Standardizing a Four-Marker Basophil Activation Test: Influence of Storage Time, Temperature, and IgE Levels

Background: Allergic diseases affect 25% of the global population, yet current diagnostics like specific IgE (sIgE) and skin prick tests lack specificity. The basophil activation test (BAT) offers high specificity but requires methodological standardization. Objective: This study develops gating strategies and pre-analytical storage conditions to develop an analytically validated BAT protocol for Dermatophagoides farinae (Df) allergy. Methods: Seventy-nine adults (Df-sensitized and controls) were enrolled. Basophil activation was assessed by CD63 expression following Df extract, anti-IgE, or fMLP stimulation. Evaluations included gating strategies, correlations with serological markers, and storage stability. Results: The three-marker gating strategy (CD123 + CD203c + CD45 + SSC low ) achieved analytical equivalence to a four-marker gating approach with 25% cost reduction, and improved phenotypic purity over a two-marker method (97.2% vs. 91.8% HLA-DR negativity). Df-induced BAT results correlated moderately with Df sIgE (r=0.4352) and membrane-bound IgE (mIgE) (r=0.6264– 0.6659) (r 2 < 0.4), leaving > 60% of variability unexplained. Quantitatively, CD63 MFI declined significantly with storage. Qualitatively, storage at 2– 8°C better preserved Df responsiveness up to 48– 52 h, while room temperature (RT) was superior for fMLP. Conclusion: This study demonstrates that a three-marker gating strategy within a four-color BAT panel reduces costs while maintaining analytical equivalence to a four-marker gating reference. Stimulus-specific storage patterns support flexible processing for research applications, and BAT outcomes provide complementary functional information beyond IgE levels. Clinical utility requires prospective validation.