177-P: ONE IS NOT ENOUGH: POTENTIAL IMPACT OF DISCONNECTS BETWEEN SOLID-PHASE ASSAYS

Abstract

Aim Single antigen beads (SABs) are becoming the preferred, and in some instances the sole, methodology to identify HLA antibodies. The aim of this study was to investigate cases in which SABs did not agree with the FlowPRA® screening assay. Methods Sera from three patients awaiting renal transplant were tested for Class I and II HLA antibodies using the FlowPRA® Screening beads, LabScreen® SABs, and FlowPRA® Specific phenotype beads (One Lambda). Results In the first case, the Class I FlowPRA was negative but the SABs revealed an A∗36 specificity at low levels, which was confirmed by FlowPRA Specific beads. This explains the negative FlowPRA screen since A∗36 is not represented in this bead set. In case #2, the Class I FlowPRA was positive (∼10% of the beads); however, SAB testing was negative as were FlowPRA Specific beads. Further testing revealed an A∗02:07 antibody: an allele that is only represented on the FlowPRA platform. In case #3, the Class II FlowPRA was positive (∼10%); however, SAB testing was negative. Testing by FlowPRA Specific beads revealed the presence of an antibody to DRB1∗01:03. This result is consistent with the positive screening assay, which has two representations of DRB1∗01:03 on the bead set but fails to explain why the DRB1∗01:03 SAB was negative. Conclusions Ideally, all solid phase assays (Flow or Luminex based) should uniformly detect HLA antibodies. However, as clearly shown here, assay results can be discordant. In 2 of the 3 cases, discrepancies were due to differences in antigen representation between products. In the third case, we speculate that the failure to identify antibodies on the SAB platform was either due to a dilutional (spreading) effect wherein a weak antibody produced an insufficient signal or due to loss of an epitope on the SAB target as the result of denaturation during the manufacturing process. Until the limitations of each assay are addressed, the strategy of a single methodology for detection of HLA antibodies is flawed.