P083 Tools of the trade: How antigen density stacks up on single antigen beads, a comparison of two vendors

Abstract

Aim Single antigen bead (SAB) solid phase assays have become a key tool in HLA antibody (Ab) assessment. These beads are made by two vendors and each provide antigen density (AgD) data but in different formats. We measured the AgD on class I and II SABs and compared the results. Methods SABs from both vendors (One Lambda and Immucor) were counted to ensure comparable Ab/antigen ratio for AgD testing. One vendor uses 40uL of beads; these were spun and resuspended in 5 uL. HLA AgD was detected using mouse monoclonal Abs (class I: W6/32, class II: WR18). Dilutions of each Ab were tested to achieve maximum saturation of SABs as well as lower bead saturation. Staining protocols were unified to 5 uL of beads and 20 uL of Ab. A PE labelled anti-mouse conjugate was used. All mean fluorescence intensity (MFI) values were exported to Excel and AgD MFI values were analysed. Common alleles were compared with some remaining differences in the class II α / β allele combinations. A ratio was created to the lowest ranked bead for each vendor and compared to the company’s published AgD data. Results The saturated SAB AgD is comparable between vendors (Figure 1). The lower dilutions of W6/32 and WR18 yields higher AgD staining with one vendor over the other with the exception of several C locus beads. The DQ SABs have the most variability within and between vendors. Our lowest ranked bead ratios are similar but not identical to either vendor’s results and varied depending on bead saturation. Conclusions We are increasingly reliant on SABs and AgD likely plays a role in interpretation of patient Ab levels. When AgD staining protocols are standardized for both vendors, AgD is similar overall. However, we have also tested patient and proficiency sera using both SABs and routinely see differences in MFI values that may be due to patient sera testing protocol dissimilarities. Our laboratory performs SAB AgD testing as part of our lot# pre-use testing and uses this information, particularly for DQ SABs to aid in patient result interpretation. Figure 1: Antigen density testing MFI results. Download : Download high-res image (394KB) Download : Download full-size image