– HLA Compatibility Supervisor, University of Cincinnati / Hoxworth Blood Center, United States
Aim: Understanding intra-laboratory test variability is critical in clinical and research settings, as it impacts the reproducibility and interpretation of test results. Intra-laboratory variability can exceed 50% for single antigen bead (SAB) analysis secondary to operator technique or environmental factors like temperatures or pH. Interpretation of fluctuations in bead-specific mean fluorescent intensities (MFI) can be further muddled when factoring in differing assay vendors, antigen sources, and test reagents. While existing literature on variability in HLA SAB studies predominantly focuses on manual techniques, this study explores intra-laboratory variability in the setting of robotic-assisted workflows.
Methods: We evaluated SAB results from an “in-house” source positive control sera across manual methods and two robotic platforms (LabXpress and HLAPRO). Coefficients of variation (%CV) were calculated across multiple assay runs for each bead using Thermo Fisher, One Lambda LS1A04, LS2A01, and MICA beads. Additionally, we assessed %CV for SAB results using Werfen LSA Class 1 and Class 2 beads run manually using the same positive control sera.
Results: Coefficients of variation (%CV) were calculated across multiple assay runs. The LabXpress consistently outperformed manual and HLAPRO workflows in precision, with average %CVs of 11.11% (LabXpress), 16.33% (Manual), and 16.35% (HLAPRO). While automation reduces operator-driven variability, residual inconsistencies were still observed.
Conclusion: SAB assays are particularly sensitive to technologist-dependent actions (including pipetting, washing, vortex speeds, centrifuging, etc.), which influence assay performance and the reported MFI value. Our findings demonstrate that despite robotic assistance, variability persists. We attribute this residual variability to factors such as instrument calibration, environmental conditions, and sample handling protocols upstream of automation.
