A known, seemingly paradoxical, phenomenon of the HLA system is the high overall degree of homology among different HLA alleles, despite vast polymorphism in the population. This was noted first by serologic methods and referred to as Cross Reactive Groups (CREGs), and later by molecular methods, which documented amino acid sequence similarities between different alleles. One implication of this is the complexity of interpreting antibody (Ab) reactivity patterns, as one Ab can recognize targets that are shared by multiple alleles. The single antigen bead (SAB) assay provides a partial solution, but if an Ab recognizes a target that is present on several beads, the MFI-per-bead value can fail to represent the true strength of the Ab. This can lead to inaccurate prediction of a virtual XM. The HLA field is only now starting to appreciate this inherent constraint of the multiplexed SAB assay. We investigated the frequency of unexpected positive physical XMs for our patients that could be retroactively traced to artificially low MFI values due to a “shared epitope” phenomenon.

We systematically reviewed all consecutive physical XM for heart and kidney patients during a one-year period (9/2016-9/2017). All unexpected positive XM were compared to the raw MFI values of DSA in the context of the potential donor HLA typing. We identified at least 29 XMs that were significantly more positive than expected given the MFI value of the DSA. In all cases, Ab reactivity was deemed “Negative” to “Weak” by MFI, but analysis indicated a “stacking” of antigens bearing a shared epitope which could explain the XM result. For example, for a patient with only weak DSA against DR11 (MFI 2436) and DR52 (MFI 1938), the B cell XM result was almost 4 times higher than the positive cutoff value. This result is not in line with the Ab strength by the DSA MFI values alone. Analysis of SAB binding patterns revealed the presence of an Ab that likely recognizes all DR52-associated specificities. Additional tests (surrogate XMs and High Definition Flow Beads) confirmed our hypothesis that the positive XM was a result of an Ab recognizing a “shared epitope.”

In conclusion, in our cohort an average of 2.5 XM assays per month were “unexpectedly” positive. Nevertheless, careful analysis of Ab reactivity patterns and the proactive use of additional investigational tools such as surrogate XMs can help identify the presence of an Ab to a “shared epitope,” which can better direct assignment of Unacceptable Antigens in UNET and prevent futile XMs.

CITATION INFORMATION: Pinelli D., Tambur A. Can We Expect the “Unexpected”? Am J Transplant. 2017;17 (suppl 3).

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