Aim: Adequate bead counts are required for the single antigen bead (SAB) assay to ensure test validity. Samples affected by low bead counts (LBC) must be retested, increasing turnaround time and cost. We have previously observed LBC problems in highly sensitized patients, primarily affecting beads with high MFI, which led to our hypothesis that LBC may be related to bead agglutination by IgM in sera. Specifically, rheumatoid factor (RF), a predominantly IgM anti-IgG, may crosslink beads coated by anti-HLA IgG and cause agglutination of high MFI beads. Here, we explored the interaction between SAB results and the IgM-reducing agent, DTT, in six index samples with LBC. We also examined the potential impact of RF levels on SAB results through a retrospective chart review.
Methods: Six clinical samples with LBC during SAB testing (LABScreen) were retested with DTT treatment. MFI and bead counts from the Fusion software were analyzed using the Pearson correlation; r2 values were compared between testing conditions using a Wilcoxon test. Next, we identified a cohort of patients at our medical center from 2020-2023 who had anti-HLA antibody testing with cPRA ≥ 90% and quantitative RF testing within six months of each other. MFIs and bead counts were plotted and evaluated for their correlation. r2 values were compared between groups of patients with RF below and above the limit of quantitation (LOQ) using a Mann-Whitney test.
Results: MFI and bead counts were inversely correlated without DTT treatment, and DTT pretreatment resolved LBC and significantly reduced the r 2 in all samples (p < 0.001) (Figure 1). Twenty patients with cPRA ≥ 90% and RF results were identified. MFI-Bead count r2 values were significantly higher in patients with RF above the LOQ overall, but not when Class I and II were analyzed separately (Figure 2).
Conclusion: Highly reactive samples with LBC may demonstrate an inverse correlation between MFI and bead count that can be reduced with DTT treatment. The correlation is stronger overall in patients with detectable RF. These findings support that RF may contribute to IgM-mediated agglutination of high-MFI beads, reducing their counts. Anti-IgM secondary antibodies and eplet analysis could further characterize bound IgM in affected samples.
