Reclassifying Assay-Intrinsic HLA Reactivity Expands Donor Availability Without Short-Term Allograft Risk

Accurate interpretation of HLA antibody reactivity is essential for virtual crossmatching (vXM) and organ allocation, particularly for sensitized kidney transplant candidates. Single-antigen bead (SAB) assays based on Luminex technology are widely used for this purpose but are susceptible to assay-intrinsic reactivity that can inflate calculated panel reactive antibody (cPRA) and unnecessarily restrict donor availability. Here, we assess the clinical and immunological impact of orthogonal SAB testing using Immucor Lifecodes in candidates initially evaluated with One Lambda assays. Cross-platform testing revealed reproducible bead-specific intrinsic reactivity affecting distinct HLA Class I and Class II specificities, as identified in a large reference cohort of non-immunized males. Orthogonal testing reclassified a substantial fraction of One Lambda–positive unacceptable antigens as assay intrinsic reactivity, resulting in marked reductions in cPRA and increased donor compatibility across sensitized candidates. Longitudinal post-transplant follow-up demonstrated rapid attenuation or disappearance of antibodies to reclassified antigens, without increased de novo donor-specific antibody formation, impaired allograft function, altered immune cell dynamics, or evidence of subclinical graft injury. Building on these findings, we developed a predictive model integrating assay intrinsic reactivity frequency, cross-platform discordance, and post-transplant antibody kinetics to identify bead specificities prone to assay-driven overcalling, with strong discriminative performance. Together, these results demonstrate that orthogonal SAB testing corrects systematic overestimation of HLA sensitization, reduces cPRA inflation without compromising short-term graft safety, and suggests a quantitative framework for harmonizing HLA antibody interpretation across transplant allocation systems.