P150 A case of a shared epitope

Abstract

A 69-year-old female Caucasian with chronic obstructive pulmonary disease awaiting lung transplant was assessed for her allosensitization status. FlowPRA assay demonstrated significant class II antibodies (0% Class I, 85% Class II) with a strong positive shift suggesting the presence of strong antibodies. In discordance, the single antigen bead (SAB) assay detected only weak Class II antibodies. Patient serum displayed a pan-DR antibody pattern characterized by top-ranking beads representing DR51- and DR52-associated HLA-antigens (550–2590 MFI). As part of our strategy to rule out inhibition, dilution studies were run resulting in a complete diminution of the antibodies observed and indicating absence of inhibition. One limitation of the SAB assay is the fact that it is a multiplex assay. In the presence of an antibody recognizing a target that is present on multiple beads (shared-epitopes), a bead-dependent “dilution” effect may be observed. This is an alarming scenario as the real strength of an antibody may be missed and the virtual crossmatch (vXM) may not accurately predict the results in a physical XM. However, this limitation can be circumvented by implementing rigorous analysis of specificity patterns and taking into account results from additional assays. In this particular case we chose to run a surrogate Flow cytometric XM (FCXM) to test whether the DR52-associated antigen stacking was a result of epitope-sharing and indicating that the resulting MFI values were an under-representation of much stronger reactivity. Surrogate FCXMs were run using two donors. Surrogate [A] was a DR52 homozygous donor (DR12,52) and surrogate [B] was a DR53 homozygous donor (DR4,7,53). The patient was typed as DR1,7,53. FCXM results for surrogate A showed negative T cell and very strong positive B cell, whereas surrogate B had a negative FCXM. This strong positive B cell result was “unexpected” based on the MFI values associated with the surrogate DR antigens in the SAB assay. Should a transplant have taken place solely based on vXM, the patient may have experienced an aggressive AMR response. This case highlights a limitation of reporting MFI values without rigorous analysis and potentially verifying SAB results by other methodologies. Additional routes to interrogate potential “epitope-sharing” will be discussed.