Presence Of Donor-specific HLA Antibodies Research Articles

Immunized Patients Face Reduced Access to Transplantation in the Eurotransplant Kidney Allocation System.

The presence of donor-specific HLA antibodies before transplantation is associated with poor transplantation outcomes. Unacceptable antigens can be assigned for Eurotransplant kidney transplant candidates to prevent kidney offers against which the candidate has developed clinically relevant HLA antibodies. This retrospective cohort study aimed to assess to what degree unacceptable antigens affect access to transplantation in the Eurotransplant Kidney Allocation System (ETKAS). Candidates who underwent kidney-only transplantation between 2016 and 2020 were included (n = 19 240). Cox regression was used to quantify the relationship between the relative transplantation rate and virtual panel-reactive antibodies (vPRAs), which is the percentage of the donor pool with unacceptable antigens. Models used accrued dialysis time as the timescale; were stratified by country and blood group of patient and were adjusted for nontransplantable status, patient age, sex, history of kidney transplantations, and prevalence of 0 HLA-DR-mismatched donors. Transplantation rates were 23% lower for vPRA 0.1% to 50%, 51% lower for vPRA 75% to 85%, and decreased rapidly for vPRA of >85%. Prior studies showed significantly lower ETKAS transplantation rates only for highly sensitized patients (vPRA of >85%). The inverse relationship between transplantation rate and vPRA is independent of Eurotransplant country, listing time, and 0 HLA-DR-mismatched donor availability. Results were similar when quantifying the relationship between vPRA and attainment of a sufficiently high rank for an ETKAS offer, suggesting lower transplantation rates for immunized patients are due to current ETKAS allocation. Immunized patients face lower transplantation rates across Eurotransplant. The current ETKAS allocation mechanism inadequately compensates immunized patients for reduced access to transplantation.

Donor-Specific HLA Antibodies Are Associated with Graft Failure and Delayed Hematologic Recovery after Unrelated Donor Hematopoietic Cell Transplantation

Graft failure (GF) is one of the major concerns after allogeneic hematopoietic cell transplantation (allo-HCT) and remains a significant cause of morbidity and mortality. Although earlier reports have associated the presence of donor-specific HLA antibodies (DSAs) with increased risk of GF after unrelated donor allo-HCT, recent studies have failed to confirm this association. We sought to validate the presence of DSAs as a risk factor for GF and hematologic recovery in the unrelated donor allo-HCT setting. We retrospectively evaluated 303 consecutive patients who underwent their first unrelated donor allo-HCT at our institution from January 2008 to December 2017. DSA evaluation was performed using 2 Single Antigen Beads (SAB) assays, DSA titration with 1:2, 1:8, and 1:32 dilutions, C1q-binding assay, and absorption/elution protocol to assess possible false-positive DSA reactivity. The primary endpoints were neutrophil and platelet recovery and GF, whereas the secondary endpoint was overall survival. Multivariable analyses were performed using Fine-Gray competing risks regression or Cox proportional hazards regression models. The median patient age was 14 years (range, 0-61 years), 56.1% were male, and 52.5% were transplanted for nonmalignant diseases. Eleven patients (3.63%) were DSA-positive. Of them, 10 had preexisting DSAs, and one showed post-transplant de novo DSA. Nine patients had 1 DSA, 1 had 2 DSAs, and 1 had 3 DSAs, with a median MFI of 4334 (range, 588-20,456) and 3581 (range, 227-12,266) in LABScreen and LIFECODES SAB assays, respectively. Overall, 21 patients experienced GF. Of them, 12 had primary graft rejection, 8 had secondary graft rejection, and 1 had primary poor graft function. The cumulative incidences of GF at 28, 100, and 365 days were 4.0% (95% CI, 2.2%-6.6%), 6.6% (95% CI, 4.2%-9.8%), and 6.9% (95% CI, 4.4%-10.2%), respectively. In the multivariable analyses, DSA-positive patients had significantly delayed neutrophil (subdistribution hazard ratio [SHR] = 0.48; 95% CI, 0.29-0.81; P = .006) and platelet recovery (SHR = 0.51; 95% CI, 0.35-0.74; P = .0003) than patients without DSAs. In addition, only DSAs were significant predictors of primary GF at 28 days (SHR = 2.78; 95% CI, 1.65-4.68; P = .0001). The Fine-Gray regression also demonstrated that the presence of DSAs was strongly associated with a higher incidence of overall GF (SHR = 7.60; 95%CI, 2.61-22.14; P = .0002). DSA-positive patients with GF had significantly higher median MFI values than DSA-positive patients who achieved engraftment in LIFECODES SAB assay using neat serum (10,334 vs. 1250; P = .006) and in LABScreen SAB at 1:32 dilution (1627 vs. 61; P = .006). All 3 patients with C1q-positive DSAs failed to engraft. DSAs were not predictive of inferior survival (hazard ratio = 0.50; 95% CI, 0.20-1.26, P = .14). Our results validate the presence of DSAs as a significant risk factor for GF and poor hematologic recovery after unrelated donor allo-HCT. Thus, careful pre-transplant DSA evaluation may optimize unrelated donor selection and improve allo-HCT outcomes.

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.

Data-driven Derivation and Validation of Novel Phenotypes for Acute Kidney Transplant Rejection using Semi-supervised Clustering.

Over the past decades, an international group of experts iteratively developed a consensus classification of kidney transplant rejection phenotypes, known as the Banff classification. Data-driven clustering of kidney transplant histologic data could simplify the complex and discretionary rules of the Banff classification, while improving the association with graft failure. The data consisted of a training set of 3510 kidney-transplant biopsies from an observational cohort of 936 recipients. Independent validation of the results was performed on an external set of 3835 biopsies from 1989 patients. On the basis of acute histologic lesion scores and the presence of donor-specific HLA antibodies, stable clustering was achieved on the basis of a consensus of 400 different clustering partitions. Additional information on kidney-transplant failure was introduced with a weighted Euclidean distance. Based on the proportion of ambiguous clustering, six clinically meaningful cluster phenotypes were identified. There was significant overlap with the existing Banff classification (adjusted rand index, 0.48). However, the data-driven approach eliminated intermediate and mixed phenotypes and created acute rejection clusters that are each significantly associated with graft failure. Finally, a novel visualization tool presents disease phenotypes and severity in a continuous manner, as a complement to the discrete clusters. A semisupervised clustering approach for the identification of clinically meaningful novel phenotypes of kidney transplant rejection has been developed and validated. The approach has the potential to offer a more quantitative evaluation of rejection subtypes and severity, especially in situations in which the current histologic categorization is ambiguous.

P045 Kidney transplant with historic but no current DSA – A case study

Presence of donor-specific HLA antibodies (DSA) at the time of transplant is associated with poor outcome. DSA may be present in historic serum samples but not at the time of a kidney offer. Going forward with the transplant holds the risk of a memory B-cell response upon re-exposure to a specific HLA antigen or epitope. A patient with X-linked Alport syndrome was investigated for a kidney transplant. Anti-HLA class II antibodies were observed following pregnancy, which were specific to her husband’s mismatched DR and DQ antigens. A year later, she received a blood transfusion and had an episode of aseptic peritonitis while on dialysis. Class I HLA antibodies appeared but were observed over a period of 7 months. Thereafter, anti-HLA antibody screening using FlowPRA beads and identification using Luminex single antigen beads showed disappearance of class I antibodies but persistence of a single anti-HLA-DR antibody developed after prior pregnancy. Three years later, a kidney from a DCD donor was offered and transplanted despite presence of historic DSA against 2 HLA-B antigens. A CDC crossmatch proved negative using a current serum whereas a flow cytometric crossmatch using 3 DSA-containing historic sera gave positive but inconsistent results (T + B+, T-B+, T-B-). One week after transplant, historic DSA reappeared (3000 MFIs). Despite absence of biopsy-proven rejection, short-term treatment comprising plasmapheresis, rituximab and low-dose IVIg was initiated. DSA quickly disappeared from circulation with only an HLA-DR-specific reactivity from the previous pregnancy. Without any further desensitization treatment, the patient has not demonstrated re-appearance of DSA nor experienced antibody-mediated rejection (AMR) as demonstrated upon for-cause and protocol biopsies, up to 8 months posttransplant at the time of abstract submission. This case demonstrates that, although DSA may reappear following re-exposure to a sensitizing HLA antigen/epitope, acceptable transplant outcome can be achieved without AMR. Time between DSA development and transplant could be a factor in identifying acceptable HLA mismatches.

Assessing Antibody Strength: Comparison of MFI, C1q, and Titer Information

The presence of donor-specific HLA antibodies before or after transplantation may have different implications based on the antibody strength. Yet, current approaches do not provide information regarding the true antibody strength as defined by antigen-antibody dissociation rate. To assess currently available methods, we compared between neat mean fluorescence intensity (MFI) values, C1q MFI values, ethylenediaminetetraacetic acid (EDTA)-treated samples, as well as titration studies and peak MFI values of over 7000 Luminex-based single-antigen HLA antibody data points. Our results indicate that neat MFI values do not always accurately depict antibody strength. We further showed that EDTA treatment (6%) does not always remove all inhibitory factors compared with C1q or titration studies. In this study of patients presenting with multiple antibody specificities, a prozone effect was observed in 71% of the cohort (usually not affecting all antibody specificities within a single serum sample, though). Similar to titration studies, the C1q assay was able to address the issue of potential inhibition; however, its limitation is its low sensitivity and inability to detect the presence of weak antibodies. Titration studies are the only method among the approaches used in this study to provide information suggesting antigen-antibody dissociation rates and are, therefore, likely to provide better indication of true antibody strength.

Looking Behind the Curtain: Pre-Transplant Mismatched Eplet Load May Better Predict Increases in Post-Transplant DSA.

Purpose: Patients receiving renal transplants in the presence of donor specific HLA antibodies (DSA) are at increased risk for antibody mediated rejection (AMR) as well as decreased graft survival. For highly sensitized patients, transplanting in the presence of low/moderate DSA may be the only viable option. We looked at which characteristics of pre-existing DSA are associated with poor post-transplant(tx) outcomes. Methods: We identified 11 patients (mean cPRA=87+7%) with DSAs from a cohort of 165 consecutive renal txs performed between 10/2012 and 12/2013. Patients were desensitized using plasmapheresis(PP) and IVIg and received standard induction and maintenance therapy post-tx. DSA titers and MFI were determined by solid phase assays (pre-Tx, day 3, weekly 1-4, months 2, 3, 6, 9, 12). Donor-recipient eplet mismatches (MM) in the DSA-associated HLA antigens were determined using HLA Matchmaker. Results: Post-tx, 5 of the 11 (45.5%) desensitized patients no longer had clinically significant DSA. Increased DSA were found in the remaining 6 (55.5%) within 2 weeks (MFI=3469-15000, titers 1:16->1:1024) and 5 developed de novo DSA. No association between pre-tx MFI values, positive xmatch (T or B), or previous tx and increased post-tx DSA was found. In contrast, patients with negative DSA post-tx averaged 6.4+3.6 eplet MM in pertinent HLA antigen, whereas those with increasing DSA post-tx averaged 16.8+5.6 MM eplets.Expression of Cytosolic DoubleFigure: No Caption available.Increased DSA were successfully reversed in 2 patients with PP and IVIg and in 1 with increased basal immunosuppression. The remaining 3, after treatment, have DSA levels that are stable or slightly lower with surviving but sub-optimal graft function. Conclusions: Renal transplants may be performed successfully in presence of DSA, but remain at increased risk for graft failure or poor function. Our data suggest that mismatched eplet load is predictive of clinically significant post-tx DSA and may identify patients in need of frequent monitoring and aggressive early intervention to minimize DSA load and improve outcomes.

Complement Binding (C1q) and MFI of DSA at Time of Initial C4d+ Only Biopsies Are Not Associated With Progression of Subclinical Antibody Mediated Rejection in Heart Transplant.

Considering complement-binding (C1q+) donor-specific HLA antibodies (DSA) was reported to improve stratification of patients at risk for graft lose in kidney transplantation. In addition MFI values generated by Luminex-based solid phase assays of HLA antibody detection are commonly used semi-quantitatively as a surrogate marker of HLA antibody strength with high MFI values associated with increased risk of rejection. In this study we investigate HLA antibody characteristics at time of initial C4d+ (Subclinical AMR) in association with conversion to concomitant C4d+/C3d+ in heart biopsies that has been correlated with circulating DSA and graft dysfunction. Of 1589 transplants, 610 were screened for AMR in the last 6 years. We included patients with stored serum specimens collected ± one day of the biopsy date. Three groups were investigated including asymptomatic patients with C4d+ biopsies till last follow up (n=29), symptomatic patients with C4d+C3d+ biopsy (n=28) and patients who started as C4d+ then converted to C4d+C3d+ (n=11). We investigated HLA antibody characteristics including Class I vs. II, breadth (cPRA), presence of DSA, MFI of the highest reacting DSA, and C1q results. We also compared pre-transplant DSA, cPRA and crossmatch results. No significant differences were noted among groups in pre-transplant mean cPRA, incidence of DSA, or positive crossmatch. In contrast, at time of initial biopsy, significant differences in incidence of DSA and mean cPRA were noted (table 1).Table: No Caption available.C4d+ converters had significantly higher incidence of DSA (more dominantly class II) and higher mean cPRA compared to patients who remained C4d+ (P 0.2 for both). Having DSA at time of initial biopsy was associated with C4d+ conversion with high negative predictive value (1.0) but low positive predictive value (0.38). Interestingly, C4d converters had lower mean MFI (p NS) compared to those who remained C4d+ only and none of the converters was associated with C1q+ DSA. Some HLA antibody characteristics such as absence of DSA and lower cPRA correlate with C4d+/C3d+ patterns and may be relevant in determining which subclinical C4d+ patients may not progress to clinical AMR however C1q and MFI were not predictive.

Therapeutic apheresis in renal transplantation; current practices.

Apheresis is an important treatment modality for the removal of pathologic antibodies and circulating proteins in kidney transplantation. The use of apheresis has been shown to be a necessary preconditioning component in ABO incompatible kidney transplant. Removal of pathologic anti-A and anti-B antibodies has been accomplished with a variety of apheresis modalities including plasma exchange, fractional plasma exchange, and immunoabsorption techniques. Using these modalities in conjunction with potent modern immunosuppression, ABO incompatible kidney transplants have achieved graft and patient survivals similar to that seen in ABO compatible transplants. Apheresis has also been an important modality in the removal of anti-human leukocyte antigen (HLA) antibodies both for the purposes of desensitization and treatment of antibody mediated rejection of the kidney. Although good randomized controlled trials are lacking in the treatment of acute antibody mediated rejection, most treatment regimens include the use of apheresis as an essential component for reduction of anti-HLA antibody titers. Similarly, a variety of desensitization protocols have been developed to allow highly sensitized kidney transplant candidates to be successfully transplanted in the presence of donor-specific HLA antibodies. Most of these protocols involve apheresis to improve the removal of pathologic antibodies. Finally, aphereis has been used with mixed success for the treatment of recurrent focal segmental glomerulosclerosis. Evidence indicates that in some cases a circulating factor exists which apheresis can remove and ameliorate the nephrotic proteinuria.

Red cell and human leukocyte antigen alloimmunization in candidates for renal transplantation: a reality

Red cell and human leukocyte antigen alloimmunization in candidates for renal transplantation: a reality

Development of Donor-Specific HLA-Antibodies after Kidney Transplantation Is Associated with Inferior Renal Function

Introduction: The clinical relevance of donor-specific HLA antibodies (DSA) detected in highly sensitive solid phase assays such as Luminex prior to kidney transplantation has been discussed controversially. Furthermore, the impact of de novo HLA antibodies occuring posttransplant detected with the same method on graft outcome is unclear. The aim of our study was to investigate to which extent preexisting as well as de novo donor-specific and non-donor-specific HLA antibodies (non-DSA) influence renal allograft function after kidney transplantation. Patients and methods: We retrospectively analyzed 1278 patients with negative complement-dependent cytotoxicity crossmatches who received a kidney transplantation within the last 15 years. Patient sera obtained prior of transplantation as well as at 2 time points thereafter (approximately 2 and 4 years) were investigated for the presence of HLA antibodies. HLA class I and II antibodies were specified by means of a solid phase assay (Luminex, Genprobe). The results were correlated with renal allograft function at the two given time points. To estimate renal function serum creatinine levels were measured within the same samples. Results: The mean age in our study population was 48 ±12 years, 62% were male recipients. The majority (90.5%) of recipients had received an organ from a cadaveric donor. One hundred sixty of 1278 patients (12.5%) were retransplanted. Only 9.4% (120/1278) of our patients were already immunized before transplantation as indicated by the presence of DSA in the sera obtained pretransplantat. Notabley, renal function did not differ in patients with DSA as compared to patients without DSA at 2 and 4 years after transplantation (serum creatinine 1.5mg/dl vs 1.6mg/dl and 1.7mg/dl vs 1.6 mg/dl, respectively). However, 3.3% of recipients (42/1278) developed de novo DSA at 2 years after transplantation and 7.3% (93/1278) at 4 years after transplantation. Furthermore, de novo non-DSA could be detected in the sera of 5.0% (64/1278) and 4.5% (58/1278) recipients at the 2 time points. The presence of de novo DSA correlated with higher serum creatinine levels at any investigated time point suggesting that the development of DSA negatively influences renal function, while the presence of non-DSA did not impact renal function. Renal function was significantly impaired in patients with de novo DSA (serum creatinine 2.4 ± 0.8 mg/dl) as compared to those with de novo non-DSA (1.9 ± 0.7 mg/dl) and patients without HLA antibodies (1.6 ± 1.0 mg/dl) 4 years after transplantation (p< 0.05). Conclusion: Our data suggest, that the development of de novo donor-specific antibodies is detrimental for the outcome after kidney transplantation, while the existence of DSA prior to transplantation as well as the development of non-DSA after transplantation seem to be less harmfull. A periodically defined post TX monitoring for de novo HLA antibodies should therefore be taken into account to guide further therapeutic considerations.

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Many Histocompatibility and Immunogenetics laboratories across the world are increasingly using the tool of Virtual crossmatching to predict positive XM based on the information about the presence of donor-specific HLA antibody (HLA-DSA) in patients. However, lab to lab variation in quantifying HLA-DSA is the major limitation to determine a cutoff concentration of DSA that can predict a positive XM. Here, we address this issue by assessing a cutoff value for HLA-DSA detected by solid-phase immunoassay (represented as mean fluorescence intensity, MFI) in a retrospective analysis that can virtually predict a positive CDC-AHG XM. We performed a retrospective study for 61 XM performed for 53 renal transplant recipients. Availability of results for HLA antibodies in patients, HLA typing of donor and patient and result of CDC-AHG XM were the inclusion criteria. The MFI values for DSA in patients were compared with the results of CDC-AHG XM. HLA class I and/or class II DSA were identified in 34 renal transplant patients. DSA strength (MFI value) significantly correlated with the CDC-AHG XM outcome (r = 0.82; p < 0.0001). An MFI value of 11500 showed 100% specificity, 93% sensitivity, 100% positive predictive value and 88% negative predictive value for predicting a positive T cell and/or B cell CDC XM. Our results showed that HLA-DSA strength correlated well the XM outcome. A precise appraisal of HLA-DSA strength (i.e. a cutoff MFI value) can significantly enhance the predictability of virtual crossmatching. The clinical significance of HLA-DSA in cases where DSA is present in strength lower than the cutoff is critical in patient management and requires assessment.

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Aim In renal transplant (tx) recipients, diagnosis of acute cellular rejection or allograft nephropathy is based on serum creatinine (Cr) level and invasive biopsies. However, serum Cr level usually increases with the onset of any type of allograft injury, and biopsies do not always indicate graft injury. On the other hand, urine is produced and excreted by the kidneys may have a potential to reflect renal allograft injury. We have investigated the presence of osteoprotegerin (OPG), monokine induced by interferon-γ (MIG) and interferon- γ induced protein of 10 kDa (IP-10) in the urine of renal tx patients with acute or chronic cellular or humoral rejection or nephrotoxicity. Methods 51 renal tx recipients who were biopsied for possible rejection were included. Donor-specific HLA-antibodies (DSA) and biomarkers were measured by solid phase immunoassays. Results All patients with clinical and biopsy proven cellular rejection had significantly elevated level of all three OPG, MIG and IP-10 chemokines. OPG alone was elevated in patients with humoral, glumerulonephropathy or tubular injury. Increase level of IP-10 appeared to be more specifically correlated with acute cellular rejection. No correlation was noted with humoral rejection, diagnosed by the presence of DSA and/or C4d staining. Interestingly, all three markers were in the normal range in four patients with serum Cr>2.4, but no indication of cellular rejection. The latter patient’s biopsies showed tubular injuries with no clear cut indication. Conclusions Discussion: This study shows that quantitation of chemokines biomarkers in the urine of renal allograft recipients may be an effective method for detection of cellular rejection. This non-invasive method may be used as an alternative method to reduce the need for invasive biopsies which may cause clinical complications such as bleeding or infection.

Donor-specific human leukocyte antigen antibodies of the immunoglobulin G3 subclass are associated with Chronic rejection and graft loss after liver transplantation

In a previous study, we found that 92% of patients with chronic rejection had donor-specific human leukocyte antigen antibodies (DSAs), but surprisingly, 61% of comparator patients without rejection also had DSAs. We hypothesized that immunoglobulin G (IgG) subclasses were differentially distributed between the 2 groups. A modified single-antigen bead assay was used to detect the presence of individual IgG subclasses against human leukocyte antigen in 39 chronic rejection patients and 66 comparator patients. DSAs of the IgG1 subclass were most common and were found in 45% of all patients; they were followed by IgG3 DSAs (21%), IgG4 DSAs (14%), and IgG2 DSAs (13%). The percentage of patients with multiple IgG subclasses was significantly higher in the chronic rejection group versus the comparator group (50% versus 14%, P < 0.001). Patients with normal graft function in the presence of DSAs mostly had isolated IgG1, whereas patients with chronic rejection had a combination of IgG subclasses. Patients who developed DSAs of the IgG3 subclass showed an increased risk of graft loss (hazard ratio = 3.35, 95% confidence interval = 1.39-8.05) in comparison with patients with DSAs of other IgG subclasses or without DSAs. Although further study is needed, the determination of the IgG subclass in DSA-positive patients may help us to identify patients with a higher risk of chronic rejection and graft loss.

Prevalence, Course and Impact of HLA Donor-Specific Antibodies in Liver Transplantation in the First Year

The presence of preformed donor-specific HLA antibodies (DSA) in liver transplant recipients is increasingly recognized; however, the prevalence of DSA and their impact on early allograft function remains unknown. We prospectively followed serum DSA levels of 90 consecutive liver transplant recipients from baseline to 4 months. Twenty recipients (22.2%) had preformed DSA. No antibody-targeting treatments were undertaken. Seven days after transplantation, DSA levels decreased markedly in all but three patients. Day 7 protocol biopsies showed diffuse C4d deposition along the portal stroma, central vein, subendothelial and stromal space in the patients with persistent high DSA levels. The rate of acute cellular rejection was not significantly different in patients with DSA. The transaminase and bilirubin levels remained comparable during the first year despite the presence of DSA. The three patients with persistently high DSA levels continue to have normal allograft function. We conclude that in most cases, DSA disappear after liver transplant, however in rare instances where they persist, there is evidence of complement activation in the liver allograft, without significant clinical impact in the first year.

Donor-Specific HLA Antibodies in a Cohort Comparing Everolimus With Cyclosporine After Kidney Transplantation

Donor-specific HLA antibodies (DSA) have a negative impact on kidney graft survival. Therefore, we analyzed the occurrence of DSA and antibody-mediated rejection (AMR) in patients from two prospective randomized trials in our center. At 3-4.5 months posttransplant 127 patients were randomized to continue cyclosporine or converted to everolimus therapy. The presence of DSA was prospectively assessed using Luminex assays. AMR was defined according to the Banff 2009 classification. Antibody screening was available in 126 patients with a median follow-up of 1059 days. Seven out of 65 (10.8%) patients on cyclosporine developed DSA after a median of 991 days. In comparison, 14/61 patients (23.0%) randomized to everolimus developed DSA after 551 days (log-rank: p = 0.048). Eight patients on everolimus compared to two patients on cyclosporine developed AMR (log-rank: p = 0.036). Four of 10 patients with AMR-all in the everolimus group-lost their graft. A multivariate regression model revealed everolimus, >3 mismatches and living donor as significant risk factors for DSA. Acute rejection within the first year, >3 mismatches, everolimus and living donor were independent risk factors for AMR. This single center analysis demonstrates for the first time that everolimus-based immunosuppression is associated with an increased risk for the development of DSA and AMR.

Clinical Relevance of Pretransplant HLA Class II Donor-specific Antibodies in Renal Transplantation Patients with Negative T-cell Cytotoxicity Crossmatches

BackgroundWe evaluated the clinical relevance of pretransplant donor-specific HLA antibodies (DSA) in renal transplantation patients who had negative T-cell cytotoxicity crossmatches.MethodsFrom 328 consecutive renal transplant recipients, we selected 28 patients who had positive pretransplant (historical or at the time of transplantation) flow cytometry crossmatches, but negative T-cell cytotoxicity crossmatches at the time of transplantation. The presence of DSA and its level at the time of transplantation were retrospectively tested using Luminex single antigen assays.ResultsDSA was present in 16 (57.1%) of 28 patients. Biopsy-proven acute rejection (9 patients) occurred more frequently in patients with DSA than in those without DSA (56.3% vs. 0.0%; P=0.003). The positivity rate of class II DSA was significantly higher in patients with antibody-mediated rejection (AMR) than in those without AMR (100% vs. 21.7%; P=0.003). However, the positivity rate of class I DSA was not different between the two groups (40% vs. 40.9%). Among patients with class II DSA, those with AMR tended to have higher antibody levels (median fluorescence intensity, MFI) than those without AMR (16,359 vs. 5,910; P=0.056). A cut-off MFI value of 4,487 for class II DSA predicted the occurrence of AMR with good sensitivity and specificity (100% and 87.0%).ConclusionsIn patients with negative T-cell cytotoxicity crossmatches, the presence of class II DSA and its level at the time of transplantation were associated with the occurrence of AMR. Pretransplant DSA measurement with Luminex single antigen assay would be useful in renal transplantation.

The role of HLA antibodies in allogeneic SCT: is the ‘type-and-screen’ strategy necessary not only for blood type but also for HLA?

The role of HLA antibodies in SCT has drawn increasing attention because of the significantly increased number of patients who receive HLA-mismatched SCT, including cord blood transplantation, haploidentical SCT and unrelated SCT. Technical advancements in the methods of HLA Ab testing have realized rapid, accurate and objective identification, as well as quantification of specific HLA antibodies. Recent clinical studies have suggested that the presence of donor-specific HLA antibodies (DSA) in patients is associated with graft failure in HLA-mismatched SCT when the above-listed stem cell sources are used and results in different impacts. Of note, most of the 'HLA-matched' unrelated SCT actually involve HLA mismatches in HLA-DP and the presence of antibodies against this locus has been reported to be associated with graft failure. Thus, HLA Ab should be examined as a work-up for all patients who undergo SCT from 'alternative donors.' The simplest route for preventing HLA Ab-mediated graft failure in Ab-positive patients is to avoid donors who possess the target Ag of HLA antibodies. If SCT from such donors must be performed, treatment for DSA before SCT may improve the chances of successful donor engraftment.

Effect of Pretransplant Human Leukocyte Antigen Antibodies Detected by Solid-Phase Assay on Heart Transplant Outcomes

Introduction The significance of pretransplant human leukocyte antigen antibodies (HLA-Abs), especially donor-specific HLA-Abs (DSA), as detected by single antigen bead assay (SAB), is not well characterized in cardiac transplantation (CTX). We analyzed the significance of DSA detected by SAB in predicting crossmatch (XM) results and post-transplant rejection. Materials and methods We performed a retrospective study of 85 CTX with negative cytotoxicity XM. We tested pretransplant sera collected within 24 hours of transplantation by flow cytometric XM (FXM) and SAB. DSA identified by SAB were utilized to perform a virtual crossmatch (VXM). Positive VXM was defined as the presence of DSA at mean fluorescence intensity (DMFI) > 1500. Additionally, to analyze the significance of low-level DSA weakly positive VXM was DMFI 300 to 1500. We defined a negative VXM as MFI < 300. VXM results were correlated with FXM results and with posttransplant rejection. Results Patients in the weakly positive and negative VXM had similar posttransplant rejections. DMFI > 1500 correlates well with FXM results (accuracy = 90%). Patients with DMFI > 1500 had a higher incidence of antibody-medicated rejection (AMR; P = .0052), AMR grade I ( P < .0001), cell-mediated rejection (CMR) grade > 1R/1A ( P = .018), and CMR grade > 2R/3A ( P = .057). Similarly patients with positive FXM had a higher incidence of AMR ( P = .091), AMR grade 1 ( P < .0001), CMR grade > 1R/1A ( P = .05), and CMR grade > 2R/3A ( P = .56). Conclusions In conclusion, SAB defined DMFI > 1500 can be used as a surrogate for FXM. Recipients with DMFI > 1500 pretransplant and positive FXM have significantly higher rates of AMR and CMR compared to recipients with DMFI < 1500 or negative FXM.

Clinical relevance of circulating donor-specific HLA antibodies

For a long period of time complement-dependent cytotoxicity was the standard assay to demonstrate clinically relevant HLA antibodies in the serum of patients before transplantation. The introduction of more sensitive solid-phase assays to detect HLA antibodies had led to a lot of discussion on the clinical relevance of the presence of these antibodies before transplantation and the appearance of these antibodies after transplantation. This review discusses the current controversies. The old dogma that the presence of donor-specific HLA antibodies before transplantation is a contra-indication for transplantation does not exist anymore. The correlation between the presence of HLA antibodies and clinical outcome is less strict although the presence of circulating donor-specific antibodies, both before and after transplantation, should be considered a risk factor. It is clear, however, that the opinion on the clinical relevance of donor-specific HLA antibodies is very different amongst the transplant centers. The clinical relevance of the presence of donor-specific HLA antibodies before transplantation and the appearance of these antibodies after transplantation is a controversial issue. The lack of consensus between different centers is partly due to the heterogeneity of the HLA antibodies involved. Standardization is essential and future studies must focus on the further characterization of the antibody titers, the immunoglobulin (sub)classes of the antibodies and the epitopes recognized. It remains to be established in which cases the HLA antibodies are the direct cause of or just associated with (chronic) graft failure.