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 <or= 0.002) with a trend towards adverse 5-year graft survival (76% and 76% vs. 90%; P <or= 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.