Parameters that can influence the results of the basophil activation test
Various factors can affect the results of the BAT, for instance: time between blood collection and the performance of BAT, medication that the patient being tested may be on, material used for basophil stimulation, antibodies used for staining of key markers and flow cytometry analyses.
Blood basophils are best used fresh, ideally on the same day or up to 24 hours of blood collection28. It is possible to obtain a positive result after two days28,29; however, that result may not be the same to that obtained with fresh blood basophils28. Individuals being tested on BAT should stop treatment with oral steroids three weeks before the test30. Antihistamines and topical treatments do not influence the result of BAT30.
Ideally, standardized extracts, recombinant or purified allergens or parenteral drug preparations should be used for the BAT, and, if necessary, the patient can bring the relevant allergen with them (Peppys principle)3. An allergen the patient brings can be solubilized according to standard methods3, and should be used at concentrations not toxic to blood. Typically, less than 1% w/v is a high concentration that can be tolerated, and response to more than four sequential log dilutions of allergen should be determined. Allergen extracts should be clearly defined in terms of protein and major allergen concentration. Molecular allergens should be defined in mole of allergen. Basophil testing is surprisingly resilient to selection of fluorochrome-conjugated antibodies used, and the method of warming up the cells to respond31.
Activated basophils are identified by measuring the percentage of CD63 positive cells and the fold change in CD203c MFI compared to negative control. During the gating analysis, it is important to have the same threshold set on a negative control at the same level of reactivity. When diagnosing drug allergy, a threshold of 2.5% CD63+ basophils in the unstimulated condition gives the best results31. Methods to use automated data analyses have been developed and have the advantage of being more standardised and objective compared to manual gating, which is still considered the gold-standard32.