63-P: QUALITY CONTROL IN THE HLA LABORATORY: THE EFFECT OF INTER-ASSAY VARIABILITY ON SINGLE ANTIGEN BEAD ASSAYS (SABs)

Abstract

Luminex®-based SAB testing is often used as a semi-quantitative assay to monitor HLA antibody levels pre- and post-transplant. However, the SAB assay is predominantly a manual and non-standardized method that exhibits a high degree of variability. The aim of this study was to determine whether a normalization algorithm using standard controls could be used to correct inter-assay variation. Over 9 months, MFI values for the internal negative (NC) and positive (PC) control beads tested with negative (NS) and positive (PS) control sera (n=264 SAB batches), were plotted. Batches in which the NC and PC MFIs exceeded ±3SD limits were then identified. Informative patient samples (⩾3 sequential sera over ⩽90 days) from these batches were selected and specificities near the positive threshold (2000 MFI) were compared against previous or subsequent runs within 90 days (sequential sera) where NC and PC MFIs were within 3SD. The inter-assay coefficient of variation (CV) was 46% for the NC and 9% for the PC, clearly demonstrating a proportional error. Among 12 batches that exceeded ±3SD limits, sera from 4 informative patients were examined. A normalization algorithm could not be derived because there was no correlation between changes in specific bead MFIs and changes in the NC or PC MFIs within the same batch. The inter-assay variability of SABs complicates and, perhaps, invalidates using MFI values to monitor a patient’s antibody levels over time or following desensitization therapies. These data reveal that SABs are subject to proportional error more pronounced at low MFI levels, making normalization difficult if not impossible. Unless and until the variability of SABs can be reduced through standardization and automation, antibody levels of sequential sera can only be reliably compared when sera are tested in parallel (intra-assay). This strategy, however, comes with a price, namely increased costs of antibody testing.