Donor specific antibodies (DSA) directed against HLA arising de novo post-kidney transplant are a major cause of graft loss, termed antibody mediated rejection (AMR). Currently, solid phase bead (SAB) assays have the ability to detect even low levels of HLA antibodies, but potentially lack the ability to distinguish between clinically relevant or irrelevant DSA. It is now reported that the C1q assay can discriminate between such antibodies. Here we test the clinical significance of a different test assessing C3d binding. 75 kidney transplant recipients, with protocol or for-cause biopsies and detection of de-novo DSA (at least weak antibodies, by One Lambda (OL), at the time of biopsy were studied. Those same serum samples were retrospectively analyzed, using the Immucor SAB assays, to detect either IgG or C3d binding HLA antibodies. Median fluorescence intensity (MFI) value of 1000 was arbitrarily used to assign DSA. The biopsy and clinical presentation of the 75 patients tested were segregated as follows: 16 AMR, 11 cell-mediated rejection (CMR), 10 mixed rejection (AMR + CMR), 4 patients had some pathologic finding other than rejection, such as BK virus, and 34 had normal biopsies. C3d DSA were detected in 12/75 patients with the majority having AMR in 9/12 patients (75%) compared to 1/12 patients (8%) with CMR, none in patients with a mixed rejection, and 2/12 patients (16%) with negative biopsies. Interestingly, these 2 negative biopsy patients had strong MFI values for Immucor IgG DSA and strong titers on the OL platform; however, both of these patients went on to have AMR. Immucor IgG assay detected 24/75 patients with DSA, in which, the majority of C3d positive DSA (11/12) being Immucor IgG DSA-positive (MFI range 2814-18294). Immucor IgG positive DSA with negative C3d results had lower MFI ranging between 1059-6791. All the C3d positive assays corresponded to high titer OL results (titers ≥1:64), although three samples had titers ≥1:128 were not C3d positive. Also, 3/4 patients with clinical AMR and C4d positive kidney biopsies were C3d DSA negative in comparison to Immucor IgG assay detecting DSA (MFI range 1059-5589). Our data indicate a strong correlation between the presence of positive C3d binding assays and AMR. Similar to previous data for C1q binding, C3d-binding antibodies seem to be restricted to stronger antibodies with at least moderate level titer. Further studies are required to substantiate out results.

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