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.