Introduction
Food allergies have become a significant global health concern. For some countries a prevalence as high as 10% has been reported, with mostly young children being affected . Peanut allergy, in particular, is one of the most common conditions and often manifests as severe, potentially life-threatening reactions . The burden of food allergies not only impacts affected individuals but has also broader implications for their families, healthcare system, and the food industry . To effectively manage this debilitating disease, easily implementable diagnostic tools that are accurate, safe, and reliable are urgently required .
While the thorough assessment of a patient’s clinical history builds the fundamental basis of a food allergy diagnosis, the oral food challenge (OFC) test in which the patient ingests the culprit allergen under professional supervision is still considered the diagnostic gold standard to achieve optimal diagnostic accuracy. However, access to OFCs is limited, as they require significant resources, and the procedure may be associated with major discomfort, anxiety, and the risk to develop a systemic allergic reaction. Therefore, the guidelines of food allergy testing suggest a stepwise diagnostic workup in which a less elaboratein vivo diagnostic tool, the skin prick test (SPT), in conjunction with the serological quantification of allergen-specific IgE (sIgE) should initially be performed . SPT and sIgE have both been reported to feature high sensitivity but rather low specificity . While these tests can be helpful to confirm a suspicion of IgE-mediated food reactions, they are often not sufficient to diagnose a food allergy on their own and diagnosis solely based on these tests would lead to overdiagnosis . Especially sIgE measurement as a non-functional assay quantifies the level of sIgE antibodies in the patient’s blood without providing further information on the potential of these antibodies to trigger an allergic reaction. Previous work has indicated that affinity and epitope specificity of sIgE antibodies might be more important for cellular functionality than their absolute serum concentration . While sIgE quantification lacks such qualitative information it also omits the detection of potentially protective serum factors including allergen-specific IgG. If not carefully interpreted serological assays can thus lead to a false positive diagnosis, causing unnecessary food avoidance strategies for patients.
Functional cell-based allergy tests, which evaluate the activation of allergic effector cells including basophils or mast cells, have been emerging as promising alternatives to clinical in vivo and serological in vitro approaches. Among these, the basophil activation test (BAT) has gained recognition as a practical tool for diagnosing food allergies, including peanut , milk and egg allergy, in the clinical setting . Our recently published results from the markers of nut allergy study (MONAS) have demonstrated that the BAT shows high accuracy in the diagnosis of peanut and tree nut allergies . The BAT has even been reported to be superior to other diagnostic tests in discriminating between peanut allergy or tolerance and to reduce the need for OFCs . Despite all these promising features including the possibility to obtain objective and quantifiable results, broad implementation of the BAT has been hampered by several limitations. These mainly include the variability between donor reactivity among which the issue of so-called non-responders (i.e. lack of basophil activation upon FcεRI-dependent stimulation) is most problematic as well as logistical challenges that arise from the necessity for fresh blood samples to be processed within maximally 24 hours . Mast cell activation tests (MAT) have more recently gained a lot of traction as potential diagnostic tool to overcome such limitations . Most importantly, the MAT is based on patient serum, which can be used to passively sensitizein vitro cultured mast cells, eliminating the need for fresh blood samples, allowing prospective as well as retrospective analysis of samples, and simultaneously reducing donor variability. Two studies using different mast cell sources (i.e. LAD2 mast cell line or peripheral blood stem cell derived mast cells) have previously reported promising diagnostic results in detecting peanut allergy , confirming the utility of using patient sera to passively sensitize cultured mast cells. So far, the major challenge of these MAT strategies was that i) the LAD2 cell line grows very slowly and ii) the generation of blood stem cell derived mast cells takes 8-10 weeks resulting in a limited number of cells available for testing. The introduction of a novel MAT technology, using genetically engineered mouse mast cell progenitors that stably express the human high-affinity IgE receptor, FcεRIα, and that are conditionally immortalized with the homeobox B8 gene (Hoxb8 MCs), has further facilitated the implementation of the MAT as diagnostic tool . In suspension culture, these cells feature a doubling time of approximately one day at the progenitor stage and can be differentiated on demand into mature mast cells within only 5 days, allowing the generation of a virtually unlimited number of cells to be used in a diagnostic setting. Similar to the BAT, the Hoxb8 MAT relies on the detection of a cell surface activation marker (i.e. CD107a; LAMP-1), which is exposed during the degranulation process triggered upon allergen challenge. Using flow cytometry, the percentage of activated cells can be reliably quantified. Preliminary studies have demonstrated that the new Hoxb8 MAT is a promising approach to provide standardized and robust diagnostic results for various allergens including those found in foods .
In this study, we aimed to assess the clinical utility of the Hoxb8 MAT in diagnosing peanut allergies, using serum samples from clinically confirmed allergic and non-allergic children and adolescents. For this purpose, we took advantage of the existing Toronto site MONA study cohort which allowed us to assess the diagnostic performance of the Hoxb8 MAT assay in a blinded fashion compared to SPT, sIgE measurements, BAT and OFC outcomes. This approach enabled us to comprehensively assess the prospective diagnostic potential of the Hoxb8 MAT in a real-world setting.