Donor-specific Antibodies Mean Fluorescence Intensity Research Articles

Impact of donor-specific antibody with low mean fluorescence intensity on allograft outcomes in kidney transplant

BackgroundImmune-mediated rejection is the most common cause of allograft failure in kidney transplant (KT) patients. Exposure to alloantigen, including human leukocyte antigen (HLA), results in the production of donor-specific antibodies (DSA). There are limited data about low levels of mean fluorescence intensity (MFI) DSA, especially post-transplantation. This study evaluated allograft outcomes in KT patients with low MFI DSA. MethodsFrom January 2007 to December 2021, KT patients who were tested for post-transplant DSA at Ramathibodi Hospital, Bangkok, Thailand, with the DSA MFI ≤ 1000 were evaluated. These KT patients were categorized into two groups: very low DSA (VLL; MFI = 1–500) and low DSA (LL; MFI = 501–1000). All KT patients were evaluated for the primary outcomes, such as the incidence of acute rejection, serum creatinine levels at one and five years after transplantation as well as allograft and patient survivals. ResultsAmong 36 KT patients 25 were included as those with VLL and 11 as those with LL. The LL group had significantly higher T-cell mediated allograft rejection (TCMR) than the VLL group (45% vs. 12%, P = 0.04). In addition, 10 patients, 5 in the VLL group and 5 in the LL group developed antibody-mediated allograft rejection (ABMR). Both TCMR and ABMR were confirmed by biopsy results. There was a trend toward higher MFI in KT patients with ABMR than without ABMR (P = 0.22). At 5 post-transplant years, serum creatinine, allograft and patient survivals were comparable between these two groups. Furthermore, the univariate and multivariate analyzes revealed that the LL group was a high risk for rejection. ConclusionLow MFI DSA values after transplantation may be associated with a higher incidence of rejection, but this finding did not show differences in allograft and patient survival in this study's analysis.

Immunology Status of Patients During Kidney Transplant.

The immunology status of a patient has a crucial role in kidney transplant. We investigated the effectiveness of a desensitization protocol, guided by the immunology status of patients, for kidney transplant candidates. Antibody screening for human leukocyte antigens was conducted with the Luminex single-antigen microsphere bead assay method for 34 patients from June 2021 to June 2022. Donor human leukocyte antigen genotypes at 8 loci (A*, B*, С*, DRB1*, DQA1*, DQB1*, DPA1*, and DPB1*) were determined, to correlate the specificities of recipient human leukocyte antigen antibodies with donor antigens and identify unacceptable donor antigen combinations. Specialized immunology studies measured panel reactive antibody levels and human leukocyte antigen class I and class II antibodies. A crossmatch compatibility test using complementdependent cytotoxicity was conducted. Of the 34 patients, 10 completed all 3 stages of the desensitization therapy. Most patients experienced decreased sensitization to human leukocyte antigen class I and class II antibodies. Two patients achieved complete clearance of A1 and DQ5 antibodies, respectively, whereas 1 patient exhibited an increase in donor-specific antibody mean fluorescence intensity. Prior to desensitization therapy, the crossmatch compatibility test yielded positive results with T and B lymphocytes. After completing the therapy, the crossmatch test showed negative results in 4 cases with T lymphocytes and positive results with B lymphocytes. Plasmapheresis sessions effectively reduced circulating antibodies. However, the combination of rituximab and plasmapheresis alone did not achieve a negative crossmatch test required for kidney transplant. It is crucial to assess the reduction of donor-specific antibody quantity, considering both the percentage and the mean fluorescence intensity. To avoid false-positive results in crossmatch analysis, drug half-life must be considered. Laboratories should have various crossmatch techniques, such as flow cytometry and single-antigen microsphere bead assay technology, available for research and urgent cases that require crossmatch analysis.

Donor-Specific Antibody Mean Fluorescence Intensity Threshold Predicts Platelet Transfusion Response in HLA-Alloimmunized Patients

Introduction:Patients with platelet transfusion refractoriness (PTR) due to HLA-alloimmunization respond to HLA-matched platelets. However, 4/4 HLA-matched platelets or platelets without donor-recipient antigenic mismatches are rarely available. Further, in severe HLA-alloimmunization, it is difficult to find donor products which do not contain antigens to which the recipient has developed antibodies ( i.e. antibody specificity prediction, ASP method). Hence, studies have evaluated platelet transfusion responsiveness in the presence of ≥1 donor specific antibodies (DSAs) to widen donor pools, a strategy termed permissive mismatching (PERMM). Using established semi-quantitative techniques for HLA-antibody detection, we sought to identify a cumulative DSA threshold which predicted platelet corrected count increment (CCI) failure despite PERMM. Methods:We retrospectively reviewed platelet transfusions among patients with HLA-alloimmunization and suspected PTR at the University of Washington/Fred Hutchinson Cancer Center from 2021-2023. HLA Class I antibodies were detected using a solid-phase single antigen bead assay (One Lambda) on the Luminex platform with individual antibody strengths measured by the mean fluorescence intensity (MFI). The calculated panel reactive antibodies (cPRA) were defined as the proportion of donors against whom recipients had antibodies. Transfusions were categorized as HLA matched, PERMM (DSA MFI>0), or random donor platelets (HLA-type unknown). CCI was calculated as described by Cohn ASH 2020. Results: Of 420 patients suspected to have PTR, 75 had cPRA > 50% and received ≥3 HLA-selected platelet transfusions. Of the 2726 transfusions these patients received, 1198 were excluded due to unavailability of post-transfusion platelet counts within 2 hours or due to multiple transfusions occurring between platelet counts. Ultimately, 1528 transfusions were analyzed (Panel A). In 1031 PERMM transfusions, the cumulative MFI was a stronger predictor of CCI at 2 hours than cPRA or the number of mismatched DSA (coefficients of determination or R2 of 7.3%, 2%, and 0.4%, respectively). We then examined the spectrum of cumulative MFIs to identify a threshold MFI predicting transfusion success/failure among the PERMM products. PERMM platelet transfusions in recipients with DSA MFIs of up to 5000 generated CCIs similar to HLA-matched platelets. However, CCI was significantly different between MFI <10,000 (median CCI 17,783) and MFI ≥10,000 (median CCI 3753) (Panel B). We then stratified data by splenomegaly, a significant non-immunologic cause of PTR (23.2% of transfusions, R2 15.8% for CCI prediction). Of the transfusions not affected by splenomegaly, 9.6% did not generate an adequate platelet response (defined as a CCI >5000) across all MFIs. This failure rate increased to 33.3%, 40.8%, and 52.8% among transfusions in recipients with MFIs >5000, >7500, and >10,000, respectively. After multiple testing correction, MFI of several individual DSAs (A1, A2, A24, A68, B8, B37, B39, B44, B51, B57, and B62) were also found to be significant predictors of CCI using linear regression ( n ranging 32-193, R2 ranging 6-33%). Conclusions: This is the largest study to date evaluating optimal MFI thresholds of DSAs for permissive HLA mismatching in the context of platelet transfusions. Cumulative DSA MFIs demonstrated a dose response effect with lower MFIs generating better CCIs. In our patient cohort overall, and when stratified by splenomegaly, we identified a threshold for cumulative DSA MFIs of 10,000, under which >50% of transfusions resulted in an adequate CCI. This MFI threshold (>10,000) has been implicated in HLA mismatched solid organ transplant rejections (Kallon, Transf. Med. 2020) as well as in a pooled analysis of smaller HLA-selected platelet transfusion studies conducted by our group (unpublished). If incorporated into a transfusion strategy, this MFI threshold may perform better than units chosen based on the number of mismatched DSAs alone or random donor platelets and widen the donor pool. Identifying the impact of specific HLA antibodies on CCIs needs further study. Studies are also underway to correlate cumulative DSA MFIs with complement activation (C1q levels) as a possible mechanistic explanation for the differences noted across the MFI threshold of 10,000 in our study.

Primary Graft Dysfunction Is Associated With Development of Early Cardiac Allograft Vasculopathy, but Not Other Immune-mediated Complications, After Heart Transplantation.

We investigated associations between primary graft dysfunction (PGD) and development of acute cellular rejection (ACR), de novo donor-specific antibodies (DSAs), and cardiac allograft vasculopathy (CAV) after heart transplantation (HT). A total of 381 consecutive adult HT patients from January 2015 to July 2020 at a single center were retrospectively analyzed. The primary outcome was incidence of treated ACR (International Society for Heart and Lung Transplantation grade 2R or 3R) and de novo DSA (mean fluorescence intensity >500) within 1 y post-HT. Secondary outcomes included median gene expression profiling score and donor-derived cell-free DNA level within 1 y and incidence of cardiac allograft vasculopathy (CAV) within 3 y post-HT. When adjusted for death as a competing risk, the estimated cumulative incidence of ACR (PGD 0.13 versus no PGD 0.21; P = 0.28), median gene expression profiling score (30 [interquartile range, 25-32] versus 30 [interquartile range, 25-33]; P = 0.34), and median donor-derived cell-free DNA levels was similar in patients with and without PGD. After adjusting for death as a competing risk, estimated cumulative incidence of de novo DSA within 1 y post-HT in patients with PGD was similar to those without PGD (0.29 versus 0.26; P = 0.10) with a similar DSA profile based on HLA loci. There was increased incidence of CAV in patients with PGD compared with patients without PGD (52.6% versus 24.8%; P = 0.01) within the first 3 y post-HT. During the first year after HT, patients with PGD had a similar incidence of ACR and development of de novo DSA, but a higher incidence of CAV when compared with patients without PGD.

The Halifax flow Crossmatch Protocol Results According to the HLA Class and MFI of the DSA

Introduction: The main strategy to reduce the immunogenicity of transplanted grafts is to seek maximum compatibility between alloantigens in the donor-recipient pair. It is understood that the virtual crossmatch (VXM) can be a good tool in the evaluation of the donor / recipient pair and that a broader application of these protocols will improve the pre-transplant immunological risk assessment. The aim of this study was to correlate the physical flow crossmatches results, looking at different mean fluorescence intensity (MFI) values of the donor specific antibodies (DSA), and the probability of a positive crossmatch. We also aimed to validate this tool using a well standardized flow crossmatch protocol. Methods: We performed a total of 15,217 FCXM between 2015 and 2019. All were tested by the Halifax Flow Cytometer Crossmatch (FCXM) protocol, with cells from deceased donors and serum from renal recipients. For this analysis we selected only samples that had one or two DSA per locus (N = 1,081), when the MFI was above 1,000, and they were divided according to the allelic group recognized by the antibody (anti-HLA-A, B, C, DR or DQ) looking at the probability of a positive crossmatch with different MFI values of the DSAs. Combinations among them were also analyzed in the same way (N=175). Results: In the presence of an exclusive DSA against the allelic groups A, B and DR, with an index MFI above 5,000, all the FCXM were positives. With exclusively antibodies against groups C or DQ, all cases with DSAs above 15,000 MFIs were positives. With two or more DSAs anti A and/or B, and/or DR, when their MFIs sums exceeded 5,000, all FCXM results against B cells were positive. The presence of anti-Class I DSAs (A, B and A+B), regardless of the MFI value, was responsible for 71% (N=424 of 601) of the T cell positivity and 77% (N=460 of 661) in the B cell crossmatches. The presence of only anti-Class II DSAs (DR and DQ) accounted for 55% (N=168 of 303) of FCXM positivity in B cells. The overall mean of the MFI of the DSAs was higher in the FCXM positive group when compared to negative crossmatch. The group with sum of DSAs A (N=217) showed that when the sum was 4,000 MFI or higher, there was a 24 times higher probability for a positive B cell crossmatch when compared with lowers MFIs. Conclusions: Our results show a strong association between the DSA MFI and the FCXM result. The data here presented confirm the results of our previous studies, justifying the VXM standard used by our center, proving to be a good tool to streamline the selection of transplant recipients and facilitate the sharing of organs from national donors.

Correlation of Luminex-Based Single Antigen Based Results With Complement-Dependent Cytotoxicity Crossmatch and Flow Cytometry Crossmatch Results: A Single-Center Experience From Istanbul

This study aimed to retrospectively investigate the correlation of mean Class I donor-specific antibody (DSA) intensity values detected in Luminex-based techniques with the results of complement-dependent cytotoxicity crossmatch (CDC-XM) and flow cytometry crossmatch (FC-XM) results. A total of 335 patients with kidney failure and their living donors whose CDC-XM, FC-XM, and single antigen based (SAB) tests were studied between 2018 and 2020 for transplant preparation from living donor candidates were included in the study. Patients were divided into 4 groups according to their mean fluorescence intensity (MFI) values of SAB assay. Anti-HLA antibodies (class I and/or class II) were detected using SAB in 91.6% patients included in the study (MFI >1000). Class I DSA was positive in 34.8% of patients with anti-HLA antibodies. When CDC-XM and FC-XM results were evaluated in the 4 groups separated according to MFI values, 3 patients with DSA MFI <1000 had negative CDC-XM and T-B-FC-XM results. Of 32 patients with DSA-MFI between 1000 and 3000, 93.75% (n=30) had T-B-FC-XM or CDC-XM-negative results, and 6.25% (n=2) had B-FC-XM-positive results. The CDC-XM, T, and B-FC-XM were negative in all 17 patients with DSA-MFI between 3000 and 5000. Our results showed that MFI >5834 DSA values were significantly correlated with positive T-FC-XM (P < .001), and MFI >6016 values were significantly correlated with positive CDC-XM (P=.002). In addition, MFI values >5000 were associated with both CDC-XM and FC-XM in our study. The MFI values >5000 correlated with both CDC-XM and FC-XM.

Desensitization using IVIG alone for living-donor kidney transplant: impact on donor-specific antibodies.

Sensitization to human leukocyte antigen is a barrier to. Few data have been published on desensitization using polyvalent human intravenous immunoglobulin (IVIG) alone. We retrospectively reviewed the of 45 patients with a positive complement-dependent cytotoxicity crossmatch (CDCXM) or flow cytometry crossmatch (FCXM) against living donors from January 2003 to December 2014. Of these, 12 were excluded. Patients received monthly IVIG infusions (2 g/kg) only until they had a negative T-cell and B-cell FCXM. During the 33 patients, 22 (66.7%) underwent living donor kidney transplantation, 7 (21.2%) received a deceased donor graft, and 4 (12.1%) did not undergo transplantation. The median class I and II panel reactive antibodies for these patients were 80.5% (range 61%-95%) and 83.0% (range 42%-94%), respectively. Patients (81.8%) had a positive T-cell and/or B-cell CDCXM and 4 (18.2%) had a positive T-cell and/or B-cell FCXM. Patients underwent transplantation after a median of 6 (range 3-16). The median donor-specific antibody mean fluorescence intensity sum was 5057 (range 2246-11,691) before and 1389 (range 934-2492) after desensitization (p = 0.0001). Mean patient follow-up time after transplantation was 60.5 (SD, 36.8) months. Nine patients (45.0%). Death-censored graft survival at 1, 3, and 5 years after transplant was 86.4, 86.4, and 79.2%, respectively and patient survival was 95.5, 95.5, and 83.7%, respectively. Desensitization using IVIG alone is an effective strategy, allowing successful transplantation in 87.9% of these highly sensitized patients.

The impact of pre-transplant donor specific antibodies on the outcome of kidney transplantation – Data from the Swiss transplant cohort study

BackgroundPre-transplant donor specific antibodies (DSA), directed at non-self human leukocyte antigen (HLA) protein variants present in the donor organ, have been associated with worse outcomes in kidney transplantation. The impact of the mean fluorescence intensity (MFI) and the target HLA antigen of the detected DSA has, however, not been conclusively studied in a large cohort with a complete virtual cross-match (vXM).MethodsWe investigated the effect of pre-transplant DSA on the risk of antibody-mediated rejection (ABMR), graft loss, and the rate of eGFR decline in 411 DSA positive transplants and 1804 DSA negative controls.ResultsPre-transplant DSA were associated with a significantly increased risk of ABMR, graft loss, and accelerated eGFR decline. DSA directed at Class I and Class II HLA antigens were strongly associated with increased risk of ABMR, but only DSA directed at Class II associated with graft loss. DSA MFI markedly affected outcome, and Class II DSA were associated with ABMR already at 500-1000 MFI, whereas Class I DSA did not affect outcome at similar low MFI values. Furthermore, isolated DSA against HLA-DP carried comparable risks for ABMR, accelerated eGFR decline, and graft loss as DSA against HLA-DR.ConclusionOur results have important implications for the construction and optimization of vXM algorithms used within organ allocation systems. Our data suggest that both the HLA antigen target of the detected DSA as well as the cumulative MFI should be considered and that different MFI cut-offs could be considered for Class I and Class II directed DSA.

The clinical utility and thresholds of virtual and Halifaster flow crossmatches in lung transplantation

Immune sensitization, defined as the presence of alloreactive donor-specific antibodies (DSA), is associated with increased wait-times and inferior transplant outcomes. Identifying pretransplant DSA with a physical cell-based assay is critical in defining immunological risk. However, improved solid phase antibody detection has provided the potential to forgo this physical assay. Here, we evaluated the association between DSA mean fluorescence intensity (MFI) and the recently introduced Halifaster Flow cytometry crossmatch (FXM) to determine if MFI could predict the outcome of FXM and whether a virtual crossmatch (VXM) would provide an accurate risk assessment. Sera from 134 waitlisted lung patients was retrospectively assessed by Halifaster FXM against lymphocytes preparations from 32 donors, resulting in 265 FXMs. HLA typing was performed to 2-field allelic level and Luminex single antigen beads (SAB) used to identify DSA. The association between FXM and Luminex MFI was calculated using ROC analysis. MFI threshold accuracy was confirmed using a separate validation cohort (174 recipient sera and 34 donors), whereby both VXM and FXMs were compared. From the 265 FXM performed, 48 (18%) T-cell (TFXM) and 56 (21%) B-cell (BFXM) were positive. In the evaluation cohort, MFI thresholds of 2000 for HLA-A, B, DRB1, and > 4000 for DQB1, were predictive of a positive FXM. The validation cohort of 233 paired FXM and VXM confirmed these MFI thresholds for both TFXM and BFXM with an accuracy of 91.4% and 89.3%, respectively. A positive VXM, defined with HLA-specific MFI thresholds predicts Halifaster FXM reactivity, and can potentially expedite organ allocation, by minimizing the need for the more time-consuming FXM.

Correlation between microvascular inflammation in endomyocardial biopsies and rejection transcripts, donor-specific antibodies and graft dysfunction in antibody-mediated rejection

The pathology-based diagnosis of antibody-mediated rejection (AMR) following heart transplantation relies on the 2013 Working Formulation in which microvascular inflammation (MVI) is considered as present or absent regardless of its extent. This work assessed the biological and clinical value of a semiquantitative evaluation of the extent of MVI in endomyocardial biopsies (EMB). We retrospectively graded the extent of MVI in 291 EMB from 291 patients according to a 4-point scale where MVI scores of 0, 1, 2 and 3 represented 0%, 1–10%, 11–50% and > 50% of the myocardial area, respectively. We analyzed the association between the MVI score and tissue rejection molecular activity assessed by micro-arrays or reverse transcriptase multiplex ligation-dependent probe amplification, current pathology classification (pAMR), circulating anti-HLA donor-specific antibodies (DSA) and graft dysfunction. Overall, 172 (59.1%), 33 (11.4%), 42 (14.4%) and 44 (15.1%) EMB were given MVI scores of 0, 1, 2 and 3, respectively. pAMR1(H+) and pAMR2/3 categories were found to be heterogenous in terms of MVI score. Acute cellular rejection grades did not influence the MVI score. In both molecular approaches, we observed a stepwise increase in the expression of AMR-related transcripts with increasing MVI scores, independently of the C4d or CD68 status ( Fig. 1 , P < 0.001). Both the frequency and mean fluorescence intensity of DSA gradually increased with the MVI score ( P < 0.001). Acute graft dysfunction was more frequent in MVI score 3 ( P < 0.001). The intensity of MVI in EMB, based on a semiquantitative evaluation of its extent, has biological and clinical importance.

Predictive Potential of Flow Cytometry Crossmatching in Deceased Donor Kidney Transplant Recipients Subjected to Peritransplant Desensitization.

Recipient sensitization is a major risk factor of antibody-mediated rejection (ABMR) and inferior graft survival. The predictive effect of solid-phase human leukocyte antigen antibody testing and flow cytometry crossmatch (FCXM) in the era of peritransplant desensitization remains poorly understood. This observational retrospective single-center study with 108 donor-specific antibody (DSA)-positive deceased donor kidney allograft recipients who had undergone peritransplant desensitization aimed to analyze variables affecting graft outcome. ABMR rates were highest among patients with positive pretransplant FCXM vs. FCXM-negative (76 vs. 18.7%, p < 0.001) and with donor-specific antibody mean fluorescence intensity (DSA MFI) > 5,000 vs. <5,000 (54.5 vs. 28%, p = 0.01) despite desensitization. In univariable Cox regression, FCXM positivity, retransplantation, recipient gender, immunodominant DSA MFI, DSA number, and peak panel reactive antibodies were found to be associated with ABMR occurrence. In multivariable Cox regression adjusted for desensitization treatment (AUC = 0.810), only FCXM positivity (HR = 4.6, p = 0.001) and DSA number (HR = 1.47, p = 0.039) remained significant. In conclusion, our data suggest that pretransplant FCXM and DSA number, but not DSA MFI, are independent predictors of ABMR in patients who received peritransplant desensitization.

Diagnostic value of donor-derived cell-free DNA to predict antibody-mediated rejection in donor-specific antibody-positive renal allograft recipients.

SummaryCirculating donor‐specific antibodies (DSA) do not necessarily indicate antibody‐mediated rejection (ABMR). Here, we evaluated the diagnostic value of donor‐derived cell‐free DNA (dd‐cfDNA) as an add‐on to DSA detection. The study included two independent cohorts of DSA+ kidney allograft recipients, 45 subclinical cases identified by cross‐sectional antibody screening (cohort 1), and 30 recipients subjected to indication biopsies (cohort 2). About 50% of the DSA+ recipients had ABMR and displayed higher dd‐cfDNA levels than DSA+ABMR− recipients (cohort 1: 1.90% [median; IQR: 0.78–3.90%] vs. 0.52% [0.35–0.72%]; P < 0.001); (cohort 2: 1.20% [0.82–2.50%] vs. 0.59% [0.28–2.05%]; P = 0.086). Receiver operating characteristic (ROC) analysis revealed an area under the curve (AUC) of 0.89 and 0.69 for dd‐cfDNA, and 0.88 and 0.77 for DSA mean fluorescence intensity (MFI), respectively. In combined models, adding dd‐cfDNA to DSA‐MFI or vice versa significantly improved the diagnostic accuracy. Limited diagnostic performance of dd‐cfDNA in cohort 2 was related to the frequent finding of other types of graft injury among ABMR− recipients, like T cell‐mediated rejection or glomerulonephritis. For dd‐cfDNA in relation to injury of any cause an AUC of 0.97 was calculated. Monitoring of dd‐cfDNA in DSA+ patients may be a useful tool to detect ABMR and other types of injury.

A virtual crossmatch-based strategy for perioperative desensitisation in lung transplant recipients with pre-formed donor-specific antibodies: 3-year outcome.

Pre-formed donor-specific antibodies (DSAs) are associated with worse outcome after lung transplantation (LTx) and might limit access to LTx. A virtual crossmatch-based strategy for perioperative desensitisation protocol has been used for immunised LTx candidates since 2012 at Foch Hospital (Suresnes, France). We compared the outcome of desensitised LTx candidates with high DSA mean fluorescence intensity and those with low or no pre-formed DSAs, not desensitised. For all consecutive LTx recipients (January 2012 to March 2018), freedom from chronic lung allograft dysfunction (CLAD) and graft survival were assessed using Kaplan-Meier analysis and Cox multivariate analysis. We compared outcomes for desensitised patients with high pre-formed DSAs (n=39) and those with no (n=216) or low pre-formed DSAs (n=66). The desensitisation protocol decreased the level of immunodominant DSA (class I/II) at 1, 3 and 6 months post-LTx (p<0.001, p<0.01 and p<0.001, respectively). Freedom from CLAD and graft survival at 3 years was similar in the desensitised group as a whole and other groups. Nevertheless, incidence of CLAD was higher with persistent high-level DSAs than cleared high-level (p=0.044) or no DSAs (p=0.014). Conversely, graft survival was better with cleared high DSAs than persistent high-level, low-level and no pre-formed DSAs (p=0.019, p=0.025 and p=0.044, respectively). On multivariate analysis, graft survival was associated with cleared high DSAs (hazard ratio 0.12, 95% CI 0.02-0.85 versus no DSAs; p=0.035) and CLAD with persistent DSAs (3.04, 1.02-9.17 versus no pre-formed DSAs; p=0.048). The desensitisation protocol in LTx recipients with high pre-formed DSAs was associated with satisfactory outcome, with cleared high pre-formed DSAs after desensitisation identified as an independent predictor of graft survival.

Apheresis Efficacy and Tolerance in the Setting of HLA-Incompatible Kidney Transplantation.

Nearly 18% of patients on a waiting list for kidney transplantation (KT) are highly sensitized, which make access to KT more difficult. We assessed the efficacy and tolerance of different techniques (plasma exchanges [PE], double-filtration plasmapheresis [DFPP], and immunoadsorption [IA]) to remove donor specific antibodies (DSA) in the setting of HLA-incompatible (HLAi) KT. All patients that underwent apheresis for HLAi KT within a single center were included. Intra-session and inter-session Mean Fluorescence Intensity (MFI) decrease in DSA, clinical and biological tolerances were assessed. A total of 881 sessions were performed for 45 patients: 107 DFPP, 54 PE, 720 IA. The procedures led to HLAi KT in 39 patients (87%) after 29 (15–51) days. A higher volume of treated plasma was associated with a greater decrease of inter-session class I and II DSA (p = 0.04, p = 0.02). IA, PE, and a lower maximal DSA MFI were associated with a greater decrease in intra-session class II DSA (p < 0.01). Safety was good: severe adverse events occurred in 17 sessions (1.9%), more frequently with DFPP (6.5%) p < 0.01. Hypotension occurred in 154 sessions (17.5%), more frequently with DFPP (p < 0.01). Apheresis is well tolerated (IA and PE > DFPP) and effective at removing HLA antibodies and allows HLAi KT for sensitized patients.

Continuous Intermittent Intravenous Immunoglobulin in Heart Transplant Recipients with Elevated Donor-Specific Antibody Levels

<h3>Purpose</h3> Donor-specific antibodies (DSA) develop following heart transplantation (HT), and are associated with antibody-mediated rejection (AMR). However, treatment of persistently elevated DSA levels remains unclear. We aimed to evaluate the use of intermittent intravenous immunoglobulin (IVIg) for the treatment of elevated DSA. <h3>Methods</h3> We identified all adult HT recipients receiving monthly IVIg infusions between September 2019 and October 2020. Patient information including serial DSA levels, as well as hemodynamic and echocardiographic characteristics, were collected from the electronic health record. <h3>Results</h3> The cohort included 17 patients with a mean age of 57 +/- 15 years, 65% were male, & median time from HT was 48 months. 7 patients had prior biopsy-proven AMR, 7 had high mean fluorescence intensity (MFI) DSA alone, and 3 had allograft dysfunction without proven etiology. The average left ventricular ejection fraction (LVEF) prior to IVIg was 50% +/- 14%. Prior to intermittent IVIg 12 patients (71%) received an additional immunosuppressive agent (thymoglobulin, bortezomib, rituximab, belatacept), while 11 of them (65%) also received plasmapheresis. The mean duration of IVIg following pAMR was 7.6 +/- 7 months. DSA MFI decreased by 27% in patients with confirmed diagnosis of AMR and by 10% in those with elevated DSA alone after initiating intermittent IVIg (figure). Both LVEF and cardiac index improved in all three groups following initiation of IVIg infusions. Pulmonary capillary wedge pressure (PCWP) decreased in the elevated DSA and allograft dysfunction groups while it increased in the AMR group. Seven patients were admitted for suspected rejection after starting treatment. <h3>Conclusion</h3> DSA MFI declined and LVEF improved following initiation of intermittent IVIg infusions. Further study is needed to determine whether IVIg can prevent <i>de novo</i> or recurrent AMR.

Are Those with Primary Graft Dysfunction More Likely to Have Acute Cellular Rejection or Donor-Specific Antibodies after Heart Transplantation?

<h3>Purpose</h3> Primary graft dysfunction (PGD) is a leading cause of early morbidity and mortality following heart transplantation (HT). We sought to determine the association between PGD and incidence of acute cellular rejection (ACR) and development of <i>de novo</i> donor specific antibodies (DSA) within two years post-HTx. <h3>Methods</h3> Consecutive adult HT recipients (n=351) from 1/2010 to 7/2018 at a single center were included. Multi-organ and re-transplants were excluded. Definition of moderate and severe PGD was based on the 2014 ISHLT consensus, which includes dependence on extracorporeal membrane oxygenation or intra-aortic balloon pump. The primary outcome was the incidence of ACR (ISHLT grade 2R or 3R) and the development of <i>de novo</i> DSA (mean fluorescence intensity >500) within 2 years post-HTx. <h3>Results</h3> The cohort was predominantly male (70.6%), with a mean age of 54.1±12.5 years; 34.6% had a durable left ventricular assist device as a bridge to HTx. There were 33 (9.1%) cases of moderate or severe PGD. HLA was assessed in a mean of 15 and 24 samples per patient in those with and without PGD respectively. At two years, patients with PGD had lower incidence of development of <i>de novo</i> DSA against HLA-C (1.6 vs. 3.2%, p=0.047), DP (1.3 vs. 4.3%, p=0.002), DQ (3.3% vs. 11.8%, p<0.001), DR (4.9 vs. 12.4%, p<0.001) compared to patients without PGD (Table 1). In patients with and without PGD, there were 7 and 8 endomyocardial biopsies performed per patient, respectively within two years post-HTx. Patients with PGD had similar incidence of ISHLT 2R or 3R ACR compared to those without PGD (5.3% vs. 3.1%, p=0.07). Finally, there was no difference in PGD among patients who received empiric treatment for ACR without positive endomyocardial biopsy results (3.1% in PGD vs. 3.7% in no PGD, p=0.47). <h3>Conclusion</h3> Development of <i>de novo</i> DSA was lower in the PGD group and there was no difference in overall incidence of ACR in patients with and without PGD at 2 years post-HTx.

Lung Transplantation Outcomes after Crossing Low Level Donor Specific Antibodies without Augmented Immunosuppression

<h3>Purpose</h3> Lung transplant recipients with pre-transplant donor specific antibodies (DSA) are often treated with augmented immunosuppression. It is unknown whether some DSA can be safely crossed without additional therapies. We implemented a protocol allowing transplant when crossing select low level DSA (defined as mean fluorescence intensity (MFI) 1000-5000) without planned augmented immunosuppression. <h3>Methods</h3> This was a single center retrospective cohort study between 4/1/2015-8/31/2020. All recipients received solumedrol and basiliximab induction without desensitization. Presence of low level pre-transplant DSA was recorded. All post-transplant DSA was monitored within 14 days of transplant and then at routine intervals. The primary study outcomes were overall survival, definite CLAD≥1-free survival, and definite antibody mediated rejection (AMR), all defined according to ISHLT consensus guidelines. <h3>Results</h3> Of the 453 recipients, 36 (7.9%) had a low-level pre-transplant DSA crossed at transplant (Table 1). 13 had Class I antibodies and 26 had Class II. The median historical DSA MFI was 1800 (IQR 1300-2400) and the median most recent MFI was 1200 (IQR 950-2000). Among recipients where pre-transplant DSA was crossed, 17 (47.2%) had persistent post-transplant DSA with a median peak MFI of 7400. Class II antibodies were more likely to be detected post-transplant than Class I (57.7% vs 15.4%, p=0.02). There was no statistical difference in definite AMR in recipients where pre-transplant DSA was and was not crossed (8.3% vs 3.1%, p=0.11). With a median follow-up time of 2.4 years, there were no differences in adjusted overall survival (HR=1.14, 95% CI=0.57-2.32, p=0.71) or CLAD≥1-free survival (HR=0.82, 95% CI=0.44-1.54, p=0.54). <h3>Conclusion</h3> Lung transplantation in the presence of low level DSA without planned augmented immunosuppression was not associated with worse overall or CLAD-free intermediate-term survival but may be associated with increased AMR.

Assessment of Rapid Optimized 96-well Tray Flow Cytometric Crossmatch (Halifax-FCXM) with Luminex Single Antigen Test

IntroductionFlow cytometric crossmatch assay (FCXM) is a sensitive cell-based method for evaluating the presence of donor-specific antibodies (DSA) before transplantation. Recently, 96-well tray FCXM protocol (Halifax FCXM) with improved test efficiency has been introduced. The objective of the present study was to assess the performance of Halifax FCXM by correlating with DSA results based on single antigen bead (SAB) assays (virtual crossmatch, VXM). MethodsA total of 341 FCXMs were evaluated for the detection of HLA-DSA. A positive VXM was defined as having at least one HLA - DSA (HLA-A, B, Cw, DR, DQB1) with ≥ 1000 MFI (mean fluorescence intensity) identified by SAB assay. ResultsOf a total 341 cases, 113 showed class I VXM (+) with class I DSA MFI ≥ 1000 exclusively against one or more donor HLA class I antigens (HLA-A, B, Cw), 72 had class I-/II + DSA, and 156 had VXM(-). Halifax T-FCXM showed a sensitivity of 87.6% (99/113) and a specificity of 98.2% (224/228) for detecting class I VXM (+). The concordance between T-FCXM and class I VXM was 94.7% (323/341). Halifax B-FCXM showed a sensitivity of 58.3% (42/72) and a specificity of 98.7% (154/156) for detecting class I-/II + DSAs. The concordance between B-FCXM and class I-/II + VXM was 86.0% (196/228). When we separately analyzed data, B-FCXM detected HLA-DR (+) (68.8%) and HLA-DQ (+) DSAs (71.0%) similarly (P > 0.05). T-FCXM detected 87.6%, 97.2%, and 98.2% of class I DSA-positive cases with MFI values (sumDSA) ≥ 1000, ≥ 3000, and ≥ 5000, respectively. B-FCXM detected 58.3% of class I-II + DSA -positive (≥1000) cases, but detected 76.7% (33/43) and 89.2% (33/37) of class I-II + DSAs if MFI values of sumDSA and immunodominant DSA (iDSA) were above 5000, respectively. Halifax FCXM had sensitivities of 91.5% and 96.2% for detecting VXM (+) having MFI values above 5000 for class I or class II sumDSA and iDSA, respectively. ConclusionHalifax FCXM showed a good correlation, especially with SAB assay-based high MFI DSA or sumDSA. Concurrent application of FCXM with VXM can improve pre-transplant risk assessment and progress organ allocation efficiency.

Assessment of Carfilzomib Treatment Response in Lung Transplant Recipients With Antibody-mediated Rejection.

All patients treated with CFZ for AMR from 2014 to 2019 were included. The primary outcome was a positive response to CFZ was defined as: (1) loss of DSA C1q-fixing ability after last CFZ dose; (2) clearance of de novo DSA; or (3) decrease in de novo DSA mean fluorescence intensity of >3000. Twenty-eight patients with 31 AMR episodes were treated with CFZ. A positive response was observed in 74.4% of AMR episodes and 82.1% of patients. This response was driven by loss of complement 1q fixation (70.6%), elimination of class I DSAs (78.6%), and reduction in both classes I (median 2815, 79.5% reduction from baseline) and II DSA mean fluorescence intensity (3171, 37.1%). CFZ shows potential for ameliorating AMR; however, additional studies are needed to define optimal time of administration.

Effects of Reoptimization of Immunosuppressive Treatment on Donor-specific HLA Antibodies after Liver Transplantation

Introduction: Donor-specific antibodies (DSAs) have negative effects on short- and long-term outcomes after organ transplantation. DSAs are prevalent in patients with low immunosuppression; thus, optimized immunosuppression is preferable even in patients with stable condition after liver transplantation. However, the effect of implementing immunosuppression reoptimization for patients with low immunosuppression remains unclear. In this study, we investigated long-term changes in DSA status and the effect of reoptimizing immunosuppression on DSA status. Methods: We retrospectively reviewed DSA status in 66 patients after liver transplantation in our center. Results: The median duration between first and second DSA evaluation was 50 months. Of the 66 patients, 43 were positive for class II DSAs in the first evaluation. Of these patients, 30 were found to have an insufficient dose of calcineurin inhibitor, or were immunosuppression free at the time of the first evaluation. Reoptimization of immunosuppression was then conducted for 20 of the 30 patients. Among the 20 patients, DSAs detected in the first evaluation became negative in 7 patients and mean fluorescence intensity (MFI) decreased in 9 patients. Compared with patients with sustained low immunosuppression, DSA levels significantly decreased in patients with reoptimized immunosuppression (p=0.005). Conclusion: The results of this study indicate that post-transplant reoptimization of immunosuppression improved DSA status after liver transplantation. Reoptimization of immunosuppression is considered to be especially preferable in patients with de novo DSAs, although the clinical significance of DSA negative conversion and/or MFI reduction needs to be further investigated.