D3.06 - Comparative Evaluation of Raw, Blanched, Individual and Composite Allergen Preparation for Basophil Activation Testing

Basophil Activation Testing (BAT) is increasingly used as a functional assay to assess IgE-mediated reactivity. However, BAT performance is highly dependent on the quality and composition of allergen extracts. Recently, “low-toxin” (lotox) raw extracts have been introduced to reduce non-specific activation. Physic-chemically processed extracts or individual allergens can also be used to better assess the source of reactivity. Additionally, composite mixtures of individual allergens are also proposed to improve standardization. Yet, their functional comparability remains to be documented.

Using fresh whole blood samples from allergic donors, this study aimed to compare basophil activation profiles obtained with different types of allergenic extracts. These included individual purified natural allergens, composite mixtures of individual allergens, and both blanched or raw extracts of peanut, egg and cow’s milk.

Whole blood samples from allergic donors to cow’s milk, egg and peanut were tested with a 96-well plate-based approach to BAT using dry and ready-to-use reagents incorporating both fluorochrome-conjugated antibodies and allergenic extracts. Blood was activated in parallel with serial dilutions of raw and blanched extracts, composite mixtures, and individual allergens. Basophil were identified as SSlowCD45+CD3-HLA-DR-CD123+ and their activation was quantified based on CD63 and CD203c upregulation.

Across all tested allergens, basophil activation for a given patient differed substantially depending on the extract used. Composite extracts generally produced the strongest activation responses, frequently reflecting the combined activation profiles of their individual allergen constituents. In comparison to lotox extracts, blanched extracts (when available) typically triggered lower basophil activation, certainly due to the loss of some allergenic epitopes during the blanching process. Finally, individual allergens captured specific component-resolved responses of each patient.

This study highlights the importance of allergen extract 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.