ABSTRACT
AIMS: Several modes of assay interference common to immunoassays affect solid-phase single-antigen bead-based immunoassays (SAB) used to detect antibodies against human leucocyte antigens (HLA). Best practice recommendations include methods to address assay interference, though the clinical impact and optimal approaches are undefined. We sought to evaluate assay interference in HLA SAB to identify an efficient approach for avoiding erroneous results. METHODS: Retrospective analysis of 14 059 patient samples tested for anti-HLA antibodies was performed. This included 4685 samples tested prior to implementation of serum pretreatment with EDTA and 4982 samples tested with routine EDTA treatment using the same testing algorithm. An algorithm for efficiently identifying and processing samples with suspected interference was evaluated in a separate cohort of 4392 EDTA-treated samples. RESULTS: EDTA serum pretreatment reduced assay interference, but did not eliminate all modes of interference. A protocol for identification and testing of samples with suspected interference facilitated efficient detection of interference while reducing the amount of additional testing required. CONCLUSIONS: Our data indicate that a single-method approach is insufficient to address all sources of interference in HLA SAB. A multimodal approach with a proactive screening is a more effective way to minimise risk of erroneous results.
ABSTRACT
The ability to identify specific HLA molecules against which a patient has alloantibodies has revolutionized assessment of immunologic compatibility. Anti-HLA antibodies are typically evaluated as reactive against well-defined serologic antigen groups. Thus, donor HLA genotyping is aimed at defining HLA at the serologic split-antigen level to avoid incompatible antigen-antibody combinations. However, anti-HLA antibodies can have reactivities not accurately described by well-defined serologic antigens. While existence of these antibodies is acknowledged, their precise impact on clinical practice is not clear. We performed a single-center review of 2 years of pre-and post-transplant anti-HLA antibody testing data combined with high-resolution HLA genotyping data for living and deceased organ donors to evaluate the clinical impact of anti-HLA antibodies with reactivities outside of commonly defined serologic antigen groups. We find approximately 15% of patients awaiting transplantation have alloantibodies with differential reactivity for HLA proteins encoded by specific alleles within a serologic antigen group. Allele-specific antibodies are associated with positive cellular crossmatches not accurately predicted by standard donor HLA genotyping and can manifest as post-transplant donor-specific antibodies. Our data highlights the importance of evaluating anti-HLA antibodies at the allele-level and provides evidence supporting utility for high-resolution HLA genotyping in solid organ transplantation.
