is presented. Adult kidney transplant recipients with AMR, diagnosed by Banff criteria, received a bortezomib-based regimen as the primary therapy. Bortezomib therapy was administered per package insert with plasmapheresis performed immediately before each bortezomib dose, and a single rituximab dose (375 mg/m2) given with the first bortezomib dose. DSA were quantitated using single-antigen beads on a Luminex platform. Two patients underwent bortezomib-based therapy for acute AMR occurring within the first 2 weeks after transplantation. High DSA levels and positive C4d staining of peritubular or glomerular capillaries were present at the time of diagnosis. Both patients experienced prompt AMR reversal and elimination of detectable DSA within 14 days of bortezomib-based therapy. Renal function remains excellent with normal urinary protein excretion at 5 and 6 months after AMR diagnosis. One patient experienced a repeated elevation of DSA (including two new human leukocyte antigen specificities) 2 months after initial bortezomib therapy, but without C4d deposition or histologic evidence of AMR. Retreatment with bortezomib provided prompt, complete, and durable DSA elimination. Proteasome inhibitor-based combination therapy provides a potential means for rapid DSA elimination in early acute AMR in renal transplant recipients.

319: Antibody Mediated Rejection (AMR) Predicted by New C1q Assay

HLA antibodies (Ab) that fix complement are essential for AMR. Current single antigen bead assays (SABs) do not distinguish binding Ab from complement fixing Ab. We developed the C1q assay to detect the presence of complement fixing Ab and examined it's utility to predict early AMR after pediatric heart tx.

Comparative Outcome Analysis of ABO-Incompatible and Positive Crossmatch Renal Transplantation: A Single-Center Experience

ABO-incompatible (ABOi) and positive crossmatch (XM) renal transplants pose special immunologic challenges. It is important to compare outcomes, study resource utilization, and attempt to risk stratify patients in these higher risk transplant settings. We compared apheresis utilization and transplant outcomes in ABOi, XM, and combined ABOi-XM renal transplants. We also analyzed multiple parameters, including patient and laboratory variables, to identify predictors of transplant outcome. Incidences of early (< or =30 days posttransplant) antibody-mediated rejection (AMR) and acute cellular rejection (ACR) were similar among the three incompatible groups whereas they differed in allograft rejection for late (>30 days posttransplant) AMR and ACR. Notably, there were no episodes of late AMR among ABOi patients. Patients treated with more than four pretransplant plasmapheresis/intravenous immunoglobulin (PP/IVIg) had a greater likelihood of experiencing early AMR. The median number of posttransplant PP/IVIg treatments was greater than twofold higher in ABOi-XM and XM patients compared to ABOi patients. Patients who required more than five posttransplant PP/IVIg procedures and those with one or more prior renal transplants had higher incidences of late ACR. Our analysis aids in defining apheresis resource utilization and helps in risk stratification of incompatible renal transplantation. It also aids in predicting allograft rejection and provides an opportunity for preemptive monitoring and treatment.

Plasmapheresis and Intravenous Immunoglobulin in Early Antibody‐Mediated Rejection of the Renal Allograft: A Single‐Center Experience

Antibody-mediated rejection (AMR) is a rare complication which often results in the loss of the kidney graft. The objective of this retrospective single center study was to evaluate two different approaches to AMR. We retrospectively evaluated data files from 936 patients who had undergone renal transplantation in 2002-2006. In 2002-2003, patients with AMR were treated with five plasmapheresis sessions (PP group, N = 13), and in 2004-2006 they received five plasmapheresis session along with intravenous immunoglobulin 0.5 g/kg (PP+IVIg group, N = 11). Twelve months of follow-up data was analyzed. First year graft survival was significantly higher in the PP+IVIg group than in the PP group (90.9% vs. 46.2%; P = 0.044); similarly, patient survival was higher in the PP+IVIg group (100% vs. 76.9%; P = 0.056). The incidence of infectious complications was similar in both groups. In re-biopsies, patients in the PP group often suffered from cellular rejection. The deposition of C4d complement was similar in re-biopsies in both groups. In this large single center study we proved the superiority of plasmapheresis with intravenous immunoglobulin administration in the treatment of early AMR of renal allografts.

Incidence and Prediction of Early Antibody-Mediated Rejection due to Non-Human Leukocyte Antigen-Antibodies

Antibody-mediated rejection (AMR) is responsible for a large proportion of early allograft losses. While preformed donor-specific human leukocyte antigen (HLA)-antibodies (HLA-DSA) are accountable for the majority of these episodes, non-HLA-DSA are also involved. However, data on the incidence of early AMR due to non-HLA-DSA are currently lacking. This study evaluated (i) the incidence of early AMR due to non-HLA-DSA -- defined by exclusion of circulating HLA-DSA detected by flow beads -- and (ii) the association with donor-specific major histocompatibility complex class I chain-related gene (MICA)-antibodies (MICA-DSA) and angiotensin-receptor antibodies. A retrospective cohort (n=279) risk stratified by complement-dependent cytotoxicity crossmatches (CDC-XM era) and a prospective cohort (n=154) risk stratified by virtual crossmatching using flow beads (virtual-XM era) were investigated. In the CDC-XM era 25/279 patients (9%) developed early AMR, but only 3/154 patients (2%) in the virtual-XM era (P=0.004). The incidence of early AMR due to HLA-DSA was significantly higher in the CDC-XM era than in virtual-XM era (18/279 patients [6.5%] vs. 0/154 patients [0%]; P=0.0005). However, the incidence of early AMR presumably due to non-HLA-DSA remained unchanged in these two cohorts (7/279 patients [2.5%] vs. 3/154 patients [2%]; P=1.0) consistent with a persisting gap in the ability to identify preformed DSA. Overall, 10/433 patients (2.3%) experienced early AMR presumably due to non-HLA-DSA. None of these 10 patients had angiotensin-receptor antibodies, at most 3/10 patients had MICA-DSA, while the antibodies remained unexplained in 7/10 cases. Early AMR due to non-HLA-DSA is a rare event, which is still difficult to predict by currently available assays.