The ABCs (DRDQDPs) of the prozone effect in single antigen bead HLA antibody testing: Lessons from our highly sensitized patients

Accurate identification of HLA antibodies using the single antigen bead (SAB) assay is critical for assessment of pre/post-transplant immunological risk and successful virtual crossmatching. Unfortunately, high titer HLA antibodies can be missed or underestimated in the SAB assay as a result of interference with the detection of IgG. This so called prozone effect has been attributed to both complement- and IgM-dependent mechanisms and can be minimized with serum dilution or treatment with heat, EDTA, or DTT. In this study we describe the frequency, nature, and degree of prozone in a cohort of highly sensitized patients (cPRA ≥ 95%), in whom accurate detection of HLA antibodies and virtual crossmatching is of paramount importance. Sera were tested by the SAB assay ± EDTA treatment, ±1:10 dilution to identify the prozone effect. The relative contribution of complement vs IgM to prozone was assessed using anti-C3d and anti-IgM reporter antibodies, respectively. We found that prozone was very frequent in highly sensitized patients (80%), especially those with a history of previous transplantation (87%). Class I HLA specificities were more commonly affected than class II and the susceptibility to prozone was locus dependent with HLA-A(31%), -B(29%) and -DQ(26%) being affected more frequently than HLA-DP(17%), -C(16%) and -DR(5%) antigens. Interestingly, the presence of prozone could be predicted by C3d positivity (MFI ≥ 4000; sensitivity = 95.2%, specificity = 97.2%) and the degree of prozone correlated directly with the extent of C3d deposition. The role of IgM was less clear. However, serum dilution studies suggested that IgM may contribute to interference in a small subset of prozone positive specificities. Our study underscores the importance of serum treatment to inhibit complement activation and minimize prozone in the SAB assay, especially in highly sensitized patients.