D3.94 - Good Practice Recommendations for Basophil Activation Test (BAT) Data Analysis in Routine and High-Background Settings

The European Academy of Allergy and Clinical Immunology task force consensus report (2023) defined harmonized protocols for the basophil activation test (BAT), including basophil identification (low SSC, CCR3/CD193) and activation assessment using CD63 with a 97.5th percentile-based threshold. The Flow CAST assay (BÜHLMANN Laboratories AG) follows these recommendations and specifies a patient-specific background threshold of 2.5% CD63+ resting basophils. However, real-world data indicate that deviations from recommended gating strategies and increased spontaneous background activation (e.g. in chronic spontaneous urticaria or persistent allergen exposure) may compromise threshold definition and result interpretation. We aimed to define good practice recommendations for BAT data analysis in both standard and high-background settings.

We evaluated routine BAT datasets generated with Flow CAST, focusing on (i) basophil gating according to consensus criteria (low SSC, CCR3+) and (ii) CD63 threshold definition based on unstimulated controls. Cases with elevated background activation were identified by increased CD63 expression in negative controls and distribution skewness. We assessed conventional fixed-threshold approaches (2.5%) and algorithm-supported strategies (quantile-based and Gaussian fitting methods) to define activation thresholds in the presence of spontaneous activation.

In standard cases with low background activation, adherence to consensus-recommended gating and a 2.5% patient-specific CD63 threshold provided robust and reproducible results. In contrast, samples with elevated spontaneous activation showed overlapping resting and activated populations, rendering fixed thresholds insufficient. Algorithm-supported threshold definition enabled reliable discrimination of activated basophils across a broad activation range and reduced the risk of false-negative interpretation. Skewness analysis proved useful for identifying samples requiring adapted thresholding.

Strict adherence to consensus-based basophil gating and patient-specific threshold definition ensures reliable BAT interpretation in routine cases. In samples with increased background activation, adapted or algorithm-supported threshold strategies are recommended to maintain diagnostic accuracy. Implementation of standardized and automated analysis workflows may improve robustness, reproducibility, and external quality assurance of BAT in clinical practice.