(605) Patterns of Non-Specific Reactivity on Single Antigen Bead Testing can Preclude Identification of HLA Antibody

Body: Single Antigen Bead (SAB) testing is the current gold standard for assessing the presence of HLA antibodies in transplant candidates. However, interpretation can be precluded by the presence of non-specific (N-S) patterns detected by SAB. Patients with N-S patterns can also elicit suspected false-positive physical crossmatches, further hindering HLA antibody interpretation. To determine whether dilution of sera clarifies reactivity, we assessed serum from a patient with a common Class II N-S pattern mixed with a serum with DR53 HLA antibody. This N-S pattern has been associated in patients with autoimmune disease (SLE), but has also been identified in patients without SLE or known autoimmune disease.
To predict the mean fluorescence intensity (MFI) values of the N-S serum and the DR53+ serum after mixing together at a 1:1 dilution, the N-S serum and DR53+ serum were diluted 1:1 in negative control serum. The MFI values of the predicted N-S and DR53+ serum were compared to the 1:1 mix of the two sera (Table 1, bolded text). This 1:1 mix was then diluted at 1:4 and 1:8 to determine if the N-S pattern and DR53 reactivity similarly diluted.
In the 1:1 mixture, the two DR53 beads increased in MFI and moved to the left of the histogram of the N-S pattern, indicating that the DR53 antibody present was able to bind the DR53 beads to enable detection. Upon dilution, the DR53 and N-S pattern did not dilute similarly in that the DR53 beads further separated and did not stay at the left of the histogram. Interestingly, the N-S pattern exhibited resistance to dilution compared to the DR53+ sera.

Conclusion: These data indicate that HLA antibody present at lower MFIs than the peak N-S pattern may be hidden in the N-S reactivity and not interpretated as present. Although this represents a single case of one N-S pattern and one HLA antibody, findings here warrant further investigation. HLA antibodies with various affinities and avidities may behave differently upon dilution than the one tested here. Additionally, the same N-S pattern in various patients may behave differently, and different N-S patterns will likely behave differently. Further study of N-S patterns is critical to accurately assess histocompatibility of patients that present with these patterns.