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.