Abstract
Background The LABScreen single antigen bead (SAB) luminex assay is routinely used for HLA antibody detection. Compared to flow or ELISA based assays, SAB assay allows for relatively rapid testing and analysis of multiple patient samples. Despite this, SAB assay is time intensive and not optimal for use in urgent cases. The goal of this study was to develop a rapid SAB protocol without compromising assay sensitivity. Methods Initially, positive control (PC) sera were used to test the effects of varying centrifugation time (1 vs 5 min), serum incubation (15 vs 30 min), IgG -PE incubation (5 vs 30 min), serum volume and IgG-PE concentration on SAB assay results. Based on this data, the Rapid Optimized SAB (ROB) protocol (70% assay time savings) was developed and compared to the standard SAB (SS) assay by parallel testing of 26 sera (17 pos/9 neg) in Halifax Lab. Protocols were compared with respect to MFI (Pearson correlation coefficient) and HLA antibody assignment (2000 MFI cutoff). Pilot evaluation with 3 ABH proficiency testing (PT) sera was performed in Santa Casa Lab. Results Reducing centrifugation from 5 to 1 min had no impact on SS assay results. When incubation times with serum, IgG-PE, or both were reduced, mean PC MFI were decreased compared to the SS protocol (by 25%, 26% and 47% respectively). In contrast, mean PC MFI were significantly enhanced (> 50%) when higher IgG-PE concentration was used. Next, the ROB protocol was formulated and compared to the SS protocol using 26 sera. HLA antibody reactivity patterns were similar with both protocols for all sera with excellent MFI correlation ( r 2 = 0.99). The ROB protocol had higher mean MFI (by 10.3%) compared to the SS protocol resulting in slight increase in positive rxns (1007/3167 vs 985/3167; 31.8% vs 31.1%; MFI > 2000). ROB+/SS- rxns ( n = 33; 0.97/ panel) had SS MFI range of 1122–1980, while ROB-/SS + rxns ( n = 10; 0.29/panel) had ROB MFI range of 1565–1703. No false positive rxns were seen in 9 antibody negative sera. ROB vs SS MFI correlation with 3 ABH PT sera in Santa Casa Lab was excellent ( r 2 = 0.99) with a single ROB+/SS- rxn (2612 vs 1978 MFI). Conclusion ROB protocol correlates well with the standard LABScreen protocol and reduces assay time by 70%. ROB protocol implementation will expedite HLA antibody testing in urgent cases and improve turn-around time for routine testing.
Published Version
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