Major pitfalls in BAT performance may be caused by gating protocols and CD63% cut off evaluation

Using a CD45dim/CD123bright/HLA-DRneg Phenotyping Protocol to Gate Basophils in FC for Airway Allergy. CD123 Does Not Decrease

“…This should not be a fundamental concern in a CD123-based gating strategy, as previously reported [4], but it may have an effect if the gating marker is widely shared with non-basophil cells. Levels and brightness of fluorochromes in different companies might also cause differences in the observed effects by different research groups and in conclusion expression of basophil markers and BAT performance depend on a good gating approach [2].…”

“…This issue represents the main technical concern of a BAT. A first reason is because researchers are endeavoured in finding stable and highly specific markers in order to phenotype basophils and separate them from other cells in a complex milieu such as the whole blood, second because in a BAT basophils perfectly distinguished from other cells have to be followed up by some highly specific markers that should show better than other ones the cell dynamics of the allergy activation [2]. In few words, to "see" (phenotype) basophils in flow cytometry, researchers needs stable membrane markers, which do not change their expression during the cell activation.…”

Basophil Activation Tests (BAT): Wheat and Chaff

“…The Authors stressed on the commonly recognized role exerted by CD63 and CD203c markers in BAT, while existing further suggestions [2,3] but they never addressed the critical issue of basophil electronic capture in flow cytometry (FC), i.e. cell phenotyping protocols [4,5]. Advantages of BAT in allergy diagnosis are closely related to the simple fact that this assay is a cell based investigation procedure [6] and therefore, while its major value is the ability in diagnosing either an IgE-or non IgE-mediated allergy, immunotherapy and anaphylaxis and to prevent the ethical issue associated to skin tests [1], a main concern is represented by the interpretation of activation markers in a good phenotyping FC approach [7].…”

“…However, it is particularly difficult to diagnose a non releaser subject through the performance of a BAT, mainly because of the many issues related to CD63 evaluation [5,7], besides to effects caused by the differential expression of membrane FceRI/IgE complexes, circulating IgEs and relationships with FcgRs [11], downregulation and intracellular recycling of Fc receptors upon activation [12,13], doses and strength of polyclonal anti-IgEs or serum IgEs in inducing an activation response from basophils in a BAT. Non releaser subjects, unless an intracellular FC evaluation of Syk, might be misled among people performing different causes to their basophils responsiveness.…”

“…How much current BATs are able to help physicians to address a medical decision? The evaluation of CD63%, based on an arbitrary non responding threshold, is particularly cumbersome, despite its apparent feasibility [5,7] and may oblige operators to add further activation markers or interpretation algorythms in BATs [7]. Very few physicians are experienced with BATs and this represents a main education concern in laboratory and clinics.…”