Refining cPRA calculation to improve HLA compatibility assessment in organ transplantation: A detailed picture of the Paris waiting list.

The determination of panel reactive antibodies (cPRA) scores plays a critical role in assessing the immunological compatibility between organ transplant recipients and potential donors. Traditional cPRA methods focus on a limited number of HLA loci using physical cytotoxicity tests. However, advancements such as the Luminex single antigen (LSA) assay, which uses mean fluorescence intensity (MFI) of individualised HLA antigens for antibody evaluation, provide a foundation for a more precise assessment. We developed cPRAdictor, a novel cPRA calculation tool using a large series of HLA-type individuals in France with NGS. cPRAdictor was applied to a cohort of 5962 kidney transplant candidates in Paris. We analysed how extending the range of HLA specificities could affect cPRA values. Implementing cPRAdictor revealed and allowed quantification of the significant discrepancies in cPRA values that appeared when HLA loci C and DP, and antigen-specific antibodies were taken into account. Notably, over 43% of the immunised transplant candidates showed an increase in calculated cPRA values when considering C/DP loci and antigen-specific antibodies, negatively impacting their eligibility and prioritisation in the transplantation programme. These findings highlight the necessity of revisiting cPRA calculation methodologies to include a broader spectrum of immunological data, as more exhaustive and precise information regarding anti-HLA antibodies in patients' sera and donor and recipient HLA typing are available prospectively. This will strongly improve both accuracy and equity at the organ allocation step, especially for highly sensitised candidates for whom organ offers are very limited in number.

Effect of angiotensin II type 1 receptor antibodies on graft function and survival in paediatric kidney transplant recipients.

HLA donor specific antibodies (DSA) are implicated in antibody-mediated rejection (AMR), graft dysfunction and failure in kidney transplant (KT) recipients. Non-HLA antibodies including angiotensin II type 1 receptor (AT1R) may also play a role in AMR, impact graft function and survival. Data is limited in paediatric KT cohorts. We aimed to assess the prevalence and effect of pre-transplant AT1R antibodies on rejection, graft function and survival in paediatric KT recipients. This was a retrospective cohort study conducted across two paediatric centres including KT recipients with a pre-transplant AT1R antibody level. Outcomes included rejection, de novo DSA formation, graft function, failure, proteinuria and hypertension. Of 71 individuals, 72% recorded a positive pre-transplant AT1R Ab level (≥17 U/mL). Over a median follow-up of 4.7 years, AT1R Ab positivity demonstrated a trend towards increased risk of rejection however was not statistically significant (HR 3.45, 95% CI 0.97-12.35, p-value 0.06). Sensitivity analysis with AT1R Ab levels of ≥25 U/mL (HR 2.05 95% CI 0.78-5.39, p-value 0.14) and ≥40 U/mL (HR 1.32, CI 95% 0.55-3.17, p-value 0.53) validated this. De novo DSA formation occurred more frequently with AT1R Ab positivity (41% vs. 20%, p-value 0.9). AT1R Ab was not associated with hypertension, proteinuria, graft failure or dysfunction. In conclusion, this cohort study demonstrated a high prevalence of pre-transplant AT1R Ab positivity (72%). AT1R Ab positivity demonstrated a trend towards increased risk of rejection and de novo DSA formation however did not meet statistical significance. There was no association between AT1R Ab and hypertension, proteinuria, graft failure or dysfunction.

Calcium-dependent HLA-DQ epitope revealed by EDTA mediated inhibition of antibody reactions in the Luminex single antigen bead assay.

Complement mediated interference with the detection of antibodies targeting HLA is a known limitation of the single antigen bead (SAB) Luminex assay. Ethylenediaminetetraacetic acid (EDTA) is currently the serum treatment of choice in most histocompatibility laboratories to block complement activation by chelating calcium. The purpose of this study was to investigate a serum with an antibody reactivity to HLA-DQ6, 7, 8 and 9 molecules, in the Luminex SAB assay, that was inhibited by treatment with EDTA. Serum was from a 55-year-old highly sensitised female renal transplant candidate that contained, among others, antibodies to an epitope containing the 74EL eplet, shared by HLA-DQ6, DQ7, DQ8 and DQ9 molecules. Serum samples were treated with EDTA, dithiothreitol (DTT), or heat prior to testing by SAB assay. EDTA-treated serum was also tested after the addition of calcium chloride (CaCl2). HLA-DQ-specific antibodies were isolated by adsorption/elution method using three informative donor cells and were tested in the absence or presence of EDTA. The antibody reactivity against HLA-DQ6, DQ7, DQ8 and DQ9 in the SAB assay was significantly inhibited by treating serum and eluates with EDTA and was restored by addition of CaCl2. The study represents the first description of a calcium-dependent epitope in HLA molecules. The relevance of this finding is that the treatment of sera with EDTA could lead to false-negative reactions in the SAB assay, which may compromise virtual crossmatching.

A simple predictor for donor-specific anti-HLA antibody desensitisation in haploidentical haematopoietic stem cell transplantation.

Donor-specific HLA antibody (DSA) has been recognised as an independent risk factor for graft failure in patients undergoing haploidentical haematopoietic stem cell transplantation (HID HSCT). Therapeutic plasma exchange (TPE), as a first-line strategy for DSA desensitisation, can promptly reduce serum DSA levels. This study aimed to investigate DSA characteristics and identify a biomarker predicting the efficacy of DSA desensitisation in patients proceeding to HID HSCT. We retrospectively enrolled 32 patients with DSA from April 2021 to January 2024, and analysed the mean fluorescence intensity (MFI) value of DSA at the different time points of desensitisation treatment. Compared with baseline DSA level before TPE, the median MFI of HLA class I DSA was reduced from 8178.6 to 795.3 (p < 0.001), and HLA class II DSA decreased from 6210.9 to 808.8 (p < 0.001) after TPE. The DSA level in 1:16 diluted pre-TPE serum correlated well with DSA value in post-TPE serum (class I, r = 0.85, p < 0.0001; class II, r = 0.94, p < 0.0001), predicting TPE efficacy in 84.4% of patients. Based on the degree of DSA reduction after TPE, patients were divided into complete responders (decreased by >70%), partial responders (decreased by 30 to 70%) and non-responders (decreased by <30%) and the percentages were 43.8%, 25% and 31.2%, respectively. Non-responders receiving aggressive immunotherapy had longer overall survival compared to those receiving standard strategies (p < 0.05). The 1:16 diluted pre-TPE serum may predict the efficacy of TPE and allow for more rational immunotherapy strategy for patients with DSA proceeding to HID HSCT.

Nipocalimab in Early-Onset Severe Hemolytic Disease of the Fetus and Newborn.

BACKGROUND: In early-onset severe hemolytic disease of the fetus and newborn (HDFN), transplacental transfer of maternal antierythrocyte IgG alloantibodies causes fetal anemia that leads to the use of high-risk intrauterine transfusions in order to avoid fetal hydrops and fetal death. Nipocalimab, an anti-neonatal Fc receptor blocker, inhibits transplacental IgG transfer and lowers maternal IgG levels. METHODS: In an international, open-label, single-group, phase 2 study, we assessed treatment with intravenous nipocalimab (30 or 45 mg per kilogram of body weight per week) administered from 14 to 35 weeks' gestation in participants with pregnancies at high risk for recurrent early-onset severe HDFN. The primary end point was live birth at 32 weeks' gestation or later without intrauterine transfusions as assessed against a historical benchmark (0%; clinically meaningful difference, 10%). RESULTS: Live birth at 32 weeks' gestation or later without intrauterine transfusions occurred in 7 of 13 pregnancies (54%; 95% confidence interval, 25 to 81) in the study. No cases of fetal hydrops occurred, and 6 participants (46%) did not receive any antenatal or neonatal transfusions. Six fetuses received an intrauterine transfusion: five fetuses at 24 weeks' gestation or later and one fetus before fetal loss at 22 weeks and 5 days' gestation. Live birth occurred in 12 pregnancies. The median gestational age at delivery was 36 weeks and 4 days. Of the 12 live-born infants, 1 received one exchange transfusion and one simple transfusion and 5 received only simple transfusions. Treatment-related decreases in the alloantibody titer and IgG level were observed in maternal samples and cord blood. No unusual maternal or pediatric infections were observed. Serious adverse events were consistent with HDFN, pregnancy, or prematurity. CONCLUSIONS: Nipocalimab treatment delayed or prevented fetal anemia or intrauterine transfusions, as compared with the historical benchmark, in pregnancies at high risk for early-onset severe HDFN. (Funded by Janssen Research and Development; UNITY ClinicalTrials.gov number, NCT03842189.).

[Analysis of the causes of incompatible cross-matching induced by two Rh blood group antibodies and corresponding laboratory treatment].

Objective To analyze the serological characteristics and clinical significance of IgG anti-D and anti-C combined antibodies and anti-E and anti-c combined antibodies in patients negative for RhDC and RhEc antigens. Methods The clinical data and laboratory results of 12 cases with two types of irregular antibodies were recorded and analyzed, including age, sex, history of blood transfusion/pregnancy, ABO and RhD blood group identification, Rh antigen typing, irregular antibody screening, antibody-specific identification, absorption-elution tests, antibody titer determination and cross-matching tests. Results Among the 12 patients, the mean age was 51.4±16.9 years. Nine patients had a history of blood transfusion; eight patients had a history of pregnancy; five patients had both. Serological tests showed positive antibody screening and incompatible cross-matching. The results of antibody-specific identification and absorption-elution tests showed the presence of both IgG anti-D and anti-C antibodies in three patients, with anti-D titers at 16-32, and anti-C titers at 8-16. Nine patients had both IgG anti-E and anti-c antibodies, with the titers of anti-E and anti-c antibodies at 8-16. From the patients with combined anti-D and anti-C antibodies, suspended red blood cells of ABO identical type, RhD negative and other Rh antigens as ccee were selected. From patients with combined anti-E and anti-c antibodies, suspended red blood cells of ABO identical type, RhD positive and other Rh antigens as CCee were selected. Cross-matching blood test results showed no agglutination or hemolysis in saline, polycoagulant and anti-human globulin media. Conclusion Blood transfusion and/or pregnancy are the primary causes of irregular antibodies in two Rh systems, leading to positive antibody screening and cross-match incompatibility. Routine compatibility transfusion of Rh antigens, based on ABO homotypic blood transfusion, is of great value and significance for the safety of clinical blood transfusion and the prevention of hemolytic disease of the newborn.

Perspective for Donor-Derived Cell-Free DNA in Antibody-Mediated Rejection After Kidney Transplantation: Defining Context of Use and Clinical Implications.

Antibody-mediated rejection (AMR) is a major cause of graft failure limiting long-term graft survival after kidney transplantation. Current diagnostic strategy to detect AMR is suboptimal and requires further improvement. Previously suggested treatment regimens for AMR could not demonstrate efficacy, however novel therapeutic agents are currently under investigation. Donor-derived cell-free DNA (dd-cfDNA) is a novel non-invasive biomarker for allograft injury, that has been mainly studied in the context of rejection. Its short-half-life in circulation and injury-dependent release are its key advantages that contribute to its superior diagnostic accuracy, compared to traditional biomarkers. Moreover, previous studies showed that dd-cfDNA-release is well-linked to histological and molecular features of AMR, and thus able to reflect real-time injury. Further observations suggest that dd-cfDNA can be used as a suitable screening tool for early detection of AMR in patients with donor-specific-anti-HLA-antibodies (DSA), as well as for monitoring AMR activity after anti-rejection treatment. The weight of evidence suggests that the integration of dd-cfDNA in the graft surveillance of patients with AMR, or those suspicious of AMR (e.g., due to the presence of donor-specific anti-HLA-antibodies) has an added value and might have a positive impact on outcomes in this specific cohort.

Introduction of the donor centre virtual crossmatch in Eurotransplant.

On 24 January 2023, Eurotransplant has introduced the virtual crossmatch for kidney and pancreas allocation as a better alternative for the physical Complement Dependent Cytotoxicity (CDC) crossmatches at the donor centre, which were associated with a longer cold ischaemia time and false positive reactions. For the time being, the physical CDC crossmatch at the recipient centre will remain in place as the final histocompatibility check. While Eurotransplant is certainly not the first organ allocation organisation to introduce virtual crossmatching, several novel aspects have been introduced, such as calculation of the virtual panel reactive antibody (vPRA) on 11 loci at the second-field level in addition to the serological broad and split level, electronic HLA typing data transmission using Histoimmunogenetics Markup Language (HML) file format, and the actual virtual crossmatch based on ambiguous, second-field HLA typing of the donor on all 11 loci. This short communication will focus on these novel aspects of the virtual crossmatch in Eurotransplant.

Large deletions and small insertions and deletions in the factor VIII gene predict unfavorable immune tolerance induction outcome in people with severe hemophilia A and high-responding inhibitors.

INTRODUCTION: Hemophilia A is an inherited bleeding disorder caused by pathogenic variants in the factor VIII gene (F8), which leads to factor VIII (FVIII) deficiency. Immune tolerance induction (ITI) is a therapeutic approach to eradicate alloantibodies (inhibitors) against exogenous FVIII in people with inherited hemophilia A. Few studies have evaluated the role of F8 variants on ITI outcome. MATERIAL AND METHODS: We included people with severe hemophilia A (FVIII ˂ 1 international units/dL) and high-responding inhibitors (≥ 5 Bethesda units/mL lifelong) who underwent a first course of ITI. Socio-demographic, clinical and laboratory data were collected. ITI outcomes were defined as total, partial successes, and failure. Detection of intron 1 and 22 inversions was performed by polymerase-chain reaction, followed by F8 sequencing. RESULTS: We included 168 people with inherited hemophilia A and high-responding inhibitors, median age 6 years at ITI start. Intron 22 inversion was the most prevalent variant (53.6 %), followed by nonsense (16.1 %), small insertion/deletion (11.3 %), and large deletion (10.7 %). In comparison with intron 22 inversion, the odds of ITI failure were 15.5 times higher (odds ratio [OR] 15.50; 95 % confidence interval [95 % CI] 4.59-71.30) and 4.25 times higher (95 % CI, 1.53-12.3) among carriers of F8 large deletions and small insertions and deletions, respectively. CONCLUSION: F8 large deletions and small insertions/deletions predicted ITI failure after a first course of ITI in patients with severe hemophilia A and high-responding inhibitors. This is the first study to show F8 large deletions and small insertions/deletions as predictors of ITI failure.

Characteristics of Early Antibody Mediated Rejection in Antibody Incompatible Living Donor Kidney Transplantation.

Antibody incompatible transplantation (AIT) may be an only option for highly sensitized patients. Severe form of early antibody mediated rejection (AMR) adversely affects graft survival after AIT. The aim of this study was to identify individuals at risk of AMR. We analyzed 213 living donor AITs performed at our center. Among 120 ABOi, 58 HLAi and 35 DSA + FCXM-negative cases, the rates of early AMR were 6%, 31%, and 9%, respectively (p < 0.001). On multivariate analysis for graft loss, early AMR had a HR of 3.28 (p < 0.001). The HLAi group had worse death-censored graft survival (p = 0.003). In the HLAi group, Patients with aggressive variant AMR (AAMR) had greater percentage of C3d complement fixing DSA, higher baseline class I and total DSA MFI levels and B-cell FCXM RMF. C1q and C3d complement fixing DSA and strong positivity of baseline B- or T-cell FXCM as predictors of AAMR had 100% sensitivity. Early AMR is of significant clinical concern in AIT as it results in poor graft survival and is not well described in literature. An aggressive variant is characterized by massive rise in DSA levels at rejection. Baseline DSA, C1q, and C3d and baseline FCXM values can be used to risk-stratify candidates for AIT.

Donor-derived cell-free DNA predicted allograft rejection and severe microvascular inflammation in kidney transplant recipients.

Introduction: The aim of this study is to investigate the clinical validity of donor-derived cell-free DNA (dd-cfDNA) in comparison with that of donor specific anti-HLA antibody (DSA) for predicting biopsy-proven rejection (BPR)and severe microvascular inflammation (severe MVI) in kidney transplant recipients (KTRs). Methods: In this prospective observational investigation, 64 KTRs who underwent the indicated biopsies were included. Blood samples collected prior to biopsy were tested for dd-cfDNA and DSA. Biopsy specimens were classified by a renal pathologist according to the Banff classification. The predictive performance of dd-cfDNA and DSA for histological allograft diagnosis was assessed. Results: KTRs were categorized into the high and low dd-cfDNA groups based on a level of 0.4%. Eighteen patients (28.1%) had positive DSA at biopsy, exhibiting higher dd-cfDNA levels than the DSA-negative patients. BPR and severe MVI incidences were elevated in the high dd-cfDNA group (BPR: 42.9% vs. 3.4%, P <0.001; severe MVI: 37.1% vs. 3.4%, P = 0.001). Also, elevated glomerulitis and MVI scores were observed in the high dd-cfDNA group. DSA showed the highest predictive value for BPR (AUC = 0.880), whereas dd-cfDNA alone excelled in predicting severe MVI (AUC = 0.855). Combination of DSA and dd-cfDNA (>0.4%) yielded sensitivities of 80.0% and 50.0% with specificities of 90.7% and 88.0% for antibody-mediated rejection and severe MVI detection, respectively. Conclusion: The dd-cfDNA test is a predictive tool for BPR and severe MVI, and it can improve the performance, especially when combined with DSA for BPR.

A Randomized Trial Comparing Imlifidase to Plasmapheresis in Kidney Transplant Recipients With Antibody-Mediated Rejection.

BACKGROUND: Antibody-mediated rejection (ABMR) poses a barrier to long-term graft survival and is one of the most challenging events after kidney transplantation. Removing donor specific antibodies (DSA) through therapeutic plasma exchange (PLEX) is a cornerstone of antibody depletion but has inconsistent effects. Imlifidase is a treatment currently utilized for desensitization with near-complete inactivation of DSA both in the intra- and extravascular space. METHODS: This was a 6-month, randomized, open-label, multicenter, multinational trial conducted at 14 transplant centers. Thirty patients were randomized to either imlifidase or PLEX treatment. The primary endpoint was reduction in DSA level during the 5 days following the start of treatment. RESULTS: Despite considerable heterogeneity in the trial population, DSA reduction as defined by the primary endpoint was 97% for imlifidase compared to 42% for PLEX. Additionally, imlifidase reduced DSA to noncomplement fixing levels, whereas PLEX failed to do so. After antibody rebound in the imlifidase arm (circa days 6-12), both arms had similar reductions in DSA. Five allograft losses occurred during the 6 months following the start of ABMR treatment-four within the imlifidase arm (18 patients treated) and one in the PLEX arm (10 patients treated). In terms of clinical efficacy, the Kaplan-Meier estimated graft survival was 78% for imlifidase and 89% for PLEX, with a slightly higher eGFR in the PLEX arm at the end of the trial. The observed adverse events in the trial were as expected, and there were no apparent differences between the arms. CONCLUSION: Imlifidase was safe and well-tolerated in the ABMR population. Despite meeting the primary endpoint of maximum DSA reduction compared to PLEX, the trial was unsuccessful in demonstrating a clinical benefit of imlifidase in this heterogenous ABMR population. TRIAL REGISTRATION: EudraCT number: 2018-000022-66, 2020-004777-49; ClinicalTrials.gov identifier: NCT03897205, NCT04711850.

Induction immunosuppression strategies and outcomes post-lung transplant: A single center experience.

PURPOSE: Currently 80% of lung transplant centers use induction immunosuppression. However, there is a lack of standardization of induction protocols within and across lung transplant centers. This study explores the association of two different induction immunosuppression strategies used at our center [single dose rabbit antithymocyte globulin (rATG) vs. alemtuzumab] compared to no induction with immunologic and clinical outcomes after lung transplantation. METHODS: A total of 174 consecutive lung transplant recipients (LTR) between 2016 and 2019 were included in the analysis. Twenty nine LTR (16.7%) received no induction, 22 LTR (12.6%) received alemtuzumab, 123 LTR (70.6%) received a single dose of rATG; 1.5 mg/kg within 24 h of transplant for induction. All LTR had a negative flow cytometry crossmatch on the day of the transplant. All LTR were assessed for de novo HLA donor-specific antibodies (DSA) development and clinical outcomes, including the risk of acute cellular rejection (ACR), antibody-mediated rejection (AMR), chronic lung allograft dysfunction (CLAD), and overall survival post-transplant. RESULTS: The median lung allocation score (LAS) was significantly higher in LTR that did not receive Induction immunosuppression (76; range = 35.3-94.3) compared to induction with rATG (41.6; range = 31.6-91) and alemtuzumab (51; range = 33.1-88.2) (p < 0.001). De novo HLA DSA were detected in 50/174 (28.7%) LTR within 12 months post-transplant. They were detected in 13/29 (44.8%) LTR without induction immunosuppression compared to 28/123 (22.8%) and 9/22 (40.9%) LTR with rATG and alemtuzumab induction, respectively (p = 0.02). The percent freedom from ACR rates between LTR who received alemtuzumab induction was significantly higher compared to LTR who received rATG or no induction at 1 (p = 0.02), 2 (p = 0.01) and 3 (p = 0.05) years post-transplant. In addition, the overall 1-year survival rates were significantly higher in LTR who received rATG or alemtuzumab induction compared to LTR without induction immunosuppression (p = 0.02). CONCLUSION: Induction immunosuppression strategies utilizing rATG or Alemtuzumab have unique and contrasting benefits in LTR. Combination of alemtuzumab induction and a lower dose of maintenance immunosuppression may reduce the incidence of ACR in LTR. Single-dose rATG or alemtuzumab induction immunosuppression may also improve the 1 year overall LTR survival compared to no induction.

Application of RhD Blood Group to Simulate Antibody Identification Test in Immunohematology Education.

BACKGROUND: Immunohematology skill education is an important part of the transfusion medicine professional training. We tried to solve the difficulty of obtaining suitable and sufficient positive samples in the immunohematology education. METHODS: Different identification panels and panel cells were created by RhD-positive red blood cells (RBCs) and RhD-negative RBCs, according to the underlying antibodies. Diluted anti-D reagent was used as simulated plasma for identification. RESULTS: The antibody identification of single antibody with dose-effect and two antibodies present at the same time were successfully simulated. CONCLUSIONS: It is a practical and cheap method for antibody identification training to use RhD blood group, especially when positive samples are short.

Intravenous Immunoglobulins Alone for the Desensitization of Lung Transplant Recipients with Preformed Donor Specific Antibodies and Negative Flow Cytometry Crossmatch.

BACKGROUND: The lack of evidence regarding optimal desensitization strategies for lung transplant candidates with preformed donor specific anti-human leukocyte antigen antibodies (DSAs) has led to varying approaches among centers towards this patient group. Our institution's desensitization protocol for recipients with preformed DSAs and negative flow cytometry crossmatch (FCXM) consists of intravenous immunoglobulin (IVIG) as the sole therapy. The study aimed to determine outcomes using this approach. METHODS: This retrospective study included adults who underwent lung-only transplantation for the first time between January 2015 and March 2022 at a single center. We excluded patients with positive or missing FCXM results. Transplant recipients with any DSA ≥ 1000 MFI on latest testing within three months of transplant were considered DSA-positive, while recipients with DSAs <1000 MFI and those without DSAs were assigned to the low-level/negative group. Graft survival (time to death/retransplantation) and chronic lung allograft dysfunction (CLAD)-free times were compared between groups using Cox proportional hazards models. RESULTS: Thirty-six out of 167 eligible patients (22%) were DSA-positive. At least 50% of preformed DSAs had documented clearance (decrease to <1000 MFI) within the first 6 months of transplant. Multivariable Cox regression analyses did not detect a significantly increased risk of graft failure (aHR 1.04 95%CI 0.55-1.97) or chronic lung allograft dysfunction (aHR 0.71 95%CI 0.34-1.52) in DSA-positive patients compared to patients with low-level/negative DSAs. Incidences of antibody-mediated rejection (p = 1.00) and serious thromboembolic events (p = 0.63) did not differ between study groups. CONCLUSION: We describe a single-center experience of administering IVIG alone to lung transplant recipients with preformed DSAs and negative FCXM. Further studies are required to confirm the efficacy of this strategy against other protocols.

Allosensitisation in NHP results in cross-reactive anti-SLA antibodies not detected by a lymphocyte-based flow cytometry crossmatch.

Xenotransplantation is a potential option for individuals for whom an acceptable human allograft is unavailable. Individuals with broadly reactive HLA antibodies due to prior exposure to foreign HLA are potential candidates for a clinical xenotransplant trial. It remains controversial if allosensitisation results in the development of cross-reactive antibodies against SLA. This may require increased histocompatibility scrutiny for highly sensitised individuals prior to enrollment in a clinical trial. Serum samples were obtained from non-human primates sensitised via serial skin transplantation from maximally MHC-mismatched donor, as reported. Sera from pre- and post-allosensitisation timepoints were assessed in a flow crossmatch (FXM) for IgM and IgG binding to pig splenocytes with or without red blood cell adsorption. Xenoreactive antibodies were eluted from pig splenocytes and screened on a single antigen HLA bead assay. A MHC Matchmaker algorithm was developed to predict potential conserved amino acid motifs among the pig, NHP, and human. Our sensitised NHP model was used to demonstrate that allosensitisation does not result in an appreciable difference in xenoreactive antibody binding in a cell-based FXM. However, antibody elution and screening on single antigen HLA beads suggest the existence of potential cross-reactive antibodies against SLA. The cross-reactive IgG after allosensitisation were predicted by comparing the recipient Mamu alleles against its previous allograft donor Mamu alleles and the donor pig SLA alleles. Our study suggests that allosensitisation could elevate cross-reactive antibodies, but a more sensitive assay than a cell-based FXM is required to detect them. The MHC Matchmaker algorithm was developed as a potential tool to help determine amino acid motif conservation and reactivity pattern.

Functionalized magnetic nanoparticles remove donor-specific antibodies (DSA) from patient blood in a first ex vivo proof of principle study.

The presence of donor-specific antibodies (DSA) such as antibodies directed against donor class I human leucocyte antigen (e.g., HLA-A) is a major barrier to kidney transplant success. As a proof of concept, functionalized magnetic nanoparticles have been designed to eliminate DSA from saline, blood and plasma of healthy donors and sensitized patients. Specific HLA-A1 protein was covalently bound to functionalized cobalt nanoparticles (fNP), human serum albumin (HSA) as control. fNP were added to anti-HLA class I-spiked saline, spiked volunteers' whole blood, and to whole blood and plasma of sensitized patients ex vivo. Anti-HLA-A1 antibody levels were determined with Luminex technology. Antibodies' median fluorescent intensity (MFI) was defined as the primary outcome. Furthermore, the impact of fNP treatment on blood coagulation and cellular uptake was determined. Treatment with fNP reduced MFI by 97 ± 2% and by 94 ± 4% (p < 0.001 and p = 0.001) in spiked saline and whole blood, respectively. In six known sensitized anti-HLA-A1 positive patients, a reduction of 65 ± 26% (p = 0.002) in plasma and 65 ± 33% (p = 0.012) in whole blood was achieved. No impact on coagulation was observed. A minimal number of nanoparticles was detected in peripheral mononuclear blood cells. The study demonstrates-in a first step-the feasibility of anti-HLA antibody removal using fNP. These pilot data might pave the way for a new personalized DSA removal technology in the future.

Successful A2 to O Simultaneous Liver and Kidney Transplantation in the Setting of Pre-operative Positive HLA Crossmatch: A Case Report.

Simultaneous liver and kidney transplantation (SLKT) is possible for patients with high donor-specific HLA antibodies or with A2 donors to O recipients with high A2 titers. We report the first case of SLKT in a highly sensitized O recipient with organs from an A2 donor. The recipient is a 59-year-old woman with chronic kidney disease and liver failure due to autoimmune hepatitis and drug-induced liver injury. Immune work-up 8 days pre-transplant demonstrated a negative crossmatch and no HLA antibody (calculated panel reactive antibodies = 0%). Anti-A2 IgG levels were 512. The donor was a deceased 24-year-old man. One day before transplantation, serum from the recipient showed a significant increase in antibody reactivity (calculated panel reactive antibodies = 100%) attributable to blood product transfusion and memory response from previous pregnancies. Consequently, a crossmatch was positive for T and B cells with two newly detected HLA antibodies against the donor's antigens. On the day of surgery, the liver was transplanted first. Six hours and 37 minutes later, a repeat flow crossmatch was negative; donor-specific antibodies (DSAs) fell below the positive threshold, and anti-A2 IgG titer fell to 256. Thus, the kidney was transplanted after basiliximab induction therapy. Seven days post-transplant, non-donor-specific HLA antibodies were present but DSAs remain negative. The patient was discharged on postoperative day 57 with no signs of rejection at 4 months. This case illustrates a rapid and prolonged reduction in antibody titers (HLA and ABO) after SLKT. SLKT is feasible in patients with both DSA and high anti-A2 titer.

Pre-transplant crossmatch-negative donor-specific anti-HLA antibody predicts acute antibody-mediated rejection but not long-term outcomes in kidney transplantation: an analysis of the Korean Organ Transplantation Registry.

Background: Pre-transplant donor-specific anti-human leukocyte antigen antibody (HLA-DSA) is a recognized risk factor for acute antibody-mediated rejection (ABMR) and allograft failure. However, the clinical relevance of pre-transplant crossmatch (XM)-negative HLA-DSA remains unclear. Methods: We investigated the effect of XM-negative HLA-DSA on post-transplant clinical outcomes using data from the Korean Organ Transplantation Registry (KOTRY). This study included 2019 living donor kidney transplant recipients from 40 transplant centers in South Korea: 237 with HLA-DSA and 1782 without HLA-DSA. Results: ABMR developed more frequently in patients with HLA-DSA than in those without (5.5% vs. 1.5%, p<0.0001). Multivariable analysis identified HLA-DSA as a significant risk factor for ABMR (odds ratio = 3.912, 95% confidence interval = 1.831-8.360; p<0.0001). Furthermore, the presence of multiple HLA-DSAs, carrying both class I and II HLA-DSAs, or having strong HLA-DSA were associated with an increased incidence of ABMR. However, HLA-DSA did not affect long-term clinical outcomes, such as allograft function and allograft survival, patient survival, and infection-free survival. Conclusion: Pre-transplant XM-negative HLA-DSA increased the risk of ABMR but did not affect long-term allograft outcomes. HLA-incompatible kidney transplantation in the context of XM-negative HLA-DSA appears to be feasible with careful monitoring and ensuring appropriate management of any occurrence of ABMR. Furthermore, considering the characteristics of pre-transplant XM-negative HLA-DSA, the development of a more detailed and standardized desensitization protocol is warranted.