Optimized In-House Basophil Activation Test for Pediatric Egg Allergy: Agreement with a Structured Composite Clinical Classification

Background: Accurate diagnosis of IgE-mediated egg allergy in young children remains challenging. While basophil activation test (BAT) offers a functional readout, methodological heterogeneity and limited real-world pediatric validation have hindered its standardized use. Purpose: To develop and evaluate an optimized in-house BAT protocol for pediatric egg allergy, and to assess its agreement with structured clinical classification and its exploratory adjunctive role in diagnostically challenging cases. Methods: Children aged 0– 6 years with suspected food allergy were prospectively enrolled in a cross-sectional agreement study without longitudinal follow-up and classified by a structured composite reference standard integrating clinical history, egg-specific IgE (sIgE), documented tolerance, and expert panel consensus as egg-allergic (EA, n=28), egg non-allergic (NEA, n=68), or diagnostically indeterminate (IND, n=6). BAT was performed using stimulation with egg extract. The stimulation index (SI) was defined as the ratio of median fluorescence intensity after stimulation to that of the unstimulated control. Marker-specific thresholds were defined as the 95th percentiles of the corresponding parameters in the NEA group. Agreement with the EA/NEA classification, utility in the IND group, and correlation with sIgE were measured and evaluated. Results: The conjunctive CD63 SI and CD203c SI rule was selected as the primary BAT interpretation rule. In 96 EA and NEA cases, BAT showed 88.5% overall agreement as evaluated against the structured composite clinical classification (positive percent agreement 64.3%, negative percent agreement 98.5%; Cohen’s κ= 0.694, 95% CI 0.530– 0.858). Among 6 IND children with suggestive histories but undetectable egg-specific IgE, BAT was positive in 3 cases; however, this finding was not confirmatory of clinical allergy without oral food challenge (OFC). BAT activation parameters correlated moderately with sIgE in the overall cohort (r=0.4238– 0.5126, all p < 0.001), but not within the EA group alone. Conclusion: This optimized in-house BAT, using a CD63 SI and CD203c SI conjunctive rule, showed substantial agreement with the structured composite clinical classification and provided functional evidence in seronegative, clinically suspected cases. However, without OFC confirmation, a positive BAT cannot establish clinical egg allergy. BAT may serve as an adjunctive functional test when first-line assessments are discordant or inconclusive, but should not be considered a replacement for OFC.