A novel human mast cell activation test for peanut allergy

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A novel human mast cell activation test for peanut allergy
To the Editor: Peanut allergy (PA) has a significant effect on patients' lives, and therefore an accurate diagnosis is extremely important. Peanutspecific IgE (P-sIgE) is associated with false-positive results and overdiagnosis. 1 Measurement of Ara h 2-specific IgE is more accurate but is associated with false-negative results. Thus a considerable proportion of patients need to undergo an oral food challenge (OFC), the current gold standard to diagnose food allergy. 2 OFCs carry the risk of causing allergic reactions, including anaphylaxis. With the advent of new treatments for PA, use of reliable in vitro tests rather than OFCs to identify eligible patients and monitor clinical response to treatment is desired.
Previously, we showed that the basophil activation test (BAT) is highly discriminative between children with PA and children with peanut sensitization but not allergy (PS children) and can reduce the number of OFCs. 3 Because the BAT requires fresh blood and 10% to 15% of individuals have uninterpretable BAT results caused by nonresponding basophils (ie, basophils that do not respond to IgE-mediated but only non-IgE-mediated stimulants), 4,5 we investigated whether the ability to elicit peanut-induced cell activation could be transferred by passive sensitization of LAD2 mast cells 6 with patients' plasma.
Children being assessed for PA (n 5 174), including 73 children with PA, 60 PS children and 41 nonsensitized nonallergic (NA) children, underwent clinical assessment, skin prick tests, blood collection for immunoglobulin measurement (by using Immuno-CAP; Thermo Fisher Scientific, Waltham, Mass), and OFCs to peanut, as previously described. 3,7 Participants were grouped as patients with PA, PS patients, or NA subjects. The allergic reaction severity was classified according to the method of Ewan and Clark, 8 and the threshold dose was determined as the total amount of peanut protein ingested during the OFC. The study was approved by the South East London Research Ethics Committee 2. Whole blood BATs and mast cell activation tests (MATs) to peanut were performed, as previously described. 3,9 Statistical analyses were performed with SAS 9.4 software (SAS Institute, Cary, NC) and JMP Pro software, Version 13.2.1. Depending on data distribution, nonparametric Wilcoxon tests or normality-based t tests were used, where specified. Optimal cut points were estimated from receiver operating characteristic analyses based on logistic regression models. Relationships between mechanistic outcomes were analyzed by using stratified linear models; cubic splines were used to allow for more linear curve relationships between variables. When relationships appeared linear, Pearson correlation coefficients were reported and visualized with simple linear models and 95% CIs. Plasma samples from children with PA, PS children, and NA children (see Table E1 in this article's Online Repository at www. jacionline.org) were tested in the MAT. Activation of mast cells sensitized with plasma from children with PA after stimulation We analyzed the utility of the MAT to diagnose PA and to identify allergic patients at risk of severe reactions by using receiver operating characteristic curve analyses (Table I and  MAT results were strongly correlated with BAT results to peanut (R s 5 0.808, P < .001; see Fig E8 in this article's Online Repository at www.jacionline.org). BATs showed greater diagnostic accuracy 3 compared with MATs, particularly because of their greater sensitivity; conversely, MATs provided a conclusive result for subjects with nonresponding basophils. Twelve children with PA had positive BAT and negative MAT results; these were patients with relatively low P-sIgE levels (median, 0.72; interquartile range, 0.27-2.79). Patients with nonresponding basophils all showed good response to anti-IgE and ionomycin and had an MAT result to peanut consistent with their allergic status.
The data reported here support the use of MATs to diagnose PA, namely in cases with equivocal P-sIgE levels, and also validate the application of the MAT as a biomarker of PA. The MAT discriminated children with PA from PS children and overcame the main limitations of the BAT because the MAT did not require fresh blood cells from the patient, thus allowing deferred testing, and provided conclusive results for all subjects with nonresponding basophils (2 of whom had PA).
Both the BAT and MAT had very high specificity when used to diagnose PA. Although the sensitivity of the BAT was superior, the enhanced specificity is the key added value of cellular tests compared with conventional serologic tests when diagnosing food allergy. The MAT can be used to diagnose PA in a sequential way when conventional tests fail, similar to what we proposed for the BAT 3 and when it is either not possible to perform the BAT or the patient has nonresponding basophils.
Apart from its use for diagnostics, the MAT identified patients at risk of severe allergic reactions during OFCs. The sensitivity and negative predictive value of the MAT's optimal cutoff for severity was particularly high, with relatively lower specificity and positive predictive value, indicating that having a MAT result of greater than the cutoff does not necessarily mean the patient will have a severe reaction but that these patients would benefit from more intense educational measures and closer follow-up.
The MAT and the inhibition of MAT results 9 can facilitate further study of the underlying mechanisms that determine peanut reactivity versus tolerance. This is because the MAT can be used to assess the function of allergen-specific IgE antibodies in their ability to elicit mast cell degranulation and therefore allergic symptoms, as well as the ability of antibodies of other isotypes to interfere with this effect, either by inhibiting, as shown previously for IgG 4 , 9 or contributing to the activation of mast cells and basophils after allergen stimulation. However, this needs to be explored further. Both the BAT and the MAT are useful to test samples with equivocal P-sIgE levels to confirm PA and relay the performance of OFCs that would otherwise have positive results. Because the MAT uses plasma, which can be stored at low temperatures for long periods of time, it allows testing samples collected far from the laboratory or in the past.
The MAT is likely applicable to other food allergens. With the advent of new treatments for food allergy being approved for marketing, the MAT might prove to be a useful in vitro assay to monitor treatment response over time and to explore the mechanisms underlying the observed clinical changes during immunomodulatory treatments.   (Fig E1, A), FcgRI (Fig E1, B), FcgRII (Fig E1, C), and FcgRIII (Fig E1, D)   .0385 BAT to peanut (% CD63 1 basophils at 10-100 ng/mL peanut extract), n 5 157