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

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.

This study develops gating strategies and pre-analytical storage conditions to develop an analytically validated BAT protocol for Dermatophagoides farinae (Df) allergy.

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.

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) (r²<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.

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.