Donor-specific Antibodies Testing Research Articles

Donor-Specific Antibody Testing is an Effective Surveillance Strategy for High-Risk Antibody Mediated Rejection in Heart Transplant Patients in the Contemporary Era.

Pathologic antibody mediated rejection (pAMR) evaluation and donor specific antibody (DSA) testing are recommended in the first year after heart transplantation (HTx) in adult patients. Whether DSA testing adds prognostic information to contemporary pAMR surveillance has not been fully studied. This was a single center study of consecutive endomyocardial biopsies (EMB) performed between November 2010 and February 2023 in adult HTx patients. The primary objective was to evaluate whether DSA testing contributes additional information to pAMR surveillance to better predict overall survival. Secondary endpoints included cardiac allograft dysfunction and loss. A total of 6,033 EMBs from 544 HTx patients were reviewed for the study. The pAMR+/DSA+ group had significantly lower overall survival versus the pAMR-/DSA- group (hazard ratio [HR] = 2.63; 95% confidence interval [CI], 1.35-5.11; pc = 0.013). In the pAMR+/DSA+ group, patients with cardiac allograft dysfunction, compared to those without allograft dysfunction, had significantly lower overall and cardiac survival (pc < 0.001 for both). In contrast, pAMR+/DSA+ and pAMR-/DSA- patients without cardiac allograft dysfunction showed no difference in overall and cardiac survival. Primary graft dysfunction (PGD) was a novel risk factor for development of de novo DSAs (dnDSA) three weeks post-HTx (p = 0.007). DSA testing as the primary surveillance method can identify high-risk pAMR+/DSA+ patients. Surveillance pAMR testing in the contemporary era may need to be reevaluated. Earlier DSA testing at 10-14 days post-HTx should be considered in PGD patients.

Progress in Noninvasive Surveillance for Acute Rejection in Pediatric Heart Transplant Recipients: A Real-World Analysis of Donor-Derived Cell-Free DNA-Based Surveillance Protocol.

Acute cellular (ACR) and antibody-mediated (AMR) rejection are risk factors for allograft loss in heart transplant (HT) recipients. Endomyocardial biopsy (EMB), although considered the gold standard for rejection surveillance, is invasive and has high interobserver variability. Noninvasive donor-derived cell-free DNA (dd-cfDNA) sampling has a high negative predictive value (NPV) for rejection in adults and is increasingly used in pediatrics. This single center study aimed to test the performance of dd-cfDNA in screening for acute rejection (AR) and donor-specific antibodies (DSAs) in pediatric HT recipients. Blood samples for dd-cfDNA were obtained per clinical protocol for all eligible HT recipients in our center from July 1, 2022 to December 31, 2023. Primary endpoints were episodes of AR, pathology grading of EMBs temporally related to ddcfDNA sampling (0-150 days), and presence of DSAs. There were 471 interpretable samples, in 192 unique patients. Of those, 199 dd-cfDNA tests were paired with EMB ± DSA in 152 patients. Abnormal dd-cfDNA (> 0.2%) was found in 77 samples (median 0.48%, range 0.21%-11%) and led to EMB, where one sample was positive for ACR (grade 2R), 13 for AMR, yielding an NPV of 97% for AMR. After excluding abnormal ddcfDNA testing associated with AR, 65 abnormal dd-cfDNA tests were paired with DSA testing. The NPV of the test for detection of DSAs was 93%. Implementation of noninvasive rejection surveillance with dd-cfDNA in a pediatric cohort demonstrates high NPV for AR and high DSAs, making it an ideal screening tool for long-term monitoring of allograft health in pediatrics.

Identification of liver transplant biopsy phenotypes associated with distinct liver biological markers and allograft survival

The intricate association between histologic lesions and circulating antihuman leucocyte antigen donor-specific antibodies (DSA) in liver transplantation (LT) requires further clarification. We conducted a probabilistic, unsupervised approach in a comprehensively well-annotated LT cohort to identify clinically relevant archetypes. We evaluated 490 pairs of LT biopsies with DSA testing from 325 recipients transplanted between 2010 and 2020 across 3 French centers and an external cohort of 202 biopsies from 128 recipients. Unsupervised archetypal analysis integrated all clinico-immuno-histologic parameters of each biopsy to identify biopsy archetypes. The median time after LT was 1.17 (interquartile range, 0.38-2.38) years. We identified 7 archetypes distinguished by clinico-immuno-histologic parameters: archetype #1: severe T cell-mediated rejection (15.9%); #2: chronic rejection with ductopenia (1.8%); #3: architectural and microvascular damages (3.5%); #4: (sub)normal (55.9%); #5: mild T cell-mediated rejection (4.9%); #6: acute antibody-mediated rejection (6.5%); and #7: chronic rejection with DSA (11.4%). Cell infiltrates vary in the archetype. These archetypes were associated with distinct liver biological markers and allograft outcomes. These findings remained consistent when stratified using the patient’s age or indications for LT, with good performance in the external cohort (mean highest probability assignment = 0.58, standard deviation ± 0.17). In conclusion, we have identified clinically meaningful archetypes, providing valuable insights into the intricate DSA-histology association, which may help standardize liver allograft pathology classification.

Evaluation of serial monitoring of donor-specific antibodies in pediatric and adult intestinal/multivisceral transplant recipients.

The study purpose was to add to limited literature assessing anti-HLA donor-specific antibody (DSA) appearance, clearance, specificity, and impact in intestinal/multivisceral (MV) transplant as well as the value of serial monitoring following an institutional protocol shift implementing serial monitoring. This single-center retrospective review included intestinal/MV recipients transplanted 1/1/15-9/31/17 with completed DSA testing. Patients were divided into groups based on DSA presence post-transplant. The primary outcome was biopsy-proven acute rejection (BPAR). Secondary outcomes included graft loss and death. Descriptive analysis of DSA was completed. Of the 35 intestinal/MV recipients (60% pediatric) with DSA testing, 24 patients had post-transplant DSA. Fifteen patients in the DSA(+) group had T-cell-mediated BPAR versus five in the DSA(-) group (63% vs 45%, p = .47). Days to BPAR were 25 [IQR 19-165] (DSA(+) group) versus 232 [IQR 25.5-632.5] (DSA(-) group) (p = .066). There were no differences between groups for graft loss or death. One hundred and five DSA were identified in the DSA(+) group with 63% being class II, and 54% cleared during follow-up. DSA were directed against 50 different HLA alleles, with the most common being directed against HLA- DQ (35%). Time to first DSA and to clearance did not differ between class I and II. Findings confirm previous data that suggest post-transplant DSA in this population may lead to increased BPAR or shorter time to BPAR, although not statistically significant. Most DSA were identified within the first month after transplant, and ahead of rejection identification on biopsy. DSA therefore may have utility as an early rejection biomarker and use may be considered in place of early protocol biopsies, particularly in pediatric patients. We identified novel findings of DSA directed against a large breadth of HLA in intestinal/MV patients.

Evidence for Alloimmune Sinusoidal Injury in De Novo Nodular Regenerative Hyperplasia After Liver Transplantation.

Posttransplant nodular regenerative hyperplasia (NRH) mostly remains unexplained. Microvascular injury due to antibody-mediated rejection (AMR) is suspected, but lack of donor specific antibody (DSA) testing makes it difficult to prove. Centered around a 1-year period of routine DSA testing, concomitant protocol, and indicated posttransplant liver biopsies (LB), recipients with NRH (n = 18) were compared with a matched control group (n = 36). All index, previous, and subsequent LB were reviewed. Both groups were similar in terms of demographics, timing of index LB, and DSA. In the index LB, the NRH group had higher sinusoidal C4d positivity (p = 0.029) and perisinusoidal fibrosis (p = 0.034), both independently associated with NRH (p = 0.038 and 0.050, respectively). Features of "possible" chronic AMR were detected in 28.5% of the NRH group without a known cause and 0% of the control group (p = 0.009). The NRH group had more preceding indicated LB with increased incidence of rejection and biliary obstruction pattern. In the follow-up histology, overall, sinusoidal and portal C4d positivity, sinusoidal microvasculitis, and perisinusoidal fibrosis were also higher (all p < 0.050). In conclusion, we provide evidence towards the hypothesis that some cases of posttransplant NRH are related to preceding active and persistent AMR. Large multicenter studies with protocol DSA testing are required to confirm.

Has the Time Come for Widespread MHC Class I MICA Genotyping in Solid Organ Transplantation?

Has the Time Come for Widespread MHC Class I MICA Genotyping in Solid Organ Transplantation?

Combining donor-derived cell-free DNA and donor specific antibody testing as non-invasive biomarkers for rejection in kidney transplantation

Donor specific anti-HLA antibodies (DSA) and donor-derived cell-free DNA (dd-cfDNA) have lead to substantial progress in the non-invasive monitoring of the renal allograft by being able to detect or rule out subclinical rejection and guide immunosuppressive changes. In this study we sought to analyze the clinical, de novo DSA (dnDSA) and histological determinants of dd-cfDNA levels. The study included a cohort of stable renal function kidney transplant (KT) recipients who underwent anti-HLA dnDSA and dd-cfDNA testing between September 2017-December 2019. Statistical models were constructed to detect association with predictors of dd-cfDNA levels and other clinical characteristics. 171 renal allograft recipients were tested for dd-cfDNA and dnDSA at a median 1.06 years posttransplant (IQR: 0.37–4.63). Median dd-cfDNA was 0.25% (IQR: 0.19–0.51), 18.7% of patients having a dd-cfDNA ≥ 1%. In a multivariate linear regression model the presence of dnDSA MFI ≥ 2500 was the best independent determinant of dd-cfDNA level (p < 0.001). Among patients tested, 54 had concurrent dd-cfDNA determination at the time of an allograft biopsy. dd-cfDNA had an AUC of 0.82 (95% CI 0.69–0.91; p < 0.001) and of 0.96 (95% CI 0.87–0.99) to discriminate any rejection and ABMR, respectively. After multivariate adjustment, the models that included ABMR (R = 0.82, R2 = 0.67, p < 0.001), or ptc (R = 0.79, R2 = 0.63, p < 0.001) showed the best correlation with dd-cfDNA level. We are confirming a strong association of dd-cfDNA with dnDSA and underlying alloimmune-mediated injury in renal allograft recipients in a cohort of patients with unsuspecting clinical characteristics for rejection and excellent allograft function. Our findings support the need for noninvasive biomarker surveillance in KT recipients and we propose that dd-cfDNA may complement dnDSA screening.

Cell-Free DNA to Distinguish High Risk Donor Specific Antibodies in Heart Transplantation

<h3>Purpose</h3> Donor specific antibodies (DSA) develop in a significant fraction of heart transplant recipients (HTR). Enhanced characterization of high-risk DSA may permit early treatment and prevent downstream antibody-mediated rejection (AMR) and allograft failure. We hypothesize that high risk DSA is associated with allograft injury. Here, we compared allograft injury between high and low risk DSA using donor-derived cell-free DNA (ddcfDNA), a sensitive marker shown to detect AMR ∼3 months earlier than biopsy. <h3>Methods</h3> Using the prospective, multicenter study, Genomic Research Alliance for Transplantation (GRAfT), 81 HTR who underwent surveillance and clinically indicated endomyocardial biopsy, and DSA testing were analyzed. <b>Measurement:</b> Serially collected plasma samples (n=860) were assessed for ddcfDNA by shotgun sequencing. <b>Analysis:</b> DSA were categorized as "high risk" if they were associated with AMR or were otherwise considered "low risk". DSA was further categorized by specificity as class II vs. class I. We compared ddcfDNA between groups with generalized mixed linear models; excluding ddcfDNA before Day 14 post-transplant to reduce transplant surgical contribution. <h3>Results</h3> 47% HTR (n=38) developed DSA at a median 24 days (IQR = 6 - 141 days) post-transplant; and include 28 <i>de novo</i> and 11 preformed DSA. 15.8% (n=6) were categorized as high risk DSA and 84.2% (n=32) were low risk DSA. High risk DSA showed 4X higher ddcfDNA levels compared to low risk DSA (mean [95% CI] 0.30[0.22% - 0.39%] vs. 0.07[0.06%-0.10%], <i>p</i> <0.01, Figure A). As compared to patients without DSA, low risk DSA showed similar ddcfDNA (p=0.52, Figure A). We also found higher ddcfDNA in class II than in class I DSA (Figure B). <h3>Conclusion</h3> High risk DSA show higher allograft injury as measured by ddcfDNA. Similarly, <i>de novo</i> and class II DSA are associated with higher allograft injury than class I or preformed DSA. We provide a novel concept that allows for use of ddcfDNA to stratify high-risk DSA in heart transplantation.

Crossmatch, Donor-specific Antibody Testing, and Immunosuppression in Simultaneous Liver and Kidney Transplantation: A Review.

Since the introduction of simultaneous liver-kidney transplantation (SLKT) in the 1960s, the potential for immunological protection from the liver allograft to a simultaneously transplanted kidney has been recognized. Due to expanded indications and changes in allocation policies, there has been increased utilization of SLKT. Despite growing experience, a lack of consensus exists regarding the extent of the immunological privilege of the liver the role for donor-specific HLA antibody (DSA) and crossmatch testing, and appropriateness of modern immunosuppression protocols in SLKT recipients. This review provides a detailed analysis of SLKT outcomes in the context of these factors, suggesting that although the liver can reduce the incidence of antibody-mediated rejection, attention should be given to liver allograft function, previous failed transplants, and other risk factors in pretransplant risk assessment. Current methods of DSA and crossmatch testing in SLKT are also discussed, and the role of specific DSA (high mean fluorescence intensity antibody, C1q+ binding) and their potential importance in posttransplant risk assessment are examined. Finally, trends in SLKT immunosuppression are discussed, including the use of nondepleting agents for induction and de-escalating use of steroids for maintenance immunosuppression. Ongoing research, including multicenter or randomized trials, will be necessary to optimize immune-related outcomes in SLKT recipients.

Antibody-Mediated Rejection of the Lung in Patients with Short Telomeres

PurposeShort telomeres are associated with interstitial lung disease (ILD) and immune dysregulation. Prior studies have suggested that lung transplant recipients with short telomeres experience high rates of renal dysfunction, cytopenias, and infections and may have reduced survival and time to onset of chronic lung allograft dysfunction. It is unknown whether immune dysfunction related to short telomeres contributes to post-transplant complications such as antibody-mediated rejection (AMR). We investigated whether leukocyte telomere length (LTL) is a risk factor for developing AMR.MethodsThis is a single-center retrospective cohort study of 114 ILD patients who underwent lung transplantation between January 1, 2008 and December 31, 2017. Pre-transplant leukocyte telomere length (LTL) was measured using qPCR. Age-adjusted telomere length was calculated by comparing against a reference population, and dichotomized above and below the 10th percentile. Each patient underwent serial testing for donor-specific antibodies (DSA) and transbronchial biopsies per institutional protocol. AMR was graded according to the 2016 ISHLT consensus report and graded as definite and probable AMR. The acute cellular rejection (ACR) score was computed as the sum of the total numeric Grade A ACR in the follow up period. Multivariable logistic regression was used to assess potential predictors of AMR and included age, sex, ACR score, transplant type and LTL as covariates.ResultsThere were 35 (30.7%) patients with age-adjusted LTL < 10th percentile. DSA was positive in 67 (58.8%) patients. Definite and probable AMR occurred in 11 (9.6%) and 16 patients (14.0%), respectively. Development of AMR was not associated with LTL < 10th percentile (p=0.274). After adjustment, LTL < 10th percentile does not increase the odds of AMR (OR 0.70; 95% CI, 0.24-2.04; P=0.516). However, ACR score was associated with AMR (OR 1.25; 95% CI 1.04-1.51; P=0.020).ConclusionPre-transplant LTL < 10th percentile does not appear to be a risk factor for development of AMR. Short telomeres are associated with interstitial lung disease (ILD) and immune dysregulation. Prior studies have suggested that lung transplant recipients with short telomeres experience high rates of renal dysfunction, cytopenias, and infections and may have reduced survival and time to onset of chronic lung allograft dysfunction. It is unknown whether immune dysfunction related to short telomeres contributes to post-transplant complications such as antibody-mediated rejection (AMR). We investigated whether leukocyte telomere length (LTL) is a risk factor for developing AMR. This is a single-center retrospective cohort study of 114 ILD patients who underwent lung transplantation between January 1, 2008 and December 31, 2017. Pre-transplant leukocyte telomere length (LTL) was measured using qPCR. Age-adjusted telomere length was calculated by comparing against a reference population, and dichotomized above and below the 10th percentile. Each patient underwent serial testing for donor-specific antibodies (DSA) and transbronchial biopsies per institutional protocol. AMR was graded according to the 2016 ISHLT consensus report and graded as definite and probable AMR. The acute cellular rejection (ACR) score was computed as the sum of the total numeric Grade A ACR in the follow up period. Multivariable logistic regression was used to assess potential predictors of AMR and included age, sex, ACR score, transplant type and LTL as covariates. There were 35 (30.7%) patients with age-adjusted LTL < 10th percentile. DSA was positive in 67 (58.8%) patients. Definite and probable AMR occurred in 11 (9.6%) and 16 patients (14.0%), respectively. Development of AMR was not associated with LTL < 10th percentile (p=0.274). After adjustment, LTL < 10th percentile does not increase the odds of AMR (OR 0.70; 95% CI, 0.24-2.04; P=0.516). However, ACR score was associated with AMR (OR 1.25; 95% CI 1.04-1.51; P=0.020). Pre-transplant LTL < 10th percentile does not appear to be a risk factor for development of AMR.

Donor Derived Cell Free DNA as a Risk Factor for Initiating De-Novo Donor Specific Antibodies in Heart Transplantation

PurposeWe hypothesize that subclinical inflammation, resulting in molecular injury to the cardiac allograft, may contribute to sensitization of the recipients’ immune system. Antigenic presentation of donor-derived nucleic acids, as reflected by elevated donor derived cell free DNA (dd-cfDNA) levels, may lead to leukocyte activation, promoting cytokine release and donor specific antibody (DSA) formation, with elevated dd-cfDNA levels seen prior to detection of de-novo DSAs in kidney and lung transplantation. We aimed to assess the relationship between DSA and dd-cfDNA in cardiac transplantation.MethodsThis was a retrospective cohort analysis examining a subset of the Donor‐Derived Cell‐Free DNA‐Outcomes AlloMap Registry (D‐OAR) data, focusing on dd‐cfDNA and DSA results. 67 patients were identified, providing 284 samples, with corresponding dd-cfDNA (AlloSure) and DSA results. Samples that had associated rejection were excluded.ResultsOf the 67 patients, 28 (42%) had negative DSA, with a median dd-cfDNA = 0.06% (IQR 0.03% - 0.08%), 27 patients (40%) had pre-formed DSA with a median dd-cfDNA = 0.09% (IQR 0.06% - 0.18%) and 12 patients (18%) had de-novo DSA formation, median dd-cfDNA = 0.34%, (IQR 0.11% - 0.45%) [Figure]. DSA negative patients had significantly lower dd-cfDNA levels than patients who were DSA positive (p< 0.001), while patients with de-novo DSA had significantly higher dd-cfDNA than patients who had pre-formed DSA. (p= 0.001). Elevations in dd-cfDNA appeared a median of 20 days (range 2-28) prior to DSA testing. Multivariate logistic regression analysis identified dd-cfDNA an independent predictor of de-novo DSA formation, when controlling for recipient race, age, donor demographics and HLA mismatch (1-6) (p < 0.001).ConclusionHigher dd-cfDNA levels are associated with de-novo DSA formation. Further studies are required to determine whether this is a causal relationship and if dd-cfDNA acts as a trigger for DSA formation. We hypothesize that subclinical inflammation, resulting in molecular injury to the cardiac allograft, may contribute to sensitization of the recipients’ immune system. Antigenic presentation of donor-derived nucleic acids, as reflected by elevated donor derived cell free DNA (dd-cfDNA) levels, may lead to leukocyte activation, promoting cytokine release and donor specific antibody (DSA) formation, with elevated dd-cfDNA levels seen prior to detection of de-novo DSAs in kidney and lung transplantation. We aimed to assess the relationship between DSA and dd-cfDNA in cardiac transplantation. This was a retrospective cohort analysis examining a subset of the Donor‐Derived Cell‐Free DNA‐Outcomes AlloMap Registry (D‐OAR) data, focusing on dd‐cfDNA and DSA results. 67 patients were identified, providing 284 samples, with corresponding dd-cfDNA (AlloSure) and DSA results. Samples that had associated rejection were excluded. Of the 67 patients, 28 (42%) had negative DSA, with a median dd-cfDNA = 0.06% (IQR 0.03% - 0.08%), 27 patients (40%) had pre-formed DSA with a median dd-cfDNA = 0.09% (IQR 0.06% - 0.18%) and 12 patients (18%) had de-novo DSA formation, median dd-cfDNA = 0.34%, (IQR 0.11% - 0.45%) [Figure]. DSA negative patients had significantly lower dd-cfDNA levels than patients who were DSA positive (p< 0.001), while patients with de-novo DSA had significantly higher dd-cfDNA than patients who had pre-formed DSA. (p= 0.001). Elevations in dd-cfDNA appeared a median of 20 days (range 2-28) prior to DSA testing. Multivariate logistic regression analysis identified dd-cfDNA an independent predictor of de-novo DSA formation, when controlling for recipient race, age, donor demographics and HLA mismatch (1-6) (p < 0.001). Higher dd-cfDNA levels are associated with de-novo DSA formation. Further studies are required to determine whether this is a causal relationship and if dd-cfDNA acts as a trigger for DSA formation.

Preemptive HLA Antibody Screening Prior to Episodic Transplant Renal Biopsy Enables Early Diagnosis and Therapeutic Response in Asymptomatic Chronically Active Antibody-Related Rejection: A Case Report

Common management of renal transplant recipients includes episodic renal biopsy based on clinical findings such as an increase in proteinuria or serum creatinine. When antibody-related rejection is suspected from the renal biopsy, subsequent testing for donor-specific antibodies (DSAs) is performed. We instead performed preemptive screening of asymptomatic post–renal transplant recipients for DSAs prior to renal biopsy. In this case, a 30-year-old woman with a secondary transplant was positive for 61 anti-HLA antibodies of class I and class II, among which DQ2 was a DSA with a mean fluorescence index of 2039. The patient had a living kidney transplant 9 years earlier. She had never been diagnosed with rejection, her serum creatinine was around 1.0 mg/dL, and her proteinuria was negative. Following the positive DSA result, a renal biopsy was performed, and she was diagnosed as C4d-negative chronic-active antibody-mediated rejection (CAABMR) with a Banff score of cg1b, (g + ptc) ≥ 2, and C4d 0. Intravenous steroid pulse, deoxyspagarin, antithymocyte globulin, rituximab, and oral everolimus were administrated. The treatment resulted in a gradual decrease in the DSA, which became negative 1 year later. The patient’s serum creatinine remains around 1.0 mg/dL, and proteinuria remains negative. Treatments for advanced CAABMR are often expensive and ineffective. Our present case suggests that early detection and treatment through preemptive HLA antibody screening could improve the prognosis of renal transplants.

Lung Transplantation: DSA to AMR Trajectory

Donor specific antibodies(DSA) are a major risk factor for antibody mediated rejection (AMR) and allograft failure in lung transplant recipients (LTs). The trajectory from DSA to AMR and its association with allograft injury remains poorly defined. Thus, we detailed DSA and their trajectory to AMR. 417 LTs from three prospective cohort studies were included. LTs had pre-transplant DSA testing, post transplantation surveillance and clinically indicated DSA testing. An AMR Working Group reviewed clinical data and used ISHLT criteria to adjudicate AMR. Allograft injury was assessed by %ddcfDNA in serially collected plasma samples via shotgun sequencing. Positive DSA: preformed or de novo if DSA was first detected before or after 14 days post-transplant. The interval from DSA detection to AMR or no AMR was computed. Preformed and de novo DSA was subcategorized in +AMR and -AMR subgroups based on whether or not DSA was associated with clinical AMR. %ddcfDNA levels were compared for these subgroups. Treated LTs prior to allograft dysfunction or AMR were removed. 185 subjects had positive HLA DSA (54.73%) detected at a median of 33 days post-transplantation. (IQR 9.0-236.8) DSA was predominantly de novo (n=119, 64.3%) than preformed (n=66, 35.7%), class II than Class I (76.44% vs. 20.24%, p<0.01). Of 185 DSA+ patients, 92 developed clinical AMR, 27 no AMR, and 66 had insufficient data to adjudicate clinical AMR. AMR developed at a medium of 130 days from DSA detection. (IQR 58- 504) The prevalence of clinical AMR was 23.65%(N=92): definite (N=6, 6.5%), probable (N=16, 17.4%) and possible (N=70, 76%).De novo DSA showed a higher rate of progression to AMR than preformed DSA (62.5% vs. 32.5%, p<0.01). %ddcfDNA levels correlated with AMR risk: median %ddcfDNA for de novo DSA that lead to AMR was 2X higher than de novo DSA that did not lead to AMR (1.55 vs. 0.88 p=0.016). Preformed DSA that lead to AMR had a median %ddcfDNA of 1.47 versus 0.87 in the preformed DSA that did not lead to AMR (p=0.014). DSA that did not lead to AMR showed higher %ddcfDNA levels than DSA negative, non-rejection timepoints. (0.87 vs. 0.33, p<0.01) CONCLUSION: De novo and preformed DSA showed greater allograft injury than non-rejection DSA- timepoints. De novo DSA progresses to AMR at a higher rate than preformed DSA. Preformed or de novo DSA that progresses to AMR causes more lung injury than DSA with no AMR. %ddcfDNA could be used to identify and treat high-risk DSA.

To Treat or Not to Treat: DSA Positive Lung Transplant Recipients

Donor-specific antibodies (DSA) is a major risk factor for chronic lung allograft dysfunction (CLAD) after lung transplantation (LTx). Unfortunately, DSA management (Rx) varies significantly. To date, there is no accepted standard on when to initiate treatment. This study examines whether the timing of DSA treatment alters outcomes. 435 LTx subjects enrolled in two cohort studies were included. They underwent surveillance and clinically-indicated HLA DSA testing. DSA+patients were treated with a Rituximab or Bortezomib-based regimen either before they showed any clinical or histological signs of AMR (Early Rx) or when they developed these signs of AMR (Late Rx); choice was based on provider's practice. Time from transplant to CLAD-free survival, defined as being alive and CLAD-free. CLAD was adjudicated by a committee. Allograft injury at time of Rx was quantified via plasma donor-derived cell-free DNA (ddcfDNA), a validated biomarker. Baseline characteristics, allograft injury, DSA clearance and CLAD-free survival were compared between treatment groups and controls (DSA- patients). Median follow-up was 10 (IQR = 11 - 37) months. 58% of patients developed HLA DSA, which was predominantly Class II than class I (85% vs. 15%). DSA+patients were treated either early (n = 49) or Late (n = 97). The remaining 32% of DSA+patients lacked relevant clinical data or were not treated. Baseline characteristics and DSA specificities were similar between treatment groups. However, Late AMR showed higher median %ddcfDNA (4% vs 1%, p < 0.01), lower percentage of DSA clearance (40% vs 63%, p = 0.02), and a 3-fold hazard of dying or developing CLAD than Early Rx (Figure). Early Rx extended CLAD-free survival by 34 months compared to Late Rx, and showed similar CLAD-free survival as DSA-patients (p = 0.67). This study supports Early Rx of DSA over Late Rx since Early Rx shows improved CLAD-free survival. Multivariate analyses to identify other potential covariates are being planned.

Testing for donor-specific antibodies in renal transplantation: Indian perspective

The evaluation of donor-specific antibodies by Luminex single-antigen bead assay (in addition to other crossmatch tests) to assess pretransplant immunological risk should be performed in recipients (especially with a history of prior sensitization) even in resource-constrained settings as this approach can help in better risk stratification, to decide on transplant eligibility, selection of immunologically favorable donor, to plan desensitization protocol and induction therapy that can lead to the reduction of posttransplant rejection rates and better graft survival. Despite the cost, it is justified to use these sensitive assays in selected cases even in a cost-limited setting as this enables earlier and better-matched transplant, and avoidance of morbidity and poor graft survival.

Exploring pre-surgery donor-specific antibodies in the context of organ shortage in liver transplant.

There is a growing disparity between the number of liver transplant (LT) candidates and availability of suitable liver allografts. Antibody-mediated rejection (AMR), secondary to positive donor-specific antibodies (DSA), remains a concern in liver transplantation. This study aimed to correlate expression of DSA on pre-transplant screening and outcomes of LT, specifically development of AMR in liver allografts and liver function profile in the post-operative period. Data of consecutive patients undergoing orthotopic LT (OLT) at the South Australian Liver Transplant Unit was analysed. All patients underwent DSA testing pre-transplant. Within a cohort of 96 patients, over a post-OLT median follow-up of 849 days, only 2 patients (2%) developed AMR. While both patients had a positive DSA test preoperatively, overall DSA positivity was noted in 31% patients, with a specificity for prediction of AMR of 0.708. No significant association was noted between AMR (p = 0.092), T cell-mediated rejection/TCMR (p = 0.797) or late hepatic artery thrombosis/LHAT (p = 0.521). There was no significant interaction effect between DSA positivity and serum bilirubin or transaminases over a period of 100 days. AMR following LT is uncommon. A positive DSA pre-transplant does not imply a definite risk of AMR. Also, there does not exist a significant interaction in time between DSA expression and serum bilirubin or transaminase levels. Until there emerges evidence to the contrary, it appears reasonable to consider DSA-positive donors within the broad context of marginal donors in the context of a worldwide shortage of LT donor allografts.

Histological picture of antibody-mediated rejection without donor-specific anti-HLA antibodies: Clinical presentation and implications for outcome.

In this cohort study (n=935 transplantations), we investigated the phenotype and risk of graft failure in patients with histological criteria for antibody-mediated rejection (ABMR) in the absence of circulating donor-specific anti-human leukocyte antigen (HLA) antibodies (DSA), and compared this to patients with definite ABMR and HLA-DSA-positivity. The histological picture did not differ between HLA-DSA-positive (n=85) and HLA-DSA-negative (n=123) cases of ABMR histology, apart from increased complement split product 4d (C4d) deposition in the peritubular capillaries in HLA-DSA-positive cases. Histology of ABMR without HLA-DSA was more transient than DSA-positive ABMR, and patients with ABMR histology without HLA-DSA had graft survival superior to that of HLA-DSA-positive patients, independent of concomitant T cell-mediated rejection (38.2%) or borderline changes (17.9%). Multivariate analysis showed that the risk of graft failure was not higher in patients with histological picture of ABMR (ABMRh ) in the absence of HLA-DSA, compared to patients without ABMRh . Despite an association between C4d deposition and HLA-DSA-positivity, using C4d deposition as alternative for the DSA criterion in the diagnosis of ABMR, as proposed in Banff 2017, did not contribute to the prognosis of graft function and graft failure. We concluded that biopsies with ABMRh but without detectable HLA-DSA represent a distinct, often transient phenotype with superior allograft survival.

Donor-derived Cell-free DNA Identifies Antibody-mediated Rejection in Donor Specific Antibody Positive Kidney Transplant Recipients.

Elevated levels of donor-derived cell-free DNA (dd-cfDNA) in the plasma of renal allograft recipients indicates organ injury and an increased probability of active rejection. Donor-specific antibodies (DSA) to HLA antigens are associated with risk of antibody-mediated rejection (ABMR). This study assessed the combined use of dd-cfDNA and DSA testing to diagnose active ABMR. Donor-derived cell-free DNA was assayed in 90 blood samples with paired DSA and clinically indicated biopsies from 87 kidney transplant patients. Sixteen cases met criteria for active ABMR. Performance characteristics of dd-cfDNA for diagnosis of active ABMR were determined for samples with prior or current positive DSA (DSA+, n = 33). The median level of dd-cfDNA (2.9%) in DSA+ patients with active ABMR was significantly higher than the median level (0.34%) in DSA+ patients without ABMR (P < 0.001). The median level of dd-cfDNA in DSA- patients was 0.29%. The positive predictive value of dd-cfDNA (at 1%) to detect active ABMR in DSA+ patients was 81%, whereas the negative predictive value was 83%. The positive predictive value for DSA+ alone was 48%. The combined use of dd-cfDNA and DSA testing may improve the noninvasive diagnosis of active ABMR in kidney transplant patients. Patients with dd-cfDNA+/ DSA+ results have a high probability of active ABMR.

P126 Comparison study of CDC and flow crossmatch vs. donor specific antibodies in transplantation

Aim Our approach to transplant highly sensitized patients has been to perform CDC crossmatch (XM) when the results of flow xm are above 200 mean channel shifts (MCS) with donor specific antibodies (DSA). The aim of our study was to determine if the flow crossmatch and solid phase antibody testing can be used as the only measures for decisions to transplant. Methods Results of flow xm, CDC xm and solid phase DSA testing were analyzed for 44 donor and recipient pairs. In all cases, the flow xm were positive with greater than 200 MCS values. Undiluted patient sera were tested by Luminex single antigen (LSA) test for HLA antibodies (ABS). The ABS detected by this method were grouped into weak (MFI 2500–5000) moderate (MFI 5000–7500), and strong (MFI 7500-greater than 17000). The CDC xm was performed with and without DTT treatment using the same sera for flow xm. Results In 70% of this group with positive T and/or B CDC xm at least one DSA detected was in the strong binding range above 17,000 MFI. In 30% of cases multiple DSA detected in weak to moderate range below 7500 MFI values. In 50% of neg T cell and 96% of neg B cell CDC crossmatches, the MFI of DSA detected was in moderate to strong range. In 90% of negative CDC xms the MCS values for flow T was below 300 and flow B below 400 MCS. The range of flow MFI values for positive CDC B cells xm was 156–596 and 238–671 for T cells. Conclusions While the CDC xm test is the classical standard to exclude transplant, it is not always a reliable test. This assay is quite subjective and dependent on quality and strength of complement, technical skills of the laboratory technician and donor cell viability. This assay is also not reliable when patient is under desensitization therapy. There is a need to develop this assay using flow cytometer platform. Use of solid phase testing methods and determination of standards for decision to transplant based on number, type and strength of antibodies can be a better method

The association of de novo anti-HLA-DPB1 donor-specific antibody formation and primary graft failure after allogeneic hematopoietic cell transplantation

Despite advances in HLA matching, graft failure remains a serious complication after allogeneic hematopoietic cell transplantation (HCT). Pre-formed anti-HLA donor-specific antibodies (DSAs) have been associated with graft failure, but the impact of newly developing (de novo) anti-HLA antibodies on HCT outcomes remains unknown. Here, we present the first reported case of graft failure associated with de novo anti-HLA-DPB1 DSA after a 10/10 matched unrelated donor HCT. Based on this observation, testing for DSA should be considered in case of unexplained pancytopenia after allogeneic transplantation, especially after reduced intensity conditioning.