HLA incompatible (HLAi) transplantation after desensitisation is associated with a higher incidence of antibody mediated rejection (AMR). We previously found that in recipients with a positive donor cell-based flow cytometric cross match (FXM+) prior to desensitisation, the presence of C1q+antibodies pretransplant is indicative of an increased risk of AMR, with acute AMR occurring in 100% of patients with C1q+antibodies and in 25% of the patients with C1q-antibodies pretransplant. All pre-transplant C1q- patients who developed acute AMR had detectable C1q+antibodies at the rejection episode. We performed ultrastructural analysis of follow-up biopsies in our retrospective cohort of 17 HLAi patients who had IgG anti-HLA donor specific antibody (DSA) with FXM+ prior to desensitisation. We compared patients with C1q+ antibodies (9 patients; 4/9 C1q+DSA; 5/9 C1q+HLA (non DSA)) to those with no C1q activating antibodies (8 patients, C1q-) pretransplant. All but 2 of the 17 patients had more than 1 biopsy and the last biopsy available to date in 15/17 was assessed for features of chronic AMR (cAMR). Amongst C1q+ patients, 6/8 had features of cAMR by 35.3±8.33 (median 37.5) months post transplantation. In 3/6, cAMR was diagnosed by transplant glomerulopathy (TG) on light microscopy (LM). In 3/6, cAMR was only visible on electron microscopy (EM) (cg 0.5 in 1, ptcbml in 1, cg0.5+ptcbml in 1). In C1q- patients, 4/7 had features of cAMR by 42.3±27.9 (median 33.5) months post transplantation. In 1/4 cAMR was diagnosed on LM (TG). In 3/4 cAMR was only visible on EM (cg 0.5 in 1, ptcbml in 2). The C1q- patient with TG had developed a C1q+activating antibody post-transplantation, but the other 3 had not. Ultrastructural analysis increases detection of early features of cAMR in patients with antiHLA antibodies. In this analysis of a small cohort of patients, ultrastructural findings of early cAMR are present in both C1q+ and C1q- patients. Longer follow up times are necessary to see whether all progress to TG and/or long-term graft loss. DISCLOSURES:Lawrence, C.: Other, OneLambda, Has recieved honoraria and speakers fees.

Transplant glomerulopathy (TG) is a manifestation of chronic AMR with a poor prognosis and no specific treatment. Although the association between TG and anti HLA antibodies [Abs] is well known, there are few studies linking the nature of these Abs to outcome. 55 patients with TG (33M, 22F, mean age 47.5±12.1 yrs, mean time to TG diagnosis 9.32±8.37 yrs, mean follow up 26.6±18.0 months) were studied. Stored serum samples from the time of TG diagnosis were analysed for the presence of IgG and IgM HLA, DSA and Complement fixing antibodies. 52/55 (94.5%) patients were HLA Ab+, 3/55 were IgG HLA Ab- and 1 patient was IgM HLA Ab+. 27/55 (49.1%) had IgG HLA DSAs, 2/27 had class I alone, 15/27 had class II alone and 10/27 had both class I+II. Overall 39/55 (69.1%) patients had Abs directed against DQ (21/39 were DSAbs, 18/39 were HLA). 24/55 (43.6%) patients had C1q+ antibodies; 2/24 (8.3%) had a C1q+ class I DSA, 16/24 (66.7%) had a C1q+ class II DSA, 3/24 (12.5%) had a C1q+ class I non DSA HLA and 3/24 (12.5%) had a C1q+ class II non DSA HLA. 16/24 (66.7%) of patients had C1q+ antibodies against DQ. Overall allograft survival was 70.8%, 35.3%, 27.2% and 15.5% at 12, 36, 48 and 60 months respectively, mean 26.4±17.7 months. Allograft survival was significantly worse in IgG DSA+ patients compared with IgG DSA- patients.Figure: No Caption available.(see Figure 1, log rank test p=0.04). The IgG DSA MFI had no effect on allograft survival. There was no difference in allograft survival in C1q+patients (p=0.88) although the presence of C1q+ Ab was associated with the presence of C4d on allograft biopsy (p=0.01).This study shows that the presence of IgG DSAs is associated with inferior allograft survival in patients with TG. This group of patients may benefit from more aggressive therapy. DISCLOSURES:Lawrence, C.: Other, Honoraria from OneLambda, speakers fee from OneLambda.

Background. Donor-specific anti-HLA antibodies (DSA) are a major cause of alloimmune injury. Transplant recipients with negative complement-dependent cytotoxic crossmatch (CDC-XM) and donor cell-based flow cytometric crossmatch (flow-XM) but low level DSA (i.e., by Luminex) have worse outcomes compared with nonsensitized patients. The aim of this study was to establish whether complement-activating ability in this low-level DSA, present before transplantation, as determined by this technique is important in dictating pathogenicity. Methods. We retrospectively studied 52 patients with preformed DSA detected by single-antigen flow cytometric fluorescent beads (SAFBs). Patients were transplanted using a steroid-sparing regimen consisting of alemtuzumab induction, 1 week of corticosteroids and tacrolimus monotherapy.Fifteen (29%) of 52 patients experienced antibody-mediated rejection (AMR), whereas 37 (71%) patients did not. There were no demographic differences between patients with AMR and those without. Pretransplant sera were retested using a modified (SAFB) assay, which detects the presence of the complement fragment C4d as a result of DSA-induced complement activation. Results. C4d+DSA were detected in 10 (19%) of 52 patients. Biopsy-proven AMR occurred in 7 (70%) of the 10 patients with C4d+DSA and in 8 (19%) of 42 patients with C4d-DSA. AMR-free survival was worse in patients with C4d+DSA (P<0.001). Conclusions. The ability of preformed, low-level, DSA to trigger C4d fixation in vitro in patients with negative conventional crossmatch tests is predictive for AMR. C4d SAFB is potentially a powerful tool for risk stratification prior to transplantation and may allow identification of unacceptable donor antigens, or patients who may require enhanced immunosuppression.