Donor-specific anti-HLA Antibodies Levels Research Articles

Impact of Immunosuppression Intensified Conditioning Regimen for Patients with Strong Positive Pre-Transplantation Donor Specific Anti-HLA Antibodies (DSAs) Undergoing Haploidentical Hematopoietic Stem Cell Transplantation

Objective: Immunosuppression intensified conditioning regimen has been applied for 10 patients with strong positive pre-transplantation Donor specific Anti-HLA Antibodies (DSAs) undergoing Haploidentical Hematopoietic Stem Cell Transplantation (HSCT), its effects on engraftment of hematopoietic stem cell were examined. Methods: Characteristics of 10 consecutive patients with strong positive pre-transplantation DSAs were retrospectively analyzed, including gender, age, diagnosis, disease status pre-transplantation, presence of DSA, conditioning regimens, engraftment, graft versus host disease (GVHD) incidence and survival. Results: Ten patients [3 males vs 7 females, median age 53.5(36-64)] received immunosuppression intensified conditioning regimen: Fludarabine plus busulfan (FluBu) combining total body irradiation and cyclophosphamide (TBI-Cy). DSA levels gradually decreased in all patients after preparative conditioning, and the mean fluorescence intensity (MFI) values dropped significantly (P＜0.003). The average MFI values dropped 40% at day 7 post-HSCT, 8 cases acquired hematopoietic reconstitution, the median time of neutrophil engraftment was 15.6(10-16) days, and 19(13-22) days for platelet engraftment. One case had primary graft failure and another one had poor graft function. After a median follow-up of 12.5(1.5-27) months, 7 cases remained in disease remission state with negative DSA assay, including 2 cases with MFI values higher than 20000 pre-transplantation. Conclusions：Immunosuppression intensified conditioning regimen can efficiently decrease the Donor specific Anti-HLA Antibodies (DSA) level, which leads to lowered chances of primary graft failure.

Double Filtration Plasmapheresis for Desensitization during Haploidentical Stem Cell Transplantation in Patients with Positive Donor-Specific Anti-HLA Antibodies

Background:Donor-specific anti-HLA antibodies (DSA) are a major cause of engraftment failure in patients receiving haploidentical stem cell transplantation (HaploSCT). Effective treatments are needed for these patients, who often have no other donor options and/or are in need to proceed urgently to transplantation. Plasma exchange (PE) is an effective treatment widely used for DSA desensitization. However, a large volume of plasma is required. Double filtration plasmapheresis (DFPP) is a semi-selective blood purification modality derived from the PE modality. DFPP avoids the unnecessary loss of plasma proteins, reduces the need for plasma and increases the efficiency of purification. To define the efficacy of DFPP for DSA-positive patients in HaploSCT, we conducted a nested case-control study. Methods: Patients who underwent HaploSCT in the Bone Marrow Transplantation Center of the First Affiliated Hospital of Zhejiang University School of Medicine from March 2017 to November 2021 were enrolled in the study cohort. Thirty-two patients with positive DSA (MFI≥2000) were treated with desensitization protocol including DFPP. Ninety-six patients (1:3) with negative DSA were randomly matched according to gender, age and transplantation time as the control group. DSA positive patients were given alternate-day DFPP twice before starting one week before the beginning of transplant conditioning. DSA was retested after DFPP treatment. A single dose of rituximab (375 mg/m2) was administrated after completion of DFPP. Results: A total of 32 HaploSCT patients with DSAs were included in the study. The majority of patients in the DSA group were female (75%) and a mean age of 41 (13-63) years . 96 DSA negative patients were selected as controls. The two groups had equivalent subject and donor characteristics, including age, gender, underlying diseases, conditioning regimens and GVHD prophylaxis.20 (62.5%) patients presented anti-HLA class I DSAs (4 of them with > 10000 MFI), 9 (28.1%) presented anti-HLA class II DSA (2 with > 10000 MFI) and 3 (9.3%) presented both anti-HLA class I and II DSA (1 with > 10000 MFI). After DFPP treatment, levels of DSA decreased significantly. The mean MFI values before and after treatment were 7189.56 ±4294.01 vs. 2869.57±3454.55 (P < 0.001), respectively. All patients in DSA group achieved hematopoietic reconstitution within 28 day of transplantation. The mean neutrophils and platelets engraftment time was 13.44±2.42 days and 14.03±3.78 days respectively, which were comparable to control group. In the DSA and control groups, the incidence of grade III-IV aGVHD was 9.37% vs. 10.41% (P < 0.05), the 2-year cumulative recurrence rate was 26.35% vs. 15.62% (P < 0.05) and the overall survival (OS) was 77.20% vs. 81.20% (P < 0.05), respectively. Conclusion: DFPP can significantly reduce the level of DSA in patients before transplantation. Treatment with DFPP and rituximab is effective in promoting engraftment for DSA-positive patients in HaploSCT.

Impact of Immunoglobulin M-Type Donor-Specific Human Leukocyte Antigen-Antibody Levels in Supernatants from Cultured Peripheral Blood Mononuclear Cells as Predictors of Antibody-Mediated Rejection.

Background: Antibody-mediated rejection (AMR) is a crucial barrier in the long-term prognosis of transplant recipients. Methods: Peripheral blood mononuclear cells (PBMCs) were collected from kidney allograft recipients (N = 41) and cultured in vitro for 1 week. Furthermore, the supernatants of the cultured PBMCs were analyzed by Luminex single-antigen beads. Results: Analyses using Luminex single-antigen beads revealed the presence of immunoglobulin (Ig) G donor-specific anti-HLA antibodies (DSAs) was detected in the supernatants of cultured PBMCs collected more frequently than IgM in de novo DSA-sensitized patients with AMR, and IgM were detectable in patients with stable graft function mainly and several IgM DSAs were detectable in the supernatants of the cultured PBMCs before detecting the IgG levels in sera. We also found that the DSA-specific IgM-secreting memory B cells (mBCs) were more sensitive to the chronic use of immunosuppressive agents than to the IgG-secreting mBCs. Conclusions: In the transplant recipients, the assessment of supernatants of cultured PBMCs provide more details of immune reactions than the commonly used method that directly measures IgG DSA levels in patient sera and some IgM DSA detection may be a better predictor of IgG DSAs production, which may cause AMR and enable early intervention, in initial stages of AMR development.

Antithymocyte Globulin is Associated with a Lower Incidence of De Novo Donor-Specific Antibody Detection in Lung Transplant Recipients: A Single-Center Experience

De novo donor-specific anti-HLA antibodies (DSA) have been associated with significantly increased risk for antibody-mediated rejection (AMR) and lung allograft dysfunction. Induction immunosuppression has improved the long-term outcome of lung transplantation. However, the impact of Induction immunosuppression on the development of de novo DSA in lung transplant recipients is not well defined METHODS: We assessed the relationship between using rabbit Antithymocyte Globulin (rATG) for induction immunosuppression and the development of de novo DSA in a cohort of consecutive lung transplant recipients (n = 67) that were followed for 12 months post-transplant. All recipients had negative flow cytometry crossmatch on the day of transplant. Anti-HLA antibodies in the serum were assessed at 1, 3, 6, 12 months post-transplant. DSA were defined as having a Mean Fluorescence Intensity (MFI) > 1000 RESULTS: 41/67 lung transplant recipients (LTR) received a single dose rATG (1.5 mg/kg) within 24 hours of transplant. De novo DSA were detected in 10/41 (24.3%) compared to 13/26 (50%) ATG and no ATG groups respectively at 12 months post-transplant. However, de novo DSA were detected within 1 month post-transplant in 5/10 (50%) compared to 3/13 (23.3%) rATG and no rATG groups respectively. There was no significant difference in the de novo DSA MFI levels between the rATG group (median MFI = 1379 & 2124 for class I & II respectively) compared to the no rATG group (median MFI = 2186 & 2388 for class I & II respectively) at 12 months post-transplant. De novo DSA prevalence is shown in Table 1. Induction with a single dose of rATG is associated with a significant decrease in the prevalence of HLA de novo DSA in LTR at 12 months post-transplant. De novo DSA were detected earlier in the rATG group compared to no rATG group. These findings warrant investigating in a larger study the impact of rATG induction on de novo DSA levels and lung allograft dysfunction.

Desensitization Therapy for Patients with DSA Receiving Haploidentical (Haplo) Stem Cell Transplantation

<h3>Introduction</h3> The effect of desensitization therapy on patients with Donor-specific Anti-HLA Antibodies (DSA) for patients receiving Haplo transplantation are unknown. <h3>Methods</h3> Data of all 51 patients with DSA receiving Haplo transplantation between 11/2010-1/2019 at MD Anderson Cancer Center (N=41) and City of Hope (N=10) were included. Patients received desensitization with plasma exchange (PE), rituximab (R), and IVIg +/- buffy coat (BC) infused on day-1, as previously described by us (Ciurea SO. BBMT.2018:53:521). We analyzed transplant outcomes for DSA treated patients vs. those without DSA receiving Haplo transplant in the same period of time (control; N=345). <h3>Results</h3> The median age of patients with DSA was 51 years (range 19-69) and 47 years (range 18-72) in control group (p=0.15). There were no significant differences in diagnosis, remission status at transplant, DRI and ABO matching between 2 groups. RIC conditioning was used in 28% and 43% in DSA and control group, respectively (p=0.03) whereas 67% and 82% respectively (p=0.02) received marrow stem cell. Median levels of DSA on single antigen assay before desensitization was 5556 MFI (range 2360-28688). Of 51 patients with DSA, 37 (72.5%) DSA patients received desensitization with PE, R and IVIg, and 27/37 (73%) patients also received BC infusion before transplant. Twenty of 37 (54%) desensitized patients had DSA >5000 MFI and 14 out of them had complement binding DSA suggested by a positive C1q assay. Cumulative incidence (CI) of neutrophil engraftment at 28 days for patients who had DSA>5000 MFI and received desensitization was 75% compared with 91% in those without DSA (p=0.12) (Figure 1A) while 1-year NRM was 32% vs. 29% (p=0.57) (Figure 1B) and 1-year OS was 54% vs. 58%, respectively (p=0.18) (Figure 1C). The engraftment rate is significantly improved with desensitization treatment as our previous study demonstrated that only 46% of patients with DSA>5000 MFI achieved engraftment (Ciurea SO. BBMT.2015;21:1392). Remarkably, patients with DSA 2000-10000 who received treatment had 83% engraftment vs. 91% (p=0.86). Among 14 patients who had C1q+ DSA, 8 patients had persistently positive C1q after desensitization and only 4 engrafted, 2 had primary graft failure and 2 died before engraftment with CI of engraftment at 28 days of 43% (p=0.09 compared with control group). Persistent C1q positivity was associated with significantly higher NRM (37% vs. 29% at 1 year, p=0.01) and lower OS (33% vs. 58% at 1 year, p=0.002) compared with control group (Figure 1D). These significances persisted after adjusting for other factors in multivariable analysis. <h3>Conclusions</h3> Treatment with PE, R, IVIg and BC infusion is an effective strategy to desensitize patients with DSA before haploidentical transplantation. Patients who remain C1q+ at transplantation have high risk for engraftment failure and should not proceed to transplantation.

Donor-Specific Anti-HLA Antibodies in Haploidentical Stem Cell Transplantation with Post-Transplantation Cyclophosphamide: Risk of Graft Failure, Poor Graft Function, and Impact on Outcomes.

The presence of donor-specific anti-HLA antibodies (DSA) is associated with a 10-fold increased risk of graft failure in haploidentical stem cell transplantation (haplo-SCT). Consensus guidelines from the European Society for Blood and Marrow Transplantation set a mean fluorescence intensity (MFI) >1000 as a cutoff for DSA positivity. In the absence of an alternative donor, it is recommended that patients undergo desensitization therapy, especially with high DSA levels (>5000 MFI). The aim of this study was to analyze the impact of DSA on risk of graft failure and poor graft function, as well as on major outcomes in a consecutive cohort of patients who were systematically screened for DSA before haplo-SCT. A total of 141 consecutive patients were candidates for unmanipulated haplo-SCT with post-transplantation cyclophosphamide (PT-Cy) at our center between January 2012 and January 2018, and 135 were analyzed for the presence of HLA antibodies. Of these 134 patients underwent haplo-SCT. HLA antibodies were detected in 40 patients, including 19 with DSA and 21 without DSA. Ten of the 19 patients with DSA underwent transplantation using that donor, whereas 2 underwent a desensitization program before transplantation. Only 2 patients experienced primary graft failure (1.4 %), both of whom were without DSA. Twenty patients developed a poor graft function (15%). The 3-year overall survival (OS), 3-year progression-free survival (PFS), and 1-year nonrelapse mortality (NRM) were analyzed according to the presence or absence of DSA. No statistically significant difference was found. No impact of the presence of DSA on the risk of developing graft failure and poor graft function was revealed. Major outcomes of transplantation were analyzed separately in patients with poor graft function and those with good graft function. The 3-year OS, 3-year PFS, and 1-year NRM in good graft function and poor graft function populations were 62% versus 20% (P < .0001), 53% versus 20% (P < .0001), and 12% versus 40% (P = .009), respectively. The presence of low-level DSA in the absence of desensitization did not correlate with the risk of developing graft failure and poor graft function. Patients who experienced poor graft function had worse outcomes than patients with good graft function.

Donor Specific Antibody Shows Different IgG Subtype in Early versus Late Antibody Mediated Renal Allograft Rejection

Background Early and late antibody mediated rejection (EAMR/LAMR) are immunologically distinct entities and represent major risk factors for allograft survival in renal transplantation. The aim of this study was to describe the nature of donor-specific anti-HLA antibodies (DSA) that mediate EAMR and LAMR, and to correlate these data with DSA mean fluorescence intensity (MFI) response to proteasome-inhibitor (PI) therapy. Methods DSA levels (Day 0 and Day 50 therapy) were determined in 35 (11 EAMR + 24 LAMR) patients who received PI-based AMR therapy (1 or two bortezomib cycles +/- a single rituximab dose (375mg/m2)). All patients had at least 9 months of follow-up after AMR therapy. EAMR was defined as occurring within 6 months after transplantation. All donors and recipients had HLA-A,-B,-C,-DRB1, DRB3/4/5,-DQB1, DQA1 and DPB1 typing by molecular methods, and all cases were transplanted after negative prospective flow T- and B-cell crossmatches. AMR was diagnosed using Antibody Working Group and Banff criteria. Anti-HLA donor-specific antibody were identified by Luminex single-antigen beads (One Lambda) using both the generic IgG and IgG1-4 subtype-specific mAb (PE-conjugated, Southern Biotech). Epitope fingerprints were used to assess the therapeutic response (figures). Results 33 DSA were detected in EAMR (11 class I, 22 class II) and 52 DSA in LAMR (11 class I, 41 class II). DSA Class distribution was 43 anti-HLA class I, 27 anti-HLA DR, 50 anti-HLA DQ and 4 anti-HLA DP. Anti-HLA DQ DSAs were found in 3/11 EAMR vs 18/24 LAMR (p=0.01). IgG1 was detected in all patients, IgG2 was detected in 3/11 EAMR and 5/24 LAMR, IgG3 was present in 15/24 LAMR versus 0/11 EAMR (p=0.007), and IgG4 was detected in 2/11 EAMR vs 6/24 LAMR. There were 4/11 EAMR versus 16/24 LAMR patients with multiple IgG subtypes. The DSA MFI reduction at 50 days after PI therapy was 79±22% in EAMR versus 30±26% in LAMR (p=0.00001).. Summary Late AMR demonstrated: 1) a greater number of de novo DSAs; 2) a greater proportion of anti-HLA class II DSA, especially anti-HLA DQ; 3) a higher proportion of IgG3, the subtype with the highest affinity for complement (C1q) binding; 4) a higher degree of multiple IgG subtypes. 5) lesser response in DSA reduction with PI therapy.

HLA Haploidentical Stem Cell Transplant with Pretransplant Immunosuppression for Patients with Sickle Cell Disease

Allogeneic stem cell transplantation (HCT) is curative in patients with severe sickle cell disease (SCD), but a significant number of patients lack an HLA-identical sibling or matched unrelated donor. Mismatched related (haploidentical) HCT with post-transplant cyclophosphamide (PTCY) allows expansion of the donor pool but is complicated by high rates of graft failure. In this report we describe a favorable haploidentical HCT approach in a limited cohort of SCD patients with significant comorbidities. To reduce the risk of graft failure we administered the conditioning regimen of rabbit antithymocyte globulin, busulfan, and fludarabine preceded with 2 courses of pretransplant immunosuppressive therapy (PTIS) with fludarabine and dexamethasone. Graft-versus-host disease (GVHD) prophylaxis consisted of PTCY on days +3 and +4 followed by tacrolimus and mycophenolate mofetil starting on day +5. Four patients (ages 13, 19, 19, and 23 years) received T cell–replete haploidentical stem cell infusion. All patients engrafted with 99.9% to 100% donor chimerism, and all patients continued with stable engraftment at the last follow-up (5 to 11 months post-transplant). Time to neutrophil engraftment was 14 to 26 days. Two patients had high levels of donor-specific anti-HLA antibodies, which required the implementation of an antibody management protocol. This facilitated neutrophil engraftment on day +16 and day +26, respectively. One patient developed grade I acute GVHD, which resolved. Three patients developed mild, limited skin GVHD that responded to conventional immunosuppressive therapy. Human herpesvirus-6 viremia was detected in 3 patients but resolved without treatment. One patient developed asymptomatic cytomegalovirus viremia that responded appropriately to standard therapy with ganciclovir. The prompt, stable engraftment and low toxicity in the post-transplant period makes PTIS with haploidentical transplant a promising option for patients with SCD.

Unique molecular changes in kidney allografts after simultaneous liver-kidney compared with solitary kidney transplantation

Kidney allografts transplanted simultaneously with liver allografts from the same donor are known to be immunologically privileged. This is especially evident in recipients with high levels of donor-specific anti-HLA antibodies. Here we investigated the mechanisms of liver's protective impact using gene expression in the kidney allograft. Select solitary kidney transplant or simultaneous liver-kidney transplant recipients were retrospectively reviewed and separated into four groups: 16 cross-match negative kidney transplants, 15 cross-match positive kidney transplants, 12 cross-match negative simultaneous liver-kidney transplants, and nine cross-match-positive simultaneous liver-kidney transplants. Surveillance biopsies of cross-match-positive kidney transplants had increased expression of genes associated with donor-specific antigens, inflammation, and endothelial cell activation compared to cross-match-negative kidney transplants. These changes were not found in cross-match-positive simultaneous liver-kidney transplant biopsies when compared to cross-match-negative simultaneous liver-kidney transplants. In addition, simultaneously transplanting a liver markedly increased renal expression of genes associated with tissue integrity/metabolism, regardless of the cross-match status. While the expression of inflammatory gene sets in cross-match-positive simultaneous liver-kidney transplants was not completely reduced to the level of cross-match-negative kidney transplants, the downstream effects of donor-specific anti-HLA antibodies were blocked. Thus, simultaneous liver-kidney transplants can have a profound impact on the kidney allograft, not only by decreasing inflammation and avoiding endothelial cell activation in cross-match-positive recipients, but also by increasing processes associated with tissue integrity/metabolism by unknown mechanisms.

Complement-Binding Donor-Specific Anti-HLA Antibodies and Risk of Primary Graft Failure in Hematopoietic Stem Cell Transplantation

Detection of donor-specific anti-HLA antibodies (DSA) has been associated with graft rejection in all forms of transplantation. The mechanism by which DSA increase the risk of graft failure remains unclear. We hypothesized that complement-binding DSA are associated with engraftment failure in hematopoietic stem cell transplantation (HSCT) and analyzed 122 haploidentical transplant recipients tested prospectively for DSA. Retrospective analysis to detect C1q binding DSA (C1q+DSA) was performed on 22 allosensitized recipients. Twenty-two of 122 patients (18%) had DSA, 19 of which were women (86%). Seven patients with DSA (32%) rejected the graft. Median DSA level at transplant for patients who failed to engraft was 10,055 mean fluorescence intensity (MFI) versus 2065 MFI for those who engrafted (P = .007). Nine patients with DSA were C1q positive in the initial samples with median DSA levels of 15,279 MFI (range, 1554 to 28,615), compared with 7 C1q-negative patients with median DSA levels of 2471 MFI (range, 665 to 12,254) (P = .016). Of 9 patients who were C1q positive in the initial samples, 5 patients remained C1q positive at time of transplant (all with high DSA levels [median, 15,279; range, 6487 to 22,944]) and experienced engraftment failure, whereas 4 patients became C1q negative pretransplant and all engrafted the donor cells (P = .008). In conclusion, patients with high DSA levels (>5000 MFI) and complement-binding DSA antibodies (C1q positive) appear to be at much higher risk of primary graft failure. The presence of C1q+DSA should be assessed in allosensitized patients before HSCT. Reduction of C1q+DSA levels might prevent engraftment failure in HSCT.

Impact of alloantibody strength in crossmatch negative DSA positive kidney transplantation

ObjectivesThe clinical relevance of pre-transplant “low-level” donor specific anti-HLA antibodies (DSAs) in crossmatch negative kidney transplant recipients remains unclear. To determine what level of DSA associates with antibody mediated rejection (AMR) could be the way to measure the clinical relevance of pre-transplant “low-level” donor specific anti-HLA antibodies (DSAs) in crossmatch negative kidney transplant recipients. Design and methodsA retrospective analysis of 221 patients from October 2008 to December 2009 was included in this study. Sera were obtained pre-transplant and two weeks post-transplant and tested for DSA using LABScreen single antigen beads. ResultsAmong the 221 patients, 11 experienced AMR within 200days after transplant (5%). Pre-transplant DSA was associated with AMR at multiple mean fluorescence intensity (MFI) cutoffs (500, 1000, 2000, 3000, 5000; p=0.003, 0.001, 0.007, 0.003, and 0.003, respectively). No correlation was seen between acute T-cell mediated rejection (CMR) and pre-transplant DSA at any of the same MFI cutoffs. There was an increased risk of AMR with higher levels of pre-transplant DSA. Finally, an increase in DSA MFI from pre- to two weeks post-transplant was indicative of a higher probability of AMR. ConclusionOverall, this data supports using the single antigen bead to detect “low-level” DSA both pre- and post- as having a positive and persistent DSA may be predictive of higher AMR rates and poorer graft survival.

Five-Year Outcomes in Living Donor Kidney Transplants With a Positive Crossmatch

Renal transplant candidates with high levels of donor-specific anti-HLA antibodies have low transplantation rates and high mortality rates on dialysis. Using desensitization protocols, good short-term outcomes are possible in "positive crossmatch kidney transplants (+XMKTx)", but long-term outcome data are lacking. The aim of the current study was to determine actual 5-year graft outcomes of +XMKTx. We compared graft survival and the functional and histologic status of 102 +XMKTx to 204 -XMKTx matched for age and sex. Actual 5-year death-censored graft survival was lower in the +XMKTx group (70.7% vs. 88.0%, p < 0.01) and chronic injury (glomerulopathy) was present in 54.5% of surviving grafts. Graft survival was higher in recipients with antibody against donor class I only compared with antibody against class II (either alone or in combination with class I) (85.3% vs. 62.6%, p = 0.05) and was similar to -XMKTx (85.3 vs. 88.0%, p = 0.64). Renal function and proteinuria ranged across a wide spectrum in all groups reflecting the different histological findings at 5 years. We conclude that when compared to -XMKTx, +XMKTx have inferior outcomes at 5 years, however, almost half of the surviving grafts do not have glomerulopathy and avoiding antibodies against donor class II may improve outcomes.

HLA Antibodies Detected by Luminex-Single Antigen Beads and Virtual Crossmatch in Renal Transplantation

Introduction: The most important immunologic consideration for kidney transplantation is the presence of preformed donor-specific anti-HLA antibodies (DSA). Solid-phase assays, such as Luminex-Single Antigen Beads (LSA), allow an accurate detection and characterization of pre-existing DSA in transplant candidates. But the ability of LSA in detecting low level of antibodies makes hard to correctly predict crossmatch results in donor selection, that is to perform a virtual-crossmatch (v-XM). In this study was to retrospectively analyze the accuracy of our v-XM protocol in predicting the results of actual-crossmatch (a-XM) in renal transplantation. We also investigated correlation between a-XM negative results and strength/specificity of pre-formed DSA. Methods: We retrospectively analyzed the correlation between v-XMs and a-XMs performed within January 2007 and June 2011 at Regional Transplant Center of Lazio (Italy). In carrying out v-XM, we considered as “unacceptable mismatches” the donor HLA class I and class II molecules against which the patients showed LSA-detected DSA with normalized MFI ≥5000. The LSA-DSA showing MFI lower than the established cutoff were defined as “acceptable DSA” for v-XM. All donors were routinely typed for HLA-A, -B, -DR and -DQB1 molecules by SSP methods. Consequently, in performing v-XM, we didn’t consider anti-HLA-C, -DQA and -DP DSA. On the basis of a negative v-XM, we performed 328 a-XMs between sera from 194 sensitized renal transplant candidates (%PRA class I = 47±39, %PRA class II = 59±32) and T/B lymphocytes from 219 cadaver donors using both cytotoxic-crossmatch (CDCXM) and flow-cytometry-crossmatch (FCXM). Results: The v-XM results predicted by LSA-DSA showed good correlation with both CDC and FC aXMs (96% and 90% respectively; Sensitivity = 100%). The high sensitivity of v-XM was related to the lack of false-negative DSA results. The limited specificity with both the XM techniques (CDCXM = 78%, FCXM = 81%) was due to the presence of “acceptable DSA” and/or anti-DQA/-DP DSA in sera used to perform 86 a-XMs. Ten of these a-XMs gave positive results by both CDC and FC assays, 20 gave positive results by FCXM only and 56 were negative by both the assays. During the study period, 104 (54%) of the 194 sensitized patients received a kidney transplant. Twenty-eight of the 104 (27%) transplanted patients had “acceptable” DSA and/or anti-DQA/-DP DSA. Late antibody mediate rejection due to preformed DSA was observed in 2 patients only; anti-DP DSA at “unacceptable” levels (MFI>5000) were present in these patients. Conclusion: Our v-XM protocol showed high sensitivity in predicting donor-recipient immunologic compatibility. Data presented here demonstrate the importance to evaluate DSA strength for implementing v-XM results in selection of recipient for kidney transplantation. Nevertheless prediction based on LSA assay feels the amount of antigen on beads and may not be representative of the amount found on cells. The findings of anti-DPB/-DQA DSA highlight the importance to define all the donor HLA molecules to perform an accurate v-XM. Finally, the good outcome of transplants carried out in patients with low levels of DSA and negative XM underlines the importance to discard discarding weak and clinically irrelevant DSA in performing v-XM.

Use of Eculizumab for Rescue Therapy of a Severe Antibody-Mediated Rejection in a Living Kidney Transplant

Introduction: treatment options for antibody-mediated rejection in kidney transplant recipients showing donor specific anti-HLA antibodies (DSA), include splenectomy, plasmapheresis or immunoadsorption, high dose IVIG, anti-CD20 antibody. In few cases, as a rescue therapy, in various associations, the new drugs eculizumab and bortezomib have been reported with variable success and with significant drug-related comorbidities. We describe the case of a living donor kidney transplant recipient with DSA, showing a severe antibody-mediated rejection despite pre-transplant desensitization, successfully treated with eculizumab, plasmapheresis (PP) and low-dose CMV IgG (100 mg/kg). Methods: The PRA of the 39 year-old female recipient was 65% as a result of sensitization from two prior pregnancies. The complement-dependent cytotoxicity crossmatch (CDCXM) with her blood type compatible donor (husband) was negative and the Luminex was positive to the donor's HLA-B7 (M FI 6200) e HLA-B55 (MFI 4200). The desensitization protocol included 2 PP/CMVIgG prior to transplant and 2 PP/CMVIgG after the transplant; the intake of immunosuppressive therapy Tacrolimus 0,2 mg/kg and e MPA 1440 mg/die was started on day -5. At the time of transplantation DSA levels were < 2000 for B7 and B55. Total Thymoglobulin dose at induction was 8.3 mg/kg. The allograft had prompt function and serum creatinine rapidly fell to 94 umol/l. On postoperative day (POD) 5, because of worsening graft function, a kidney allograft biopsy was performed showing severe acute humoral rejection ATN-like, with C4d deposition (+3) and strong C5b9 positivity. DSA showed an increase in intensity of anti-HLA-B7, HLA-B55 and appearance of anti-HLA-DR15 and NDSA with MFI> 15000. Daily sessions of PP/CMVIgG and pulses of steroids failed to improve renal function, therefore on POD 10 rescue therapy with Eculizumab was initiated at a dose of 1200 mg, followed by 4 doses of 900 mg weekly, 2 additional doses of 600 mg after each PP weekly, and 2 doses of 1200 mg on POD 47 and on POD 55. Results: Forty eight hours after eculizumab infusion, urine output began to rise and renal function improved. A protocol biopsy performed at 7 weeks after transplantation showed no signs of rejection, the decrease in C5b-9 deposition and C4d negative. At 6 months, the patient had satisfactory kidney function (serum creatinine 160 μmol/L), and histological evaluation revealed no evidence of sustained graft damage. Conclusion: This is the first case reported on the utilization of eculizumab as a rescue therapy for a severe antibody-mediated rejection without splenectomy, anti-CD20 or proteasome inhibitor therapy. Eculizumab proved to be effective in protecting against complement-mediated renal injury while DSA were removed through PP. The entire treatment was well tolerated without infectious complications.

HLA Antibodies Detected by Luminex-Single Antigen Beads and Virtual Crossmatch in Renal Transplantation

Introduction: The most important immunologic consideration for kidney transplantation is the presence of preformed donor-specific anti-HLA antibodies (DSA). Solid-phase assays, such as Luminex-Single Antigen Beads (LSA), allow an accurate detection and characterization of preexisting DSA in transplant candidates. But the ability of LSA in detecting low level of antibodies makes hard to correctly predict crossmatch results in donor selection, that is to perform a virtual-crossmatch (v-XM). In this study was to retrospectively analyze the accuracy of our v-XM protocol in predicting the results of actual-crossmatch (a-XM) in renal transplantation. We also investigated correlation between a-XM negative results and strength/specificity of pre-formed DSA. Methods: We retrospectively analyzed the correlation between v-XMs and a-XMs performed within January 2007 and June 2011 at Regional Transplant Center of Lazio (Italy). In carrying out v-XM, we considered as “unacceptable mismatches” the donor HLA class I and class II molecules against which the patients showed LSA-detected DSA with normalized MFI ≥5000. The LSA-DSA showing MFI lower than the established cutoff were defined as “acceptable DSA” for v-XM. All donors were routinely typed for HLA-A, -B, -DR and -DQB1 molecules by SSP methods. Consequently, in performing v-XM, we didn't consider anti-HLA-C, -DQA and -DP DSA. On the basis of a negative v-XM, we performed 328 a-XMs between sera from 194 sensitized renal transplant candidates (%PRA class I = 47±39, %PRA class II = 59±32) and T/B lymphocytes from 219 cadaver donors using both cytotoxic-crossmatch (CDCXM) and flow-cytometry-crossmatch (FCXM). Results: The v-XM results predicted by LSA-DSA showed good correlation with both CDC and FC aXMs (96% and 90% respectively; Sensitivity = 100%). The high sensitivity of v-XM was related to the lack of false-negative DSA results. The limited specificity with both the XM techniques (CDCXM = 78%, FCXM = 81%) was due to the presence of “acceptable DSA” and/or anti-DQA/-DP DSA in sera used to perform 86 a-XMs. Ten of these a-XMs gave positive results by both CDC and FC assays, 20 gave positive results by FCXM only and 56 were negative by both the assays. During the study period, 104 (54%) of the 194 sensitized patients received a kidney transplant. Twenty-eight of the 104 (27%) transplanted patients had “acceptable” DSA and/or anti-DQA/-DP DSA. Late antibody mediate rejection due to preformed DSA was observed in 2 patients only; anti-DP DSA at “unacceptable” levels (MFI>5000) were present in these patients. Conclusion: Our v-XM protocol showed high sensitivity in predicting donor-recipient immunologic compatibility. Data presented here demonstrate the importance to evaluate DSA strength for implementing v-XM results in selection of recipient for kidney transplantation. Nevertheless prediction based on LSA assay feels the amount of antigen on beads and may not be representative of the amount found on cells. The findings of anti-DPB/-DQA DSA highlight the importance to define all the donor HLA molecules to perform an accurate v-XM. Finally, the good outcome of transplants carried out in patients with low levels of DSA and negative XM underlines the importance to discard discarding weak and clinically irrelevant DSA in performing v-XM.

Antibody-mediated rejection

The diagnosis of antibody-mediated rejection (AMR) has been revolutionized in recent years by advances in the detection of human leukocyte antigen (HLA) and non-HLA antibodies and by the standardized classification of histologic and immunologic changes in endomyocardial biopsies. The treatment of AMR is also undergoing significant development with targeted use of therapies directed at antibody production and function. The diagnosis of AMR achieved greater standardization after a consensus conference in 2010. By the proposed classification, endomyocardial biopsies are now graded on the basis of the severity of histologic and immunologic changes. Management of AMR is often complicated, as patients may have persistent symptoms after treatment, including a reduction in ejection fraction, restrictive physiology leading to recurrent heart failure, and accelerated progression of transplant coronary artery disease. We often rely on therapies to reduce the levels of donor-specific anti-HLA antibodies, including rituximab and bortezomib, as well as photopheresis to alter the function of T cells, although such patients may go on to require redo transplantation. AMR offers challenges of diagnosis and management, but recent advances in the detection of antibodies, the interpretation of endomyocardial biopsies, and the use of therapies directed toward antibody production offer great promise. In the future, standardization of management across transplant centers will allow for clinical trials of the effectiveness of these therapies.

Bortezomib as the Sole Post‐Renal Transplantation Desensitization Agent Does Not Decrease Donor‐Specific Anti‐HLA Antibodies

Persistence of donor-specific anti-HLA antibodies (DSA) associated with antibody-mediated graft injuries following kidney transplantation predicts evolution toward chronic humoral rejection and reduced graft survival. Targeting plasma cells, the main antibody-producing cells, with the proteasome inhibitor bortezomib may be a promising desensitization strategy. We evaluated the in vivo efficacy of one cycle of bortezomib (1.3 mg/m(2)x 4 doses), used as the sole desensitization therapy, in four renal transplant recipients experiencing subacute antibody-mediated rejection with persisting DSA (>2000 [Mean Fluorescence Intensity] MFI). Bortezomib treatment did not significantly decrease DSA MFI within the 150-day posttreatment period in any patient. In addition, antivirus (HBV, VZV and HSV) antibody levels remained stable following treatment suggesting a lack of efficacy on long-lived plasma cells. In conclusion, one cycle of bortezomib alone does not decrease DSA levels in sensitized kidney transplant recipients in the time period studied. These results underscore the need to evaluate this new desensitization agent properly in prospective, randomized and well-controlled studies.