Detection of Sensitization Profiles with Cellular In Vitro Tests in Wheat Allergy Dependent on Augmentation Factors (WALDA)

Abstract

Wheat allergy dependent on augmentation factors (WALDA) is the most common gluten allergy in adults. IgE-mediated sensitizations are directed towards ω5-gliadin but also to other wheat allergens. The value of the different in vitro cellular tests, namely the basophil activation test (BAT) and the active (aBHRA) and passive basophil histamine-release assays (pBHRA), in the detection of sensitization profiles beyond ω5-gliadin has not been compared. Therefore, 13 patients with challenge-confirmed, ω5-gliadin-positive WALDA and 11 healthy controls were enrolled. Specific IgE (sIgE), skin prick tests, BATs, aBHRA, and pBHRA were performed with allergen test solutions derived from wheat and other cereals, and results were analyzed and compared. This study reveals a distinct and highly individual reactivity of ω5-gliadin-positive WALDA patients to a range of wheat allergens beyond ω5-gliadin in cellular in vitro tests and SPT. In the BAT, for all tested allergens (gluten, high-molecular-weight glutenin subunits, α-amylase/trypsin inhibitors (ATIs), alcohol-free wheat beer, hydrolyzed wheat proteins (HWPs), rye gluten and secalins), basophil activation in patients was significantly higher than in controls (p = 0.004-p < 0.001). Similarly, significant histamine release was detected in the aBHRA for all test substances, exceeding the cut-off of 10 ng/mL in all tested allergens in 50% of patients. The dependency of tests on sIgE levels against ω5-gliadin differed; in the pBHRA, histamine release to any test substances could only be detected in patients with sIgE against ω5-gliadin ≥ 7.7 kU/L, whereas aBHRA also showed high reactivity in less sensitized patients. In most patients, reactivity to HWPs, ATIs, and rye allergens was observed. Additionally, alcohol-free wheat beer was first described as a promising test substance in ω5-gliadin-positive WALDA. Thus, BAT and aBHRA are valuable tools for the identification of sensitization profiles in WALDA.

Keywords: IgE; WALDA; WDEIA; basophil activation test; basophil histamine-release assay; food allergy; gluten; hydrolyzed wheat proteins; rye; wheat allergy dependent on augmentation factors.

Figure 1

Overview of positive and negative skin prick tests to different allergens in WALDA patients and controls. Data are shown as wheal diameter in mm exceeding the negative control. Histamine dihydrochloride (10%) solution was used as a positive and isotonic sodium chloride solution as a negative control. Abbreviations: ATI, α-amylase/trypsin inhibitors; HMW-GS, high-molecular-weight glutenin subunits; HWPs, hydrolyzed wheat proteins; LMW-GS, low-molecular-weight glutenin subunits; sIgE, specific IgE.

Figure 2

Overview of the in vitro basophil tests, BAT, aBHRA, and pBHRA, in WALDA patients. For the BAT, data are shown as maximum values of % CD63⁺ basophils in any concentration of the respective test substance (%CD63⁺ max). Anti-FcɛRI monoclonal antibodies and N-formyl-methionine-leucyl-phenylalanine (fMLP) were used as positive controls, with two blank determinations as negative controls (mean value shown in the figure). For the aBHRA, the values are presented as maximum histamine release in ng/mL, and anti-IgE (aIgE) was used as the positive control. For the pBHRA, the values are shown as maximum histamine release in ng/mL exceeding the negative control. The color scheme used is purely indicative.

Figure 3

BAT measurement of a WALDA patient (patient 4) with alcohol-free wheat beer in a dilution of 1:100: (A) The cells were gated based on their granularity and size using side scatter (SSC-A) and forward scatter (FSC-A). (B) Basophil identification was performed using side scatter and the CCR3 identification marker, labeled with an anti-CCR3-phycoerythrin monoclonal antibody. (C) The quantification of CD63-positive cells within the total basophil population was performed using CD63 as the basophil activation marker, labeled with anti-CD63-fluorescein-isothiocyanate monoclonal antibodies; in this patient, 81% of 502 counted basophils were CD63⁺ upon stimulation with alcohol-free wheat beer (dilution 1:100).

Figure 4

(A) Correlation between sIgE against ω5-gliadin and the basophil activation expressed as the maximum %CD63⁺ basophils with HMW-GS (high-molecular-weight glutenin subunits) allergen test solution in the BAT; (B) correlation between the basophil activation (expressed as maximum %CD63⁺ basophils) induced by ATIs (α-amylase/trypsin inhibitors) and alcohol-free wheat beer in the basophil activation test.

Figure 5

Histamine release (ng/mL) in the aBHRA with HMW-GS (high-molecular-weight glutenin subunits) test solution in different concentrations (in µg/mL) in a patient with WALDA (patient 13).