Discrepant Antibody Testing Results: Which One to Believe?

The impact of solid-phase immunoassay for HLA antibody detection on the field of transplantation has been extremely significant by providing the most sensitive and precise method for characterization of HLA antibodies. However, despite all the benefits, technical limitations and inherent artifacts represent significant challenges, particularly with Luminex-based single-antigen bead (SAB) assay. Discordant results between antibody detection (screening assay) and identification (SAB) is not uncommon. Positive SAB assay in the context of negative screening testing is well documented and attributed to altered tertiary structure of HLA molecules exposing new epitopes or detection of naturally occurring antibodies. However, there are few reports that addressed the opposite scenario when negative SAB appeared in the context of positive screening assay. In such discrepant results, unmissed HLA antibody has to be excluded with certainty by other tests; however, with the availability of variable assays it may be difficult to choose the best combinations that clarify discrepancy without adding more confusion. Here we describe the results of correlation between 2 antibody screening solid-phase immunoassays (LABScreen Mixed using Luminex and FlowPRA Screen) on conventional flow cytometry and compare their outcomes with SAB and crossmatch results.

The dilemma of DQ HLA-antibodies.

Accurate identification of antibody reactivity against HLA-DQ antigens was difficult by using the old serological assays because of the strong linkage disequilibrium between HLA-DR and HLA-DQ (the usual inheritance of a certain HLA-DR molecule that ties together with the same DQ molecule within a racial group). The accurate and precise identifications of anti-HLA-antibodies of DQ specificities were made possible with the introduction of multiplex-bead arrays (Luminex), using single antigen bead (SAB) assay. The SAB assay is also considered today to be the most sensitive and specific method for alloimmunization assessment even for the low titer anti-HLA-antibodies. However, it is becoming clear that the detection of the HLA antibodies by SAB is not absolutely perfect due to the variation in densities, conformations and orientations of the antigen coated beads. Unlike HLA-DR, the HLA-DQ antigens are made of two polymorphic chains, both (alpha and beta chains) can contribute to the process of immunization individually or jointly. Routine SAB testing approach, which assigns the specificities based on beta chains and ignores the contribution of the DQα chains, can lead to erroneous DQ-antibody assignments. Therefore, it is important to recognize both the peculiarity of the HLA-DQ antigens as well as the nature of the assay format used in order to reach the correct antibody assignments. Erroneous donor specific antibodies (DSA) assignment may lead to denial of an otherwise immunologically compatible organ transplant, or exposing transplant recipients to unnecessary investigations or immunosuppression. The following two patients presented with HLA-antibodies against DQ antigens (anti-DQ-Abs) highlight these two scenarios.