Detection Of Donor-specific Antibodies Research Articles

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.

Immunopathology of Corneal Allograft Rejection and Donor-Specific Antibodies (DSAs) as Immunological Predictors of Corneal Transplant Failure.

Despite tremendous developments in the field of laboratory testing in transplantation, the rules of eligibility for corneal transplantation still do not include typing of human leukocyte antigens (HLAs) in the donor and recipient or detection of donor-specific antibodies (DSAs) in the patient. The standard use of diagnostic algorithms is due to the cornea belonging to immunologically privileged tissues, which usually determines the success of transplantation of this tissue. A medical problem is posed by patients at high risk of transplant rejection, in whom the immune privilege of the eye is abolished and the risk of transplant failure increases. Critical to the success of transplantation in patients at high risk of corneal rejection may be the selection of an HLA-matched donor and recipient, and the detection of existing and/or de novo emerging DSAs in the patient. Incorporating the assessment of these parameters into routine diagnostics may contribute to establishing immune risk stratification for transplant rejection and effective personalized therapy for patients.

Human leukocyte antigen compatibility and incidence of donor-specific antibodies in pediatric liver transplant recipients.

Antibody-mediated rejection following liver transplantation (LT) has been increasingly recognized, particularly with respect to the emergence of de novo donor-specific antibodies (DSAs) and their impact on graft longevity. While substantial evidence for adult populations exists, research focusing on pediatric LT outcomes remains limited. To investigate the prevalence of human leukocyte antigen (HLA) mismatches and DSA and evaluate their association with rejection episodes after pediatric LT. A cohort of pediatric LT recipients underwent HLA testing at Santa Casa de Porto Alegre, Brazil, between December 2013 and December 2023. Only patients who survived for > 30 days after LT with at least one DSA analysis were included. DSA classes I and II and cross-matches were analyzed. The presence of de novo DSA (dnDSA) was evaluated at least 3 months after LT using the Luminex® single antigen bead method, with a positive reaction threshold set at 1000 MFI. Rejection episodes were confirmed by liver biopsy. Overall, 67 transplanted children were analyzed; 61 received grafts from living donors, 85% of whom were related to recipients. Pre-transplant DSA (class I or II) was detected in 28.3% of patients, and dnDSA was detected in 48.4%. The median time to DSA detection after LT was 19.7 [interquartile range (IQR): 4.3-35.6] months. Biopsy-proven rejection occurred in 13 patients at follow-up, with C4d positivity observed in 5/13 Liver biopsies. The median time to rejection was 7.8 (IQR: 5.7-12.8) months. The presence of dnDSA was significantly associated with rejection (36% vs 3%, P < 0.001). The rejection-free survival rates at 12 and 24 months were 76% vs 100% and 58% vs 95% for patients with dnDSA anti-DQ vs those without, respectively. Our findings highlight the importance of incorporating DSA assessment into pre- and post-transplantation protocols for pediatric LT recipients. Future implications may include immunosuppression minimization strategies based on this analysis in pediatric LT recipients.

DONOR-SPECIFIC ANTIBODIES AS A PREDICTOR OF GRAFT REJECTION AFTER LIVER TRANSPLANTATION

The main reason for graft loss is the rejection of the donor organ, which may occur at different time after transplantation and may be caused by the recipient’s organism reaction against donor’s human leukocyte antigen (HLA) proteins. Donor-specific antibodies (DSA) are produced in patient’s organism as a response to foreign HLA antigens. Aim. The purpose of our study was to evaluate the effects of already existed and/or de novo generated DSAs in liver transplantation as predictors of graft rejection and to establish an interconnection between blood biochemical parameters (Alanine aminotransferase (ALT), aspartate aminotransferase (AST), total bilirubin level) with the level of DSA in patients with liver transplant. Methods. xMAP-Luminex next generation flow cytometry technology and LABScreen Single antigen beads reagent (Onelambda, USA) were used for antiHLA determination. Total bilirubin level was detected photometrically. The activity of ALT and AST was determined spectrophotometrically on the automatic analyzer COBAS C 111 (Roche, Switzerland) in accordance with the manufacture’s instruction. Results. Detection of DSA and PRA was important at the same level as measurement of classical biochemical parameters of liver function (ALT, AST etc.) for monitoring of graft status and prevention of acute or chronical rejection and choosing correct immunosuppression protocol. Conclusions. The DSA and PRA levels as well as total bilirubin and ALT and AST activity corresponded to each other and could be used for comprehensive both pre- and post-transplantation screening of patients requiring liver transplantation or re-transplantation. Detection of DSA and PRA was important at the same level as measurement of classical biochemical parameters of liver function (ALT, AST etc.) for monitoring of graft status and prevention of acute or chronical rejection and choosing correct immunosuppression protocol.

Long-term outcomes of two-dose alemtuzumab induction in pediatric kidney transplantation.

Alemtuzumab is a lymphocyte depleting agent used for induction in kidney transplant, but long-term information on its use in pediatric recipients remains sparse. We performed a single-center retrospective cohort study of 57 pediatric kidney transplant recipients receiving alemtuzumab 20 mg/m2/dose ×2 doses for induction immunosuppression. All patients underwent surveillance biopsies, and 91.3% underwent steroid withdrawal by day 4 post-transplant. Outcomes of interest included graft survival, development of donor specific antibodies (DSA), incidence of viremia and PTLD, and duration of lymphopenia. Median follow-up time was 7.9 years (IQR 5-13.6 years). Median graft survival was 16.5 years (95% CI 11.6-unknown). DSA developed in 36.5% at a median of 944 days (IQR 252-2113 days). Incidences of BK polyomavirus DNAemia (BKPyV-DNAemia), CMV DNAemia, and EBV DNAemia were 38.6%, 22.8%, and 14%, respectively; one patient developed PTLD at 13.3 years post-transplant. Median duration of lymphopenia was 365 days (IQR 168-713 days); 19.3% of patients remained lymphopenic at 3 years post-transplant. There was no association between duration of lymphopenia and graft survival, rejection, DSA detection, or viremia. A two-dose alemtuzumab induction protocol can have excellent outcomes with a steroid-free maintenance immunosuppression regimen. More comprehensive, multicenter, comparative studies of pediatric kidney transplant are needed to improve long-term outcomes.

(699) - Ten-Year Results of an IgA- and IgM-Enriched Human Immunoglobulin-Based Therapy for Early Detectable Anti-HLA Donor Specific Antibodies After Lung Transplantation

(699) - Ten-Year Results of an IgA- and IgM-Enriched Human Immunoglobulin-Based Therapy for Early Detectable Anti-HLA Donor Specific Antibodies After Lung Transplantation

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

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

Tracking Circulating HLA-Specific IgG-Producing Memory B Cells with the B-Cell ImmunoSpot Assay.

Donor-specific antibodies (DSA) against human leukocyte antigen (HLA) molecules are a major risk factor for rejection of transplanted organs (in antibody-mediated rejection [ABMR]), particularly in patients who have prior sensitization or receive insufficient immunosuppression through minimization or noncompliance. These DSA are measured routinely in the serum of patients prior to transplantation mainly using bead-based technologies or cell-based assays. However, the absence of detectable serum DSA does not always reflect the absence of sensitization or histologically defined ABMR, and so it has been proposed that the detection and measurement of memory B cells capable of secreting antibodies against donor HLA antigens could be carried out using B-cell ImmunoSpot, to better inform the degree of immune sensitization of transplant patients prior to as well as after transplantation. Such an assay is described here.

Natural killer cell functional genetics and donor-specific antibody-triggered microvascular inflammation

Antibody-mediated rejection (ABMR) is a leading cause of graft failure. Emerging evidence suggests a significant contribution of natural killer (NK) cells to microvascular inflammation (MVI). We investigated the influence of genetically determined NK cell functionality on ABMR development and activity. The study included 86 kidney transplant recipients subjected to systematic biopsies triggered by donor-specific antibody detection. We performed killer immunoglobulin-like receptor typing to predict missing self and genotyped polymorphisms determining NK cell functionality (FCGR3AV/F158 [rs396991], KLRC2wt/del, KLRK1HNK/LNK [rs1049174], rs9916629-C/T). Fifty patients had ABMR with considerable MVI and elevated NK cell transcripts. Missing self was not related to MVI. Only KLRC2wt/wt showed an association (MVI score: 2 [median; interquartile range: 0-3] vs 0 [0-1] in KLRC2wt/del recipients; P = .001) and remained significant in a proportional odds multivariable model (odds ratio, 7.84; 95% confidence interval, 2.37-30.47; P = .001). A sum score incorporating all polymorphisms and missing self did not outperform a score including only KLRC2 and FCGR3A variants, which were predictive in univariable analysis. NK cell genetics did not affect graft functional decline and survival. In conclusion, a functional KLRC2 polymorphism emerged as an independent determinant of ABMR activity, without a considerable contribution of missing self and other NK cell gene polymorphisms.

Comparison of HLA antibody identification methods for the selection of platelet products for HLA-mediated platelet refractory patients.

In an ineffective transfusion context, solid-phase immunoassays using the Luminex platform for the detection and characterization of HLA antibodies are currently used to select HLA-compatible platelet products. A new HLA antibody identification method, the HISTO SPOT® HLA AB test (BAG Health care GmbH, Lich, Germany), based on the detection of antibodies directed against a recombinant single antigen (SA) by colored spots detected by HISTO MATCH HLA AB module software, runs fully automated on the MR.SPOT®. The aim of this study was to compare the ability of the HISTO SPOT HLA AB and C1qScreen™ (C1q SAB) assays with that of the Labscreen single antigen class I (OL SAB) assay to detect anti-HLA class I antibodies in 56 serum samples from 54 platelet refractory acute myeloid leukemia patients who received HLA mismatch platelet concentrates at a single oncohematology center. In total, 1414 class I specificities, 433 HLA-A and 981 HLA-B, were detected by the OL SAB test. The mean fluorescence intensity (MFI) was >5000 for 874 antigens and <5000 for 655 antigens. The HISTO SPOT® HLA AB and C1q SAB tests identified 85% and 79% of OL SA-detected antigens with an MFI >5000, respectively, but did not identify 34% and 44% of OL SAB-detected antigens, highlighting the lower sensitivity of these techniques. Interestingly, the donor-specific antibodies (DSAs) identified by the HISTO SPOT® HLA AB and C1q SAB assays reacted against HLA mismatch platelet concentrates with the same specificity (86%) and positive predictive (77%) value as in the OL SAB test when the MFI threshold was >2000 for DSA detection. Although the HISTO SPOT® HLA AB test is less sensitive than the OL SAB test, this test could be used for the selection of HLA-compatible platelet products.

Implementing a self-reported immunosuppression adherence questionnaire to screen for non-adherence in routine care of kidney transplant recipients.

Self-reported measures of immunosuppression adherence have been largely examined in research settings. In this single center study of 610 kidney transplant recipients, we examined if a voluntary, non-anonymous self-report measure could identify non-adherence in a routine clinic setting and how patients perceived such a measure. Non-adherence was measured using the Basel Assessment of Adherence to Immunosuppressive Medications Scale (BAASIS) and patient perception was elicited using a customized questionnaire. Non-responders to the survey (15%) were younger, more likely to be black, and less likely to have had a pre-emptive transplant. Among complete responders (n=485), 38% reported non-adherence with non-adherent patients being younger (54y vs. 60y; p=.01), less likely to have been on dialysis pre-transplant (59%vs. 68%; p=.04), further out from transplant (37vs. 22 months; p<.001) and had more rejections in the preceding year (8%vs. 3%; p=.02). Self-reported non-adherence was associated with higher calcineurin inhibitor intra-patient variability (27.4%vs. 24.5%; p=.02), but not with donor-specific antibody detection (27.8%vs. 21.2%, p=.15). Of patients providing feedback (n=500), the majority of patients felt comfortable reporting adherence (92%), that the survey was relevant to their visit (71%), and that the survey did not interfere with their clinic visit (88%). In summary, a self-reported questionnaire during clinic visits identified immunosuppression non-adherence in a significant proportion of patients and was well received by patients. Integrating self-report measures into routine post-transplant care may enable early identification of non-adherence.

Immune surveillance and humoral immune responses in kidney transplantation - A look back at T follicular helper cells.

T follicular helper cells comprise a specialized, heterogeneous subset of immune-competent T helper cells capable of influencing B cell responses in lymphoid tissues. In physiology, for example in response to microbial challenges or vaccination, this interaction chiefly results in the production of protecting antibodies and humoral memory. In the context of kidney transplantation, however, immune surveillance provided by T follicular helper cells can take a life of its own despite matching of human leukocyte antigens and employing the latest immunosuppressive regiments. This puts kidney transplant recipients at risk of subclinical and clinical rejection episodes with a potential risk for allograft loss. In this review, the current understanding of immune surveillance provided by T follicular helper cells is briefly described in physiological responses to contrast those pathological responses observed after kidney transplantation. Sensitization of T follicular helper cells with the subsequent emergence of detectable donor-specific human leukocyte antigen antibodies, non-human leukocyte antigen antibodies their implication for kidney transplantation and lessons learnt from other transplantation "settings" with special attention to antibody-mediated rejection will be addressed.

Discussion on two common problems in renal transplantation donor-specificity discrimination of anti-human leukocyte antigen antibody

Antibody-mediated rejection (AMR) is a key factor affecting the long-term survival of renal allografts. Donor-specific antibody (DSA) is the etiology of AMR. So it is very important to accurately detect DSA. The single antigen bead (SAB) method, which is widely used in clinical practice, is prone to miss DSA detection and underestimate its mean fluorescence intensity (MFI). In this paper, the probability of missed detection of two SAB reagents was calculated by comparing common HLA alleles in China population, and the in vitro effect of antibody cross reaction on MFI value of DSA was revealed. The authors emphasized the clinical significance of the above two problems, tried to manage them by using functional epitope (eplet) analysis and give some clinical examples. Finally, the limitations of this correction method were analyzed.

Significance of C4d expression in peritubular capillaries concurrent with microvascular inflammation in for-cause biopsies of ABO-incompatible renal allografts.

Pathologic diagnosis of antibody-mediated rejection (ABMR) in ABO-incompatible (ABOi) transplantation patients is often challenging because patients without ABMR are frequently immunopositive for C4d. The aim of this study was to determine whether C4d positivity with microvascular inflammation (MVI), in the absence of any detectable donor-specific antibodies (DSAs) in ABOi patients, could be considered as ABMR. A retrospective study of 214 for-cause biopsies from 126 ABOi kidney transplantation patients was performed. Patients with MVI score of ≥2 and glomerulitis score of ≥1 (n = 62) were divided into three groups: the absolute ABMR group (DSA-positive, C4d-positive or C4d-negative; n = 36), the C4d-positive group (DSA-negative, C4d-positive; n = 22), and the C4d-negative group (DSA-negative, C4d-negative; n = 4). The Banff scores, estimated glomerular filtration rates (eGFRs), and graft failure rates were compared among groups. C4d-positive biopsies showed higher glomerulitis, peritubular capillaritis, and MVI scores compared with C4d-negative specimens. The C4d-positive group did not show significant differences in eGFRs and graft survival compared with the absolute ABMR group. The results indicate that C4d positivity, MVI score of ≥2, and glomerulitis score of ≥1 in ABOi allograft biopsies may be categorized and treated as ABMR cases.

(882) Nine-Year Results of an IgA-And IgM-Enriched Human Immunoglobulin-Based Therapy for Early Detectable Anti-HLA Donor Specific Antibodies after Lung Transplantation

(882) Nine-Year Results of an IgA-And IgM-Enriched Human Immunoglobulin-Based Therapy for Early Detectable Anti-HLA Donor Specific Antibodies after Lung Transplantation

(101) Nine-Year Experience with Treatment of Early Detectable Donor Specific Anti-HLA Antibodies in Pediatric Lung Transplant Recipients

(101) Nine-Year Experience with Treatment of Early Detectable Donor Specific Anti-HLA Antibodies in Pediatric Lung Transplant Recipients

BAASIS for Monitoring Therapy Nonadherence in Clinical Transplantation: Are We There Yet?

BAASIS for Monitoring Therapy Nonadherence in Clinical Transplantation: Are We There Yet?

Donor-Specific Antibody Detection by Single-Antigen Bead Assay for Renal Transplantation: A 2-Year Experience from South India.

Recipient sensitization against donor human leukocyte antigens (HLA) plays a key role in transplant rejection, and this risk is best minimized by efficient pre transplant antibody detection. Determination of antibody specificity with the highest sensitivity and degree of resolution to the allelic antigen level is achieved by using single-antigen bead (SAB) assay. This study evaluated the correlation of Luminex cross match (LXM) with SAB assay for detection of donor-specific antibodies (DSA). A total of 2075 renal transplant patients were screened for the presence of DSA by LXM, complement-dependent cytotoxicity (CDC) cross match, and 125 patients for SAB from January 2018 to December 2019. There was a male preponderance among recipients (P < 0.0001), and the most affected age group was 21-40 years. HLA typing was done in 550/2075 by DNA PCR-reverse sequence-specific oligonucleotide probes (SSOP) method. HLA DSA by LXM was detected in 16.3% of recipients (338/2075). Majority 180/338 (53.2%) of the patients were class II DSA positive, (P < 0.0001). Among the class II DSA positive patients, 20/180 (11.1%) samples gave false-positive results by LXM. SAB for class I and class II HLA IgG antibodies was done in 125/338 renal transplant recipients, which included 20 recipients with false-positive class II Luminex DSA, to check whether the DSA detected were really donor specific or not. The results showed that although 20/125 patients had some antibodies detected in their serum, they were not against the donor HLA antigens, as per the HLA typing reports of the donors. When compared to SAB assay, LXM showed more discrepant results, particularly to class II DSA. In conclusion, LXM, if used in combination with SAB assay and HLA typing of donors if necessary for virtual cross match, will help in avoiding unnecessary exclusion of donors for renal transplant recipients and also for post transplant monitoring of recipients, especially in cadaveric donor transplants.

Chimerism Revisited: Perspective of an Immunopathologist

Introduction: Chimerism analysis is an important diagnostic tool not only for assessing the risk of relapse after allo-Haematopoietic Stem Cell Transplant (HSCT) in patients with malignant diseases, it is also the predominant method for monitoring post-transplant engraftment status in both haematological malignancies and non malignant haematological disorders. Aim: To show the chimerism data observed in post-HSCT cases of our hospital over last four years and to emphasise on timely and close monitoring of these patients in the laboratory. Materials and Methods: The present study was a retrospective study in which 40 HSCT cases comprising of both haematological malignancies and non malignant haematological disorders were monitored in the molecular laboratory of Army Hospital (Research and Referral), at regular intervals by Short Tandem RepeatsPolymerase Chain Reaction (STR-PCR) for quantification of donor chimerism. The pretransplant workup included Human Leukocyte Antigens (HLA) typing of all recipients and donors, serum Panel Reactive Antibody (PRA) testing and Single Antigen Bead (SAB) assays for detecting donor specific antibodies in all haploidentical transplants. Results: Male patients formed majority, with only nine female cases. After complete HLA matching and preconditioning, 35 patients underwent matched related stem cell transplant, two were Matched Unrelated Donor (MUD) and three haploidentical transplants. Complete donor chimerism at D+90 was reported in 19 patients (73%) of haematologic malignancies with two cases of relapse and five showing evidence of Graft Versus Host Disease (GVHD). Amongst non malignant disorders 10 patients (71.4%) showed complete donor chimerism at D+90 with two cases of GVHD. Conclusions: Post-HSCT, progressive chimerism monitoring is an essential molecular test that predicts engraftment status of the patient by verifying the dynamic relationship between recipient and donor cells.

HLA diversity in ethnic populations can affect detection of donor-specific antibodies by single antigen beads.

In solid-organ transplantation, human leukocyte antigen (HLA) donor-specific antibodies (DSA) are strongly associated with graft rejection, graft loss, and patient death. The predominant tests used for detecting HLA DSA before and after solid-organ transplantation are HLA single antigen bead (SAB) assays. However, SAB assays may not detect antibodies directed against HLA epitopes that are not represented in the SAB. The prevalence and potential impact of unrepresented HLA epitopes are expected to vary by ethnicity, but have not been thoroughly investigated. To address this knowledge gap, HLA allele frequencies from seven ethnic populations were compared with HLA proteins present in SAB products from two manufacturers to determine unrepresented HLA proteins. Allele frequencies were obtained from the Common, Intermediate, and Well Documented HLA catalog v3.0, and frequencies of unrepresented HLA types were calculated. Next-generation sequencing was used to determine HLA types of 60 deceased solid-organ donors, and results were used to determine if their HLA-A, -B, -C, and -DRB1 proteins were not present in SAB reagents from two vendors. Unrepresented HLA proteins were compared with the most similar protein in SAB assays from either vendor and then visualized using modeling software to assess potential HLA epitopes. For the seven ethnic populations, 0.5% to 11.8% of each population had HLA proteins not included in SAB assays from one vendor. Non-European populations had greater numbers of unrepresented alleles. Among the deceased donors, 26.7% (16/60) had at least one unrepresented HLA-A, -B, -C, or -DRB1 protein. Structural modeling demonstrated that a subset of these had potential HLA epitopes that are solvent accessible amino acid mismatches and are likely to be accessible to B cell receptors. In conclusion, SAB assays cannot completely rule out the presence of HLA DSA. HLA epitopes not represented in those assays vary by ethnicity and should not be overlooked, especially in non-European populations. Allele-level HLA typing can help determine the potential for HLA antibodies that could evade detection.