9-P: FACTORS THAT INFLUENCE CONSISTENCY IN HLA SINGLE ANTIGEN ASSAYS – A STUDY DISCUSSING FUTURE STANDARDIZATION STRATEGIES

Aim: Solid phase antibody detection assays are used in most clinical histocompatibility laboratories. Recent studies have shown that current methodologies can be inconsistent within and between laboratories. Understanding the source of assay inconsistency will promote standardization. In this study, we investigated secondary antibody systems and HLA integrity inconsistencies to facilitate a standardization strategy for HLA antibody detection assays. Methods: A soluble HLA-based assay was tested in a three step protocol; (1) binding of soluble HLA to a solid support, (2) incubation of the coupled HLA with sera/antibody, and (3) visualization of anti-HLA antibodies using a detection system. Nearly 200 soluble HLA molecules provided the platform content and more than 40 positive and negative controls were included to address Ig isotype specificity, HLA structural integrity on the platform, capacity of the support, and additional contributing factors. Results: Consistency challenges in anti-HLA antibody testing are multi-layered. In the secondary antibody detection system, anti-IgG PE antibodies crossreact with IgM controls resulting in non-proportional signal amplification because of IgM’s pentameric nature. Furthermore, variation in the avidity of tested secondary antibodies influences a consensus MFI. Commercial bead systems compared to soluble HLA revealed variation in bead HLA content. Structural disintegration of particular HLA impaired bead capacity by two logs, altered MFI saturation, and a linear detection range could only be achieved by testing various dilutions of sera samples. Conclusions: First, identifying assay inconsistencies and then standardizing methods, reagents, and controls to eliminate these inconsistencies will achieve concordance within and between laboratories. Here, we characterize variability in secondary antibodies, bead specificities, and HLA integrity on bead preparations. Having characterized these assay variables, options for assay standardization are discussed. [Copyright &y& Elsevier]