Single Antigen Bead Assay Research Articles

Microvascular Inflammation: An Incremental Path to Refining the Diagnosis of Antibody-mediated Rejection in Heart Transplantation

Microvascular Inflammation: An Incremental Path to Refining the Diagnosis of Antibody-mediated Rejection in Heart Transplantation

Approaching the sensitized lung patient: risk assessment for donor acceptance.

The presence of HLA antibodies is widely recognized as a barrier to solid organ transplantation, and for lung transplant candidates, it has a significant negative impact on both waiting time and waiting list mortality. Although HLA antibodies have been associated with a broad spectrum of allograft damage, precise characterization of these antibodies in allosensitized candidates may enhance their accessibility to transplant. The introduction of Luminex-based single antigen bead (SAB) assays has significantly improved antibody detection sensitivity and specificity, but SAB alone is not sufficient for risk-stratification. Functional characterization of donor-specific antibodies (DSA) is paramount to increase donor accessibility for allosensitized lung candidates. We describe here our approach to evaluate sensitized lung transplant candidates. By employing state-of-the-art technologies to assess histocompatibility and determine physiological properties of circulating HLA antibodies, we can provide our Clinical Team a better risk assessment for lung transplant candidates and facilitate a “road map” to transplant. The cases presented in this paper illustrate the “individualized steps” taken to determine calculated panel reactive antibodies (cPRA), titer and complement-fixing properties of each HLA antibody present in circulation. When a donor is considered, we can better predict the risk associated with potentially crossing HLA antibodies, thereby allowing the Clinical Team to approach allosensitized lung patients with an individualized medicine approach. To facilitate safe access of sensitized lung transplant candidates to potential donors, a synergy between the histocompatibility laboratory and the Clinical Team is essential. Ultimately, donor acceptance is a decision based on several parameters, leading to a risk-stratification unique for each patient.

Prospective multi-centric study to analyze pre-transplant compatibility algorithm for live-related donor kidney transplant in Indian setting: the “Delhi approach”!

BackgroundSince no single test is always accurate and sensitive, two or more tests are used to increase the precision of evaluation. Different algorithms have been proposed by centers in Leiden, Basel, Vienna and Minnesota, etc. With an intention to develop an optimal algorithm for India, we evaluated pre-transplant compatibility tests for live-donor kidney transplants. Three tests complement dependent cyto-toxicity cross-match (CDCXM), flow-cytometry cross-match (FCXM) and anti-HLA antibody screening (HAS) were performed and confirmed by the anti-HLA antibody identification (HAI) assay in a multi-centric trial (three transplant centers) in India. Materials and methodsAll prospective recipients (and their potential donors) underwent low-resolution HLA typing as well as CDCXM, FCXM and HAS assays. In addition, HAI {single antigen bead assay; (SAB)} was done for all recipients to identify possible anti-HLA antibodies. In a virtual cross-match (VXM), antibody specificity was mapped to donor HLA type to determine donor-specific antibodies (DSA). Only patients without DSA were cleared for the transplant. Alternatively, patients with DSA were offered an exchange in the kidney paired donation (KPD) program. The screening results (CDCXM, FCXM, and HAS) were analyzed, individually as well as in combination of screening assays (CDCXM+HAS, CDCXM+FCXM, and FCXM+HAS) and the results were compared with those from the HAI test. ResultsOut of 100 patients, 69 were males and 31 were females; 85 recipients (85%) underwent a kidney transplant. The sensitivity of CDCXM was only 12.1% and the specificity of CDCXM was 100%; whereas the sensitivity of FCXM was 84.8% and the specificity of FCXM was 89.6%. The sensitivity and specificity of class I HAS was 88.2% and 84.3%, respectively. The sensitivity and specificity class II HAS was 88.0% and 80.0%, respectively. However, when both class I/II HAS were tested together the sensitivity increased to 97.0% and the specificity to 82.1%. Similarly, the sensitivity of combined FCXM+HAS had the sensitivity of 100% and the specificity of 76.1%; CDCXM+FCXM had the sensitivity of 84.8% and the specificity of 89.6% and CDCXM+HAS assays reached 97% with the specificity of 82.1%. ConclusionsOur results showed that the algorithm of FCXM with HAS produced the best sensitivity of 100%. The specificity of 76.1% indicate that the combined FCXM+HAS assays may detect up to 24.9% false positive results. We suggest that these false-positives may be easily resolved by performing the virtual crossmatch based on HAI (SAB) results. In our reflex testing algorithmic approach only 49% patients needed HAI (SAB). Finally, our results suggested that the CDCXM assay may be discontinued in pre-transplant workup owing to its very low sensitivity (12.1%).

Antibodies to cryptic epitopes on HLA class I and class II heavy chains bound to single antigen beads: Clinically relevant?

Cell surface HLA class I consists of trimers, i.e., alpha - heavy chain, beta – 2 - microglobulin, and a peptide, termed closed conformers (CC) on non-activated lymphocytes. HLA class I and class II may also exist, respectively, as alpha-chain only or alpha and beta - chain only on activated cells termed open conformers (OC). We extend previous studies using an OC-specific monoclonal antibody that demonstrate LABScreen HLA class I and II single antigen beads (SABs) contain a mixture of open and closed conformers. LIFECODES SABs have bound CC only. More HLA class I and class II LABScreen SABs were reactive than LIFECODES SABs due to the presence of OC on LABScreen SABs. We hypothesized that antibody against OC on HLA B antigens would not be detected in cell based cross matches (XMs) with typical lymphocyte targets since anti-HLA OC antibodies would not react with native HLA CC on the cell surface. To test this hypothesis, we performed flow cytometry XM (FCXM) assays with sera of sufficient strength that most laboratories would likely predict positive FCXMs. Sera that reacted strongly with LABScreen SABs (>13,000 MFI) but weakly or not at all with LIFECODES SABs (<1000 MFI) gave negative T and B cell FCXMs. In contrast, sera that reacted with LIFECODES SABs (>13,000 MFI) but weakly with LABScreen SABs (<2100 MFI) exhibited positive FCXMs. Detection of antibodies directed against OC in SAB assays, may lead to inappropriate listing of unacceptable antigens, a decision not to XM or pre-or post – transplant desensitization procedures.

TEMPORAL RELATIONSHIP BETWEEN DIFFUSE ALVEOLAR HEMORRHAGE AND DETECTION OF DQ-DSAS IN LUNG TRANSPLANT RECIPIENTS

TOPIC: Transplantation TYPE: Original Investigations PURPOSE: After lung transplant (LTx), donor specific antibodies (DSAs) to anti-human leukocyte antigen (HLA) play an important role in antibody mediated rejection (AMR) and subsequent allograft dysfunction. DSAs amplify the immune response via complement-dependent and independent pathways leading to poor outcomes. Diffuse alveolar hemorrhage (DAH) is a life-threatening condition with dismal outcomes that is known to complicate hematopoietic stem cell transplant (HCT). Although its etiology remains unknown, lung tissue inflammation causing disruption of the alveolar-capillary membrane and cytokine release is implicated in DAH pathogenesis. DAH has not been previously linked to DSA formation in LTx; a temporal relation may exist between the two processes resulting in negative outcomes. METHODS: retrospective chart review of 100 LTx done over 3 years, revealed 3 patients with DAH post-LTx. Cases included bilateral and single LTx. All patients had induction and post-transplant immunosuppression as per standardized institution protocols. Anti-HLA antibodies were tested using Single Antigen Bead and C1q assays (One Lambda, Canoga Park, CA) and all cases had 0% cPRA class I & II and negative HLA pre-transplant. Post-transplant courses were complex, including primary graft dysfunction, pneumonia leading to respiratory failure. DAH was diagnosed clinically and via bronchoscopy with biopsy within 2 to 4 weeks of LTx. Treatment included pulse steroids and intravascular immunoglobulins infusions (IVIg). RESULTS: DSAs became positive over a period of 1 to 6 months. All patients developed class II DQ4 and DQ7 DSAs with two patients having positive C1q. They were treated with IVIg and rituximab or bortezomib. Plasmapheresis was not performed. One patient successfully cleared DSAs. Two patients died and 1 progressed to RAS-CLAD this was irrespective of DSA clearance. CONCLUSIONS: It is essential to understand the risk factors for de novo DSA formation to prevent AMR post LTx. In this case series DAH likely triggered the development of DQ-DSAs secondary to tissue damage and release of donor antigens. Once formed, DSAs caused significant morbidity and mortality. This sample is small, and all patients had other potential causes for DSA development, a temporal relationship between DAH & DSA formation likely exists. Additional investigations are needed to understand this process and develop treatment protocols. In a recent publication, DAH in HCT patients receiving modest amounts of steroids had better outcomes, this may be beneficial in LTx recipients and could be studied further. Physicians must remain vigilant in managing these patients CLINICAL IMPLICATIONS: DAH triggering DSA formation is an area that needs further study amongst all solid organ transplant recipients specifically in LTx. LTx is known to have inferior survival outcomes, and identifying contributing causes helps develop better treatment protocols. DISCLOSURES: No relevant relationships by Sana Ahmed, source=Admin input No relevant relationships by Sammar Alsunaid, source=Web Response No relevant relationships by Victoria Caputo, source=Web Response No relevant relationships by Adriana Colovai, source=Web Response No relevant relationships by Ali Mansour, source=Web Response No relevant relationships by Eros Qama, source=Web Response No relevant relationships by Kristine Stryker, source=Web Response

Desensitization Therapy in Kidney Transplantation Cases with Positive Baseline Complement-Dependent Cytotoxicity Crossmatch and High Donor-Specific Antibodies

Objective: The objective of the study is to assess the graft and patient outcome after desensitization in human leukocyte antigen incompatible kidney transplantation (KT) with positive baseline complement-dependent cytotoxic (CDC) crossmatch and high mean fluorescein intensity (MFI) of donor-specific antibodies (DSA). Methods: This was a retrospective study conducted at Jaypee Hospital, Noida. This study included highly sensitized patients who were transplanted with positive CDC and DSA &gt;10,000 MFI for single antigen or &gt;5000 MFI for multiple donor antigens. The patient's renal outcomes were documented. The desensitization protocol consisted of rituximab, therapeutic plasma exchanges (TPE), and thymoglobulin. Results: A total of five patients who had positive CDC crossmatch with very high level of preformed DSA underwent KT. Three patients had end-stage renal disease due to diabetic kidney disease while other two due to autosomal dominant polycystic kidney disease and chronic glomerulonephritis. All the patients were on dialysis. The MFI by Luminex single antigen bead assay for Class I varied from 1657 to 23440 and for Class II varied from undetectable to 11120. The mean number of pretransplant TPE sessions given per patient was 7.8 ± 2.68 and posttransplant TPE sessions per patient was 0.8 ± 0.45. The mean follow-up period was 308.2 days. Mean creatinine on the day of discharge was 0.58 ± 0.17 mg/dL. None of the patients had any postoperative infections or rejections. Conclusion: The current report showed favorable short-term patient and graft outcomes post-KT without any postoperative infections or rejections with desensitization therapy comprising of rituximab, TPE, and thymoglobulin induction.

Prospective De Novo Donor-Specific Antibody Monitoring in Renal Transplant Patients

De novo donor-specific antibodies (dnDSA) are associated with antibody-mediated rejection leading to kidney transplant failure. However, many transplant patients are stable with no signs of graft dysfunction at the time of dnDSA detection at screening test. We prospectively studied 26 kidney transplant patients for 3 years, with dnDSA detected using the Luminex single-antigen bead assay in a routine test. Proteinuria and estimated glomerular filtration rate were evaluated along with dnDSA monitoring at least annually. Graft loss associated with dnDSA occurred in 8 (31%) patients, 14 ± 11 months after the initial detection of dnDSA. These patients had more frequently multiple dnDSA (P=.004) and remarkable proteinuria, more than 1 g daily (P < .001). The remaining 18 patients were followed for 38 ± 15 months and considered stable as there was no deterioration regarding estimated glomerular filtration rate and proteinuria. In 7 (39%) of these patients, dnDSA waned and were not detected anymore at follow-up. Antibodies against HLA class I had a significantly (P < .001) lower mean fluorescence intensity (MFI) (2603 ± 1098) compared with those against HLA class II (11,109 ± 6414). In regression analysis, they were predictive of dnDSA wane (P=.043; odds ratio, 0.18; 95% confidence interval, 0.04-0.94). Despite fluctuations during follow-up, there was no significant change in MFI for those who retained the dnDSA. The presence of dnDSA is transient in a significant proportion of stable renal transplant patients, especially in those with antibodies against HLA class I and a low MFI. Multiple dnDSA and severe proteinuria adversely affect renal graft survival.

Approaches for the characterization of clinically relevant pre‐transplant human leucocyte antigen (HLA) antibodies in solid organ transplant patients

The avoidance of antibody-mediated rejection (AMR) attributed to human leucocyte antigen (HLA) antibody incompatibility remains an essential function of clinical Histocompatibility and Immunogenetics (H&I) laboratories who are supporting solid organ transplantation. Developments in HLA antibody identification assays over the past thirty years have greatly reduced unexpected positive cellular crossmatches and improved solid organ transplant outcomes. For sensitized patients, the decision to register unacceptable HLA antigen mismatches is often heavily influenced by results from solid phase antibody assays, particularly the Luminex® Single Antigen Bead (SAB) assays, although the clinical relevance of antibodies identified solely by these assays remains unclear. As such, the identification of non-clinically relevant antibodies may proportionally increase the number of unacceptable transplant mismatches registered, with an associated increase in waiting time for a compatible organ. We reflect on the clinical relevance of antibodies identified solely by the Luminex SAB® assays and consider whether the application of additional assays and/or tools could further develop our ability to define the clinical relevance of antibodies identified in patient sera. Improvements in this area would assist equity of access to a compatible transplant for highly sensitized patients awaiting a solid organ transplant.

Transfusions in Aplastic Anemia Patients Cause HLA Alloimmunization: Comparisons of Current and Past Cohorts Demonstrate Progress

Transfusions are the mainstay of supportive therapy in patients with aplastic anemia (AA) and may lead to anti- HLA alloimmunization, thereby also increasing the risk for donor-specific antibodies in the setting of HLA-mismatched transplantation. Historically, AA patients were thought to be at particularly high risk for HLA alloimmunization. In past decades, blood product manufacturing (leukoreduction) and HLA antibody testing have improved significantly by single antigen bead (SAB) technology. It is currently unknown how those developments have impacted HLA alloimmunization and treatment outcome in patients with AA. We retrospectively investigated 54 AA patients treated by immunosuppressive therapy or allogeneic hematopoietic cell transplantation after the introduction of the SAB assay at our center. We compared the HLA antibody results to a historical AA cohort (n = 26), treated before introduction of leukoreduced blood products from 1975 to 1995. HLA alloimmunization was detected in 43 of 54 (80%) recently treated patients. Past pregnancy, female gender, disease severity, age, and a history of other transfusions were significantly associated with a larger number or higher intensity (mean fluorescence intensity) of HLA antibodies. Treatment outcome including bleeding episodes, response to treatment, engraftment, graft-versus-host disease, and overall survival was not associated with HLA alloimmunization. In the historical cohort a significantly higher number of HLA antibodies (P < .01) with a higher mean fluorescent intensity (P < .01) was observed. HLA alloimmunization remains frequent in AA tested by current techniques, but it has significantly decreased since prior decades and does not affect treatment outcome.© 2021 American Society for Blood and Marrow Transplantation. Published by Elsevier Inc. All rights reserved.

IgM single antigen bead HLA-assay is affected by imlifidase through the cleavage of IgG but not IgM

AimThe aim of this study was to investigate if human IgM is a cleavable substrate for imlifidase and to explain an observed effect in anti-HLA IgM single antigen bead (SAB) assays in sensitized patients. MethodsSerum samples collected pre- and 24 h post-imlifidase administration from sensitized patients enrolled in a phase II trial were investigated for anti-HLA IgG and IgM using SAB assays, with and without in vitro IgG depletion using a CaptureSelect™ affinity matrix. In addition, pre-dose samples and purified human IgM samples were treated with imlifidase in vitro and evaluated by SDS-PAGE, Western blot (PE-conjugated anti-human IgM) and SAB (IgG, IgM) assays. ResultsBy comparing the mean fluorescence intensity (MFI) of HLA-beads, pre- and post-imlifidase administration, three IgM-related patterns were observed; IgM-specific HLA-SABs with an increased MFI post-imlifidase, IgM-specific HLA-SABs with a decreased MFI post-imlifidase, and IgM-specific HLA-SABs with a marginal MFI difference between the pre- and post-imlifidase administration. These IgM signal patterns were observed despite neither purified IgM nor serum IgM could be cleaved by imlifidase. After removing IgG, the effects observed on anti-HLA IgM was largely eliminated with the biggest differences seen in patients with very high anti-HLA IgG in pre-dose samples. ConclusionWe demonstrate that imlifidase does not cleave human IgM, including HLA-specific IgM antibodies from highly sensitized subjects. Observed decreases of SAB-HLA IgM signals after imlifidase treatment may result from the cleavage of IgG-IgM complexes which are bound to SAB-HLA. Serum analysis of patients with high levels of anti-HLA IgG will result in a more accurate SAB-HLA IgM reading after IgG depletion.

Luminex Multiplex Bead Assay Monitoring HLA IgG Antibodies in Sensitized Pre- and Post-transplant Patients: Clonality of the Detection Antibody Impacts Specificity and Sensitivity

The number and the binding affinity, measured as the mean fluorescent intensity (MFI) of HLA-specific IgG antibodies, formed in the sera of end-stage organ disease patients and allograft recipients, referred to as sensitization, may restrict the availability of a donor organ and/or lead to graft failure after transplantation. The MFI of HLA Abs in sera is monitored with the Luminex-based single-antigen bead (SAB) immunoassay. The following two factors may impact the reliable measurement of MFI: one, the HLA structural variants on the SAB, namely, trimeric HLA (closed conformers, CC) and monomeric heavy chains (open conformers, OC); and two, the nature of the detection Abs, namely, IgG heavy-chain binding polyclonal-Fab (IgHPolyFab) or Fc-binding monoclonal-IgG (FcMonoIgG). Anti-CC Abs correlate with positive flow cross-matches, and are considered to be pathogenic and damaging to the graft, whereas anti-OC Abs appear to have little relevance to graft attrition. The presence of both CC and OC on beads may impair the reliability of monitoring the nature and MFI of pathogenic Abs. Our objective is to compare the MFI of the HLA Abs in the sera of 20 sensitized patients in two different SAB assays, with the two detection Abs. Our data reveal that the admixture of OC with CC on beads will affect the reliability of the measurement of the pathogenic Abs, and that FcMonoIgG is the more sensitive and specific detection Ab for the accurate assessment of HLA sensitization.

Causes of Positive Pretransplant Crossmatches in the Absence of Donor-Specific Anti-Human Leukocyte Antigen Antibodies: A Single-Center Experience.

Pretransplant crossmatch (XM) testing is widely used for detecting preformed donor-specific antibodies (DSAs) against human leukocyte antigen (HLA). However, in some cases, there is a positive XM result in the absence of HLA-DSAs, the cause of which was rarely identified. We reviewed the causes of sequential positive XM results at a single center and analyzed the presence of non-HLA antibodies in patients with an unexplained positive pretransplant XM result. Among 251 patients with T-cell/B-cell complement-dependent cytotoxicity (CDC) or flow cytometric crossmatch (FCXM) positivity, HLA-DSAs were confirmed in 88 (35.1%) by a single antigen bead (SAB) assay, 150 (59.8%) used rituximab (anti-CD20), and 13 (5.2%) had neither HLA-DSAs nor a desensitization history. Anti-angiotensin II type 1 receptor IgG and 33 non-HLA antibodies were tested in the 13 patients with an unexplained positive pretransplant XM result, and more than one non-HLA antibody were revealed in all these patients; 11 patients had non-HLA antibodies reported to be associated with graft rejection, and two patients experienced rejection episode after kidney transplantation. Our study suggests considering non-HLA antibodies testing when a CDC or FCXM test is positive without a definite cause. Assessing non-HLA antibodies might be useful for interpreting XM results and evaluating immunologic risk in transplant recipients.

Genomic and Biochemical Proteasomal Adaptations in Carfilzomib-Resistant Plasma Cells That Limit HLA-Desensitization in Transplantation Candidates

Bone marrow plasma cells (BMPCs) are the source of long-term humoral immunity including HLA antibodies, which promote organ rejection. End-stage renal disease (ESRD) affects more than 660,000 patients per year in the U.S. and is associated with significant morbidity and mortality. Renal transplantation remains the optimal treatment for a substantial proportion of ESRD patients and dramatically improves patient overall survival and quality-of-life. However, preformed human leukocyte antigen (HLA)-specific antibodies are associated with reduced transplantation rates, graft loss and poorer outcomes. Proteasome inhibitor (PI) therapies that target BMPCs to reduce HLA-antibody levels represent an effective means of enhancing transplantation outcomes but are limited by resistance mechanisms. Single antigen bead assays determined that the spectrum of antibody production by HLA-sensitized patient (BMPCs) was remarkably similar to that detected in matching serum samples, supporting the role of BMPCs as the primary source of circulating HLA antibodies. Desensitization therapy with the irreversible proteasome inhibitor carfilzomib reduced PCs in patient BM by ~70% and was associated with a profound reduction in HLA antibodies. We then used bulk RNA-seq transcriptomics to explore potential drug resistance mechanisms within carfilzomib-resistant BMPCs. Network-based pathway analysis revealed that genes encoding the immunoproteasome, a specialized proteasome variant with unique catalytic subunits, and the proteasome activator PA28 were upregulated in BMPCs that survived carfilzomib therapy. Native gel electrophoresis and in vitro functional studies confirmed that BMPCs isolated after carfilzomib therapy contained greater levels of immunoproteasomes as well as increased immunoproteasome-specific peptide-hydrolyzing activity relative to BMPCs from healthy donors or untreated HLA-sensitized patients. Supervised hierarchical clustering analysis (Fig. 1A) and western blotting (Fig. 1B) indicated that PSMB8, which encodes an immunoproteasome catalytic subunit, and PSME1 and PSME2 which encode the proteasome PA28 activator, were significantly upregulated in BMPCs isolated from patients following carfilzomib therapy. Assays then demonstrated that BMPCs that survived carfilzomib therapy were resistant to the FDA-approved PIs bortezomib, carfilzomib and ixazomib but were sensitive to an immunoproteasome-specific inhibitor KZR-616. Thus, we have uncovered proteasomal adaptations in BMPCs as a resistance mechanism underlying carfilzomib therapy and suggest novel strategies that could improve desensitization regimens and reduce antibody-mediated rejection in organ transplantation. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

MO926RAPID VS. LATE RE-TRANSPLANTATION FOR EARLY KIDNEY GRAFT LOSS

Abstract Background and Aims Early graft failure (EGL) is a devastating complication of kidney transplantation. Patients with EGL have an increased risk of mortality of up to twelve times compared to patients who received grafts that survive beyond 30 days. Moreover, they may have become sensitized to antigens from the failed graft and that human leukocyte antigen antibodies (anti-HLA), identified on single antigen bead assays, may not be reliable until several weeks after transplantation. Thus, if rapid re-transplantation occurs, there is no certainty regarding the recipient's immunological status. Hence, there could be an increased immunological risk with the consequent disturbance of the new graft's survival. Method We performed a retrospective single-center observational study in re-transplanted patients with EGL (defined as graft loss before 30 days from transplant) between January 1977 and November 2019 from our center to analyze the outcomes of rapid re-transplantation (occurred within 30 days of EGL) vs late re-transplantation (occurred beyond those 30 days). Results: T here were 82 re-transplants after EGL. The median overall patient survival after re-transplantation was 32 years. Eight patients died within the first year. Among the mortality causes, there were four septic shocks, one cardiogenic shock, one massive pulmonary thromboembolism, one myocardial infarction, and one unknown cause. When analyzed for periods, death censored graft survival was 89% at one and five years after re-transplantation. One graft was lost at eight days due to antibody-mediated rejection (AMR), while there was one death with a functioning graft three months after re-transplantation secondary to a pulmonary embolism. Seventy-three late re-transplants occurred. When analyzed for periods, death censored graft survival was 81% and 69% at one and five years after re-transplantation, respectively. The median patient survival after late re-transplantation was 32 years. There were fewer deaths after rapid re-transplantation than late re-transplantation, but given the small number of cases in the former, this difference did not reach statistical significance (p = 0.3). There was no association between the timing of re-transplantation and an increased risk of graft failure (HR 0.30 [0.04 – 2.2]). While four rapid re-transplants did not share any incompatibilities between donors, four did share at least one HLA type I incompatibility, and one shared an incompatibility of HLA class I and class II. There were no T-cell mediated rejections (TCMR), and there was only one AMR in the rapid rapid re-transplantation group, whereas there were six TCMRs and fifteen AMRs in the late re-transplantation group (p = 0.03 and p = 0.4, respectively). Conclusion Rapid re-transplantation appears to be safe and does not entail increased rejection risk, nor it diminishes long-term graft survival when compared to late re-transplantation.

Impact of C1q fixing donor-specific antibodies on renal transplant outcome.

Not all anti-HLA donor-specific antibodies (HLA-DSAs) are detrimental to renal allograft. In this context, the C1q complement activating ability of antibodies appears to be an important parameter to distinguish clinically inert versus detrimental DSAs. We evaluated sera of 206 consecutive primary live donor renal transplant recipients before transplant and at post-operative day 7, 30, 90, 180 and at the time of graft dysfunction for quantifying HLA-DSAs using single antigen bead assay on a Luminex platform. Patients positive for these antibodies with an MFI >500 were further screened for C1q fixing nature of DSA. Fourteen of the 18 antibody-positive patients had C1q fixing DSA with MFI value >5000. Only 4 antibody-positive patients did not have C1q fixing DSA. The MFI values of DSA detected by C1q assay were generally higher at least by 25% than those detected by the conventional IgG-SAB assay. Twelve of the 14 patients (85.71%) with C1q+ DSA developed antibody-mediated rejection during the mean follow-up period of 21.43±8.03months as compared to none of the four C1q-negative DSA (85.71% vs 0%; P=.001). These results suggest deleterious effect of C1q+ DSA vis-à-vis C1q-negative DSA on renal allograft.

Single antigen testing to reduce early antibody-mediated rejection risk in female recipients of a spousal donor kidney

Female recipients of a spousal donor kidney transplant are at greater risk of donor-specific pre-immunization, which may increase the risk of acute antibody-mediated rejection (ABMR). We assessed the incidence of early ABMR (within two weeks after transplantation), risk factors for ABMR and graft function in 352 complement-dependent cytotoxicity test-negative LURD transplant recipients, transplanted between 1997 and 2014 at the Leiden University Medical Center in The Netherlands. Risk factors for immunization were retrieved from the health records. As methods to screen for preformed donor-specific antibodies (pDSA) have developed through time, we retrospectively screened those with ABMR for pDSA using pooled-antigen bead (PAB) and single-antigen bead (SAB) assays. The cumulative incidence of rejection in the first six months after transplantation was 18% (TCMR 15%; early ABMR 3%). Early ABMR resulted in inferior graft survival and was more common in women who received a kidney from their spouse (10%) than in other women (2%) and men (<1%). The SAB assay retrospectively identified pDSA in seven of nine cases of early ABMR (78%), while the PAB detected pDSA in only three cases (33%). Seeing that early ABMR occurred in 10% of women who received a kidney from their spouse, a SAB assay should be included in the pre-transplant assessment of this group of women, regardless of the result of the PAB assay.

Prospective Follow-up of Panel Reactive Antibodies From Diagnosis Until Completion of Therapy Including Allogeneic Stem Cell Transplantation Among Patients With Acute Leukemia.

Existence of panel reactive antibodies is the limiting step in both solid-organ and hematopoietic stem cell transplantation. There are hypotheses related to panel reactive antibody formation, but there is no knowledge about its formation in acute leukemia at diagnosis and during the chemotherapy period, in which there is a strong myelosuppression and immunosuppression. We aimed to determine the panel reactive antibodies positivity in acute leukemia patients at diagnosis and during the entire therapy period, including stem cell transplantation. In this single-center prospective study, we enrolled 35 patients with acute leukemia (8 with acute lymphoblastic leukemia, 27 with acute myeloid leukemia). Serum samples were obtained before induction therapy and every 3 months thereafter until the last follow-up or death, for a median of 369 days (minimum-maximum, 9-725 days). Panel reactive antibodies were defined with single-antigen bead assays on a Luminex platform. A total of 10 patients (29%) were found to have panel reactive antibodies at any time point. At diagnosis, 5 patients (14.3%) had antibodies of either class I (n = 2) or II (n = 1) or both (n = 2), and in 4 patients these persisted during median follow-up of 168 days (minimum-maximum, 9-322 days). Among the remaining 30 patients, an additional 5 (17%) developed de novo antibodies. Incidence rate of development of de novo antibodies was 5.5 per 10 000 person-days. There was no effect of transfusion load on the development of panel reactive antibodies. Differences in percentages in males versus females, blood type mismatch, and graftversus-host disease were higher in patients who had de novo antibodies after transplantation. Positivity at any time had no statistically significant effect on overall survival (P = .71). Panel reactive antibodies do not occur frequently in the acute leukemia setting despite intensive transfusions.

Intensity of de novo DSA detected by Immucor Lifecodes assay and C3d fixing antibodies are not predictive of subclinical ABMR after Kidney Transplantation.

De novo donor-specific antibodies (dnDSA) are associated with antibody-mediated rejection (ABMR) and allograft loss. We tested Immucor* (IM) Luminex Single-antigen beads (LSAB) assay and C3d-fixing antibodies in the setting of dnDSA and subclinical (s) ABMR. This retrospective multicentric study included 123 patients biopsied because of the presence of subclinical de novo DSA detected by One Lamda* Labscreen (MFI > 1000). In 112 patients, sera of the day of the biopsy were available and tested in a central lab with IM Lifecodes LSAB and C3d fixing antibodies assays. In 16 patients (14.3%), no DSA was detected using Immucor test. In 96 patients, at least one DSA was determined with IM. Systematic biopsies showed active sABMR in 30 patients (31.2%), chronic active sABMR in 17 patients (17.7%) and no lesions of sABMR in 49 KT recipients (51%). Intensitity criteria (BCM, BCR and AD-BCR) of DSA were not statistically different between these 3 histological groups. The proportion of patients with C3d-fixing DSA was not statistically different between the 3 groups and did not offer any prognostic value regarding graft survival. Performing biopsy for dnDSA could not be guided by the intensity criteria of IM LSAB assay. C3d-fixing DSA do not offer added value.

False-positive reactivity of anti-human leukocyte antigen antibodies detected using the single-antigen bead assay

The single-antigen bead assay (SABA) demonstrates high sensitivity and specificity for detecting anti-human leukocyte antigen (HLA) antibodies. However, SABA may produce false-positive results for anti-HLA antibodies. Herein, we analyzed the data of patients with complement-dependent cytotoxic crossmatch−/flow cytometric crossmatch−/SABA+/− results to determine false-positive results for anti-HLA antibodies. We also determined the prevalence of false-positive results by comparing false-positive data from our laboratory and national allele frequency data obtained with high-resolution HLA typing. For HLA-A, -B, -C, and -DR, a ratio of positive frequency to allele frequency of ≥3 in our laboratory was considered a false-positive result. For HLA-DQA1/DQB1 and HLA-DPA1/DPB1, we considered the positive frequency of ≥3 as a false positive result due to lack of haplotype frequency data. SABA results from 284 patients (78.0%) demonstrated false reactivity. The antibody against HLA-C*17:01 displayed the highest frequency ratio (298.3). If false-positive reactivity is suspected, results should be confirmed using different methods. If confirmation tests are unfeasible, comparing the allele frequency with the positive rate of detected anti-HLA antibodies and using a ratio ≥3 may facilitate the interpretation of SABA results. The positive rate of anti-HLA antibodies can be validated using the HLA allele frequency of the population to determine false-positive results.

Allosensitization in Heart Transplantation: The Importance of a Comprehensive Approach

<h3>Introduction</h3> HLA sensitization presents a major barrier to transplantation yet sensitive techniques such as the Luminex Single Antigen Bead (SAB) assay, when used in isolation, may inappropriately limit the donor pool. We report a case of apparent robust HLA sensitization following HBV vaccination and describe our approach to the assessment of HLA reactivity leading to successful heart transplantation (HTx). <h3>Case Report</h3> A 50 year old Caucasian male with long standing ARVD developed recurrent VT and worsening RV dysfunction leading to HTx evaluation. There was no history of transfusion, surgery, or mechanical circulatory support. The Luminex SAB assay at listing and serially thereafter was negative. Unexpectedly, broad class II HLA sensitization (cPRA 98%) was suddenly detected by SAB assay after HBV vaccination concerning for either a heterologous immune response or false reactivity to the beads. Multiple antibodies (Abs) persisted at 1:8 dilution and included Abs against the recipients own HLA (autologous flow XM negative). At the time when a donor became available, 4 DSA were identified on the most recent Luminex SAB. Given his high cPRA in the absence of a strong allosensitizing event, we combined assessment with an alternative solid phase assay (LABScreen PRA), 4-digit donor typing, and prospective crossmatches by Flow Cytometry (FC) and Complement Dependent Cytotoxicity (CDC) to determine the safety of proceeding with HTx (Figure 1). Notably, no HLA Abs were detected using the LABScreen PRA and both FC and CDC crossmatches were negative. The patient underwent successful HTx with no rejection and normal graft function. <h3>Summary</h3> HLA allosensitization portends worse outcomes after transplant. However, the high sensitivity of the Luminex SAB and potential for false positivity may result in inappropriately limiting access to HTx. This case highlights the importance of comprehensive assessment of HLA sensitization in situations of uncertainty to help identify pathogenic HLA Abs and allow for successful HTx.