Performance of basophil activation testing (BAT) is highly dependent on the allergen extracts. Recently, physico-chemically processed extracts, individual allergens, or composites of individual purified allergens, with defined allergen compositions, have been introduced to improve standardization and reduce non-specific activation. Yet, their functional comparability remains to be documented.

This study aimed to compare basophil activation profiles obtained with different types of allergenic reagents. These included individual purified allergens, composites of individual purified allergens, and extracts of egg, cow's milk, and peanut (blanched and roasted).

Whole blood samples from allergic donors to cow's milk, egg and peanut were tested with a dry 96-well plate-based approach to BAT incorporating both fluorochrome-conjugated antibodies and serial dilutions of allergenic extracts. Basophil were identified as SSlowCD45 + CD3-HLA-DR-CD123+ and their activation was quantified based on CD63 and CD203c upregulation.

Basophil activation differed depending on the allergen preparation. Composites of purified allergens generally produced the strongest activation responses, reflecting the combined profiles of their individual allergen constituents. Blanched extracts (when available) triggered lower basophil activation, certainly due to the creation of new and/or accessibility of existing epitopes upon roasting and/or loss of some allergenic epitopes during the blanching process. Finally, individual purified allergens captured specific component-resolved responses.

This study highlights the importance of allergen selection for BAT experiments and provides various perspectives to adapt BAT-based allergy testing to pursued objectives. These findings emphasize the need for careful extract standardization in BAT workflows and support further systematic comparisons to guide clinical and research applications.