Complement-dependent Cytotoxicity Crossmatch Research Articles

#681 Bw4/6 mismatch or missing donor Bw6 allele is a risk of early acute rejection in panel reactive antibodies negative kidney transplant recipients

Abstract Background and Aims Epitope and antibody diversity allows Bw4+ or Bw6+ patients to produce antibodies to Bw4 or Bw6 epitopes without breaking self-tolerance. This study assessed the prevalence of mismatched Bw4/6 or missing donor Bw6 allele in kidney transplant (KT) recipients with early acute rejection and its relation to patients and graft outcomes. Method A retrospective study included 40 live-related KT recipients divided into 2 equal groups. Group A: 20 KT recipients with early acute rejection in 1st 3 months post-KT and group B: 20 KT recipients with no rejection. Bw4/6 mismatches or missing donor Bw6 were examined in all patients with an assessment of the type of rejection, patients, and graft outcomes. Results The prevalence of Bw4/6 mismatch or missing donor Bw6 allele in patients with early acute rejection was 70% (14 patients). Bw4/6 mismatch represented 25% (5 patients) while missing donor Bw6 allele was 45% (9 patients) while the prevalence of Bw4/6 mismatch or missing donor in patients with no rejection was 10% (2 patients) with significant p-value (0.0001). The analysis of demographic data of Bw4/6 mismatch or missing donor Bw6 allele in patients with rejection (N=14) (Fig. 1); mean age was 27.92 years, 71.4% were males (10 patients), panel reactive antibodies (PRA) was positive in 7.1% (1 patient), Donor specific antibodies (DSA) and complement-dependent cytotoxicity (CDC) cross-match were negative in all patients. The most common type of rejection in these patients was acute antibody-mediated rejection (ABMR) 71.4% (10 patients). Patients and graft outcomes; mean serum creatinine after treatment of rejection was 2.16 mg/dl, mortality was 14.3% (2 patients) with graft loss, and survival with functioning graft was 85.7% (12 patients). Conclusion Bw4/6 mismatch or missing donor Bw6 allele is a risk of early acute rejection in PRA-negative kidney transplant recipients and is linked to negative patients and graft outcomes.

Histocompatibility Testing: A Fundamental Aspect of Renal Transplant Workup

Histocompatibility testing is pivotal in any renal transplantation workup, aimed at enhancing prospective donor recipient compatibility and improving transplant outcomes. The evolution and advancement of histocompatibility testing, particularly HLA typing, have significantly improved its precision. This study outlines the historical progression from serologic to DNA-based HLA typing, emphasizing the role of HLA proteins in immune response. Anti-HLA antibodies, targeting HLA proteins, pose challenges in renal transplantation. Monitoring and managing these antibodies are critical for renal transplant success. Complement-dependent cytotoxicity crossmatch and flow cytometry crossmatch are essential techniques for assessing donor–recipient compatibility. Panel-reactive antibody assesses antibodies against a panel of donor antigens, often HLA. Higher PRA levels (percentage) complicate donor matching, requiring specialized protocols. Virtual crossmatch evaluates recipient anti-HLA antibodies against potential donors through synthetic beads. This approach predicts crossmatch outcomes by comparing antibody profiles, offering a valuable tool for the risk assessment of renal transplantation. Despite advancements, a comprehensive understanding of alloreactive immune responses requires a combination of assays, emphasizing the importance of a multifaceted approach in histocompatibility testing. This is an attempt to compile the relevant information, providing a basis for comparison in a clear and foundational format for histocompatibility testing laboratories.

Role of Complement-dependent Cytotoxicity Crossmatch and HLA Typing in Solid Organ Transplant.

Solid organ transplantation is a life-saving medical operation that has progressed greatly because of developments in diagnostic tools and histocompatibility tests. Crossmatching for complement-dependent cytotoxicity (CDC) and human leukocyte antigen (HLA) typing are two important methods for checking graft compatibility and reducing the risk of graft rejection. HLA typing and CDC crossmatching are critical in kidney, heart, lung, liver, pancreas, intestine, and multi-organ transplantation. A systematic literature search was conducted on the internet, using PubMed, Scopus, and Google Scholar databases, to identify peer-reviewed publications about solid organ transplants, HLA typing, and CDC crossmatching. Recent advances in HLA typing have allowed for high-resolution evaluation, epitope matching, and personalized therapy methods. Genomic profiling, next-generation sequencing, and artificial intelligence have improved HLA typing precision, resulting in better patient outcomes. Artificial intelligence (AI) driven virtual crossmatching and predictive algorithms have eliminated the requirement for physical crossmatching in the context of CDC crossmatching, boosting organ allocation and transplant efficiency. This review elaborates on the importance of HLA typing and CDC crossmatching in solid organ transplantation.

Complement-dependent cytotoxicity crossmatch in solid organ transplantation: The gold standard or golden history?

Complement-dependent cytotoxicity crossmatch (CDC-XM) has been considered for many years the standard of practice for determining compatibility in solid organ transplantation (SOT). However, as this method is laborious, time intensive and lacks sensitivity and specificity, it has been replaced in many laboratories worldwide by flow cytometry crossmatch (FCXM) and/or virtual crossmatch (vXM).With this study we intend to show the relevance of performing CDC-XM in the era of virtual crossmatching. We retrospectively analyzed 1,007 consecutive T and B cell deceased donor (DD) CDC-XMs performed in parallel using non-treated and dithiothreitol (DTT) treated sera between May 2022 and January 2023 in waitlisted patients with no donor specific antibodies (DSA) against HLA-A, B and/or DR antigens. Thirty five of 1,007 (3.5%) T cell crossmatches and 132 of 1,007 (13.1%) B cell crossmatches were positive with non-treated sera. Correlation with the vXM demonstrated no DSA in any of the positive T cell crossmatches. DSA were also absent in 126/132 positive B cell crossmatches, indicating a high rate of false positive CDC-XM. Indeed, only 4/35 T cell and 13/132 B cell CDC-XM remained positive after treatment with DTT, confirming that false positive reactivity with non-treated sera is high. Class I HLA DSA against C locus antigens were present in 17/1,007 T cell crossmatches and none were detected by CDC-XM (sensitivity = 0%). Similarly, only 6/77 B cell crossmatches with DSA targeting HLA-C, DQ and/or DP antigens were CDC-XM positive (sensitivity = 7.8%). Furthermore, only 4/6 positive B cell CDC-XM were confirmed to have complement binding potential using the C1q assay, suggesting additional false positive reactivity in 2/6 of the positive CDC-XM.Our study demonstrates that CDC-XM exhibits poor sensitivity, high false positive reactivity (especially without DTT treatment) and does not meaningfully contribute to pre-transplant compatibility testing in the context of vXM based allocation. Furthermore, the use of CDC-XM can unnecessarily delay or even prevent safe and appropriate transplant allocation.

#6257 PROGNOSTIC VALUE OF A POSITIVE CROSSMATCH BEFORE DESENSITIZATION IN A COHORT OF HIGHLY-SENSITIZED PATIENTS

Abstract Background and Aims Human Leukocyte antigen (HLA) sensitization in patients is a barrier for Kidney Transplantation (KT) access, and highly-sensitized patients remain on the waiting list longer than other patients which results in a higher morbidity and mortality. Desensitization may be an option to increase their access to KT. All patients transplanted in this context have a negative Complement-Dependent Cytotoxicity crossmatch (CDC-CM) the day of KT. However, the stratification of the long-term risk of graft loss according to flow cytometry crossmatch (FC-CM) and to CDC-CM during the desensitization period remains unknown. Method In this retrospective study, all highly-sensitized patients (Panel Reactive Antigen (PRA) ≥ 85) in our center were included. CDC-CM and FC-CM were performed on samples before and during the desensitization period and clinical follow-up data were retrieved. Pre-KT desensitization strategy consisted in two Rituximab infusions (375mg/m2) and a variable number of apheresis (mostly semi-specific immunoadsorption) associated with a classical immunosuppression-based regimen (tacrolimus, mycophenolate and steroids). Results A total of 183 highly-sensitized recipients received a KT between 2014 and 2022. Of these, 23 were desensitized prior to KT and among them 18 had a positive FC-CM before KT and 5 had no positive FC-CM on all assessed serums. Mean age was 52.9 ± 13 years. Mean follow-up was 5.2±4 and 3±2 years for non-desensitized and desensitized patients respectively. PRA before desensitization was higher in desensitized patients (98%) versus non-desensitized patients (95%) and the mean time on the waiting list was 5.8±4 versus 7.5±4 years respectively. Death-censored graft survival and patient's raw survival, using cox-survival analysis, were not statistically different between the desensitized kidney transplanted patients and the highly-sensitized patients without desensitization (p = 0.77 and p = 0.57 respectively). In sub-group analyses, the positivity of FC and/or CDC-CM did not add an additional risk of graft loss or patient mortality. Conclusion Desensitization allows highly-sensitized patients to access KT with an HLA-incompatible graft (with a positive CDC or FC-CM on the serum collected prior to desensitization), with the same graft survival as similarly sensitized patients transplanted with an HLA-compatible graft without desensitization.

Impact of allele-specific anti–human leukocyte antigen class I antibodies on organ allocation

Technological advances in the field of histocompatibility have allowed us to define anti–human leukocyte antigen (HLA) antibody specificity at the allelic level. However, how allele-specific antibodies affect organ allocation is poorly studied. We examined allelic specificities of class I HLA antibodies in 6726 consecutive serum samples from 2953 transplant candidates and evaluated their impact on the corresponding crossmatch and organ allocation. Out of 17 class I HLA antigens represented by >1 allele in the LABScreen single antigen bead assay, 12 had potential allele-specific reactivity. Taking advantage of our unbiased cohort of deceased donor-candidate testing (123,135 complement-dependent cytotoxicity crossmatches between 2014 and 2017), we estimated that the presence of allele-specific antibody detected using a single antigen bead assay (median fluorescence intensity, >3000) against only the rare allele was a poor predictor of a positive complement-dependent cytotoxicity crossmatch, with a positive predictive value of 0% to 7%, compared with 52.5% in allele-concordant class I HLA antibodies against A or B locus antigens. Further, we confirmed allele-specific reactivity using flow crossmatch in 3 scenarios: A11:01/A11:02, A68:01/A68:02, and B44:02/B44:03. Our results suggest that allele-specific antibodies may unnecessarily exclude transplant candidates (up to 10%) from organ offers by overcalling unacceptable antigens; incorporation of selective reactivity pattern in allocation may promote precision matching and more equitable allocation.

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

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

Is Lysate-based Human Leukocyte Antigen Crossmatch a Reliable Method to Contraindicate a Transplant?

The presence of preformed cytotoxic donor-specific antibodies (DSAs) has been associated with inferior allograft outcomes in the immediate posttransplant period. Since the 1970s, the primary method for determining the presence of DSAs has been the complement-dependent cytotoxicity crossmatch. Solid phase assays on the Luminex platform were introduced in India in 2010 in the form of human leukocyte antigen antibody screening, single antigen bead (SAB) assay and lysate-based crossmatch (LumXM) to identify low titer antibodies, which are deleterious to allograft. Instead of SAB, LumXM has been popularly used in India to identify DSAs and also called DSA crossmatch, which is not recommended or validated in International literature for denying or accepting to transplant a patient due to its fallacies. We are reporting three cases which showcase the flawed nature of this test and the implications of this test on day to day practice in transplantation.

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.

An understanding of the evaluation of histocompatibility testing for a deceased-donor transplant

Dear Editor, Several obstacles have been overcome in the recent decades to make organ transplantation an effective lifesaving treatment for many patients. However, there is a tremendous disparity between the demand for organs and the number of organs necessary around the world, which is regarded as one of the major barriers, and India is not an exception.[1] Since 2013, the rate of deceased-donor organ donation increased from 0.27 per million people (pmp) to 0.65 pmp in 2018. Whereas the rate of deceased-donor renal transplant increased from 0.43 pmp to 0.86 pmp during the same period; this is roughly a 2.4-fold increase in just 5 years.,[2] Still, a lot of support and awareness for deceased-donor organ transplant programs is needed to help patients who do not have living kidney donors. There are numerous obstacles to overcome, with persuading family members being one of the most significant. Furthermore, transportation and allocation of the cadaver are also other issues associated with deceased-donor transplants. Once a cadaver is available, a set of histocompatibility testing of the potential patients is performed to determine the transplant’s suitability. Hence, a transplant immunology laboratory’s ability to accurately report the compatibility tests (to evaluate preformed human leukocyte antigen [HLA] donor-specific antibodies) on time is critical to the success of a deceased-donor transplant program. We want to emphasize the importance of histocompatibility testing evaluation in deceased-donor transplant programs. To support the deceased-donor transplant program, the laboratory must set up an efficient operational system to determine what kind of tests will be needed before, during, and after the transplant. In addition, a functioning 24-h on-call system should be in place. So far, a standalone transplant focused laboratory in North India, accredited for ISO15189: 2012, has aided pretransplant testing workup for 62 cadaveric cases (between February 2015 and December 2022) by optimizing the appropriate and effective test for anti-HLA antibodies between complement-dependent cytotoxicity (CDC) crossmatch, flow cytometric crossmatch (FCXM), and Luminex crossmatch.Both the CDC and the FCXM are time-consuming and labor-intensive, with the potential of false results, especially in the case of deceased donations where there is no time to redo the tests. Even technological advancements have contributed to a better understanding of transplant immunology in the recent years. In the identification of HLA antibodies, the advent of Luminex cross-matching technology provides more sensitivity than CDC and FCXM. Furthermore, unlike Luminex crossmatch, CDC and FCXM are manually created assays that require manual interpretation and whose processing method varies between laboratories. Furthermore, they (CDC and FCXM) both require a high number of viable lymphocytes to complete the experiment, whereas Luminex crossmatch requires a cell lysate to process. It is difficult to isolate a high number of viable lymphocytes from a cadaver; hence, cell count is a major concern in the cadaver transplant workup. The outcome may be harmed as a result of sample quality difficulties. Due to the aforementioned constraints, these approaches are less reliable during cadaver workup, and they also take a longer time than Luminex crossmatch. Even lysate can be saved for posttransplant monitoring.[3,4] Mishra et al. also support the findings of the use of Luminex crossmatch for pretransplant workup of renal transplants.[4] Later on, data published by Mathur et al. on 342 patients supported the usefulness of Luminex lysate crossmatch in deceased donor pretransplant workup.[3] Further, some more published literature[5,6] supports these findings; hence, Luminex crossmatch (lysate) should be used as the primary compatibility investigation for deceased-donor transplants. At present, no specific pretransplant compatibility workup for deceased-donor transplants is available. In routine practice, broadly, the pretransplant compatibility workup falls into two broad categories; cell-based tests (CDC and FCXM) and HLA bead-based (solid phase) tests (such as anti-HLA antibody screening and ant-HLA antibody identification). Laboratories typically utilize testing algorithms for compatibility, which are combinations of two or more tests, since no single test, whether cell-based or HLA bead-based, is 100% accurate and sensitive. To make donor lysate Luminex crossmatch a consensus technique, transplant physicians can be taught about the benefits. Recently, some pretransplant compatibility testing algorithm in the Indian setup is published,[7,8] which necessitates the development of one for better organ transplant outcomes, particularly in cadaver instances where there are numerous hurdles (both clinical and nonclinical). For deceased donor workup, expert analysis in a quicker turnaround time is critical, and hence, the laboratory should be equipped with cutting-edge advanced histocompatibility equipment to aid in the evaluation of histocompatibility testing for deceased-donor transplants. Financial support and sponsorship Nil. Conflicts of interest There are no conflicts of interest.

Novel Flow Cytometry-Based Method for Detection of Anti-HLA Complement Activating Donor-Specific Antibodies in Renal Transplant Recipients and Its Comparison With Other Conventional Detection Methods

Presence of preformed donor specific antibodies (DSAs) detected by complement-dependent cytotoxicity (CDC-XM) is a strong contraindication for transplant. However, it has limitations including its sensitivity and its inability to distinguish between HLA-specific and other non-HLA-specific antibodies. In this study, we standardized CDC-XM by flow cytometry and determined its relevance by comparing its results with other methods of DSA detection, such as routine CDC-XM, antibody binding assay by flow cytometry (FC-XM), and Luminex-based crossmatch assays, such as Luminex crossmatch (LXM) and virtual crossmatch (VXM). A total of 79 serum samples were tested for DSAs by the flow cytometric complement-dependent cytotoxicity crossmatch assay (FC-CDC-XM) and then the results of FC-CDC-XM were compared with other detection methods such as CDC-XM, FC-XM, LXM, and VXM. We found that the FC-CDC-XM assay is more sensitive than routine CDC-XM. Out of total 79 sera, 24 sera were detected positive (T cells positive: 1 case and B cells positive: 23) by FC-CDC-XM as compared with 3 sera using CDC-XM; these 3 sera also showed positivity by FC-CDC-XM. After FC-XM assay, 23 samples were positive by FC-XM and out of these 23 samples, 13 were also positive by FC-CDC-XM. On comparing the FC-CDC-XM results with VXM and LXM, 10 sera of 24 FC-CDC-XM positive had HLA class II antibodies detected on a Luminex platform. The FC-CDC-XM is a more sensitive and specific method for detection of HLA-specific complement-fixing antibodies than CDC-XM and FC-XM. FC-CDC-XM should be used in tissue-typing laboratories after intra- and inter- laboratory validation.

Detection and management of HLA sensitization in candidates for adult heart transplantation

Heart transplantation (HT) remains the preferred therapy for patients with advanced heart failure. However, for sensitized HT candidates who have antibodies to human leukocyte antigens , finding a suitable donor can be challenging and can lead to adverse waitlist outcomes. In recent years, the number of sensitized patients awaiting HT has increased likely due to the use of durable and mechanical circulatory support as well as increasing number of candidates with underlying congenital heart disease. This State-of-the-Art review discusses the assessment of human leukocyte antigens antibodies, potential desensitization strategies including mechanisms of action and specific protocols, the approach to a potential donor including the use of complement-dependent cytotoxicity, flow cytometry, and virtual crossmatches, and peritransplant induction management.

Successful Deceased Donor Kidney Transplantation in a Highly Sensitized Patient after Reclassification of Unacceptable Antigens Based on HLA Epitope Analysis

For patients with end stage renal disease, kidney transplant offers significant survival and quality-of-life advantages compared with dialysis. But for patients seeking transplant who are highly sensitized, waiting times have traditionally been long and options limited. We present the case of a 34-year-old hypersensitized female who underwent renal retransplantation. Histocompatibility tests revealed a calculated panel-reactive antibody of 99.53% with multiple antibodies against class I and II human leucocyte antigens and an eplet analysis was performed. The donor’s potential unacceptable antigens were re-defined and the calculated panel-reactive antibody decreased to 88.38%. After one month the patient received a deceased-donor kidney transplant. Complement dependent cytotoxicity crossmatch was negative; virtual crossmatch and flow cytometry crossmatch with historical serum were positive. High-dose intravenous immunoglobulin and rituximab were added to the thymoglobulin-based induction immunosuppression. Three donor-specific antibodies were detected and plasmapheresis was performed. Renal allograft biopsy revealed no manifestations of rejection. Repeated testing observed a decrease in donor-specific antibodies median fluorescence intensity values. Four months post-transplant, the patient remained with normal graft function without proteinuria. She is receiving a standard maintenance immunosuppression regime with prednisolone, mycophenolate mofetil and tacrolimus. The careful discussion among the transplantation center and histocompatibility laboratory in association with intense immunosuppression and close laboratory monitoring allowed a successful human leukocyte antigen-incompatible deceased donor kidney transplantation in the most critical phase for the occurrence of humoral rejection. It is noteworthy that the new histocompatibility and immunogenetics methodologies provide a more affirmative and comprehensive assessment of mismatch acceptability.

RE: Imlifidase-generated Single-cleaved IgG: Implications for Transplantation.

With great interest, we read the article by Bockermann et al1 on crossmatch assays after the administration of the novel desensitization drug Imlifidase. Proteolysis by Imlifidase generates single-cleaved immunoglobulin G (IgG) in a rapid first reaction and subsequently 2 F(ab’)2 fragments and a fully separated Fc fragment in a longer second reaction.2 Bockermann et al1 demonstrated that Luminex single antigen bead (SAB) testing, and crossmatching with both flowcytometry (FCXM) and the anti-human IgG-amplified complement-dependent cytotoxicity crossmatch (CDCXM) do not fully distinguish single-cleaved IgG from intact IgG. Bockermann et al1 suggest that the nonamplified CDCXM is best capable of demonstrating relevant residual intact IgG. Our center performed the first Imlifidase reimbursed HLA-incompatible kidney transplantation in March 2022. Contrary to Bockermann et al,1 we had difficulties in the interpretation of nonamplified CDCXM after Imlifidase treatment. A 29-y-old female with 6-y dialysis vintage after 2 failed kidney transplants was running out of vascular access. Despite high-urgency status and priority allocation via the Eurotransplant Acceptable Mismatch program, she remained without kidney offers. This was caused by her virtual panel-reactive antibody status of 100%. Her historical SAB mean fluorescence intensity (MFI) levels were high with broad reactivity in B-cell memory, with current MFI levels in the lower ranges. To enable for a timely HLA-incompatible kidney transplant, we delisted all unacceptable antigens with corresponding MFI levels of <3000, provided that CDC screening was negative. By this delisting, we increased her donor frequency of 0.000%–3.95%. Eight days later, this resulted in an HLA 1-2-2 mismatched kidney offer, with 2 “delisted DR-unacceptables.” Imlifidase was administered based on a virtual crossmatch. Serum was sampled 2 and 4 h after Imlifidase administration. Nonamplified CDCXMs with donor spleen cells were performed with preblood simultaneously tested with post 2-h and post 4-h sampling. Pre-CDCXM without dithiothreitol (DTT) treatment (which dissolves the disulfide bridges of IgM tetramers) was positive, indicating autoreactivity. DTT treatment revealed a negative pre-Imlifidase CDCXM. To our surprise, both the 2- and 4-h Imlifidase CDCXMs with DTT were positive (value 35 of 100). We had to decide whether or not to proceed with transplantation based on the nonamplified CDCXMs alone, as additional FCXM and SAB testing were scheduled for the next day. Because our patient had not recently received blood transfusions, was without infectious episodes, and had a negative pre-Imlifidase CDCXM, we expected assay interference by Imlifidase and decided to proceed with transplantation. The retrospectively performed pre-Imlifidase FCXM was negative for T cells and was 1.94 MCS for B cells. Luminex SAB was positive on 2 donor DR loci with 2500 and 2200, and became negative after Imlifidase. We investigated CDCXM on frozen donor spleen cells retrospectively and noticed CDC-positivity 2 and 4 h after Imlifidase, with negative CDCXMs on day 3. The patient did experience a vascular rejection after 1 wk, without signs of AMR. SAB testing revealed HLA antibody rebound from day 4 onward; however, DSA remained negative until current follow-up of 4 mo. This case demonstrates that even in nonamplified CDCXM, an interference by Imlifidase may occur. We advise to combine different assays when interpreting desensitization results after Imlifidase treatment.

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

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

Low Incidence of Acute Antibody-Mediated Rejection after HLA Desensitization in Living Donor Kidney Transplant Recipients.

Desensitization allows the performance of human leukocyte antigen (HLA)-incompatible transplants. However, the incidence of acute rejection (AR) is high. This study aims to analyze the incidence of AR after transplantation with HLA-incompatible living donors in patients who underwent desensitization. Patients were immunosuppressed with tacrolimus, mycophenolic acid derivatives, and steroids after being desensitized with rituximab, plasma exchange, and/or immunoadsorption with specific cytomegalovirus immunoglobulins. A negative complement-dependent cytotoxicity or flow cytometry crossmatch and a donor-specific antibody titer < 1000 mean fluorescence intensity (MFI) were used to determine desensitization efficacy. A total of 36 patients underwent desensitization, and 27 (75%) were transplanted. After a follow-up of 58 ± 58 months (Min−Max: 0.13−169.5), five episodes of AR occurred: two antibody-mediated and three T-cell-mediated. No differences were found in baseline calculated panel-reactive antibodies (cPRA), class I or II MFI, number of antibodies, or Relative Intensity Scale (RIS) between AR and non-AR patients. Patients with antibody-mediated AR had higher cPRA (NS), MFI class I (p = 0.07) and class II (p = 0.006), and RIS (p = 0.01). The two patients with antibody-mediated AR and one patient with T-cell-mediated AR lost their grafts. In conclusion, the incidence of acute antibody-mediated rejection after desensitization was 7.4%, which occurred early post-transplantation in patients with high MFI and was associated with early graft loss.

The Medawar Prize Acceptance Speech 2022.

The Medawar Prize Acceptance Speech 2022.

Virtual crossmatch versus complement-dependent cytotoxicity crossmatch in deceased kidney paired donations: a single-center experience from India

Virtual crossmatch versus complement-dependent cytotoxicity crossmatch in deceased kidney paired donations: a single-center experience from India

Effects of different sensitization events on HLA alloimmunization in renal transplant cases; a retrospective observation in 1066 cases

BackgroundPatients awaiting solid organ transplantation may develop anti-HLA antibodies after sensitization events such as transfusions, pregnancies, or previous transplantations. However, the effects of a particular sensitization event on HLA alloimmunization have not been well studied in parallel using cell-based assays and solid-phase assays. In this study, we evaluated and compare how different sensitization events affect the HLA antibody screening (HLA-Ab) and donor specific antibody (DSA) status in solid renal organ transplantation patients. MethodsHLA antibody (HLA-Ab) screening tests like complement-dependent cytotoxicity crossmatch (CDC-XM), flow cytometry crossmatch (FC-XM) and Luminex panel-reactive antibody (L-PRA) were performed in all 1066 patients (635 males and 431 females). If any of these tests turned out to be positive, a Luminex single antigen bead (L-SAB) assay was performed for DSA identification. Test positive rates and antibody strengths were analyzed according to the different sensitization events and gender. ResultsIn this study, HLA-Ab screening tests positive rates (L-PRA, FC-XM and CDC-XM) were significantly higher in patients with previous transplantation (73.91%, 100% and 56.52% p < 0.001), previous pregnancy (57.46%, 70.14% and 18.85% p < 0.001) or blood transfusion (27.33%, 35.55% and 7.33% p < 0.001) compared with patients without a sensitizing event (6.17%, 13.58% and 1.09). In this study, re-transplantation group showed significantly stronger antibody strength (DSA) than non sensitized group (class I and II MFI 11418.04, 17,837.78 vs class I and II MFI 2659, 3329; P < 0.001) and those with single sensitization events of transfusion (class I and II MFI 11418.04, 17,837.78 vs class I and II MFI 5790.26, 6004.16; P < 0.001) or pregnancy (class I & II MFI 11418, 17,837 vs class I and II MFI 8631.71, 7253.29; P < 0.001). ConclusionsPregnancy and blood transfused had high allo-immunization rate for class I HLA antigens. While re-transplantation patients had high allo-immunization rate for both the HLA classes (HLA- class I and HLA- class II). Re-transplantation group showed significantly stronger antibody strength, followed by pregnancy and then transfusion.

Clinical significance of low pre-transplant donor specific antibodies (DSA) in living donor kidney recipients with negative complement-dependent cytotoxicity crossmatches (CDCXM), and negative flow cytometry crossmatches (FLXM) – A single-center experience

BackgroundIt is controversial whether all donor-specific antibodies (DSA) detected by the solid-phase single antigen bead (SAB) assay negatively affect kidney transplantation outcomes. The study aimed to evaluate the possible clinical significance of low pre-transplant DSA in living donor kidney recipients. We analyzed a group of patients with HLA-A, B, and -DR DSA reactivities below a virtual crossmatch (VXM) value of 5000 MFI but with all VXM DSA reactivities at HLA-DQ, -DP, and -Cw, which were not typed routinely for donors prior to transplantation. We also investigated the incidence of persistent and de novo DSAs in available posttransplant SAB assays. MethodsFrom the historical cohort of living donor recipients transplanted between 2014 and 2018 at our center (n = 82), 55 patients met the inclusion criteria, namely: these patients were > 18 years old with non-HLA identical sibling donors, who were not desensitized, who had available pre-transplant SAB results, and who had negative both complement-dependent cytotoxicity crossmatch (CDCXM) and flow cytometry crossmatch (FLXM) results. An additional donor HLA typing, performed for all 55 recipients, identified donor additional HLA-DQ, -DP, and -Cw DSA reactivities. These patients were then divided by SAB reactivity into three groups: 1) those with DSA-positive reactivities; 2) those with non-donor-specific anti-HLA reactivities (NDSA); and, 3) those who were anti-HLA-negative. All these recipients were followed for three years and checked for their de novo or persistent DSA. ResultsIn the studied cohort, DSA-positive, NDSA reactive, and anti-HLA negative recipients constituted 33%, 36%, and 31% of 55 patients, respectively. Non-routinely considered pre-transplant HLA-DQ, -DP, and -Cw DSA-positive reactivities were shown in as many as 78% of DSA-positive cases (group 1) with the lowest MFI value of 319 to DP4 and the highest MFI of 5767 to DQ2. Of the pre-transplant HLA-A, B, and -DR DSA reactivities, only -DR52 DSA reactivity reached the highest MFI value of 2191. These detected DSAs did not reduce the mean estimated glomerular filtration rate (eGFR) values and did not increase the incidence of proteinuria in recipients. While the 3-year graft survival was lower in the DSA-positive group (94.4%) with one recipient who lost kidney transplant, the difference was not significantly different (p = 0.7) from the NDSA (100%) and negative (100%) groups. In terms of the incidence of de novo acute antibody-mediated rejection (AMR) at three years after transplantation, no case has been reported in the cohort. This may suggest that low DSA-positive recipients do not experience higher rejection rate. However, DSA-positive recipients had a tendency for a higher frequency of C4d deposits in peritubular capillaries (PTC) and de novo DSA. ConclusionOur 3-year follow-up of patients with low pre-transplant DSA found no association with a deterioration in graft function and worse graft survival. Furthermore, we did not observe an increase in AMR in our patients with low DSA. A larger cohort and a longer follow-up period may be needed to evaluate the tendency of low DSA-positive recipients towards the higher incidence of C4d deposits in PTC and/or de novo DSA.