Geraldine Ong

Anti-angiotensin type 1 receptor antibodies associated with antibody mediated rejection in donor HLA antibody negative patients.

Background. Angiotensin type 1 receptor (AT1R) mediates most physiologic and pathophysiologic actions of its endogenous ligand, angiotensin II, with overactivity leading to vascular remodeling and hypertension. Antibodies to AT1R are implicated in several vascular pathologies. The aim of our study was to determine the impact of antibody to AT1R on clinical outcomes including antibody mediated rejection (AMR), with or without C4d deposition, in patients whose sera contained no donor human leukocyte antigen (HLA)-specific antibody (HLA-DSA). Methods. Pretransplant sera from 97 recipients and sera obtained at the time of acute rejection (AR) were tested by Luminex-based single-antigen bead assays to determine HLA-DSA and antibodies to major histocompatibility class I chain-related gene A (MICA). The presence of antibody to AT1R was determined by a cell-based ELISA method using a cutoff of 17 units to distinguish high from low binding. Results. Sera from 63 recipients were determined to have no HLA-DSA and no donor-specific MICA antibodies pretransplant and at the time of AR, and 16 of these recipients were diagnosed with AR including 7 with AMR and 9 with cellular AR (cell-mediated rejection). High-binding AT1R antibodies were identified for six of seven in the AMR+ group and zero of nine in the cell-mediated rejection+ group (P=0.0009). Conclusions. A strong association was observed between the presence of high binding to AT1R and AMR in recipients whose sera contained no antibody to donor HLA or MICA. Assessing the AT1R antibody status along with the HLA-DSA provides additional information to determine the immunologic risk for recipients.

Acceptable donor-specific antibody levels allowing for successful deceased and living donor kidney transplantation after desensitization therapy

Background. The aims of this study were to determine the level of donor-specific antibody (DSA) that allows for successful transplantation after desensitization with IVIG and rituximab and to identify patients at risk for antibody-mediated rejection (AMR). Methods. Pre- and posttransplant sera from 16 patients with DSA before desensitization were tested. Strength of DSA was determined by single antigen Luminex bead assay and results expressed as standard fluorescence intensity (SFI). T-cell flow crossmatch results were expressed as mean channel shifts (MCS). AMR was determined by biopsy and C4d deposition. Results. Six had negative pretransplant flow crossmatches with a mean DSA of 8,805 SFI. Five had positive flow crossmatches (78-192 MCS) with mean DSA of 55,869 SFI. No patients in either group had AMR. Five had positive flow crossmatches (222-266 MCS) with mean DSA of 118,063 SFI. Three experienced AMR. The MCS and DSA levels for patients with AMR were significantly higher than patients without (P≤0.001). For patients without complications (n=7), DSA remained less than 105 SFI and usually decreased to approximately 104 SFI posttransplant for both class I and II. For patients with AMR (n=3), predominant increases in class II DSA more than 105 SFI were observed. All three patients continue to have DSA approximately 105 SFI with stable creatinine after treatment for AMR. Conclusions. Approximately 63% of patients were transplanted with a positive flow crossmatch. The results show that patients with DSA more than 105and FCM more than 200 MCS are at higher risk for AMR. Treatment of AMR improves renal function without significant changes in DSA.

Increased Negative Impact of Donor Hla-specific Together With Non-hla–specific Antibodies on Graft Outcome

Background De novo donor HLA-specific (dnDSA) and non-HLA antibodies including antiangiotensin type 1 receptor antibodies (AT1R-abs) have been associated with antibody-mediated rejection (AMR) and decreased graft survival as well as cellular-mediated rejection (CMR) and early onset of microvasculopathy in heart transplantation. The aim of our study was to determine the impact of anti–AT1R-ab and anti–donor HLA–specific antibody (DSA) on clinical outcomes. Methods Pretransplant and posttransplant sera from 200 recipients transplanted between May 2007 and August 2011 were tested for DSA (Luminex-based single antigen bead assay) and AT1R-ab (enzyme-linked immunosorbent assay). Two cutoff levels (≥17 and ≥12 units) were used to define high and intermediate binding of AT1R-ab. Clinical parameters examined were 5-year AMR/CMR (≥grade 2), coronary artery vasculopathy, and survival. Results At 2 years after transplant, freedom from AMR and/or CMR was 95.4% for those with no DSA (n=175), 66.9% for those with dnDSA (n=19), and 25% for those with DSA at transplant (n=6) (P<0.0001). Neither ≥17 nor ≥12 units of pretransplant levels indicated a significant difference in freedom from AMR and/or CMR. When both dnDSA and AT1R-ab ≥17 or ≥12 units were considered, freedom from AMR and/or CMR decreased to 50% and 45% (P<0.0001), respectively. Coronary artery vasculopathy and survival were not significantly impacted. Conclusions These results show the increased negative impact of dnDSA and AT1R-ab on freedom from AMR and/or CMR and an increased hazard ratio when both parameters are considered. Both HLA- and non-HLA–specific antibodies seem to impact graft outcome in heart transplantation.

Antibody testing strategies for deceased donor kidney transplantation after immunomodulatory therapy

Background. Immunomodulatory protocols including intravenous immunoglobulin/rituximab (IVIG/R) are employed to decrease anti-human leukocyte antigen (HLA) antibody levels for patients broadly sensitized to HLA and increase chances for transplantation with a compatible deceased donor (DD). The aim of our study was to identify the optimal antibody levels allowing for selection of compatible DDs for these sensitized patients. Methods. From January 2006 to December 2009, 108 patients broadly sensitized to HLA who had reached the top of the DD waitlist were treated with IVIG/R. Antibody levels were monitored monthly by Luminex-based single-antigen bead assay. The antigens identified to produce positive complement-dependent cytotoxicity crossmatches (XMs; >200,000 standard fluorescence intensity [SFI]/10,000 median fluorescence intensity [MFI]) were determined to be unacceptable and entered into the United Network for Organ Sharing database generating the calculated panel reactive antibody (CPRA). The mean CPRA (mCPRA) for this group was more than 80. DDs were selected based on T-cell flow XMs (FXMs) less than 250 MCS and B-cell FXMs less than 300 mean channel shifts (MCS). Results. Monthly Luminex-based single-antigen assays showed that the IVIG/R therapy decreased antibody levels for a period of 30 to 120 days. Of the 108 patients treated, 80 (74%) were transplanted and 28 (26%) were not (mCPRA 96±11). Forty-two (53%) patients were transplanted with a positive FXM; 28 (35%) patients (mCPRA 84±25) were transplanted with a negative FXM; and 10 patients (12%; mCPRA 90±19) received zero HLA ABDR mismatched grafts. Conclusions. After therapy, careful selection of acceptable DD involves the antibody profiling strength and XM results. These approaches provide patients broadly sensitized to HLA with an opportunity for compatible DD transplantation.

Optimizing transplantation of sensitized heart candidates using 4 antibody detection assays to prioritize the assignment of unacceptable antigens.

BACKGROUND The virtual crossmatch relies on the assignment of unacceptable antigens (UAs) to identify compatible donors. The purpose of our study was to identify an algorithm for assignment of UAs such that a negative complement-dependent cytotoxicity (CDC) crossmatch and concomitant negative or weakly positive flow cytometric crossmatch (FXM) are obtained. METHODS We used 4 antibody methods: (1) Luminex single antigen (LSA), (2) LSA with a 1:8 serum dilution, (3) C1q LSA, and (4) CDC panel. The UAs were prioritized in the following order: (1) all C1q+/CDC+, (2) LSA 1:8 >7,500 median fluorescence intensity, and (3) LSA >10,000 median fluorescence intensity. RESULTS Of 295 heart transplants that were performed at our center, 69 (23%) recipients had detectable human leukocyte antigen specific antibody at the time of transplant. All donor specific antibodies (DSAs) were avoided for 44 of 69 (64%) (DSA-). There were 25 recipients who had DSA at the time of transplant: 12 (48%) had negative FXM (DSA+/FXM-), and 13 (52%) had positive T-cell and/or B-cell FXM (DSA+/FXM+). Lower freedom from antibody-mediated rejection was observed for the DSA+/FXM+ group compared with the DSA- group (p < 0.0001). DSA remained detectable after transplant in the sera of 14 recipients, and de novo DSA was detected in 32 recipients. Freedom from antibody-mediated rejection was comparable for both groups (p = 0.53) but was lower than the DSA- group (p < 0.0001). Survival was comparable for all groups at 1,200 days post-transplant. CONCLUSIONS Strategic prioritization of UA assignment has allowed transplantation of highly sensitized patients across the DSA barrier with survival rates comparable to DSA- heart transplant recipients.

Optimizing Transplantation of Broadly Sensitized Heart Patients: Three Antibody Profiling Strategies and Therapeutic Intervention: 932

Heart transplant candidates who are broadly sensitized to human leukocyte antigens (HLA) wait longer before a compatible donor is identified. Solid phase antibody testing has allowed for antibody specificity analysis to determine which donor HLA antigens would be unacceptable for a given patient, thereby allowing for a virtual crossmatch in lieu of a prospective crossmatch. Therapeutic treatments such as the proteosome inhibitor, bortezomib, have been used to reduce the circulating antibodies in order to shorten waiting times to transplant. Aims: The aims of our study were to compare 3 solid phase testing strategies to prioritize definition of unacceptable antigens and to assess the impact of bortezomib treatment on antibody status. Methods: The 3 methods used are: the conventional Luminex single antigen beads (L-IgG), Luminex + C1q to determine complement binding (L-C1q) and L-IgG with 1:8 dilution of serum (L-1:8) to assess prozoning and binding strength. A total of 9 patients received 5 doses of bortezomib (1.3 mg/m2) administered over 2 weeks in conjunction with plasmapheresis. Results: Antibody patterns for all 9 patients showed some fluctuation of L-IgG following treatment. L-C1q antibody patterns decreased to negative for 7 of 9 patients following the bortezomib treatment (Figure 1 shows representative results); 5 of these patients have been transplanted. In general the L-1:8 also showed a marked decrease and detected a few additional antibodies compared to the L-C1q. Antibody patterns for the 2 other patients showed a reduction to negative for class II but not class I. Nonetheless, the class I calculated PRA decreased from 84 (by L-IgG) to 44 (by L-C1q) for one patient, and from 95 to 84 for the second patient respectively. Conclusions: The 3 antibody detection methods allowed prioritization of unacceptable antigens from those that bound antigen, those with the strongest binding, and those that bound complement, presumably being the most detrimental. A marked reduction in complement binding antibodies was observed after bortezomib treatment. Both approaches used in concert allow for a comprehensive profile of the patient‘s antibody status thereby optimizing transplantation for these broadly sensitized patients.