Single Antigen Bead Research Articles

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.

Untreated Donor-Specific HLA Antibodies Are Associated With Graft Failure and Poor Survival After Haploidentical Transplantation With Post-Transplantation Cyclophosphamide in Pediatric Patients With Nonmalignant Disorders

Donor-specific HLA antibodies (DSAs) have been recognized as a major risk factor for graft failure (GF) in adult patients with malignancies undergoing haploidentical transplantation with post-transplantation cyclophosphamide (haplo-PTCy). However, the impact of DSAs after pediatric haplo-PTCy for nonmalignant disorders (NMDs) has been poorly reported. We sought to investigate whether preexisting DSAs adversely affect pediatric haplo-PTCy outcomes. We retrospectively analyzed 59 pediatric patients (≤21 years) who received their first haplo-PTCy for NMDs from January 2008 to December 2017. DSA testing was performed using single antigen beads, and mean fluorescence intensity (MFI) >1000 was considered positive, and MFI <1000 and >500 was considered potentially positive, based on HLA epitope reactivity patterns. Primary endpoints were neutrophil and platelet recovery and GF, whereas secondary endpoints included event-free and overall survival. Multivariable analyses were performed using Fine-Gray competing risk regression or Cox proportional hazards regression models. The median age was 10 years, and 66.1% were male. Main indications for haplo-PTCy were Fanconi anemia (n=33) and severe aplastic anemia (n=11). All patients received bone marrow as the graft source, and most patients (91.5%) received fludarabine-based conditioning. Overall, 15 patients (25.4%) had DSAs >500 MFI. Four patients had false-positive DSAs with median MFI of 1762. Of the 11 patients with true-positive DSA reactivity, 5 had 1 DSA, 5 had 2 DSAs, and 1 had 3 DSAs, with median MFI of 2372 (range 527-24,200). Four patients received desensitization therapy with rituximab and plasmapheresis, whereas 7 patients were untreated. All patients with treated DSAs achieved donor engraftment. In the multivariable analyses, untreated DSAs were associated with lower neutrophil recovery (subdistribution hazard ratio [SHR] = 0.15; 95% confidence interval [CI], 0.03-0.63; P=.001), increased GF (SHR=20.57; 95% CI, 6.57-64.43; P < .001), inferior event-free survival (hazard ratio [HR] = 10.09; 95% CI, 3.37-30.22; P < .001), and poor overall survival (HR 5.56; 95% CI, 1.92-16.12; P=.002). Both treated DSAs (SHR=0.26; 95% CI, 0.10-0.68; P=.006) and untreated DSAs (SHR=0.13; 95% CI, 0.04-0.37; P < .001) adversely affected platelet recovery. Our results indicate that the presence of DSAs is an independent predictor of poor outcomes after pediatric haplo-PTCy for NMDs. Therefore DSA-positive donors should be avoided whenever possible, and when a DSA-negative donor is unavailable, desensitization therapy must be performed to enhance the likelihood of donor engraftment and improve transplantation outcomes.

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.

Lung transplantation in an 18-month-old with donor specific antibodies - The use of intraoperative, targeted plasma exchange.

Sensitised patients undergoing Human Leukocyte Antigen-incompatible transplantation are at increased risk of hyperacute rejection and may be predisposed to antibody-mediated rejection, chronic lung allograft dysfunction and higher mortality. We present a case of primary lung transplantation in the setting of late identification of donor specific antibodies treated with intraoperative target plasma exchange. The patient was treated with fresh human plasma to a final volume of 1.5 times the patient's systemic circulation. From a pre-transplant mean fluorescence intensity of 5002, donor-specific antibodies were undetectable following plasma exchange on single antigen bead assay. This method represents a potential desensitisation technique for use in the intraoperative period.

Relevance of Anti-HLA Antibody Strength Underestimation in Single Antigen Bead Assay for Shared Eplets.

HLAs contain combinations of multiple eplets, sometimes shared between numerous HLA alleles. Some authors suggested that single antigen bead (SAB) assays may underestimate the signal of anti-HLA antibodies (Ab) when several beads share the targeted eplet. However, this assumption has not yet been validated experimentally. We selected 5 eplets shared by 1-24 beads of the routine SAB kits: the eplet 163LS/G; the 3 eplets 127K, 62GE, and 62GRN thereafter called cross-reactive group 2C; the 82LR eplet, well-known as Bw4; the locally called QB2A5 eplet associated with the DQA1*05:01/DQB1*02:01 combination; and the 40GR DQ eplet. We selected a dozen of sera for each eplet with Ab mean fluorescence intensity (MFI) between 1000 and 15 000 for the beads carrying the targeted eplet. We tested them with the classical SAB panel (SABp), with an isolated bead carrying the eplet (isolated SAB [SABi]) and with a mixture of both (SABp+i). No significant difference in MFI was detected among SABi, SABp, and SABp+i conditions for all the eplets. We noticed only a nonsignificant difference in the Ab MFI signal due to eplet sharing on the SAB assay. We, therefore, conclude that this phenomenon should no longer be considered as a significant risk factor during patient follow-up pre- or posttransplantation.

Misidentification of preformed anti-HLA-DP antibodies leads to antibody-mediated kidney transplant rejection: a case report

BackgroundPatients who are HLA-sensitized are at high risk for early antibody-mediated rejection (AMR) and worse outcomes. Therefore, it is crucial to detect the presence of donor-specific antibodies (DSAs) using pretransplant antibody identification and crossmatch assays. An error in antibody identification can lead to disastrous clinical outcomes. We present a case of acute AMR associated with preformed HLA-DPα and HLA-DPβ DSAs that were not identified before transplantation.Case presentationA 27-year-old woman received a second kidney transplant from a deceased donor. Her pretransplant panel-reactive antibody level was 94%. The complement-dependent cytotoxicity crossmatch was negative for T and B cells at the time of transplantation. She experienced early acute AMR proven by a kidney biopsy. Single antigen bead testing of the patient’s serum at the time of rejection as well as the pre-second transplant serum revealed strong antibodies against the DPA1*01:03 and DPB1*02:01 alleles in the second donor. These antibodies were not identified by phenotypic bead assay during the patient’s time on the waiting list. The patient was treated with plasmapheresis and anti-thymocyte globulin. However, she experienced abdominal pain on day 37 post-transplantation. Surgical exploration revealed a laceration on the transplanted kidney, which was then repaired. Subsequently, infected hematoma was suspected and the transplanted kidney was removed.ConclusionThe present case highlights the clinical significance of preformed HLA-DPα and HLA-DPβ DSAs. Accuracy in determination of HLA antibodies before transplantattion is critical for transplant outcome. HLA-DP typing and single antigen bead testing are recommended for a precise antibody interpretation, especially in highly sensitized patients. Careful interpretation of antibody testing results is essential for the success of organ transplantation.

Impact of catch-up vaccinations on anti-HLA antibody response in pediatric kidney transplant candidates.

Efforts have been concentrated on improving vaccination administration during the pretransplant evaluation period. However, concern for human leukocyte antigen (HLA) sensitization subsequent to vaccination exists. A retrospective review of pediatric kidney transplant candidates (PKTCs) ≤18 years old who had received vaccinations between February 1, 2017 and November 30, 2019 was conducted. Emergence of de novo anti-HLA antibody (HLA-Ab) 3-4 weeks postvaccinations detected by the Luminex single antigen bead assay (SAB) was evaluated. Outcomes assessed included change in the HLA-Ab mean fluorescence intensity (MFI) ≥25% from baseline, and change in preexisting HLA-Ab MFI strength, categorized as weak: 1000-2999; moderate: 3000-9999; and strong: ≥10 000. Sixty vaccinations were administered to 14 patients. Forty-one potential de novo HLA-Ab were detected in five patients. After additional antibody panel testing, 5/41 potential de novo HLA-Ab were determined to be HLA specific; the remaining 36 were deemed nonspecific. The 5 de novo HLA-Ab were observed in three patients and were deemed weak antibody (Ab). Median MFI showed a significant increase for nonspecific Ab, but not de novo HLA-Ab. Median MFI values were deemed transient at 7-10 week follow-up. No HLA-donor-specific Ab developed posttransplant in the patients who developed de novo HLA-Ab. Vaccination resulted in a transient increase in non-HLA-specific Ab. The majority of responses were non-HLA specific, hypothesized to be related to denatured antigens on single antigen beads. These data suggest limited clinical impact of vaccinations on the emergence of de novo HLA-Ab.

Prevalence of anti-HLA antibodies in highly sensitized kidney transplant candidates

Introduction: Kidney transplantation is the standard gold therapy for the treatment of the majority of end-stage renal diseases (ESRDs). Despite the general success rate of allogeneic transplantation due to immunosuppressive therapy, it is difficult to find an appropriate donor for some sensitized patients. Objectives: This study aimed to estimate the prevalence and titers of anti-HLA-class I and anti-HLA-class II antibodies in sensitized patients in a kidney transplantation center. The history of the risk factors of sensitization was studied. Patients and Methods: Twenty highly sensitized ESRD patients with a calculated panel-reactive antibody (CPRA) ≥50% were selected, and anti-HLA-I and anti-HLA-II antibodies were assessed in their sera using a single antigen bead (SAB) Luminex assay. Results: The previous history of kidney transplantation was the most critical sensitization risk factor. The results indicated that HLA A*24:02 and DQA1*02:01/DQB1*06:02 were the most frequent antibodies in class I and class II, respectively. Moreover, the mean fluorescence intensity (MFI) levels of anti-HLA class II antibodies were significantly higher than the MFI levels of anti-HLA class I antibodies. Conclusion: According to the findings of this study, matching HLA alleles, particularly class II molecules, can reduce sensitization in the first kidney transplant. A better understanding of the sensitization status of transplant candidates could be gained by examining CPRA values.

FC 112: HLA-Specific Memory B Cells and Paternal HLA Molecular Mismatch in Female Transplant Candidates Sensitized by Pregnancy

Abstract BACKGROUND AND AIMS Access to kidney transplantation (KT) may be limited by HLA sensitization. It is clinically relevant to know both level of HLA-immunization and potential sensitizing events in the history transplant candidates [1–3]. Pregnancy is a frequent sensitizing event in female transplant candidates. Women exposed to foreign paternal HLA can generate specific HLA antibodies (HLA-Ab) and memory B cells (mBc) [4, 5], which can be re-stimulated upon antigen reencounter after TR. We aimed to study the existence of mBc able to produce HLA-Ab in female KT candidates with previous pregnancies, to compare them with HLA-Ab in their serum and to analyse them in the context of molecular HLA mismatch between mother and father. METHOD We selected eight HLA sensitized women awaiting KT, who fulfilled these criteria: minimum of one successful pregnancy before KT (12–44 years before) from a single partner, HLA-Ab (HLA class I or II) and availability of a DNA sample from the father of their children. All women in the study cohort and the father of their children were HLA typed by next-generation sequencing (NGS®) for HLA–A, B, C, DRB1, DRB3/4/5, DQB1/A1 and DPB1. We used peripheral blood mononuclear cells cryopreserved before KT. We cultured them with a polyclonal stimulation cocktail consisting of a Toll-like receptor 7/8 agonist and a proleukin for 10 days. We examined concentrated culture supernatants (SN) derived from activated cells, purified IgG and identified HLA-Abs using single antigen beads (SAB) on Luminex. Then, we compared the HLA-Ab profiles of SN and serum. We also studied the paternal molecular HLA incompatibilities with the HLAMatchmaker software. RESULTS After 10-day polyclonal stimulation, mBc increased from 29.90% (day 0) to 65.29% (day 10) (Figure 1), and antibody-secreting cells (ASC) from 0.08% (day 0) to 29.37% (day 10). SAB analysis revealed 211 HLA-Abs in serum and 116 HLA-Abs in SN: 98/211 (46.5%) of total HLA-Ab were serum-exclusive [78/98 (52.9%) class I and 25/98 (34.3%) class II] and only 3/116 (2.59%) HLA-Ab were SN-exclusive, all class II. Finally, we found 113/327 (34.6%) HLA-Ab shared by serum and SN: 65/113 (57.5%) class I and 48/113 (42.5%) class II. Seven of eight women had Pregnancy-Induced Antibodies (PIA) in serum (20 HLA-Abs: 10 HLA class I and 10 HLA class II) and 4/8 women had PIA in SN (8 HLA-Abs: 2 HLA class I and 6 HLA class II) (Table 1). The HLAMatchmaker analysis showed that antibodies were reacting against incompatible paternal epitopes: in 6/8 women for HLA class I and 5/8 women for class HLA II in serum, and in 2/8 women for HLA class I and 4/8 women for HLA class II in SN. CONCLUSION We describe the study of the memory B cell compartment and the specific HLA-Abs produced by them with Luminex technology in KT female candidates with previous pregnancies. We found that the HLA-Abs secreted by these memory B cells followed a restricted pattern in number and intensity compared with serum HLA-Ab. The study of paternal HLA molecular mismatch incompatibilities explains many of these HLA-Abs decades after pregnancies. The entire value of this tool for risk stratification in HLA-sensitized patients awaiting TK is currently a matter of study.

MO1023: Effects of Bortezomib on Complement Fixation and IGM Reactivity in Late Abmr — Results of A Randomized Controlled Trial (The Borteject Trial)

Abstract BACKGROUND AND AIMS Late antibody-mediated rejection (ABMR) is a cardinal cause of kidney allograft failure. Currently, there is no proven effective treatment for this type of rejection. In a recent randomized controlled trial, we have reported no meaningful effect of plasma cell-directed treatment using the proteasome inhibitor bortezomib on the clinical course of late ABMR (BORTEJECT trial; ClinicalTrials.gov, NCT01873157; Eskandary et al. J Am Soc Nephrol, 2018; 29: 591). Treatment had also no significant effect on IgG type donor-specific antibody (DSA) patterns, possibly due to rapid reconstitution of the allospecific plasma cell repertoire. To further dissect the impact of bortezomib on donor-specific B cell alloimmunity, we here sought to investigate whether and to what extent bortezomib impacts the dynamics of specific alloreactivity patterns, in particular, the course of IgM type DSA and complement-activating capability of detected DSA. METHOD The BORTEJECT trial was a randomized, placebo-controlled parallel group trial designed to investigate whether two cycles of bortezomib (each cycle: 1.3 mg/m2 intravenously on days 1, 4, 8 and 11) were able to halt the progression of IgG-DSA-positive late ABMR. A total of 44 recipients were randomly assigned to receive bortezomib (n = 21) or placebo (n = 23). In the present re-analysis of biobanked serum samples obtained in the context of the trial, we applied modified single antigen bead assays to assess IgM-DSA and, in addition, complement (C1q) binding. To estimate antibody/complement binding, levels of mean fluorescence intensity (MFI) were recorded. IgM DSA was defined in relation to donor/recipient HLA typing, independently of IgG reactivity patterns. RESULTS At baseline, the complement-fixing capability of the immunodominant IgG DSA did not differ between groups. Thirty of 44 patients had detectable IgM DSA at baseline, and we observed a considerable overlap of IgG and IgM DSA specificities (22/30) without group differences. IgM DSA levels were also similar between groups. Bortezomib treatment did not affect the complement-fixing capability of IgG DSA over time. Similarly, there was no effect on the course of IgM-DSA. There were also no significant differences between groups when analysing percentage changes. CONCLUSION A limited course of bortezomib as a sole treatment strategy may not be sufficient to impact on DSA levels, including the evolution of IgM type DSA and the complement-binding capability of detected alloreactivity patterns. Our study results, which may mirror the lack of clinical efficacy, reinforce the need for the development of innovative new strategies to effectively target plasma cells and halt the progression of ABMR.

In-Depth Analysis of Anti-HLA Antibodies Using C1q Assay

Alloantibodies are significant biomarkers of posttransplant kidney rejection. With solid-phase assays, the presence of alloantibodies and complement-binding capacities can be tested. It has been noted that complement-binding anti-HLA (C1q) correlates well with high titers of anti-HLA antibodies (single antigen bead, SAB), but we have recently shown that lower SAB titers may be associated with complement in the complement cytotoxicity test. If so, low titers of donor-specific antibodies could be stratified for complement binding and used for better donor-recipient matching. To study this, we tested 268 patients awaiting kidney transplantation for SAB and C1q and stratified them into positive (Allo+) and negative (Allo-) cohorts. Next, all assayed specificities of SAB were matched with the corresponding C1q in order to correlate mean fluorescence intensity levels. We found a strong correlation between SAB and C1q for all HLA loci apart from HLA-DP. Moreover, there was no strict cutoff for C1q prediction on SAB mean fluorescence intensity level. In addition, an unusual laboratory phenotype was found; that is, a positive C1q result without corresponding SAB specificity. We tested this phenomenon and found positive IgM SAB. CONCLUSIONS: Simultaneous C1q and SAB testing may serve as a tool for in-depth analysis of alloantibodies before transplantation.

Determination of unacceptable antigens by summation of anti-HLA eplet antibody strength (MFI) based on single antigen bead assays: Excellent correlation with negative cell based cross matches

The reliability of single antigen bead (SAB) assays and their use in predicting a negative cell based cross match (CBXM) is essential in the era of expanded organ sharing. A wide range of accuracy (80–95%) in predicting negative CBXM has been reported. We hypothesized that in SAB assays an antibody against an HLA eplet that was common among a number of different HLA alleles would be distributed among all of the shared eplet positive SABs. This would reduce binding to the donor specific SAB resulting in an under-estimate of antibody strength. We tested this proposal in adsorption studies using, instead of lymphocytes, a novel reagent, single-SAB (sSAB). Properties of SAB assays were examined that provided a basis for conducting adsorption – elution experiments with the sSABs. We found that incubation of sera with sA*02:01 or sB*42:01 not only depleted reactivity to these alleles but also depleted reactivity to beads that shared the reactive eplet. Anti-eplet strength from SAB data (sum of the MFI of eplet positive SABs (MFI-s) was compared with CBXM out comes in two case studies and with 99 proficiency testing sera. In these validation studies, an MFI-s above 11,000 was associated with a positive FCXM. This approach was placed into clinical practice for listing unacceptable antigens that shared a common eplet. CDCXMs (n = 3261) and FCXMs (n = 1012) were performed on patients listed in UNOS for deceased donor kidneys. All CDCXMs were negative and all FCXMs except one were negative. We conclude that summation of eplet strength provides a highly reliable method of predicting prospective negative CBXMs resulting in substantial savings of time and effort. Based on shared eplet summation data, CMS/NYSDOH has accepted our bead based XM (BBXM) method (aka, virtual XM) performed prior to transplant as fulfilling the regulation that XM results be available before kidney transplantation.

Deciphering the role of the conjugate's phycoerythrin label in complement-mediated interference occurring in HLA single antigen Luminex bead assays.

Complement-mediated interference is a well described phenomenon in single antigen bead (SAB) Luminex assay that leads to falsely low or negative results for anti-HLA antibody (Ab). In a context of high amount of Ab, the enrichment of the Ab around the bead can lead to complement cascade activation and deposition, thereafter impairing Ab detection. EDTA is now routinely used to circumvent this interference. In this report, we attempted to decipher the role of the phycoerythrin (PE) label conjugated to the secondary Ab in this interference. Indeed, PE is a huge molecule (240 kDa) that could participate to limiting access of the conjugate to its Ab target on the bead. To this purpose, 22 sera displaying complement interference without pre-treatment with EDTA were compared on SAB assay with three detection strategies: the recommended PE-conjugated secondary Ab (IgGPE), an Alexa Fluor 532-conjugated Ab (IgGAF) bearing a tiny 724 Da fluorochrome, and a biotinylated Ab followed by PE-conjugated streptavidin (IgGBiot). Complement interference occurred with the three detection methods, but its depth, defined by the percentage of MFI loss with neat serum, was the highest for IgGPE. Our study highlighted the partial role of the PE fluorochrome in complement interference in SAB assays.

The clinical utility and thresholds of virtual and Halifaster flow crossmatches in lung transplantation

Immune sensitization, defined as the presence of alloreactive donor-specific antibodies (DSA), is associated with increased wait-times and inferior transplant outcomes. Identifying pretransplant DSA with a physical cell-based assay is critical in defining immunological risk. However, improved solid phase antibody detection has provided the potential to forgo this physical assay. Here, we evaluated the association between DSA mean fluorescence intensity (MFI) and the recently introduced Halifaster Flow cytometry crossmatch (FXM) to determine if MFI could predict the outcome of FXM and whether a virtual crossmatch (VXM) would provide an accurate risk assessment. Sera from 134 waitlisted lung patients was retrospectively assessed by Halifaster FXM against lymphocytes preparations from 32 donors, resulting in 265 FXMs. HLA typing was performed to 2-field allelic level and Luminex single antigen beads (SAB) used to identify DSA. The association between FXM and Luminex MFI was calculated using ROC analysis. MFI threshold accuracy was confirmed using a separate validation cohort (174 recipient sera and 34 donors), whereby both VXM and FXMs were compared. From the 265 FXM performed, 48 (18%) T-cell (TFXM) and 56 (21%) B-cell (BFXM) were positive. In the evaluation cohort, MFI thresholds of 2000 for HLA-A, B, DRB1, and > 4000 for DQB1, were predictive of a positive FXM. The validation cohort of 233 paired FXM and VXM confirmed these MFI thresholds for both TFXM and BFXM with an accuracy of 91.4% and 89.3%, respectively. A positive VXM, defined with HLA-specific MFI thresholds predicts Halifaster FXM reactivity, and can potentially expedite organ allocation, by minimizing the need for the more time-consuming FXM.

Clinical Perspectives towards Improving Risk Stratification Strategy for Renal Transplantation Outcomes in Indian Patients

Graft loss and rejections (acute/chronic) continue to remain important concerns in long-term outcomes in kidney transplant despite newer immunosuppressive regimens and increased use of induction agents. Global guidelines identify the risk factors and suggest a framework for management of patients at different risk levels for rejection; however, these are better applicable to deceased donor transplants. Their applicability in Indian scenario (predominantly live donor program) could be a matter of debate. Therefore, a panel of experts discussed the current clinical practice and adaptability of global recommendations to Indian settings. They also took a survey to define risk factors in kidney transplants and provide direction toward evidence- and clinical experience-based risk stratification for donor/recipient and transplant-related characteristics, with a focus on living donor transplantations. Several recipient related factors (dialysis, comorbidities, and age, donor-specific antibodies [DSAs]), donor-related factors (age, body mass index, type – living or deceased) and transplantation related factors (cold ischemia time [CIT], number of transplantations) were assessed. The experts suggested that immunological conflict should be avoided by performing cytotoxic cross match, flow cross match in all patients and DSA-(single antigen bead) whenever considered clinically relevant. HLA mismatches, presence of DSA, along with donor/recipient age, CIT, etc., were associated with increased risk of rejection. Furthermore, the panel agreed that the risk of rejection in living donor transplant is not dissimilar to deceased donor recipients. The experts also suggested that induction immunosuppression could be individualized based on the risk stratification.

Development of De Novo Donor Specific Antibodies Following COVID-19 Vaccination in Cardiac Transplant

PurposeSome transplant patients mount an insufficient response to standard COVID-19 vaccine (COVAX) dosing and a booster dose has now been approved. Vaccination carries the theoretical risk of stimulating antibody production to the donor. There are no prior studies surveying de novo donor specific HLA antibodies (dnDSA) after COVAX. HLA-DQ is the most common dnDSA post cardiac transplant and is associated with worse outcomes. We reviewed the development of dnDSA to HLA-DQ in cardiac transplant patients after COVAX.MethodsDSA testing (luminex single antigen bead) was performed routinely at the time of surveillance endomyocardial biopsy. We retrospectively recorded any dnDSA to HLA-DQ with MFI >1000 after COVAX. We further identified a historical cohort (pre vaccination) of 32 patients who had DSA testing a minimum of 5 months after transplant.Results32 adult patients completed COVAX (16 Pfizer-BioNTech, 8 Moderna, 8 Johnson and Johnson) 3-79 mths after transplantation. 16 patients had DSA testing 1-5 mths post COVAX (4-84 mths post transplant) (Table 1). Three patients (18.8 %) demonstrated dnDSA to HLA-DQ (MFI 6360-25,824) 10-20 weeks post COVAX and all had episodes of high grade acute cellular rejection. One additional patient had rejection post COVAX without dnDSA. All 3 patients with dnDSA had robust response to the vaccine with antibodies to spike protein RBD of > 200 U/ml (Elecsys®). Eight vaccinated patients without dnDSA had anti-RBD testing, half had titres > 200 U/ml. In the historical pre COVAX cohort, 3 patients (9 %) demonstrated dnDSA (2022-25480 MFI), one of these had cellular rejection and one developed antibody mediated rejection.ConclusionOne fifth of cardiac transplant recipients developed dnDSA in the weeks following COVAX, all associated with rejection. While no definitive conclusions may be drawn, we believe these data suggest the need for immunological surveillance after COVAX. This may be even more important after a booster dose.

Significant Reduction of Donor Specific Antibodies in Heart Transplant Recipients Treated with Proteasome Inhibitors for Antibody Mediated Rejection

<h3>Purpose</h3> Antibody-mediated rejection (AMR) results in deleterious outcomes, including coronary artery vasculopathy (CAV), graft failure, and death. Proteasome inhibitors (PI) were used in pre-transplant desensitization to reduce antibodies to Human Leucocytes Antigen (HLA). We report our experience treating heart transplant recipients with AMR using PI, plasmapheresis (PLEX), and intravenous immunoglobulin (IVIG). <h3>Methods</h3> We retrospectively analyzed all patients treated with PI, either bortezomib (BTZ) or carfilzomib (CFZ), for AMR at our institution from 2018-present. CFZ (20mg/m2) or BTZ (1.3mg/m2) was given in combination with PLEX on days 1,2, 8, 9, 15 and 16, followed with IVIG 2g/kg. Mean fluorescence intensity (MFI) for donor-specific antibody (DSA) was evaluated by Luminex single antigen beads (One Lambda). Graft dysfunction (GD) was defined by left ventricular ejection fraction (LVEF) < 40%. <h3>Results</h3> Eleven patients were treated with PI for AMR (10 with CFZ, 1 with BTZ). Patients received 1-3 cycles of PI (1 cycle n=4; 2 cycles n=4; 3 cycles n=3), based upon the response in DSA. Before treatment, all patients except one had one or more strong DSAs (MFI> 8000) at neat serum. Most of the 22 strong DSAs observed were specific for HLA class II (DQ, n=14, 64%; DR, n=5, 23%). The treatment reduced MFI by 47+/- 46% at neat serum and by 70+/- 31% at 1:16 dilution (Figure 1). Four DSAs from 3 patients did not respond to treatment at 1:16 dilutions: one patient had high-titer DSA before treatment (MFI=14027 at 1:1024 dilution), another had rebound DSA from pre-transplantation. 5/11 patients presented with GD at onset (median EF reduction by 67%); 3/5 recovered LVEF to >40% after treatment. 9/11 (81.8%) patients survived to 1 year after AMR diagnosis. <h3>Conclusion</h3> Using PI in AMR treatment resulted in significant DSA reduction in 8/11 patients. Large studies are needed to further evaluate PI in AMR treatment.

HLA Sensitization within One-Year of VAD Implantation: Prospective Study in Adults and Pediatrics

<h3>Purpose</h3> Ventricular assist devices (VADs) have improved survival to heart transplant (HTx). However, VADs have been associated with the development of human leukocyte antigen antibodies (HLA-Ab) which may limit the donor pool and increase risk for worse outcomes post-HTx. Since HLA-Ab development after VAD insertion is poorly understood, the aim of this prospective study was to quantify the incidence of and risk factors for HLA-Ab development post-VAD insertion across the age spectrum. <h3>Methods</h3> Adult and pediatric patients who were undergoing durable VAD placement between 5/2016 and 7/2020 were enrolled. HLA-Ab were assessed pre-VAD and at approximately 1, 3, and 12 months post-implant. HLA-Ab were measured by Luminex screen; all positive/borderline screens were confirmed by Luminex single antigen beads. Factors associated with HLA-Ab development post-VAD implant were explored. <h3>Results</h3> The study cohort included 41 adult and 17 pediatric patients. Following implant, 37% of adults and 41% of pediatric patients developed new HLA-Ab. The majority of patients (19/22) developed HLA-Ab within two months of implant and three patients developed HLA-Ab within four months post-implant. Of those that developed HLA-Ab, class I antibody development was most common (80% adult and 86% pediatric patients). Factors associated with HLA-Ab development within one year are listed in Table 1. <h3>Conclusion</h3> More than one-third of adult and pediatric VAD patients developed new HLA-Ab early after implant with the majority specific to class I. HLA-Ab development in adults was associated with female sex and pre-VAD HLA sensitization; these associations were not identified in pediatric patients. Further studies into the persistence of HLA-Ab over time in VAD patients and the clinical impact of HLA-Ab on long-term outcomes are needed.

Anti-HLA DQ Antibodies Are Associated with Chronic Lung Allograft Dysfunction in a Pediatric Lung Transplant Population

<h3>Purpose</h3> Chronic lung allograft dysfunction (CLAD) limits long term survival after lung transplantation. Antibody mediated rejection (AMR) has been associated with earlier CLAD development, but studies are limited in children. Clinical AMR can exist on a spectrum from subclinical to definite AMR based on the presence of anti-HLA donor specific antibodies (DSA), allograft dysfunction, and histopathologic findings, with DSA development first to appear. This study aims to describe the relationship of elevated DSA with clinical outcomes in a single center pediatric lung transplant population. <h3>Methods</h3> This was a single center, retrospective study. Pediatric bilateral lung transplant recipients were included from 2002-2020 if they had longitudinal HLA and lung function data for at least 6 months after transplantation. Preliminary analyses with chi-squared or Fisher's exact test for categorical variables and ANOVA or Kruskal-Wallis for continuous variables were used. DSA were detected with single-antigen bead arrays, and elevated DSA was defined as a mean fluorescence intensity level above 500. <h3>Results</h3> 34 patients were included: 19 with elevated DSA and 15 without. Most patients had subclinical AMR (n=10) without allograft dysfunction and two patients had definite AMR, based on the 2018 Banff consensus statement. The proportion of patients who developed CLAD was significantly higher in the elevated DSA group and had an earlier onset of CLAD. Average graft longevity and rates of acute cellular rejection, while not significant, trended towards shorter survival and higher rates of rejection in the elevated DSA group. All persistently elevated DSA were directed against HLA Class II DQ (6 of 14 patients) epitopes. Survival analysis and the significance of different HLA antigens remains ongoing. <h3>Conclusion</h3> The development of persistently elevated HLA Class II DQ DSA is associated with CLAD and likely decreased survival in the pediatric lung transplant population.

Accuracy of virtual crossmatch (VXM) prediction of physical crossmatch (PXM) results of donor specific antibody (DSA) in routine pretransplant settings–a single-center experience

BackgroundVirtual crossmatch (VXM) is a new powerful tool in pre-transplant risk assessment. However, the ability of VXM to predict physical crossmatch (PXM) results remains controversial. Our work evaluated the predictive potential of VXM results, measured by SAB (single antigen bead assay), for CDCXM (complement-dependent cytotoxicity crossmatch) and FLXM (flow cytometry crossmatch) results of DSA (donor specific antibody) in sensitized patients. MethodsIn total, 261 CDCXM and FLXM measurements were performed for 180 potential kidney transplant candidates, each with a single HLA-A, B, or -DR DSA against a potential deceased donor. Analysis was conducted with two SAB datasets of four-month distant and collected prior to and after PXM results. Optimal MFI (mean fluorescence intensity) thresholds and likelihood ratios were assigned based on low (<2000 MFI), medium (2001–5000 MFI) and high risk (>5000 MFI). The impact of VXM predictability was determined by the ROC curves comparison. In addition, inter-assay changes of MFI were evaluated. ResultsThe accuracy of VXM to predict CDCXM was inferior to that of FLXM with the AUC (area under ROC curve) of 0.644 vs. 0.849. In contrast, the initial ROC analysis showed that the VXM prediction was good for both T-FLXM with ROC value of 0.849 and by B-FLXM with ROC value of 0.706 for a single antigen of HLA-A, B, or -DR DSA. In fact, the best VXM prediction was for FLXM with good sensitivity for B-FLXM against HLA-DR-specific DSA (0.851). Similar results of VXM predictability were observed for pre- and post-crossmatch ROC curves. ConclusionVXM predictability is better for positive/negative FLXM than for positive/negative CDCXM results to evaluate a single HLA-A, B, -DR DSA disparity. This may be related to the fact that VXM and FLXM rely on binding of antibodies to beads or cells, respectively. In contrast, VXM is less predictive for CDCXM because the latter measures complement-dependent cytotoxic function. We intend expand VXM analysis to correlate their results with FLXM results to select low/medium risk patients for kidney transplantation in Poland.