C4d as a significant predictor for humoral rejection in renal allografts

OBJECTIVE: To determine the diagnostic and clinical significance of C4d accumulation in renal allografts followed by acute rejection. METHODS: A total of 158 graft biopsies performed from December 1997 to December 2002 were classified, according to the Banff-97 criteria, into hyperacute rejection (HAR, three cases), acute vascular rejection (AVR, 27), acute cellular rejection (ACR, 24), borderline rejection (BR, 38), acute tubular necrosis (ATN, five), stable graft function (SGF, 30) and baseline kidney (31). Immunohistochemical technique was used to determine the C4d deposition level. RESULTS: The percentages of C4d positive in HAR, AVR, ACR, BR, ATN, SGF and baseline kidney groups were 100% (3/3), 77.8% (21/27), 37.5% (9/24), 23.7% (9/38), 0% (0/5), 3.3% (1/30), 0% (0/31), respectively. In acute rejection patients, the peak serum creatinine (sCr) level in C4d(ptc)-positive group (41 cases) was 334.82 +/- 238.37 micromol/L, with that of C4d(ptc)-negative group (47 cases) being 220.20 +/- 176.94 micromol/L (p < 0.01). After treatment, the trough sCr level in C4d(ptc)-positive group and C4d(ptc)-negative group were 176.87 +/- 111.80 and 121.75 +/- 34.59 micromol/L (p < 0.01), respectively. In each AVR, ACR and BR subgroups, the peak sCr level, the trough sCr level, after 3 or 6 months of AR, the sCr level in C4d(ptc)-positive subgroup was higher than that of C4d(ptc)-negative subgroup. There were more resistance against steroid therapy [65.9% (27/41) vs. 36.2% (17/47), p = 0.005] and a higher rate of graft loss [29.3% (12/41) vs. 6.4% (3/47), p = 0.001] in C4d(ptc)-positive group than those of C4d(ptc)-negative group. In each C4d(ptc)-positive subgroup of AVR, ACR and BR the complete reversion was 57.1, 56 and 66.7%, respectively, it is almost same. CONCLUSION: The C4d deposition level is of great value in diagnosis of acute rejection caused by humoral immune components. It is a significant predictor of graft survival and will be of great help when treating acute rejection.     

C4d and/or immunoglobulins deposition in peritubular capillaries in perioperative graft biopsies in ABO-incompatible renal transplantation

Abstract:  We evaluated 0 h and/or 1 h graft biopsy specimens from 14 recipients in ABO-incompatible renal transplantation using immunofluorescence for C4d, IgG, and IgM. All 0 h biopsy specimens revealed negative C4d, IgG, and IgM deposition in peritubular capillaries (PTC). In contrast, 8 of 14 1 h biopsy specimens revealed a positive C4d deposition in PTC. Eight specimens revealed positive IgM staining and seven of them had both C4d and IgM depositions. Three specimens had C4d, IgM, and IgG depositions in PTC. Three of eight patients with C4d deposition and two of six patients without C4d deposition in the 1 h biopsy group suffered from acute rejection within 1 month of transplantation. These findings suggest that complement fragments and immunoglobulin deposition in PTC in ABO-incompatible renal grafts can start soon after reperfusion, although acute rejection may or may not develop.     

Histological evaluation of renal allograft protocol biopsies in the early period and 1 year after transplantation

Abstract:  We histologically evaluated protocol biopsy specimens of renal allografts obtained in the early period and 1 year after transplantation. The patients were divided into those with at least one history of acute rejection (AR group) and no history of rejection (NAR group), and the histopathological features in the two groups were compared. A total of 45 early protocol biopsy specimens were obtained from 40 patients, and 31 1-year biopsy specimens were obtained from 30 patients. Acute rejection (AR) or borderline change was observed in the early protocol biopsy specimens from 19 (45.2%) cases. AR or borderline change was observed in 12 of 19 (63.2%) in the AR group, and in 7/26 cases (26.9%) in the NAR group. The incidence of AR or borderline change in the AR group was higher than in the NAR group. Toxic tubulopathy was found in the early protocol biopsy in 16 cases (35.6%). The 1-year biopsies tended to reveal more complicated findings. Chronic rejection (CR) was seen in 8/16 cases (50.0%) in the AR group, and it was more frequent than NAR group (two cases, 13.3%). In conclusion, the incidences of both AR and CR were higher in the cases with a previous episode of AR. The early protocol biopsy was useful in screening for subclinical AR and toxic tubulopathy. The 1-year biopsy was useful for evaluating various types of chronic graft damage. We expect that adequate treatment based on protocol biopsy findings in each patient will lead to better graft survival.     

Renal allograft C4d deposition in Chinese: Hong Kong perspective

Background: Acute rejection constitutes a significant proportion of renal allograft loss. Peritubular capillary deposition of C4d has been recognized as the footprint of humoral alloimmunity and proven to be a sensitive and specific marker for humoral rejection in the appropriate clinical context. Its presence in indication biopsies is the most important independent risk factor for graft failure. Data are, however, scarce among Chinese subjects. Methods: We retrospectively reviewed all renal graft biopsies performed from 1 April 2002 to 31 March 2006 for unexplained acute renal dysfunction or delayed graft function. Renal outcomes were assessed at the time of renal biopsy and at 1 month, 3 months, 6 months and 1 year afterwards. Survival was assessed by Kaplan–Meier analysis. Multivariate analysis was used to determine if C4d positivity is an independent risk factor for poor renal outcome. Results: Fifty‐two biopsies were included, of which 16 were positive for peritubular capillary C4d. Peritubular capillary C4d was associated with lower glomerular filtration rate and higher serum creatinine at 6 and 12 months after renal biopsies. The C4d‐positive group fares worse in terms of death‐censored graft failure, doubling of serum creatinine and reaching 50% of glomerular filtration rate at the end of the study. Peritubular capillary C4d deposition was the only significant risk factor that predicts graft failure in multivariate analysis. Conclusion: Our findings confirmed the independent prognostic value of peritubular capillary C4d staining on renal allograft survival in Chinese.     

Antibody‐mediated rejection (AMR) after pancreas and pancreas–kidney transplantation

Antibody‐mediated rejection (AMR) requires specific diagnostic tools and treatment and is associated with lower graft survival. We prospectively screened C4d in pancreas (n = 35, in 27 patients) and kidney (n = 33, in 21 patients) for cause biopsies. Serum amylase and lipase, amylasuria, fasting blood glucose (FBG) and 2‐h capillary glucose (CG) were also analysed. We found that 27.3% of kidney biopsies and 43% of pancreatic biopsies showed C4d staining (66.7% and 53.3% diffuse in peritubular and interacinar capillaries respectively). Isolated exocrine dysfunction was the main indication for pancreas biopsy (54.3%) and was followed by both exocrine and endocrine dysfunctions (37.1%) and isolated endocrine dysfunction (8.6%). Laboratorial parameters were comparable between T‐cell mediated rejection and AMR: amylase 151.5 vs. 149 U/l (P = 0.075), lipase 1120 vs. 1288.5 U/l (P = 0.83), amylasuria variation 46.5 vs. 61% (P = 0.97), FBG 69 vs. 97 mg/dl (P = 0.20) and 2‐h CG maximum 149.5 vs. 197.5 mg/dl (P = 0.49) respectively. Amylasuria values after treatment correlated with pancreas allograft loss (P = 0.015). These data suggest that C4d staining should be routinely investigated when pancreas allograft dysfunction is present because of its high detection rate in cases of rejection.     

移植肾组织中补体C4d沉积在CAN的诊断和治疗中的临床意义

目的 探讨移植肾组织中补体C4d沉积在慢性移植肾肾病(CAN)的诊断和治疗中的临床意义.方法 将2000年1月至2008年8月间诊断为CAN,且已行移植肾活检,并能收集到活检后2年以上随访资料者纳入研究.符合标准者共有86例,其中男性53例,女性33例,移植时年龄18～65岁.应用免疫组织化学染色方法检测移植肾组织活检标本C4d的表达.所有患者在行移植肾穿刺诊断为CAN后均给予了常规治疗.结果 86例患者中,C4d沉积阳性者(C4d阳性组)39例,C4d沉积阴性者(C4d阴性组)47例,两组患者在性别、年龄、供肾来源、多次移植、移植时群体反应性抗体水平等各指标的比较,差异均无统计学意义(P＞0.05).活检2年后,C4d阳性组和阴性组患者的SCr水平分别为(551.5±140.4)和(443.0±133.1)μmol/L,两组比较,差异有统计学意义(P＜0.05);并且C4d阳性组患者移植肾功能丧失率(23.1%,10/39)也显著高于C4d阴性组(8.5%,4/47),两组比较,差异有统计学意义(P＜0.05).CAN治疗前,C4d阳性组发生高血压和高血脂者多于阴性组(P＜0.05);常规治疗后,剔除移植肾功能丧失者,两组间发生高血压、高血脂、高血糖和高血尿酸者差异无统计学意义(P＞0.05).结论 C4d阳性的CAN患者提示有慢性体液排斥反应的参与,临床常规治疗预后较差,若针对体液免疫反应进行干预,能够改善移植肾长期存活.     

Clinicopathological relevance of granular C4d deposition in peritubular capillaries of kidney allografts

While linear C4d staining in peritubular capillaries (PTC) is established as a marker of antibody‐mediated rejection, the significance of a distinct granular C4d deposition pattern has not yet been clarified. In this study, 329 renal allograft recipients who underwent indication biopsies were analysed for immunohistochemical C4d staining characteristics. Fifty‐six (17%) recipients showed granular C4d in PTC, without any relationship to conventional risk factors and morphological features of rejection. We found a strong association with long‐term overall graft survival (7‐year survival: 41% vs. 66% in granular C4d‐negative subjects, P = 0.001), which was mainly driven by a greater risk of mortality [hazard ratio: 3.12 (95% confidence interval: 1.23–7.94); P = 0.02]. Granular C4d was associated with delayed graft function [39% vs. 22% (C4d‐negative subjects), P = 0.007], higher 1‐year serum creatinine [median 2.1 (interquartile range: 1.7–2.6) mg/dl vs. 1.6 (1.3–2.0) mg/dl, P = 0.001] and a trend towards worse death‐censored graft survival (P = 0.07). In support of a role of capillary immune complex formation, granular C4d was associated with electron‐dense deposits in PTC basement membranes, which were occasionally accompanied by focally distributed capillary IgG deposits. In conclusion, our study suggests clinical relevance of detecting capillary granular C4d deposition. Our results point to a pathogenetic role of alloimmune‐independent immune complex deposition.     

Peritubular capillary C4d deposition and renal outcome in post-transplant IgA nephropathy

BACKGROUNDS: Immunological staining of the transplanted kidney for C4d in peritubular capillaries (C4d(PTC)) has emerged as a useful method to detect antibody-mediated rejection in situ. In this retrospective study, we evaluated the prevalence of C4d(PTC) deposition in allograft renal biopsies diagnosed of IgA nephropathy (IgAN) and analysed its clinical significance. METHOD: Sixty-six biopsy specimens of post-transplant IgAN, which were obtained to evaluate azotemia and/or heavy proteinuria, were examined by immunohistochemical staining of the paraffin sections with polyclonal antibody for C4d. RESULTS: C4d was stained positively in peritubular capillaries in 16 (24%) of the 66 cases. The C4d(PTC)-negative (n=50) and C4d(PTC)-positive groups (n=16) were not different in recipient gender, age, donor age, type of donor (living vs. cadaveric), interval from transplantation to graft biopsy (41.6+/- 21.8 vs. 48.3+/-26.1 months) and post-biopsy follow-up period (60.3+/-23.3 vs. 56.9+/-25.4 months). During the follow-up period, 12 of 50 (24%) although the incidence of graft failure was not different by the C4d deposition in peritubular capillaries, intervals from renal biopsy to graft failure tended to be shorter in C4d(PTC)-positive cases than C4d(PTC)-negative cases. In Kaplan-Meier analysis, the renal allograft function of the C4d(PTC)-positive group deteriorated more rapidly than that of the C4d(PTC)-negative group (p<0.05). Histologically, the C4d(PTC)-positive group had findings suggestive of acute cellular rejection more commonly than the C4d(PTC)-negative group (p<0.01). CONCLUSIONS: Evidence of humoral rejection, as demonstrated by C4d(PTC) deposition, was concurrently present in significant portions of post-transplant IgAN biopsy specimens and was associated with more rapid deterioration of renal function. These results suggest that C4d(PTC) positivity needs to be determined at the time of biopsy even in cases of post-transplant glomerulonephritis and immunosuppression may need to be modified accordingly.     

Complement C4d deposition in transplanted kidneys: preliminary report on long-term graft survival

Abstract:  The effects of antibody-mediated rejection on long-term graft survival have not been fully investigated. The aim of this study is to clarify the influence on long-term survival of deposition of the complement split product C4d in allografts using polyclonal anti-C4d antibody. Inclusion criteria were recipients who underwent graft biopsy during acute deterioration of graft function within the first 2 yr after transplantation. Patients whose graft did not survive more than 1 yr and who received graft from an human leucocyte antigen (HLA)-identical sibling or an ABO-incompatible donor were excluded. Among the 92 recipients investigated, 22 (23.9%) had peritubular capillary C4d deposition, 15 (16.3%) had glomerular capillary C4d deposition and seven (7.6%) had both peritubular and glomerular capillary C4d deposition. Twenty of these 22 patients revealed acute cellular rejection, including borderline changes. There was no significant relationship between pathological severity of acute rejection and presence or absence of peritubular capillary C4d deposition. Graft survival was inferior in patients with peritubular capillary C4d deposition to that in patients without C4d deposition (p = 0.0419). Graft survival in patients with glomerular C4d deposition did not differ from that in patients without C4d deposition. In conclusion, C4d deposition in peritubular capillaries has a substantial impact on long-term graft survival.     

Use of C4d as a Diagnostic Adjunct in Lung Allograft Biopsies

PURPOSE: Humoral allograft rejection is a defined mechanism for cardiac and renal graft dysfunction; C4d deposition, a stable component of complement activation, inversely correlates with graft survival. With the recent recognition of humoral rejection in lung grafts, we examined C4d's role as a prognostic adjunct in lung allografts. MATERIAL AND METHODS: Twenty-three lung recipients underwent biopsies for deterioration in clinical status or routine surveillance. Clinically unwell patients possessed acute rejection or bronchiolitis obliterans syndrome (BOS). Biopsies attributable to infection were excluded from the study. In addition to routine light microscopy, an attempt was made to correlate the clinical status and morphologic findings with the pattern of C4d deposition and also to compare these clinical and morphologic parameters with the other assessed immunoreactants. Panel reactive antibody testing was also carried out at various points in their post transplantation course whereby in 6 of the cases the samples were procured at exactly the same time as the tissue samples. RESULTS: The patients were segregated into two groups: those patients with recurrent acute rejection and those with BOS. In those patients with symptomatic acute rejection, all biopsies showed light microscopic and immunofluorescent evidence of humoral allograft rejection. The level of C4d was positively correlated with the degree of parenchymal injury, the hallmark being one of septal capillary necrosis. In addition, high and intermediate levels of C4d correlated with a clinical diagnosis of acute rejection. C4d was the strongest predictor of parenchymal injury and of the clinical status (p <.0001) compared to other the immunoreactants C1q, C5b-9 and immunoglobulin. There was no specific correlation between C4d deposition and the presence of acute cellular rejection. In those patients fulfilling clinical criteria of BOS, deposits of C4d as well as other immunoreactants were found in the bronchial wall as opposed to the rarity of this finding in bon-BOS patients. However the only statistically significant predictor of BOS was bronchial wall deposition of C1q. In no case were panel reactive antibodies at significant levels discovered post transplantation. CONCLUSIONS: In the context of acute rejection, C4d deposition correlates with clinical evidence of rejection and the degree of humoral rejection assessed pathologically; there is no association with the presence of histocompatibility related antibodies. It is a more specific predictor of allograft status compared to other immunoreactants. C4d deposition within the bronchial wall is a feature of BOS and hence may be used as a marker of chronic graft dysfunction. The antigenic target resulting in C4d deposition may not be histocompatibility related.     

C4d immunostaining is an independent predictor of cardiac allograft vasculopathy and death in heart transplant recipients

Antibody‐mediated rejection (AMR) occurs in 10–20% of patients after heart transplantation. C4d immunostaining is one parameter used in its diagnosis. This study aimed to determine whether C4d staining has prognostic significance for mortality, coronary allograft vasculopathy (CAV), cell‐mediated rejection (CMR), and graft dysfunction in patients post‐transplantation. Consecutive patients receiving an endomyocardial biopsy between 2007 and 2008 were selected. Left ventricular function, angiography, episodes of AMR/CMR, and death were noted. C4d was graded from 0 to 3 (immunostaining). Cox proportional models (recurrent events analysis) were used to evaluate C4d staining with mortality, graft dysfunction, CAV (≥grade 2), and episodes of ≥2R‐CMR. We analyzed 2525 biopsy specimens (n = 217). During a follow‐up of 4.5 ± 2 years, 35 died, 49 had graft dysfunction, seven had ≥grade 2 CAV, and 95 episodes of CMR occurred. A one‐grade increase in C4d staining was associated with an increase in mortality (HR 1.57; 95% CI 1.0–2.5), a higher risk of CAV (HR 2.4, 95% CI 1.04–5.4), and a trend toward graft dysfunction (HR 1.42; 95% CI 1.0–2.09). C4d was not associated with CMR. C4d immunostaining was a significant predictor of CAV and death but not subsequent episodes of CMR. There was also a trend toward increased graft failure.     

The Impact of C4d Pattern and Donor‐Specific Antibody on Graft Survival in Recipients Requiring Indication Renal Allograft Biopsy

We examined the pattern of PTC C4d by immunohistochemistry and DSA in 297 kidney recipients with indication biopsies, and evaluated their predictive value for graft survival. Median biopsy time was 5.1 months posttransplant. Patients were followed for 17.9 ± 9.4 months postbiopsy. An 18.5% had focal and 15.2% had diffuse C4d, with comparable graft survival (adjusted graft failure HR: 2.3, p = 0.001; HR:1.9, p < 0.02, respectively). 31.3% were DSA+, 19.5% class I and 22.9% class II DSA. Only those with class II DSA had worse outcome (adjusted HR:2.5, p = 0.001 for class II only; HR:2.7, p < 0.001 for class I/II DSA). Among patients with <10%C4d, 23.9% had DSA, compared to 68.9% with diffuse staining. For patients biopsied in first‐year posttransplant presence of DSA, regardless of C4d positivity in biopsy, was a poor prognostic factor (adjusted graft failure HR: 4.2, p < 0.02 for C4d?/DSA+; HR:4.9, p = 0.001 for C4d+/DSA+), unlike those biopsied later. We have shown that focal C4d had similar impact on graft survival as diffuse pattern. During the first‐year posttransplant either class I or II DSA, and afterward only class II DSA were associated with worse graft survival. DSA was predictive of worse outcome regardless of C4d for patients biopsied in first year and only with C4d positivity afterward, supporting the importance of assessment of both DSA and C4d pattern in biopsy.     

C1q binding is not an independent risk factor for kidney allograft loss after an acute antibody‐mediated rejection episode: a retrospective cohort study

After kidney transplantation, C4d is an incomplete marker of acute antibody‐mediated rejection (AMR) and C1q‐binding donor‐specific antibodies (DSA) have been associated with allograft survival. However, the impact on allograft survival of C1q+ DSA after clinical AMR has not been studied yet. We analysed retrospectively in clinical AMR C4d staining and C1q‐binding impact on allograft survival. We compared clinical, histological and serological features of C4d− and C4d+ AMR, C1q+ and C1q− DSA AMR and analysed C4d and C1q‐binding impact on allograft survival. Among 500 for‐cause kidney allograft biopsies, 48 fulfilled AMR criteria. C4d+ AMR [N = 18 (37.5%)] have significantly higher number class I DSA (P = 0.02), higher microvascular score (P = 0.02) and more transplant glomerulopathy (P = 0.04). C1q+ AMR [N = 20 (44%)] presented with significantly more class I and class II DSA (P = 0.005 and 0.04) and C4d+ staining (P = 0.01). Graft losses were significantly higher in the C4d+ group (P = 0.04) but similar in C1q groups. C4d+ but not C1q+ binding was an independent risk factor for graft loss [HR = 2.65; (1.11–6.34); P = 0.028]. In our cohort of clinical AMR, C4d+ staining but not C1q+ binding is an independent risk factor for graft loss. Allograft loss and patient survival were similar in C1q+ and C1q− AMR.     

体液性排斥反应患者移植物组织C4d沉积和浆细胞聚集性浸润初探

目的检测移植肝及肾组织中浆细胞的浸润和补体C4裂解产物C4d的沉积情况,分析浆细胞浸润、C4d沉积与体液性排斥反应的相关性。方法25例肝移植术后出现不同程度肝脏损害的患者的34次经皮肝脏穿刺活体组织病理检查标本与43例因排斥反应而丧失功能的移植肾组织,进行HE染色和免疫组织化学染色,对排斥反应进行病理分型,检测移植物组织中C4d和CD138的表达,分析二者之间的相关性。同时以10例非排斥反应导致移植肾功能丧失者和供肝术前标本作为对照。结果34个移植肝组织标本病理检查诊断为急性排斥反应16个,慢性排斥反应9个,非排斥反应9个。移植前供肝病理标本中均未见C4d沉积,急性排斥反应和慢性排斥反应标本中分别有9／16（56．3％）和5／9（55．6％）见到C4d沉积现象,非排斥反应标本只有1例非吻合口胆管狭窄患者C4d染色也呈阳性（11．1％）。移植肝排斥反应标本中11／25（44．0％）CD138阳性,8／25（32．0％）标本C4d和CD138均阳性。C4d的沉积与CD138的表达存在相关性（r=0．346,P〈0．05）。43例移植肾排斥反应中,超急性排斥反应5例,急性排斥反应9例,慢性排斥反应29例;43例中,C4d阳性19例（44．2％）,CD138阳性24例（55．8％）;有10例（23．3％）C4d和CD138均阳性,C4d的沉积与CD138的表达亦存在相关性（r=0．5051,P〈0．01）。10个对照标本中,C4d阳性1个,无CD138阳性标本。结论CD138与C4d的沉积存在相关性,提示移植肝和肾组织中聚集性浸润的浆细胞可能通过局部分泌抗体的方式参与体液性排斥反应。     

Compartment‐specific expression of natural killer cell markers in renal transplantation: immune profile in acute rejection

Natural killer (NK) cells have been implicated in graft dysfunction. Here, we formulated hypothesis that distinct patterns of expression NK cells markers correlated with acute rejection in kidney transplantation. Therefore, we studied the pattern of NK cell markers CD56, CD57, and CD16 in different compartments of biopsies obtained from recipients diagnosed with acute graft rejection, with or without donor‐specific antibodies (DSA). DSA‐negative biopsies‐from patients with acute T‐cell mediated rejection (aTCMR) had an increased expression of CD56+ and CD57+ cells (P = 0.004 and P = 0.001) in the interstitial compartment in comparison with DSA‐positive biopsies from patients acute antibody‐mediated rejection (aABMR) with (aABMR C4d+) and without C4d deposition (aABMR C4d‐). CD16+ cells was increased (P = 0.03) in the glomerular compartment in DSA‐positive biopsies. We assume that CD16+ expression and antibody‐dependent cellular cytotoxicity (ADCC) in microvascular injury can be associated with aABMR. IFN‐γ release from cytoplasmic granules of NK cell could be associated with aTCMR. Our findings suggest that NK cells need to be carefully evaluated because variations in NK cell marker expression might imply the involvement of different immune system pathways in graft rejection.     

Clinical relevance of preformed C4d-fixing and non-C4d-fixing HLA single antigen reactivity in renal allograft recipients

Donor-specific alloantibodies (DSA), especially those fixing complement, may pose a particular immunologic risk to transplant recipients. To assess the clinical impact of C4d- or non-C4d-fixing (IgG) HLA sensitization, pretransplant sera obtained from 338 kidney allograft recipients prescreened by FlowPRA were retrospectively evaluated by Luminex single antigen (SA) testing using a novel fluorescent-labeled anti-C4d reagent for detection of antibody-triggered C4d deposition in addition to IgG binding. Recipients with [IgG]DSA ( n  = 39) showed a substantially higher rate of C4d positive rejection (33%) than 16 patients with [IgG] non-DSA (0%) or 283 antibody-negative patients (4%, multivariate analysis excluding retransplantation because of high co-linearity: P  < 0.0001), and adversely affected 5-year death-censored graft survival  (74% vs. 81% and 90%, respectively, multivariate model: P  < 0.05). [C4d] DSA ( n  = 21) and [C4d] non-DSA ( n  = 25) increased rates of C4d positive rejections to a similar extent (24% and 28% vs. 4% in recipients without C4d-fixing reactivity; multivariate analysis: P  ≤ 0.002) with a trend towards adverse 5-year graft survival (76% and 76% vs. 90%; P  ≤ 0.2). In conclusion, Luminex-based characterization of HLA sensitization may be a useful strategy for risk stratification. Possibly as a result of intensified immunosuppression in presensitized recipients, identification of C4d-fixing DSA was not associated with a further increase of rejection and graft loss rates.