The emerging role of humoral immunity in the pathogenesis of chronic allograft damage has prompted research aimed at assessing the role of anti-HLA antibody (Ab) monitoring as a tool to predict allograft outcome. Data on the natural history of allografts in children developing de novo Ab after transplantation are limited. Utilizing sera collected pretransplant, and serially posttransplant, we retrospectively evaluated 82 consecutive primary pediatric kidney recipients, without pretransplant donor-specific antibodies (DSA), for de novo Ab occurrence, and compared results with clinical-pathologic data. At 4.3-year follow up, 19 patients (23%) developed de novo DSA whereas 24 had de novo non-DSA (NDSA, 29%). DSA appeared at a median time of 24 months after transplantation and were mostly directed to HLA-DQ antigens. Among the 82 patients, eight developed late/chronic active C4d+ antibody-mediated rejection (AMR), and four C4d-negative AMR. Late AMR correlated with DSA (p < 0.01), whose development preceded AMR by 1-year median time. Patients with DSA had a median serum creatinine of 1.44 mg/dL at follow up, significantly higher than NDSA and Ab-negative patients (p < 0.005). In our pediatric cohort, DSA identify patients at risk of renal dysfunction, AMR and graft loss; treatment started at Ab emergence might prevent AMR occurrence and/or progression to graft failure.

These authors contributed equally to this work.Alloantibody-mediated graft injury is a major cause of kidney dysfunction and loss. The complementbinding ability of de novo donor-specific antibodies (dnDSAs) has been suggested as a prognostic tool to stratify patients for clinical risk. In this study, we analyzed posttransplant kinetics of complementfixing dnDSAs and their role in antibody-mediated rejection development and graft loss. A total of 114 pediatric nonsensitized recipients of first kidney allograft were periodically monitored for dnDSAs using flow bead assays, followed by C3d and C1q assay in case of positivity. Overall, 39 patients developed dnDSAs, which were C1q + and C3d + in 25 and nine patients, respectively. At follow-up, progressive acquisition over time of dnDSA C1q and C3d binding ability, within the same antigenic specificity, was observed, paralleled by an increase in mean fluorescence intensity that correlated with clinical outcome. C3d-fixing dnDSAs were better fit to stratify graft loss risk when the different dnDSA categories were evaluated in combined models because the 10-year graft survival probability was lower in patients with C3d-binding dnDSA than in those without dnDSAs or with C1q + /C3d À or non-complement-binding dnDSAs (40% vs. 94%, 100%, and 100%, respectively). Based on the kinetics profile, we favor dnDSA removal or modulation at first confirmed positivity, with treatment intensification guided by dnDSA biological characteristics.

The recent introduction of new technologies such as Luminex has provided alternative methods to the Complement Dependent Cytotoxicity (CDC) test for HLA specific antibody detection. In this study we compared the results obtained with CDC to those obtained using a Luminex method with the aim of evaluating the impact of this new technology on antibody screening policies in our transplant setting.A total of 1,421 sera, acquired from patients on the waiting list for a kidney transplant or following transplantation, were tested by both methodologies. CDC was performed using a whole lymphocyte population comprising a panel of 52 cells. The percentage panel reactive antibodies (PRA) and antibody specificity were evaluated using Lambda Scan Analysis software. For the Luminex method sera screening and identification of antibody specificity were carried out using the LABScreen Mixed and LABScreen PRA respectively.The overall concordance between the results obtained using the CDC and the Luminex methods was 85%. HLA antibody specificity was confirmed in 96% of the sera which tested positive using the Luminex system and serum positivity corresponded with a previous sensitisation event in these individuals. Using the Luminex method 18% of patients on the waiting list were considered and managed as sensitised as compared to 7% when testing with CDC alone. The Luminex method was able to detect a number of antibody specificities significantly more frequently than the CDC method and in addition the CDC method failed to detect some of the antibody specificities detected by the Luminex system.Based on this comparison study we have incorporated the Luminex methodology into our screening strategy. q 2007 Clinical Cytometry Society

In this single-center matched-cohort study, we evaluated the phenotype of repopulating B cells and its correlation with donor-specific anti-HLA Ab development and long-term graft function in 16 renal transplant recipients and 32 age- and gender-matched controls induced with alemtuzumab or basiliximab (Bas)/low-dose rabbit anti-thymocyte globulin (rATG), respectively. Alemtuzumab, but not Bas/rATG, profoundly depleted peripheral B cells in the first 2 mo posttransplantation. Early posttransplant, naive B cells were significantly depleted, whereas Ag-experienced and memory B cells were partially spared. Transitional B cells transiently increased 2 mo posttransplant. At month 6 posttransplant, pregerminal center B cells emerged, a process promoted by increased BAFF serum levels. Thereafter, B cell counts increased progressively, mainly due to expansion of naive B cells. Conversely, Bas/rATG did not modify the B cell phenotype throughout the follow-up period. Alemtuzumab was associated with a higher incidence of de novo DSA compared with Bas/rATG. DSA development was predicted by changes in the B cell compartment and correlated with worse long-term graft function. Thus, alemtuzumab-induced B cell depletion/reconstitution may promote chronic humoral responses against the graft.