Hoxb8 MAT discriminates between peanut allergic and non-allergic children and adolescents.
All samples had been stored at -80°C for more than 12 months before they were analyzed in the previously described Hoxb8 MAT in a blinded manner and all experiments were analyzed by a biostatistician outside of the lab of the two principal investigators. The primary goal of this study was to assess the diagnostic performance of the serum based Hoxb8 MAT in comparison to the whole blood based BAT using 96 samples (69 PA; 27 NA controls). Mature Hoxb8 MCs were passively sensitized with pre-processed sera and stimulated with 6 serial 10-fold dilutions (0.01-1000 ng/mL) of peanut extract. The percentage of activated mast cells was quantified by the detection of CD107a using flow cytometry. Mature Hoxb8 MCs sensitized with samples from peanut allergic patients showed a significantly higher percentage of activated mast cells upon stimulation with 10-1000 ng/mL peanut extract as compared to those with samples of the non-allergic controls (Fig. 2a ). All PA patient sera, except five, followed an allergen dose-dependent activation curve (Fig. 2b ), while all sample in the NA population, except one, showed no activation. Interestingly, the false positive sample featured remarkably high sIgE against peanut extract (82.3 kUA/L), Ara h1 (21.2 kUA/L) and Ara h2 (54.1 kUA/L), consumes peanut regularly at relevant amounts without being on an OIT and displays, mild, intermittent skin reactivity. The very patient also displayed BAT reactivity.
Similarly, the percentage of CD63 (i.e. LAMP-3) positive basophils in whole blood of PA patients were significantly higher compared to NA patient samples upon allergen challenge at concentrations ranging from 0.1-1000 ng/mL (Fig. 2c ). Overall, the allergen dose response curves on the individual sample level showed more variability in the BAT (Fig. 2d ), with several samples in the NA group becoming activated at allergen-doses >100 ng/ml and various samples in the PA group showing inverse dose-dependency or the so-called “hook effect”. To assess and re-evaluate the diagnostic performance of the Hoxb8 MAT and the BAT in the identification of peanut allergic patients in our cohort, we performed ROC curve analyses for both assays including the data from all 96 tested samples at each individual allergen concentration (0-1000ng/ml). For the Hoxb8 MAT the AUROC curves demonstrated a consistent allergen dose-dependent increase of diagnostic accuracy which hit a plateau at the 100 ng/ml peanut allergen dose (AUROC of 0.97) and stayed at this level for 1000 ng/ml peanut allergen (Fig. 2e and Table 1 ). The diagnostic accuracy of the BAT was high at low peanut allergen concentration (e.g. 0.1 and 1 ng/ml), reached its maximum at 10 ng/ml (AUROC of 0.99) and then declined at 100 and 1000 ng/ml of peanut allergen (Fig. 2f and Table 3 ).