The single antigen bead assay is a widely-used platform for the detection of human leukocyte antigen (HLA)-specific antibodies in both pre- and post-transplant patients. Our regional organ procurement organization recommends a cutoff value of 2000 mean fluorescence intensity (MFI) for the detection of HLA antibodies using the Luminex single antigen bead assay. This cutoff has proven effective for the prevention of HLA-mediated hyperacute rejection; however, this cutoff may be too lenient for the detection of post-transplant donor-specific antibodies. Indeed, we hypothesize that some patients have donor-specific HLA antibody-mediated organ allograft rejection even with a MFI of <2000. To test this hypothesis, we performed a retrospective analysis of all biopsy-proven cases of antibody-mediated rejection (ABMR) in the Houston Methodist Hospital System. Over the period from 02/2012 to 057/2014, we identified 75 cases with findings at least suggestive of ABMR (all kidney biopsies) in our surgical pathology database. Of these, 42 had donor-specific antibody assays run at the time of the biopsy. Six out of 42 cases (14%) showed no DSAs.To define a clinically-relevant DSA MFI cutoff, we analyzed MFI values of 75 runs of a commercially available negative control serum and MFI values of 75 post-transplant patients with negative DSAs. After normalizing the data by log-transformation, we found that the negative control runs had a mean+3 standard deviation cutoff of 328, and the post-transplant negative DSA patients had a mean+3 standard deviation cutoff of 969, suggesting that a cutoff of 1000 MFI would be adequate to capture approximately 99% of all negative DSA patients.We applied the 1000 MFI cutoff to the six cases with findings suggestive of ABMR but with no DSAs identified. We found that the highest MFI for donor-specific antigens in these patients was well below the 1000 MFI cutoff. Using a cutoff of 1000 would not have changed the DSA result to positive in these patients. The overall findings suggest that a cutoff of 2000 MFI has been adequate to capture all cases of HLA-mediated antibody-mediated rejection over the period analyzed at our institution.