IgG Donor Specific Antibodies Research Articles

Assessment of IgM DSAs in Transplant Recipients: Relationship to De Novo IgG DSAs and Risk for Antibody Rejection.

The presence of anti-HLA donor-specific antibodies (DSAs) is associated with antibody-mediated rejection (AMR) and inferior graft survival. However, recent data suggest that ~50% of AMR episodes are IgG DSA negative and possibly related to non-HLA DSAs. After the initial activation of B cells to alloantigen, IgM is the first immunoglobulin produced. In addition, both IgM and IgG isotopes can activate the classic complement pathway and induce complement-dependent cytotoxicity to allograft targets. Current practices focus on the assessment of IgG DSAs with little concern for the assessment of IgM DSAs. Here, we examined anti-HLA IgM in a cohort of 22 patients who developed de novo IgG DSAs by a modified single-antigen bead-based test. We found IgM HLA DSAs developed before IgG DSAs. The median time from the detection of IgM DSAs to the appearance of de novo IgG DSAs was 461 d. Most patients had IgM DSAs against the same HLA-DQ antigens, for which IgG de novo DSAs were also later detected. IgM DSAs were detected in patients with biopsies suspected of AMR. The detection of IgM DSAs could be an early indicator of alloimmune responses to allografts before IgG de novo DSAs appear.

P477 PIRCHE (PREDICTED INDIRECTLY RECOGNIZABLE HLA EPITOPES) ALGORITHM AS A POSSIBLE PREDICTOR OF AMR (ANTIBODY MEDIATED REJECTION) FOLLOWING HEART TRANSPLANT, FROM LAB EXPERIENCE TO CLINICAL PRACTICE: A CASE REPORT

Abstract PIRCHE–II is an algorithm used to estimate the risk for developing alloreactivity towards HLA mismatches. A 35–year–old man with idiopathic dilated cardiomyopathy with several previous hospitalizations for heart failure is admitted to our unit for a new episode in August 2021. After evaluation, the patient was placed on the waiting list for transplant. The following month the patient underwent heart transplant. The donor had a compatible CDC crossmatch and was negative for anti–HLA antibodies. After 46 days, the patient was discharged at home in stable conditions after 2 negative endomyocardial biopsies (EMB): ISHLT’04 0. In January 2021, the patient was newly admitted for dyspnea and oliguria. Imaging revealed bilateral severe pleural effusion, biventricular cardiac disfunction; EMB showed acute rejection (ISHLT’04 3R). Panel–reactive antibody (PRA) screening found 93 % of class I PRA, 3 % of class II PRA with IgM and IgG donor–specific antibodies (DSA), C1q–positive. After 2 months, the patient was placed again on the transplant waitlist for a persistent severe biventricular disfunction, massive tricuspid regurgitation and diffuse myocardial fibrosis; class I PRA was decreasing (41 % in March 2022, 31 % in April 2022). Unfortunately, the patient died of infection before re–transplantation. After the event, which was recognized as AMR caused by class I DSA, a form rarely described in literature, we employed the PIRCHE (Predicted Indirectly Recognizable HLA Epitopes) algorithm to predict indirect donor–recipient alloreactivity: PIRCHE–II was 54. Is a high PIRCHE–II score a reliable predictor of AMR following heart transplant? More data and deeper understanding is surely needed. A correct after–transplant serologic monitoring is definitely needed to search for de–novo DSA.

Low Expression Loci and the Use of Pronase in Flow Cytometry Crossmatch

Pronase treatment of lymphocytes has been used to improve the specificity and sensitivity of flow cytometric crossmatch, especially B-cell flow cytometric crossmatch, due to the presence of Fc receptors on the cell surface. Some limitations have been reported in the literature: false negatives due to the reduction of major histocompatibility complex expression and false-positive T cells in HIV+ patients due to exposure to cryptic epitopes. This study aimed to evaluate the effect of pronase in our assays, using untreated and treated cells with 2.35 U/mL of pronase to improve flow cytometric crossmatch specificity and sensitivity. The study was carried out with donor-specific IgG antibodies (DSAs) to low expression loci (HLA-C, -DQ, or -DP) because, in our laboratory practice, patients with virtual crossmatch (LABScreen single antigen assays) to DSA against antigen HLA-A, B, and DR are excluded from cellular crossmatch. Our results showed that, for T-cell flow cytometry crossmatch (FCXM), a cutoff value of 1171 median fluorescence intensity (MFI), an area under the curve (AUC) of 0.926 (P < .0001), and 0.834 (P < .0001), a sensitivity of 100% and 85.7%, and a specificity of 77.5% and 74.4%, without and with pronase treatment, respectively. For B-cell FCXM without pronase treatment, the best cutoff was 2766 MFI, an AUC of 0.731 (P < .0001), a sensitivity of 69.6%, and a specificity of 66.7%, whereas for B cells treated with pronase, the cutoff value was 4496 MFI, an AUC of 0.852 (P < .0001), a sensitivity of 86.4%, and a specificity of 77.8%. Our analysis of 128 FCXM showed a better performance using the untreated lymphocytes for FCXM with the prerequisite of a higher cutoff value (≈5000 MFI) to reach a better sensitivity and specificity due to the loss of HLA expression.

Significance of Luminex-crossmatch assay and its mean fluorescence intensity - a retrospective observation in 380 renal transplant cases.

&lt;b&gt;Introduction:&lt;/b&gt; Cell-based complement-dependent cytotoxicity crossmatch (CDC-XM) and solid phase assays were introduced for assessing HLA antibodies. However, the complexity of data from cell-based and solid phase assays have led to potential confusion about how to use the results for clinical decision making. &lt;/br&gt;&lt;/br&gt; &lt;b&gt; Aim:&lt;/b&gt; Aim of this study was to compare results of cell-based assay and solid phase assay, to evaluate the usefulness of L-XM for pretransplant detection of HLA class I and II donor-specific IgG antibodies, correlate the mean fluorescence intensity (MFI) values of class I and class II L-XM assay and with CDC-XM and L-PRA (panel reactive antibodies) results. &lt;/br&gt;&lt;/br&gt; &lt;b&gt; Methods:&lt;/b&gt; In this retrospective study, 380 prospective renal transplant recipients were tested for the presence of HLA antibodies by CDC-XM, IgG-L-XM, IgG-L-PRA &amp; L-SAB screening with their corresponding donor. &lt;/br&gt;&lt;/br&gt; &lt;b&gt;Results:&lt;/b&gt; Fifty-one recipients (13.42%) had a positive CDC-XM. L-XM was positive in 125 recipients (32.89%); class I-L-XM was positive in 46 (36.80%) cases, and class II-L-XM was positive in 58 (46.4%) cases and 21 (16.8%) samples were positive for class I and class II. High background was present in 22 (5.87%) samples, the results of which were confirmed by retesting or by correlation with L-PRA and L-SAB assays. &lt;/br&gt;&lt;/br&gt; &lt;b&gt;Conclusion:&lt;/b&gt; The introduction of more sensitive approaches for the detection of anti-HLA-IgG-antibodies, such as L-XM and L-PRA assay, has allowed the identification of anti-HLA-antibodies in recipient serum which is not usually identified by CDC-XM alone. However, L-XM has some limitations; they can be overcome if we combine this assay with L-PRA.

Validation and cross-reactivity pattern assessment of monoclonal antibodies used for the screening of donor-specific IgG antibody subclasses in transplant recipients

The screening for IgG subclass donor-specific antibodies (DSAs) in allograft recipients uses IgG1-4 subclass-specific monoclonal antibodies (mAbs) that should be mono-specific. The cross-reactivity discrepancies reported for IgG subclass-specific mAbs warranted a critical cross-reactivity pattern analysis of the IgG subclass-specific mAbs most commonly used to detect DSAs.We tested the reactivity of 2 anti-IgG1-, 3 anti-IgG2-, 1 anti-IgG3-, and 2 anti-IgG4-specific PE-conjugated mAbs against microbeads coated with IgG1-4 proteins separately. Each IgG subclass protein was coated at three densities on the beads (0.5, 1, and 2 μg of protein per 106 beads), and the PE-conjugated mAbs were titrated from 0.04 μg/mL to 5 μg/mL. The IgG subclass reactivity of the sample was acquired on the Luminex multiplex platform.Among the IgG subclass-specific mAbs, only the anti-IgG3 (clone: HP6050) mAb was mono-specific. All other mAbs tested were binding to IgG subclass proteins other than their respective immunogen, thereby being cross-reactive. IgG subclass cross-reactivity patterns were dependent on the concentration of both IgG subclass-specific mAbs and IgG1–4 protein targets coated onto the beads. With the current IgG subclass mAbs available, 3 of the 15 possible combinations of IgG1–4 subclass protein could be identified. While the remaining 12 unique combinations cannot be distinguished clearly, 6 groups that corresponded to two different unique combinations of IgG1–4 subclass protein could be identified.The dilution of serum samples and IgG subclass-specific mAbs, other than the anti-IgG3 (clone: HP6050), must be further optimized before their implementation in IgG subclass DSA screening in allograft recipients.

P046 Under assessment of DSA strength in single antibody bead assays may lead to poor outcome in kidney transplantation

Solid phase anti-HLA antibody tests have become an important tool in predicting the compatibility of donor-recipient pairs for kidney transplantation. Additionally, single antigen bead (SAB) assays provide valuable information for the management of post-transplant immunotherapy. However, there can be limitations to SAB assays, for example, the presence of donor specific IgM antibodies and/or binding of high levels of compliment may block binding of donor specific IgG antibodies (DSA) to the SAB. This could possibly result in an under estimation of DSA. The following case is an example of this phenomenon. A 48 years old female patient (HLA Type: A1 A29 B53 B72 DR8 DR13) was transplanted with a deceased donor kidney (HLA Type: A2 A33 B53 B58 DR8 DR11 DQB7 DQA04 DQA05 DPB01:01 DPB04:02) in 2017. The patient was presensitized (pregnancy) to a donor antigen A2 (MFI of 4 A2 beads = 3904) with an associated epitope 144TKH (MFI = 5101, includes A68, A69). On day 3 post-transplant, there was no detectable antibodies to donor antigens due to the sponge effect of the transplanted kidney. On day 6 antibodies to A2 (MFI = 5000) and the associated epitope 144TKH (MFI = 8946) reappeared. Additionally, a low level of de novo antibodies to B*58:01 (MFI = 716) and DPB1*04:02 (MFI = 864) was detected. On day 12, antibodies were detected to only A*02:01 (MFI = 615), while a significant increase in B58 antibody was observed (MFI = 9025). At this time, a biopsy indicated antibody mediated graft rejection; creatinine was 10 mg/dl. Accordingly, the patient’s antibody was further evaluated by an AMOS one wash CDC cross match with frozen cells and sera from day 6 and 12. On day 6, CDC-XM was negative, but on day 12 the CDCXM was positive. The level of DSA to class I of MFI 9640 would typically not give a positive CDC cross match. To determine if there was interference of IgM and/or compliment, the 12-day serum was treated at 63C for 13 min to inactivate these molecules. The heat-treated serum demonstrated an MFI to A*02:01 of MFI = 20,567 compared to an MFI of 615 before heat inactivation. Unfortunately, treatment for antibody-mediated rejection was not initiated soon enough to save the kidney. Although it is rare to observe IgM and/or compliment interference with SAB assays (4 out of 3020 cases, 0.1%), post-transplant assessment of DSA should be performed with heat inactivated sera or other methods to eliminate the under estimation of the strength of IgG antibodies. A.J. Norin:2. Consultant; Company/Organization; ICON PLC, Stony Brook Pathologist. 3. Speaker’s Bureau; Company/Organization; immucor, one lambda.

Standard Induction Immunosuppression Interferes with the Role of Vascularized Bone Marrow in Vascularized Composite Allograft Protection

Introduction Clinical induction therapies have been demonstrated to interfere with the development of immunologic tolerance in experimental models. Vascularized bone marrow (VBM) has been demonstrated to provide immunologic protection to vascularized composite allografts (VCA). We utilized a non-human primate (NHP) model of facial transplantation to investigate the effects of recipient T cell depletion and steroid therapy on VBM-mediated graft protection. Materials and Methods We utilized a well-established model of heterotopic facial subunit VCA in a NHP model. Recipients (N=3) were administered 50 mg/kg of anti-thymocyte globulin (ATGAM) on Day -2, -1, and 0 and transplanted Day 0. Steroids were administered with an initial 20 mg/kg dose and tapered over 21 days and maintained on tacrolimus (Tac) and mycophenylate mofetil (MMF). Chimerism was determined using flow cytometry from peripheral blood samples and final tissues. Comparison was made to historical cohorts, VCA + VBM (n=4) and VCA (no VBM, n=3) treated with Tac/MMF. Results and Discussion Mean survival in the ATGAM with steroid taper group was 28.3 ±8.02 days, as compared to VCA + VBM group 348.2±85.9 days, and VCA (no VBM) 24.7±16.6 days (p=0.008). NHP receiving induction therapies developed early Banff 4 rejection, as well as histopathologic evidence of thrombophlebitis and muscle necrosis. In contrast to the VCA + VBM group, chimerism was undetected in peripheral blood and graft bone marrow was nearly acellular, with an average of 91.5%±2.4 (range 89.8-93.2%) of the lymphocytes present recipient in origin (n=2). Also in contrast to VCA + VBM but comparable to VCA (no VBM), the development of IgM and IgG donor specific antibodies was noted in the ATGAM with steroid taper group, with a mean of 357.5 and 180.5 MFI respectively.Conclusion Presumptive VBM cell populations that regulate immune responses are susceptible to T cell depleting and steroid therapies. The observed interference of standard induction therapies on the protective value of VBM highlights relationships between pharmacologic and biologic mechanisms of VCA graft protection. Henry Jackson Foundation, Department of Defense Award # W81XWH-13-2-0056. Astellas Pharma US, Inc.

Clinical utility of complement-dependent C3d assay in kidney recipients presenting with late allograft dysfunction.

The objective of this study was to evaluate the utility of a complement-dependent C3d assay to risk stratify donor-specific antibodies (DSA) in a multicenter cohort of kidney recipients presenting with new-onset clinical dysfunction. A total of 106 subjects with evidence of DSA at a mean period of 5.3±5.0years posttransplant underwent testing using C3d reagents. C3d positivity was strongly associated with both the peak and sum IgG DSA MFI, with 98.3% (n=57/58) of strongly reactive sera (peak MFI>10000) eliciting a positive signal. Patients with C3d+ DSA had a higher creatinine (P=.03), more significant graft fibrosis (P=.035), and a faster rate of graft loss posttest compared to those with C3d- DSA (P=.05). Subanalysis of patients with low-moderate level DSA confirmed the inferior outcome associated with C3d positivity. Despite the prognostic value of C3d as a stand-alone test, the assay did not provide independent risk prediction after incorporation of graft fibrosis in a multivariate model (P=.94). Overall, C3d offered limited discriminatory value for strong DSA with peak IgG MFI >10000 and in patients where histologic data is available, but its utilization may be considered in those with low-moderate level DSA and where an allograft biopsy is not accessible.

Decision tree and random forest models for outcome prediction in antibody incompatible kidney transplantation

Clinical datasets are commonly limited in size, thus restraining applications of Machine Learning (ML) techniques for predictive modelling in clinical research and organ transplantation. We explored the potential of Decision Tree (DT) and Random Forest (RF) classification models, in the context of small dataset of 80 samples, for outcome prediction in high-risk kidney transplantation. The DT and RF models identified the key risk factors associated with acute rejection: the levels of the donor specific IgG antibodies, the levels of IgG4 subclass and the number of human leucocyte antigen mismatches between the donor and recipient. Furthermore, the DT model determined dangerous levels of donor-specific IgG subclass antibodies, thus demonstrating the potential of discovering new properties in the data when traditional statistical tools are unable to capture them. The DT and RF classifiers developed in this work predicted early transplant rejection with accuracy of 85%, thus offering an accurate decision support tool for doctors tasked with predicting outcomes of kidney transplantation in advance of the clinical intervention.

Renal Transplant Patients Biopsied for Cause and Tested for C4d, DSA, and IgG Subclasses and C1q: Which Humoral Markers Improve Diagnosis and Outcomes?

The association between donor specific antibodies (DSA) and renal transplant rejection has been generally established, but there are cases when a DSA is present without rejection. We examined 73 renal transplant recipients biopsied for transplant dysfunction with DSA test results available: 23 patients diffusely positive for C4d (C4d+), 25 patients focally positive for C4d, and 25 patients negative for C4d (C4d−). We performed C1q and IgG subclass testing in our DSA+ and C4d+ patient group. Graft outcomes were determined for the C4d+ group. All 23 C4d+ patients had IgG DSA with an average of 12,500 MFI (cumulative DSA MFI). The C4d− patients had average DSA less than 500 MFI. Among the patients with C4d+ biopsies, 100% had IgG DSA, 70% had C1q+ DSA, and 83% had complement fixing IgG subclass antibodies. Interestingly, IgG4 was seen in 10 of the 23 recipients' sera, but always along with complement fixing IgG1, and we have previously seen excellent function in patients when IgG4 DSA exists alone. Cumulative DSA above 10,000 MFI were associated with C4d deposition and complement fixation. There was no significant correlation between graft loss and C1q positivity, and IgG subclass analysis seemed to be a better correlate for complement fixing antibodies in the C4d+ patient group.

P145 The presence of HLA-IgM donor specific antibodies and possible association with antibody mediated rejection

Aim The significance of HLA IgG antibodies in antibody mediated rejection (AMR) is well documented however the effect of HLA IgM donor specific antibodies (DSA) is relatively unknown. HLA IgM antibodies detected in crossmatching are often autoantibodies, however since the availability of beads conjugated with HLA antigens, the detection of HLA IgM antibodies is possible. Our laboratory does not routinely test for HLA IgM antibodies, but we report 4 separate cases which proceeded to transplant with no HLA IgG DSA present, however AMR resulted in graft loss in 3 cases. Method Investigations as a result of reported graft rejection were performed on both pre and post-transplant serum for the presence of HLA IgG and IgM DSA using One Lambda Labscreen Single antigen kits as per request from clinical units. Flow and Complement dependent cytotoxicity (CDC) crossmatches prospective and retrospectively were also reviewed. Results For all 4 recipients tested, we identified HLA IgM DSA pre- and post-transplant none of which were identified prior to transplant. All patients were HLA IgG DSA negative. Graft loss was observed in 3 of 4 patients and CDC and Flow crossmatches (IgG) were negative [Table 1]. Conclusions HLA IgM antibody testing is not routinely performed for patients prior to transplant. As a consequence potential IgM antibodies are not investigated until after graft rejection has already occurred. Note: Australian and state governments fund the Australian Red Cross Blood Service for the provision of blood, blood products and transplantation services to the Australian community.

Clinically-relevant threshold of preformed donor-specific anti-HLA antibodies in kidney transplantation

BackgroundPretransplant anti-HLA donor-specific antibodies (DSA) are recognized as a risk factor for acute antibody-mediated rejection (AMR) in kidney transplantation. The predictive value of C4d-fixing capability by DSA or of IgG DSA subclasses for acute AMR in the pretransplant setting has been recently studied. In addition DSA strength assessed by mean fluorescence intensity (MFI) may improve risk stratification. We aimed to analyze the relevance of preformed DSA and of DSA MFI values. Methods280 consecutive patients with negative complement-dependent cytotoxicity crossmatches received a kidney transplant between 01/2008 and 03/2014. Sera were screened for the presence of DSA with a solid-phase assays on a Luminex flow analyzer, and the results were correlated with biopsy-proven acute AMR in the first year and survival. ResultsPretransplant anti-HLA antibodies were present in 72 patients (25.7%) and 24 (8.6%) had DSA. There were 46 (16.4%) acute rejection episodes, 32 (11.4%) being cellular and 14 (5.0%) AMR. The incidence of acute AMR was higher in patients with pretransplant DSA (41.7%) than in those without (1.6%) (p<0.001). The median cumulative MFI (cMFI) of the group DSA+/AMR+ was 5680 vs 2208 in DSA+/AMR− (p=0.058). With univariate logistic regression a threshold value of 5280 cMFI was predictive for acute AMR. DSA cMFI’s ability to predict AMR was also explored by ROC analysis. AUC was 0.728 and the best threshold was a cMFI of 4340. Importantly pretransplant DSA>5280 cMFI had a detrimental effect on 5-year graft survival. ConclusionsPreformed DSA cMFI values were clinically-relevant for the prediction of acute AMR and graft survival in kidney transplantation. A threshold of 4300–5300 cMFI was a significant outcome predictor.

Role of Pretransplant Complement-fixing Donor-specific Antibodies Identified by C1q Assay in Kidney Transplantation

BackgroundKidney transplant recipients who have pretransplant donor-specific human leukocyte antigen (HLA) antibodies have greater risk for developing allograft rejection and allograft loss. However, there is a varied effect of graft injury among patients with pretransplantation donor-specific antibodies (DSA). The difference of complement activating ability may be the reason why some DSA are detrimental to kidney allograft. This study aimed to investigate the association between pretransplantation C1q-binding DSA and clinical outcomes. MethodsThis retrospective study included 48 pretransplant sera from kidney transplant recipients who had pretransplant DSA with negative complement-dependent cytotoxic (CDC) crossmatches. The IgG DSA testing and C1q testing were performed on a Luminex platform with single antigen bead assay. The clinical outcomes between C1q-positive and C1q-negative groups were compared. ResultsC1q-positive DSA were detected in 12 out of 48 patients (25%). The incidences of antibody-mediated rejection (AMR) were higher among patients with C1q-positive DSA than patients with C1q-negative DSA (66.7% vs 41.7%). Nevertheless, there were no statistically significant associations between C1q-DSA and AMR (odds ratio 2.8, 95% CI 0.68–11.6, P = .13) and between C1q-DSA and graft loss (odds ratio 0.52, 95% CI 0.09–2.89, P = .44). The C1q-positive DSA group had significantly higher IgG DSA MFI than the C1q-negative DSA group (P < .001). ConclusionC1q-binding ability of DSA in pretransplant sera of kidney recipients was not associated with antibody-mediated rejection and graft loss post-transplantation. In contrast with the clinical relevance of C1q testing in the post-transplantation setting, C1q testing in pretransplant sera has limited use for immunological risk assessment.

OR35 IgG subclass analysis in C4d+ DSA positive patients

Aim The association between donor specific antibodies (DSA) and renal transplant rejection has been generally established, but there are cases when a DSA is present without rejection. The Luminex® Single Antigen (SA) assay is the current standard for identifying HLA IgG antibody specificities and determining if they are donor specific. As a means to improve the ability to discriminate between harmful and benign antibodies, the SA C1q assay was developed to detect antibodies that are capable of fixing complement. We also have adapted the standard SA IgG assay by utilizing IgG-subclass specific monoclonal antibodies. We sought to explore the detection capacity of the three SA assays and their correlation with renal transplant rejection. Methods 23 patients were selected who had received biopsies due to graft dysfunction and all had C4d deposition on the peritubular capillaries. Out of the 23 patients who presented with graft dysfunction, 5 proceeded to graft loss. Serum samples collected within a week of the biopsy were used for Luminex SA IgG, C1q and IgG subclass analysis. Results All 23 C4d+ patients had SA IgG DSAs with an average of 12,500 MFI (cumulative DSA MFI). 25 control patients with C4d− biopsies had average DSAs less than 500 MFI. The C1q assay showed 16 positive results out of the 23 DSA and C4d+ patients. The C1q assay appeared to be less sensitive particularly when multiple low level IgG DSAs occurred. There was no significant correlation to graft loss and C1q positivity. The IgG subclass assay showed complement fixing IgG1 in 20 of 23 patients. IgG2 DSAs were found in six patients and IgG3 DSAs were found in two patients, all in conjunction with IgG1 DSAs. Interestingly, IgG4 was seen in 10 of the 23 recipients’ sera, but always along with complement fixing IgG1. The detection of IgG4 subclass did not correspond to any enhanced graft survival outcome. Conclusions Cumulative DSA’s above 10,000 MFI were associated with C4d deposition and complement fixation. In this study of patients with C4d+ biopsies, 100% had IgG DSAs, 70% had C1q+ DSAs, and 83% had complement fixing IgG subclass antibodies. We could not demonstrate any enhanced benefit of IgG4. However, in this cohort of patients, IgG4 appeared coincidently to IgG1, and we have previously seen excellent function in patients when IgG4 DSA exists alone.

P117 : MASKING OF HLA IGG ANTIBODIES BY HLA IGM ANTIBODIES IN SINGLE ANTIGEN BEAD TESTING

Aim On postoperative day (POD) 17 after deceased donor transplant, an 18 month old kidney recipient experienced acute humoral and cellular rejection with high titer HLA class 1 & 2 donor specific antibodies (DSA) present. The recipient was exposed to transfusions, but showed only DPB ∗ 04:01 DSA at an MFI of 792 in pretransplant sera. This was determined with the Luminex Single Antigen Bead (SAB) assay for IgG HLA antibodies after heat inactivation (HI) of serum. Pronased flow crossmatch produced negative results with peak PRA and current sera. The speed that DSA appeared post transplant (POD 9) suggested undetected DSA was present pretransplant. Further methods were explored in an attempt to detect the hidden antibodies. Methods HI day-of-transplant serum was tested with Flow PRA Specific Beads, and found to have both IgG and IgM HLA DSA. The serum was dialyzed to precipitate IgM euglobulin. The IgM depleted fraction was tested by the same methods. Luminex SAB testing for IgG and IgM were performed as separate assays, using PE labeled anti-IgG or anti-IgM. Flow PRA Specific Bead testing was done as a single combined test using FITC labeled anti-IgG and APC labeled anti-IgM. Results After HI, IgM antibody was detectable by Luminex SAB and Flow PRA. Dialysis was able to remove IgM antibodies. Flow PRA efficiently detected IgG and IgM in a single assay. IgG DSA that was not found by Luminex SAB was detected by Flow PRA. When IgM was removed by dialysis, multiple IgG DSA antibodies were unmasked in Luminex SAB. Adding back the re-dissolved IgM euglobulin fraction to Luminex IgG SAB decreased DSA MFI values. Conclusion Patients frequently make both IgG and IgM HLA antibodies. Heat inactivation does not effectively remove IgM antibodies. High epitope density of Luminex SAB increases sensitivity to IgG anti-HLA antibodies. It also increases the possibility of competitive inhibition from IgM anti-HLA antibodies. IgG Luminex SAB testing alone may not be sufficient for detecting all HLA antibodies that may have clinical relevance. DSA results for day of transplant serum. Donor HLA type Luminex Single Antigen IgG Luminex Single Antigen IgG Dialyzed Luminex Single Antigen IgG Dialyzed Luminex Single Antigen IgM Flow PRA Single Antigen IgG Flow PRA Single Antigen IgG Dialyzed Flow PRA Single Antigen IgG Dialyzed Flow PRA Single Antigen IgM A 01, 02 – – 01 – – 02 – – B 08, 57 – 57 8, 57 – – 57 57 – C 06, 07 – – 06, 07 – – – – – DR 07, 17 – – 07 – – 07 – – DQ 02, 09 – 02 09 – – 02 – – B345 B3 ∗ 01 – – – – – – – – DP 0101, 0401 0401 – 0101, 0401 – – 0401 – – “–” indicates no DSA present. Other antibody specificities may be present.

OR30: SUCCESSFUL OUTCOMES OF COMBINED HEART LIVER TRANSPLANTS ACROSS PRE-FORMED HIGH LEVELS OF DONOR SPECIFIC HLA ANTIBODIES

In combined heart liver (CHL) transplants (Txp), it is unclear if the heart would be protected from rejection when pre-Txp donor specific antibodies (DSA) are present since the heart Txp procedure is usually performed a few hours before the liver Txp. We describe 2 cases of a successful CHL Txp across high levels of pre-Txp HLA class I and class II DSA. Patients’ demographics, past medical history, and treatments are shown in Table 1. In case 1, the patient was desensitized pre-Txp and intra-operatively (intra-op) as described in Table 1. Pre- and post-Txp sera were analyzed for IgG and complement-binding DSAs using commercially available single-antigen bead assay kits. Both patients had excellent clinical outcomes without any episodes of antibody mediated rejection (AMR) after 1.5 and 4 years of follow up in case 1 and case 2 respectively (Table 1). In case 1, post-Txp anti-HLA antibody measurements showed a near complete depletion of HLA class II IgG DSA 1 day post-Txp, and remained negative thereafter. A significant decrease of HLA class I complement-binding (C1q) DSA was also observed while the decrease of HLA class I IgG DSA was more moderate (Fig. 1, Fig. 2). A similar depletion of HLA class I and class II DSA was also observed in case 2 (data not shown). In 2 cases, a successful CHL Txp was achieved across high levels of pre-Txp HLA class I and class II DSA. Long term outcome was excellent, without any episodes of AMR.

OR38: CARFIZOMIB FOR REFRACTORY ANTIBODY MEDIATED REJECTION IN LUNG TRANSPLANTATION THE IMPACT ON DSA

Aim Antibody mediated rejection (AMR) after lung transplantation (LTx) is associated with early and severe chronic rejection and death. Conventional therapies consisting of plasma exchange and IVIG with and without Rituximab are partially effective. Newer therapies with proteasome inhibitors (Carfizomib (CFZ) and Bortezomib (BTZ)) that target both B and plasma cells may be more effective in removing the donor specific antibodies (DSA). CFZ, unlike BTZ, irreversibly inhibits the activity of 20S proteasome and results in less neuropathic side effects. Method We evaluated the impact of CFZ therapy in 7 LTx on DSA level and function and correlated with clinical outcome as measured by pulmonary function test. All 7 patients had DSA IgG specificity and titer measured by single antigen bead Luminex assay and DSA function of complement binding by C1q-screen at baseline and at 1 month and 3 months post-treatment. Results The specificity, strengths and C1q reactivity pre- and post-therapy is summarized in Table below. All 7 patients had class II DSA and 6/7 were DQ specific with C1q reactivity. 5/7 LTx responded to initial treatment with a drop in IgG DSA level, with negative C1q DSA reactivity ( 15,000 MFI the IgG DSA was inhibited (“prozone effect”), however IgG DSA significantly increased in the presence of EDTA (LTx 3, 5, 6, 7). Conclusion In this small cohort of LTx who mostly failed conventional therapies and have persistent DSA, CFZ based approach appears to be useful, initially. However, 5/7 LTx recovered their C1q DSA reactivity upon longer follow-up suggesting that one cycle of CFZ it may not be effective to impact the rebound of circulating DSA.

IgG Donor Specific Antibodies [DSAs] in Patients With Transplant Glomerulopathy [TG] Are Associated With Inferior Allograft Survival.

Transplant glomerulopathy (TG) is a manifestation of chronic AMR with a poor prognosis and no specific treatment. Although the association between TG and anti HLA antibodies [Abs] is well known, there are few studies linking the nature of these Abs to outcome. 55 patients with TG (33M, 22F, mean age 47.5±12.1 yrs, mean time to TG diagnosis 9.32±8.37 yrs, mean follow up 26.6±18.0 months) were studied. Stored serum samples from the time of TG diagnosis were analysed for the presence of IgG and IgM HLA, DSA and Complement fixing antibodies. 52/55 (94.5%) patients were HLA Ab+, 3/55 were IgG HLA Ab- and 1 patient was IgM HLA Ab+. 27/55 (49.1%) had IgG HLA DSAs, 2/27 had class I alone, 15/27 had class II alone and 10/27 had both class I+II. Overall 39/55 (69.1%) patients had Abs directed against DQ (21/39 were DSAbs, 18/39 were HLA). 24/55 (43.6%) patients had C1q+ antibodies; 2/24 (8.3%) had a C1q+ class I DSA, 16/24 (66.7%) had a C1q+ class II DSA, 3/24 (12.5%) had a C1q+ class I non DSA HLA and 3/24 (12.5%) had a C1q+ class II non DSA HLA. 16/24 (66.7%) of patients had C1q+ antibodies against DQ. Overall allograft survival was 70.8%, 35.3%, 27.2% and 15.5% at 12, 36, 48 and 60 months respectively, mean 26.4±17.7 months. Allograft survival was significantly worse in IgG DSA+ patients compared with IgG DSA- patients.Figure: No Caption available.(see Figure 1, log rank test p=0.04). The IgG DSA MFI had no effect on allograft survival. There was no difference in allograft survival in C1q+patients (p=0.88) although the presence of C1q+ Ab was associated with the presence of C4d on allograft biopsy (p=0.01).This study shows that the presence of IgG DSAs is associated with inferior allograft survival in patients with TG. This group of patients may benefit from more aggressive therapy. DISCLOSURES:Lawrence, C.: Other, Honoraria from OneLambda, speakers fee from OneLambda.

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In combined liver kidney (CLK) transplants (Txp), the kidney is not always protected from rejection when pre-Txp donor specific antibodies (DSA) are present. We applied a C1q solid-phase assay to examine complement-fixing DSA in CLK Txps who developed accelerated antibody mediated rejection (AMR). Four CLK Txps who had high PRA and high levels of DSA pre-Txp were evaluated. Pre- and post-Txp sera were analyzed for DSAs using commercially available single-antigen bead assay kits. The MFI cut off values of IgG DSA and C1q+ DSA are >1,000 and >300 respectively. Two of these 4 recipients (case 1 and 2) experienced biopsy-proven AMR during the first 2 weeks post-txp. All 4 cases had a persistent Post-Txp IgG DSA (Table). The 2 subjects who developed AMR had very high pre-Txp C1q+ DSA levels (MFI range for single specificity: 10,800-17,500). In contrast, case #3 who had relatively lower pre-Txp C1q DSA (MFI= 4,400) did not experience AMR.[ Table 1 ] With the limitation of small number of cases, the presence of high levels of pre-Txp C1q+ DSA appears to be associated with an increased risk of AMR of the kidney. Future studies should evaluate the risk of AMR and long-term outcomes associated with the presence of C1q+ DSA during the pre- and post-Txp periods in CLK Txps.

58-P: CORRELATION OF CIRCULATING COMPLEMENT-FIXING DONOR SPECIFIC ANTIBODIES IDENTIFIED BT THE C1q ASSAY AND PRESENCE OF C4d in ENDOMYOCARDIAL BIOPSY: A SINGLE-CENTER RETROSPECTIVE STUDY

Aim Donor-specific antibodies (DSA) are associated with increased cardiac graft loss. Detection of antibody-mediated rejection (AMR) relies on graft dysfunction, C4d immunofluorescence (IF) and DSA. We applied a C1q solid-phase assay in parallel with the standard IgG SAB assay to examine the correlation of circulating complement-fixing donor-specific antibodies and presence of C4d in endomyocardial biopsy (EMB). Methods We retrospectively studied the relationship of C1q+ DSA, and C4d IF in 40 EMB from 38 cases of heart transplant recipients (The Journal of Heart and Lung Transplantation, Vol 32, No 4, April 2013). C4d evaluation was performed for cause, including (1) clinical evidence of graft dysfunction; (2) high PRA; and (3) ACR, ISHLT Grade 3R. We applied a C1q solid-phase assay in parallel with the standard IgG SAB assay to examine the DSA profiles in heart transplant patients post-transplant. The MFI cut off values of IgG DSA and C1q+ DSA are > 1,000 and >200 respectively. Results A better concordance is observed between C1q+ DSA with C4d IF+ as compared to IgG DSA with C4d IF+ (54% vs.24%, p = 0.09, paired samples T test). However, the correlation of C1q DSA with C4d IF is not statistically significant ( p = 0.12, Fisher Exact test). Importantly, C1q+ DSA is observed in 14/15 cases with C4d IF +; 17 cases had circulating C1q+ DSA without detectable C4d staining (Table), suggesting that that the presence of C1q+ DSA may precede the detection of C4d deposition in EMB and/or the development of AMR. [ Table 1 ] Conclusions In this cohort of 38 patients, no significant correlation is observed between circulating C1q DSA and C4d IF in EMB. Additional studies are needed to further evaluate the association of C1q DSA with EMB and IF C4d staining, and with long-term outcomes in heart transplant recipients.