Search

In the past two decades, we witnessed the evolution of the basophil activation test (BAT) from mainly research applications to a potential complementary diagnostic tool to document IgE-dependent allergies. However, BAT presents some technical weaknesses. Around 10%-15% of tested patients are non-responders, BAT can be negative immediately post-reaction and the use of fresh basophils, ideally analysed within 4 h of collection, restricts the number of tests that can be performed per sample. The need for fresh basophils is especially limiting when conducting batch analyses and interlaboratory comparisons to harmonize BAT methodology. These limitations significantly hinder the wider application of BAT and urge the development of alternative testing, such as the mast cell activation test (MAT). The essential difference between BAT and MAT is the heterogeneity of the starting material used to perform the assays. Mast cells are tissue-resident, so cannot be easily accessed. Current alternative sources for functional studies are generating primary human mast cells, differentiated from donor progenitor cells, or using immortalized mast cell lines. Hence, the methodological approaches for MAT are not only vastly different from BAT, but also different among MAT protocols. This review summarizes the advantages and disadvantages of BAT and MAT assays, dedicating special attention to elucidating the key differences between the cellular sources used and provides an overview of studies hitherto performed comparing BAT and MAT in the diagnosis of IgE-mediated food and drug allergies., (© 2024 John Wiley & Sons Ltd.)

Background: Effector cells assays provide an overall measure of responsiveness to allergen, but the lack of reliable and high-throughput assays limits the clinical utility. We aimed to develop a high-throughput basophil activation test based on human progenitor cell-derived basophils (PCB) and investigate the role of PCB activation test (PCBAT) in allergic diseases., Methods: Progenitor cell-derived basophils were differentiated from CD34 + progenitor cells and sensitized with sera from subjects sensitized to cat, peanut or atopic controls. Sensitized PCBs were stimulated with increasing concentrations of the corresponding allergens in vitro. Degranulation was assessed by measuring CD63 expression using flow cytometry. The correlations between PCBAT and clinical allergy were assessed., Results: Following passive sensitization of the mature PCBs with serum and allergen stimulation, an allergen specific dose-dependent increase in CD63 expression was observed. Sera from subjects sensitized to cat (n = 35, of which 17 subjects had clinical reactivity quantified using inhaled allergen challenge), peanut allergic (n = 30, of which 15 subjects had clinical reactivity validated using double blind, placebo controlled food challenges [DBPCFC]), peanut-sensitized but tolerant subjects (n = 5) were used to sensitize PCBs. PCBAT area under the curve (AUC) correlated with sIgE (r 2  = .49, p = .001) in subjects sensitized to cat (sIgE ≥ 0.35KU/L). The provocation concentration of inhaled cat allergen (PC 20 ) correlated with PCBAT AUC (r 2  = .33, p = .016). In subjects sensitized to peanut, PCBAT AUC was highly correlated with sIgE to Ara h 2 (r 2  = .59, p < .0001). Peanut threshold cumulative dose during DBPCFC was negatively correlated with PCBAT AUC (r 2  = .57, p = .001) and IgE to Ara h1 (r 2  = .55, p = .007), but not with sIgE to whole peanut or Ara h2. All peanut-sensitized but tolerant subjects showed no reaction to peanut on PCBAT., Conclusion: Progenitor cell-derived basophils activation test is a high-throughput assay, which correlates with clinical allergy and may confer a powerful alternative tool in allergy testing., (© 2022 The Authors. Clinical & Experimental Allergy published by John Wiley & Sons Ltd.)

Since the late nineties, evidence has accumulated that flow-assisted basophil activation test (BAT) might be an accessible and reliable method to explore the mechanisms governing basophil degranulation and diagnostic allowing correct prediction of the clinical outcome following exposure to the offending allergen(s) and cross-reactive structures for different IgE-dependent allergies and particular forms of autoimmune urticaria. Although the BAT offers many advantages over mediator release tests, it is left with some weaknesses that hinder a wider application. It is preferable to perform the BAT analysis within 4 h of collection, and the technique does not advance diagnosis in patients with non-responsive cells. Besides, the BAT is difficult to standardize mainly because of the difficulty to perform large batch analyses that might span over several days. This article reviews the status of flow cytometric mast cell activation test (MAT) using passively sensitized mast cells (MCs) with patients' sera or plasma (henceforth indicated as passive MAT; pMAT) using both MC lines and cultured MCs in the diagnosis of IgE-dependent allergies. In addition, this paper provides guidance for generating human MCs from peripheral blood CD34 + progenitor cells (PBCMCs) and correct interpretation of flow cytometric analyses of activated and/or degranulating cells. With the recent recognition of the mas-related G protein-coupled receptor X2 (MRGPRX2) occupation as a putative mechanism of immediate drug hypersensitivity reactions (IDHRs), we also speculate how direct activation of MCs (dMAT)-that is direct activation by MRGPRX2 agonists without prior passive sensitization-could advance paradigms for this novel endotype of IDHRs., (© 2021 John Wiley & Sons Ltd.)