Clinical utility analysis of the Hoxb8 mast cell activation test for the diagnosis of peanut allergy

Abstract

Background: Peanut allergy is among the most severe and common food allergies. The diagnosis has a significant impact on the quality of life for patients and their families. An effective management approach depends on accurate, safe, and easily implementable diagnostic methods. We previously developed a cell-based assay using Hoxb8 mast cells (Hoxb8 MCs) aimed at improving clinical allergy diagnosis. In this study, we assessed its diagnostic performance by measuring blinded sera from a prospectively enrolled and pre-validated peanut allergy cohort.

Methods: Hoxb8 MCs were passively sensitized with sera from peanut-allergic and peanut tolerant children and adolescents (n = 112). Degranulation of Hoxb8 MCs was quantified upon stimulation with dose-titrated peanut extract by means of flow cytometry, using CD107a as activation marker. The results from the Hoxb8 mast cell activation test (Hoxb8 MAT) were compared to established diagnostic assays such as the skin prick test (SPT), specific IgE (sIgE) levels, and the basophil activation test (BAT). Additionally, serum samples from BAT nonresponders were assessed with the Hoxb8 MAT.

Results: Hoxb8 MAT displayed a robust dose-dependent activation to peanut extract, with a cutoff value of ≤5.2% CD107a positive cells. The diagnostic accuracy was highest at allergen concentrations ≥100 ng/mL, with an area under the receiver operating characteristic curve (AUROC) of 0.97, 93% sensitivity, and 96% specificity, outperforming traditional SPT and sIgE tests. When compared to BAT, Hoxb8 MAT exhibited comparable diagnostic efficacy. Moreover, sera from BAT nonresponders were accurately classified into allergics and nonallergics by the Hoxb8 MAT.

Conclusions: The Hoxb8 MAT demonstrated a very good diagnostic precision in patients prospectively assessed for peanut allergy comparable to the fresh whole blood-based BAT. Additionally, it demonstrated its value for accurate classification of BAT nonresponders into allergic and nonallergic individuals. Further investigations into its utility in the routine clinical setting are warranted.

Keywords: Hoxb8 mast cell activation test; basophil activation test; oral food challenge; peanut allergy diagnosis; specific IgE.

FIGURE 1

Study population with comparison of skin prick test and allergen‐specific IgE measurement results between clinically confirmed nonallergic controls and peanut allergic patients. (A) Blinded MONA study serum samples (n = 112) from the Toronto site with available clinical status were transferred to the University of Bern. Serum samples for which BAT data was recorded in MONAS (n = 96) were included in the clinical performance analysis of the Hoxb8 MAT and in direct comparison to BAT. Amongst those SPT information for 47 samples and sIgE measurements for 67 samples has been available. (B) Wheal size measurement results from skin prick test compared between nonallergic controls (NA: N = 10; blue) and peanut allergic patients (PA: N = 37; red). (C) Comparison of allergen‐specific IgE measurements by ImmunoCAP for peanut extract, Ara h1, Ara h2, Ara h3, Ara h8, and Ara h9 between nonallergic controls (NA: N = 16; blue) and peanut allergic patients (PA: N = 51; red). Data are shown as individual data points and means ± SEMs. *p < .05, ****p < .001, ns, not significant.

FIGURE 2

Performance analysis of Hoxb8 MAT and BAT to differentiate between clinically confirmed nonallergic controls and peanut allergic patients. Passively sensitized Hoxb8 MCs (A and B) or whole blood (C and D) were stimulated with peanut allergen extract at various doses (0–1000 ng/mL). Comparison of activated Hoxb8 MCs or blood basophils in the nonallergic (NA) and peanut allergic (PA) group on a population basis is shown (A and C). Dose–response curves of activated Hoxb8 MCs and blood basophils for individual patient samples are represented in two separate subpanels (B and D). Nonallergic controls are depicted in blue, while peanut allergic patients are represented in red. (E and F) ROC curve analyses for Hoxb8 MAT and BAT results across different allergen concentrations (color coded) are depicted and AUROC values are indicated. Data are shown as individual data points and means ± SEMs. *p < .05, ****p < .001.

FIGURE 3

Correlation analysis between skin prick test or allergen‐specific IgE measurements and maximal activation signal in the Hoxb8 MAT. Wheal size results from skin prick test (A) or allergen specific IgE measurement for peanut extract (B), Ara h1 (C), and Ara h2 (D) from nonallergic controls (red dots) and allergic patients (blue dots) were plotted against the maximal activation signal from the Hoxb8 MAT and a linear correlation analysis (red line) was performed. The correlation coefficient (r) as well as the significance of the correlation (p) are indicated for each subpanel. (E) The strength of the linear relationship (r‐value) between the maximal Hoxb8 MAT activation and the disease severity according to Astier classification was plotted against the different allergen concentration used (0.01–1000 ng/mL) and a linear correlation analysis (red line) was performed.

FIGURE 4

Analysis of seven BAT nonresponder samples with the Hoxb8 MAT. Seven sera from previously identified BAT nonresponders (NR1‐7) were measured on passively sensitized Hoxb8 MCs. The clinically confirmed allergy status of the individual patient samples is color coded (peanut allergic, PA: red; nonallergic, NA: blue; no information).

FIGURE 5

Direct comparison of performance analyses between different diagnostic tests. Direct ROC curve analysis for SPT and sIgE against peanut extract compared with ROC curve analysis of logistic regression models with Hoxb8 MAT and BAT results including all peanut allergen concentrations are depicted (color coded) and AUROC values are indicated.