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Clinical Management Review
Making the Most of In Vitro Tests to Diagnose
Food Allergy
Alexandra F. Santos, MD, PhD
a,b
, and Helen A. Brough, MBBS, PhD
a,b
London, United Kingdom
INFORMATION FOR CATEGORY 1 CME CREDIT
Credit can now be obtained, free for a limited time, by reading the
review articles in this issue. Please note the following instructions.
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Date of Original Release: March 1, 2017. Credit may be obtained for
these courses until February 28, 2018.
Copyright Statement: Copyright Ó2017-2019. All rights reserved.
Overall Purpose/Goal: To provide excellent reviews on key aspects of
allergic disease to those who research, treat, or manage allergic disease.
Target Audience: Physicians and researchers within the field of
allergic disease.
Accreditation/Provider Statements and Credit Designation: The
American Academy of Allergy, Asthma & Immunology (AAAAI) is
accredited by the Accreditation Council for Continuing Medical Ed-
ucation (ACCME) to provide continuing medical education for phy-
sicians. The AAAAI designates this journal-based CME activity for
1.0 AMA PRA Category 1 CreditÔ. Physicians should claim only the
credit commensurate with the extent of their participation in the
activity.
List of Design Committee Members: Alexandra F. Santos, MD, PhD,
and Helen A. Brough, MBBS, PhD
Learning objectives:
1. To describe the different in vitro tests for diagnosing food allergy
and their diagnostic performance.
2. To analyze the results of allergy tests to determine the likelihood of
clinical allergy.
3. To explain the factors that influence the decision to perform an oral
food challenge.
Recognition of Commercial Support: This CME has not received
external commercial support.
Disclosure of Significant Relationships with Relevant Commercial
Companies/Organizations: A. F. Santos has received research support
from the Medical Research Council (MRC Clinical Research Training
Fellowship G0902018, MRC Centenary Early Career Award, MRC
Clinician Scientist Fellowship MR/M008517/1), Immune Tolerance
Network and National Institutes of Health, and the Department of
Health via the National Institute for Health Research (NIHR)
comprehensive Biomedical Research Centre award to Guy’s&St
Thomas’NHS Foundation Trust in partnership with King’s College
London and King’s College Hospital NHS Foundation Trust; has
received lecture fees from Thermo Scientific, Nutricia and Infomed;
has received travel support from European Academy of Allergy and
Clinical Immunology (EAACI), British Society of Allergy and Clinical
Immunology (BSACI), Academy of Medical Sciences, Portuguese
Society of Allergy and Clinical Immunology (SPAIC), Spanish Society
of Allergy and Clinical Immunology (SEAIC), and French Meeting of
Molecular Allergology. H. A. Brough has received consultancy fees
from MEAD Johnson Nutrition and Nutricia; has received research
support from the Food Allergy Research and Education Charity grant,
Immune Tolerance Network, and National Institutes of Health; has
received lecture fees from MEDA Pharmaceuticals, Thermo Scientific,
and Nutricia; receives royalties from Wiley Blackwell Ltd; has
received travel support from British Society of Allergy and Clinical
Immunology, MEDA Pharmaceuticals, Thermo Scientific, and Nutri-
cia; and has received research support from Thermo Scientific,
Stallergenes, and Meridien Foods.
a
Division of Asthma, Allergy and Lung Biology, Department of Paediatric Allergy,
King’s College London, London, United Kingdom
b
Children’s Allergy Service, Guy’s and St Thomas’Hospital, London, United
Kingdom
This work was funded by the Medical Research Council through MRC Clinici an
Scientist Fellowship MR/M008517/1 awarded to A.F.S.
Conflicts of interest: A. F. Santos has received research support from the Medical
Research Council (MRC Clinical Research Training Fellowship G0902018, MRC
Centenary Early Career Award, MRC Clinician Scientist Fellowship MR/
M008517/1), Immune Tolerance Network and National Institutes of Health, and
the Department of Health via the National Institute for Health Research (NIHR)
comprehensive Biomedical Research Centre award to Guy’s & St Thomas’NHS
Foundation Trust in partnership with King’s College London and King’s College
Hospital NHS Foundation Trust; has received lecture fees from Thermo Scientific,
Nutricia and Infomed; has received travel support from European Academy of
Allergy and Clinical Immunology (EAACI), British Society of Allergy and
Clinical Immunology (BSACI), Academy of Medical Sciences, Portuguese So-
ciety of Allergy and Clinical Immunology (SPAIC), Spanish Society of Allergy
and Clinical Immunology (SEAIC), and French Meeting of Molecular
Allergology. H. A. Brough has received consultancy fees from MEAD Johnson
Nutrition and Nutricia; has received research support from the Food Allergy
Research and Education Charity grant, Immune Tolerance Network, and National
Institutes of Health; has received lecture fees from MEDA Pharmaceuticals,
Thermo Scientific, and Nutricia; receives royalties from Wiley Blackwell Ltd; has
received travel support from British Society of Allergy and Clinical Immunology,
MEDA Pharmaceuticals, Thermo Scientific, and Nutricia; and has received
research support from Thermo Scientific, Stallergenes, and Meridien Foods.
Received for publication October 3, 2016; revised December 5, 2016; accepted for
publication December 19, 2016.
Corresponding author: Alexandra F. Santos, MD, PhD, Department of Paediatric
Allergy, King’s College London, 2nd floor, South Wing, St. Thomas’Hospital,
London SE1 7EH, United Kingdom. E-mail: alexandra.santos@kcl.ac.uk.
2213-2198
Ó2017 The Authors. Published by Elsevier Inc. on behalf of the American Academy
of Allergy, Asthma & Immunology. This is an open access article under the CC
BY license (http://creativecommons.org/licenses/by/4.0/).
http://dx.doi.org/10.1016/j.jaip.2016.12.003
237
Various in vitro tests assess different aspects of the underlying
immune mechanism of IgE-mediated food allergy. Some can be
used for diagnostic purposes; specific IgE to allergen extracts is
widely available; specific IgE to allergen components is used in
most specialist centers, and the basophil activation test is
becoming increasingly used clinically. IgE to allergen peptides,
T-cell assays, allergen-specific/total IgE ratios, and allergen-
specific IgG4/IgE ratios are currently reserved for research.
Different factors can modulate the likelihood of IgE-mediated
food allergy of a given allergy test result, namely, the patients’
age, ethnicity, previous allergic reaction to the identified food,
concomitant atopic conditions, and geographical location, and
need to be taken into account when interpreting the allergy test
results in the clinic. The importance of the specific food, the
clinical resources available, and patient preferences are
additional aspects that need to be considered when deciding
whether an oral food challenge is required to reach an accurate
diagnosis of IgE-mediated food allergy. Ó2017 The Authors.
Published by Elsevier Inc. on behalf of the American Academy of
Allergy, Asthma & Immunology. This is an open access article
under the CC BY license (http://creativecommons.org/licenses/
by/4.0/). (J Allergy Clin Immunol Pract 2017;5:237-48)
Key words: In vitro tests; Diagnosis; Food allergy; Specific IgE;
Basophil activation test; Component-resolved diagnosis; IgG4/
IgE ratio; Specific/total IgE ratio; Peptide microarray; T-cell
assay
Food allergy (FA) is an adverse reaction caused by an
abnormal response of the immune system to food allergens.
Food allergies are classified based on the involvement of IgE
antibodies in their pathogenesis.
1,2
This review will focus on
IgE-mediated FA. The immunologic mechanism underlying
IgE-mediated allergy is type I hypersensitivity.
3
During allergic
sensitization, food allergens are presented to T cells, a Th2-
skewed immune response commits B cells to IgE production
and allergen-specific IgE binds to the high-affinity IgE receptors
(FcεRI) on the surface of mast cells and basophils. In allergic
individuals, on subsequent exposure to the allergenic food,
multivalent allergens cross-link receptor-bound IgE leading to
mast cell and basophil activation and the release of preformed
mediators and de novo synthesis of leukotrienes and cytokines,
which contribute to the symptoms that patients experience
during allergic reactions.
Various in vitro assays reflect different aspects of the immu-
nologic mechanisms of IgE-mediated FA. For instance, the
amount of circulating allergen-specific IgE antibodies can be
determined using immunoenzymatic assays, and basophil acti-
vation and T-cell proliferation in response to allergen can be
assessed using flow cytometry (Figure 1). Some of these in vitro
assays can be used to diagnose FA and/or defer or obviate the
need for an oral food challenge (OFC). An OFC is the most
accurate test to diagnose FA but requires expensive resources,
highly trained personnel, and carries the risk of causing an acute
allergic reaction. Therefore, in clinical practice, the diagnosis of
FA is based on a combination of the clinical history and the
results of allergy tests when possible. The clinical history,
including the allergic reaction(s) to the culprit food and the
dietary history, is the cornerstone of the diagnosis of FA; it
guides the selection of allergens to be tested and the interpre-
tation of allergy test results. In this review, we discuss the main
in vitro tests for FA and how to make the most of these tests to
decide whether an OFC is required to reach an accurate diag-
nosis of FA.
IN VITRO TESTS FOR IgE-MEDIATED FOOD
ALLERGY
Specific IgE to allergen extracts
Specific IgE (sIgE) testing has been used to diagnose FA for
many years. Automated systems permit the use of enzymatic
immunoassays for a large number of samples in a standardized
way; however, levels determined with different methodology may
not be comparable.
4
IgE is quantified using kilounits per liter
(kU/L) based on the World Health Organization Reference
Standard with 1 unit equaling 2.42 ng of IgE.
5
Using the cutoff of 0.35 kU/L, sIgE testing has high sensi-
tivity but poor specificity to diagnose FA. For example, in the
case of peanut allergy (PA), sIgE to peanut has a sensitivity of
75% to 100% and a specificity of 17% to 63%.
6-13
Adopting
95% positive predictive value (PPV) cutoffs, the specificity of IgE
testing increases. Following on with the example of PA, the
cutoff of 15 kU/L
7,14
showed a specificity of 96.8% and a
sensitivity of only 28.4% in a UK study.
7
This indicates that the
95% PPV cutoffs can be useful to confirm the diagnosis of FA,
especially if there is a recent history of an immediate-type allergic
reaction. On the contrary, the cutoff of 0.35 kU/L can be useful
to exclude the diagnosis of FA as it has a high negative predictive
value (NPV). Levels of sIgE between positive and negative cut-
offs without a clear clinical history do not allow us to confirm or
exclude the diagnosis, falling in the so-called immunological gray
area.
15,16
Positive and negative cutoffs can be helpful in guiding
the clinical diagnosis of FA; however, they are not absolute and
need to be interpreted in light of the clinical history, as patients
can still be allergic or tolerant below and above 95% NPV and
95% PPV, respectively.
16
PPV and NPV decision levels have
been identified for sIgE to other foods (Table I).
Diagnostic cutoff values can vary widely in different studies.
For instance, the 95% PPV cutoff to diagnose PA was 15 kU/L
in US
14
and UK
7
studies, but was 24.1 kU/L, 34 kU/L, and
57 kU/L in studies performed in the Netherlands,
17
Australia,
18
and France,
19
respectively. These differences can result from the
patient population (eg, prevalence of FA, comorbidities) and/or
from the research study where the cutoffs were determined (eg,
inclusion criteria, reference standard against which the perfor-
mance of sIgE was compared, criteria for referring for OFC and
the OFC protocol).
20
These factors need to be taken into ac-
count when comparing studies and when extrapolating cutoffs
from published studies into daily clinical practice. When criti-
cally reviewing the literature for diagnostic decision levels for FA
one should take into consideration the limitations of studies
assessing the diagnostic utility of allergy tests (eg, small sample
size, selected sample of participants, OFC not done in all par-
ticipants, etc.).
21
Validated diagnostic cutoffs are reliable when
applied to a similar population to the population in which they
were generated. PPVs are a function of the sensitivity and
specificity of the test and the prevalence of the disease; therefore,
they are only valid for patients who have the same pretest
probability of disease as the population in which the PPV was
established. For instance, in our clinic population in London,
the cutoff of 15 kU/L for peanut sIgE had 95% PPV in 2
different studies performed approximately 10 years apart.
7,16
The
J ALLERGY CLIN IMMUNOL PRACT
MARCH/APRIL 2017
238 SANTOS AND BROUGH
Abbreviations used
BAT- Basophil activation test
CMA- Cow’s milk allergy
FA- Food allergy
kU/L- Kilounits per liter
NPV- Negative predictive value
nsLTP- Nonspecific lipid-transfer protein
OFC- Oral food challenge
PA- Peanut allergy
PFAS- Pollen-food allergy syndrome
PPV- Positive predictive value
sIgE- Specific IgE
SPT- Skin prick test
consistency of these findings indicates that the identified cutoff
can be reliably applied to our patient population in the clinic.
Specific IgE to allergen components
Conventional IgE testing uses natural extracts containing a
complex mixture of proteins. Allergen sIgE to component
allergen tests for IgE binding to single allergens, allowing more
precise profiling of the allergen-sIgE repertoire. The list of
allergenic molecules available for testing is not complete; thus
IgE assays using extracts are likely to be useful for some time.
sIgE testing to components is available for single allergens and for
multiple allergens in microarrays. Multiplex assays may introduce
concerns where they reveal sensitization to molecules with
potentially no clinical relevance as they are all tested independent
of the patient’s history. However, multiplex assays can be useful
in identifying patterns of sensitization in complex polysensitized
patients (eg, patients sensitized to pollen, plant foods, and latex
with unclear clinical relevance that are sensitized to a pan-
allergen) and in identifying the culprit allergen in patients with
recurrent anaphylaxis.
22,23
The food that has received the most research into component
allergens and their validation in terms of clinical relevance is
peanut. The number of identified peanut allergens is extensive
although not all of these are available for testing in clinical practice
(Table II). The immunodominant peanut allergen in adults and
children is Ara h 2 based on OFC, serial skin prick test (SPT)
dilutions, and basophil degranulation assays.
24-27
Secondary
sensitization to peanut occurs because of panallergens such as
nonspecific lipid-transfer proteins (nsLTPs) (eg, Pru p 3 in peach
giving rise to Ara h 9 sIgE), Bet v 1 homologs (eg, Bet v 1 in birch
pollen giving rise to Ara h 8 sIgE), and profilins (eg, Phl p 12 in
grass pollen or Bet v 2 in birch pollen giving rise to Ara h 5 sIgE).
Similar to what has been reported for peanut sIgE, diagnostic
cutoffs for Ara h 2 sIgE vary between studies (Table III).
Of the tree nuts, hazelnut has received the most extensive
evaluation leading to the identification of seed storage proteins
(eg, Cor a 9 and Cor a 14) as well as cross-reactive proteins (eg,
Cor a 8 and Cor a 1) as allergens. sIgE to whole hazelnut has a
poor predictive value for clinical reactivity due to cross-reactivity
with birch pollen. Birch pollen-associated hazelnut allergy is the
dominant phenotype, although Cor a 9 and 14 are the
allergens more commonly associated with systemic reactions
(Table III).
27-30
In Danish,
31
German,
27
and Belgian children
32
Cor a 14 was superior to Cor a 9 in predicting challenge-proven
hazelnut allergy; however, in Dutch children Cor a 9 was the best
predictor.
28
It was postulated that these differences were due to
the age of children assessed with Cor a 9 specificity decreasing
with age and Cor a 14 specificity increasing with age.
28,33
Other
2S albumins have been identified for walnut (Jug r 1),
34,35
cashew (Ana o 3),
36
and Brazil nut (Ber e 1)
37
(Table III).
Casein (Bos d 8), beta-lactoglobulin (Bos d 5), and alpha-
lactoglobulin (Bos d 4) are the major allergens in cow’s milk.
Sensitivity to various cow’smilkproteinsiswidelydistributed;
thus generally no single allergen is considered to be immunodo-
minant.
38
In some studies, Bos d 8 was the best predictor of
challenge-proven cow’smilkallergy(CMA).
39,40
In a Spanish
study, the optimum cutoffs for Bos d 8 increased with age; using 2
kU/L (13-18 months), 4.2 kU/L (19-24 months), and 9 kU/L
(24-36 months) gave a sensitivity of 95% and a specificity of
90%.
41
This observation is important with regard to cutoffs for
transient food allergies, such as cow’s milk and egg, as one would
expect that children who persist with CMA beyond 2 years would
have higher casein levels than those who have already grown out of
their CMA. In fact, IgE antibodies directed against sequential
casein epitopes are a marker of persistent CMA.
42
High casein-IgE
antibodies are predictive of baked CMA as casein is more resistant
TABLE I. Examples of diagnostic cutoffs with 95% PPV and 50%
NPV for specific IgE to food allergen extracts
14,107,125
Approximate predictive value Cow’s milk Egg Peanut Fish
95% PPV 32 kU/L 7 kU/L 15 kU/L 20 kU/L
50% NPV 2 kU/L 2 kU/L 2 kU/L*e
5 kU/L*
NPV, Negative predictive value; PPV, positive predictive value.
*The 50% NPV cutoff is different depending on the previous history of reaction: 2
kU/L if the patient reports a reaction and 5 kU/L if the patient has never had an
allergic reaction to peanut in the past.
FIGURE 1. Tests used to diagnose IgE-mediated food allergy
reflect different aspects of the underlying mechanism of this
immune-mediated disorder: the skin prick test measures the
response of skin mast cells to allergen, the basophil activation test
measures the response of circulating basophils to allergen, and
IgE tests measure the concentration of circulating IgE, either total
IgE or sIgE to allergen extracts or to individual allergen compo-
nents. Total IgE and allergen-specific IgG4 can be used to calcu-
late ratios with allergen sIgE.
J ALLERGY CLIN IMMUNOL PRACT
VOLUME 5, NUMBER 2
SANTOS AND BROUGH 239
to extensive heating.
43
Clinical decision points for a positive
challenge to baked milk have been reported (Table III).
44
The main hen’s egg allergens are ovomucoid (Gal d 1),
ovalbumin (Gal d 2), conalbumin (Gal d 3), and lysozyme (Gal
d 4).
45
Ovomucoid is considered to be the immunodominant
allergen based on OFCs to heated and ovomucoid-depleted egg
46
and serial dilutions of ovomucoid SPT and ovomucoid sIgE in
egg allergic children.
47
Ovomucoid is stable against heat and
digestion by proteinases
46
; this is why it has been evaluated in the
prediction of tolerating extensively heated egg (Table IV). IgE
antibodies to sequential epitopes of ovomucoid have been shown
to predict persistent egg allergy beyond the age of 11 years.
48
Component-resolved diagnosis (CRD) of wheat allergy has
gained interest as wheat extract IgE testing has a poor predictive
value. The major wheat allergens relevant for FA (rather than
Baker’s asthma) are glutens that can be subdivided into gliadins
(subunits
a
,
b
,
g
,and
u
) and glutenins (high molecular and low
molecular weight). The role of omega-5-gliadin (Tri a 19) in
wheat-dependent exercise-induced anaphylaxis has been shown in
several studies
49,50
; however, results for this component in the
prediction of IgE-mediated wheat allergy are conflicting. In a
Japanese population, Tri a 19 has been shown to correctly predict
challenge-proven IgE-mediated allergy to wheat,
51,52
and in a
Swedish population, Tri a 19 correlated better with OFC-proven
IgE-mediated wheat allergy than the extract-based in vitro test or
other component allergens.
53
However, the results for Tri a 19
have not been reproduced in American or German populations.
54
Allergens predictive of systemic reactions to soya include the
seed storage proteins Gly m 5, 6, and 8. Gly m 5 and 6 predicted
systemic allergic reactions to soy (with both positive Gly m 5 and
6 giving an odds ratio of 12 for severe reactions) more than the
Bet v 1 homolog Gly m 4.
55
More recently, the 2S albumin Gly
m 8 was found to be a better marker for systemic reactions to soy
than Gly m 5 and 6 (or soy extract), but it still misclassified many
patients.
56-58
It is important to note that sole reactivity to the
PR-10 protein Gly m 4 has been responsible for anaphylaxis after
consumption of unprocessed soya.
59
Ratios: allergen-specific/total IgE and allergen-
specific IgG4/IgE
To try to improve the diagnostic performance of food sIgE,
the added value of total IgE and food-specific IgG4 has been
tested in food-specific/total IgE ratios or food-specificIgG4/
IgE ratios. Some studies showed an improvement in the
prediction of OFC outcome with specific/total IgE ratios
compared with sIgE alone,
60
although other studies did not
found it to be useful.
61
The discrepancy in these findings
couldbeduetothefoodsstudied,asGuptaetal
60
found the
specific/total IgE ratio particularly useful for persistent food
allergies (eg, peanut, tree nuts, shellfish, and seeds) and the
studybyMehletal
61
focused on transient food allergies,
namely, cow’s milk, egg, and wheat allergies. Recently, in a
multicenter study of children with suspected peanut or
hazelnut allergies,
62
calculating the Ara h 2/peanut sIgE or Ara
h 2-specific/total IgE ratios did not improve the diagnostic
performance of Ara h 2 sIgE. Peanut-specific/total IgE was
also not better than Ara h 2 sIgE in diagnosing PA. Similar
results were reported for the relative diagnostic performance of
Cor a 14/hazelnut sIgE, Cor a 14-specific/total IgE, and Cor a
14 sIgE and hazelnut-specific/total IgE to diagnose hazelnut
allergy.
Food-specific IgG4/IgE ratios have been determined in
various studies, but their diagnostic utility has not been estab-
lished. Sensitized-tolerant children tend to have higher allergen-
specific IgG4/IgE ratios than allergic children. For instance,
peanut-sensitized tolerant patients have a higher peanut-specific
as well as Ara h 1-, Ara h 2-, and Ara h 3-specific IgG4/IgE
ratios compared with peanut allergics.
63
This increased IgG4 in
relation to IgE was not due to higher peanut consumption as the
majority of children had not knowingly eaten peanut before
entering the study. A higher peanut-specific IgG4/IgE ratio has
also been observed in peanut allergic patients treated with peanut
oral immunotherapy
63
and in high-risk infants who consumed
peanut early in life and developed tolerance.
64
Conversely,
allergic patients tend to show a higher food sIgE/IgG4 ratio. For
example, egg allergic patients who react to baked egg have higher
ovalbumin and ovomucoid sIgE/IgG4 ratios than egg allergic
patients who tolerate baked egg.
65
Basophil activation test
The basophil activation test (BAT) is a functional assay that
uses live basophils in whole blood to detect the ability of IgE to
mediate activation of basophils after stimulation with allergen. It
goes beyond the detection of IgE binding to allergen to test IgE
function, which depends not only on the allergen-sIgE levels but
also on IgE epitope specificity, affinity, and clonality.
66
The
basophils of allergic patients typically show a dose-dependent
expression of activation markers, such as CD63 or CD203c,
whereas the basophils of sensitized-tolerant patients do not ex-
press or have a much lower expression of activation markers after
stimulation with allergen. In a PA study, basophils of peanut
allergic patients showed higher basophil activation to peanut
compared with peanut-sensitized-tolerant even in the subgroup
where allergic and tolerant children had comparable levels of
peanut sIgE.
16
The difference in upregulation of basophil acti-
vation markers in response to allergen between allergic and
nonallergic patients forms the basis of the use of the BAT to
diagnose FA.
TABLE II. Peanut allergens described to date
126
Allergen Biochemical name
Ara h 1 Cupin (Vicillin-type, 7S globulin)
Ara h 2 Conglutin (2S albumin)
Ara h 3 Cupin (Legumin-type, 11S globulin, Glycinin)
Ara h 4 Considered an isoform of Ara h 3 and renamed to Ara h 3.02
Ara h 5 Profilin
Ara h 6 Conglutin (2S albumin)
Ara h 7 Conglutin (2S albumin)
Ara h 8 Pathogenesis-related protein 10 (PR-10, Bet v 1 homolog)
Ara h 9 Nonspecific lipid-transfer protein type 1
Ara h 10 Oleosin
Ara h 11 Oleosin
Ara h 12 Defensin
Ara h 13 Defensin
Ara h 14 Oleosin
Ara h 15 Oleosin
Ara h 16 Nonspecific lipid-transfer protein type 2
Ara h 17 Nonspecific lipid-transfer protein type 1
Allergens in bold are commercially available for clinical use.
J ALLERGY CLIN IMMUNOL PRACT
MARCH/APRIL 2017
240 SANTOS AND BROUGH
The main added value of the BAT in the diagnosis of FA
compared with tests routinely used in clinical practice, such as
SPT and sIgE to allergen extracts, is its enhanced specificity with
often conserved sensitivity. For instance, the BAT to peanut
showed 98% sensitivity and 96% specificity to diagnose PA, with
the specificity reaching 100% in a subsequent validation. The
specificity of the BAT ranged between 77% and 100% in other
studies (Table IV).
11,67-73
The BAT with single allergen com-
ponents can potentially improve its diagnostic accuracy, but
further research studies are needed.
72,74,75
The BAT has been
shown to be potentially useful in identifying the culprit allergen
in cases of pollen-food allergy syndrome (PFAS),
71,76,77
allergy to
red meat,
78
or food-dependent exercise-induced anaphylaxis.
79
As for other diagnostic tests, cutoffs determined for the BAT
can vary with the patient population, the design of the study, and
the methodology adopted for the BAT procedure and data
analyses.
20
The BAT requires fresh blood and uses flow cytometry for
which appropriate equipment and trained personnel are needed.
It is anticipated that the BAT is reserved for selected cases where
the results of routinely used tests do not allow a precise diagnosis.
Indeed, in the previously mentioned study,
16
the BAT sustained
its good performance in a subgroup of patients with equivocal
test results for SPT, peanut sIgE, and Ara h 2 sIgE with 92%
accuracy compared with its 97% accuracy in the study popula-
tion overall. Used as a second step in the diagnostic workup, the
BAT was performed in patients who would have otherwise been
referred for an OFC after standard allergy testing. A positive
BAT confirmed the diagnosis of FA and dispensed with an OFC,
whereas patients with a negative BAT or nonresponder basophils
(ie, basophils that solely responded to noneIgE-mediated and
not to IgE-mediated stimulants) needed to be referred for the
OFC. This stepwise approach ensured a 67% reduction in the
need for the OFC.
16
As any other diagnostic test, the BAT cannot be used in
isolation to diagnose FA. The results of the BAT need to be
considered in light of the clinical history. In addition to patients
with a negative BAT or nonresponder basophils, patients with
BAT results that are discordant with the clinical history require
an OFC to confirm or refute the diagnosis of FA.
IgE to allergen peptides
IgE specificity can be refined further by determining the
allergen epitopes to which IgE binds. This has been evaluated
using short linear allergen peptides of 15 to 20 amino acids bound
to a solid phase (eg, microarray or spot membrane) using
immunofluorescence. Beyer et al
80
identified 5 immunodominant
epitopes in selected peanut allergen peptides in 2003. Years later, a
microarray containing peptides of the major peanut allergens, Ara
h 1, Ara h 2, and Ara h 3, identified epitopes bound more by the
IgE of peanut allergic patients than by the IgE of peanut
sensitized-tolerant patients; this allowed the development of a
machine-learning method that markedly enhanced the diagnostic
utility of the microarray.
81
Similar methods have tested the utility of IgE to allergen
peptides in diagnosing and in predicting the resolution of other
TABLE III. Allergen components associated with clinical allergy and examples of cutoffs for specific IgE testing to main allergen
components
Foods Components associated with clinical allergy Cutoffs for specific IgE to main components
Peanut Ara h 1 Ara h 2 sIgE: 0.35 to 42.2 kU/L had 90%-95% PPV
16,24,27
Ara h 2
Ara h 3
Ara h 9 (in Southern Europe)
Hazelnut Cor a 9 Cor a 9 sIgE: 1 kU/L had 83% accuracy
28
Cor a 14 Cor a 14 sIgE: 0.72 to 47.8 kU/L had 87%-90% accuracy
27,31
Cor a 8 (in Southern Europe)
Cashew, Pistachio Ana o 3 Ana o 3 sIgE: 0.16 kU/L had 97.1% accuracy for
cashew and/or pistachio nut allergy
127
Brazil nut Ber e 1 Ber e 1 sIgE: 0.25 kU/L had 94% PPV
128
Walnut Jug r 1 Jug r 1 sIgE: 0.1 kU/L had 91% PPV
129
Jug r 3
Soya Gly m 5 Gly m 8 sIgE: 1 kU/L had 89% PPV
56
Gly m 6 Gly m 8 sIgE: 0.1 kU/L had 83% NPV
56
Gly m 8
Wheat Tri a 19 (IgE-mediated wheat allergy and WDEIA) Tri a 19 sIgE: 0.04 AU had 100% PPV and 88% NPV
for IgE-mediated wheat allergy
51,52
Tri a 14 (nsLTP involved in Baker’s asthma)
Cow’s milk Casein (for baked milk allergy and persistent cow’s
milk allergy)
Casein sIgE: 10 kU/L had 95% PPV for a positive
OFC to baked milk
44
Casein sIgE: 5 kU/L had 50% PPV for a positive
OFC to baked milk
44
Egg Ovomucoid (for cooked or baked egg allergy
and persistent egg allergy)
Ovomucoid sIgE: 3.74-26.6 kU/L had 95% PPV for
cooked egg allergy
130,131
Ovomucoid sIgE: 50 kU/L had 90% PPV and Ovomucoid sIgE:
0.35 kU/L had 90% NPV for a positive OFC to baked egg
132
nsLTP, Nonspecific lipid-transfer protein; OFC, oral food challenge; WDEIA, wheat-dependent exercise-induced anaphylaxis.
J ALLERGY CLIN IMMUNOL PRACT
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SANTOS AND BROUGH 241
TABLE IV. Basophil activation test to food extracts or to component allergens in the diagnosis of food allergy
Food extract or
allergen component Cutoffs
Diagnostic performance
S Sp PPV NPV LRD*LRL*
Cow’s milk SI CD203c 1.9
67
89% 83% 86% 86% 5.24 0.13
>6% CD63þ
117
to diagnose resolution of CMA 91% 90% 81% 96% 9.10 0.10
Casein SI CD203c 1.3
67
67% 71% 74% 63% 2.31 0.46
Egg white SI CD203c 2.4
67
to diagnose baked egg allergy 74% 62% 85% 44% 1.95 0.42
SI CD203c 1.7
67
to diagnose raw egg allergy 77% 63% 92% 33% 2.08 0.37
Ovalbumin 5% CD63þor SI CD203c 1.6 to diagnose
egg allergy
77% for
CD63
100% for CD63 Inf†0.23 for CD63
63% for
CD203c
96% for CD203c 15.75 for CD203c 0.39 for CD203c
Ovomucoid SI CD203c 1.7
67
to diagnose baked egg allergy 80% 73% 90% 53% 2.96 0.27
SI CD203c 1.6
67
to diagnose raw egg allergy 83% 83% 97% 42% 4.88 0.20
Wheat >11.1% CD203cþto diagnose wheat allergy
68
86% 58% 77% 71% 2.05 0.24
Omega-5 gliadin nTri a 19: >14.4% CD203cþto diagnose wheat
allergy
68
86% 58% 77% 71% 2.05 0.24
rTri a 19: >7.9% CD203cþto diagnose
wheat allergy
68
83% 63% 81% 67% 2.24 0.27
Peanut 4.78% CD63þ
16
98% 96% 95% 98% 24.50 0.02
Ara h 1 ND BAT to Ara h 1 was higher in peanut allergic patients compared with controls from Southern Spain
74
Ara h 2 ND 92% 77% 4.00 0.10
Ara h 3 ND There was no difference in BAT to Ara h 3 between peanut allergic and control subjects from Southern Spain
74
Ara h 6 ND There was no difference in BAT to Ara h 6 between peanut allergic and control subjects from Southern Spain
74
Ara h 8 ND There was no difference between CD-sens to Ara h 8 between patients with PFAS to peanut and patients with sIgE to Ara h 8 and
no reaction during OFC to roasted peanuts
76
Ara h 9 ND BAT to Ara h 9 was higher in peanut allergic patients compared with controls from Southern Spain
74
Hazelnut CD-sens >1.7
69
to diagnose hazelnut allergy 100% 97% 33.33 0.00
6.7% CD63þ
70
to diagnose PFAS to hazelnut 85% 80% 4.25 0.19
Peach >20% CD63þand SI CD63 >2
75
87% 69% 2.81 0.19
Pru p 3 >20% CD63þand SI CD63 >2
75
77% 97% 25.67 0.24
Apple 17% CD63þ
71
to diagnose PFAS to apple 88% 75% 3.52 0.16
Carrot 8.9% CD63þ
70
to diagnose PFAS to carrot 85% 85% 5.67 0.18
Celery 6.3% CD63þ
70
to diagnose PFAS to celery 85% 80% 4.25 0.19
BAT, Basophil activation test; CMA, cow’s milk allergy; Inf,infinity; LRþ, positive likelihood ratio; LR, negative likelihood ratio; ND, not determined; NPV, negative predictive value; OFC, oral food challenge; PFAS, pollen-food
syndrome; PPV, positive predictive value; S, sensitivity; SI, stimulation index; Sp, specificity.
*Likelihood ratios were calculated from sensitivity and specificity using the formulas LRþ¼sensitivity/(1 specificity) and LR¼(1 sensitivity)/specificity.
†Infinity, the denominator is zero.
J ALLERGY CLIN IMMUNOL PRACT
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242 SANTOS AND BROUGH
food allergies.
82-86
In a CMA study,
82
IgE binding was more
diverse and had higher affinity for cow’s milk allergen peptides in
milk allergic patients reacting to baked milk compared with
patients who reacted to unheated milk but tolerated baked milk,
suggesting that the peptide microarray could be useful in iden-
tifying different phenotypes of CMA.
T-cell assays
T-cell responses are central to the development of oral toler-
ance in nonallergic individuals and to the development of the
allergic immune response in allergic individuals. Peanut allergic
individuals have been shown to have greater proliferation of their
T cells when their PBMCs were stimulated with whole peanut or
individual major peanut allergens.
87,88
Peanut allergic patients
also showed a typical Th2-skewed response to peanut allergen
with higher levels of IL-4, IL-5, and IL-13, whereas nonallergic
controls showed a Th1-type response characterized by
IFN-gamma production.
87
Interestingly, peanut allergic and
peanut-sensitized-tolerant individuals showed higher T-cell pro-
liferation compared with nonsensitized controls; however, only
allergic patients showed a Th2-skewed response to peanut al-
lergens.
88
These findings suggest that the absence of clinical
reactivity in sensitized individuals is an active ongoing process,
whereas in nonsensitized individuals, it is a passive process,
probably due to anergy or clonal deletion.
Food allergic patients may also have impaired regulatory T-cell
function in response to specific food allergens. Dang et al
89
recently showed that egg and/or peanut allergic infants had a
reduction in the number of T regulatory cells and a lower ratio of
activated regulatory/effector T cells in vitro after in vivo allergen
exposure during the OFC. This is consistent with studies in
mouse models.
90
CLINICAL REASONING TO DIAGNOSE FOOD
ALLERGY
The tests available for routine use in the clinic can vary, with
some practices using mainly SPT, others mainly sIgE, and others
both. sIgE to allergen components is used in most specialist
centers and the BAT is becoming increasingly used clinically.
The other tests described in the previous section are reserved for
use in the research setting, namely, peptide microarrays and
T-cell assays.
Interpretation of allergy test results
The ultimate goal of the allergy test result is to determine the
probability of clinical allergy; this is then used to decide whether
an OFC is warranted.
91
The probability of clinical allergy de-
pends first and foremost on the clinical history (Table V) and
secondarily on the allergy test result (Tables I,III, and IV). For
example, if a patient consumes age-appropriate amounts of the
food regularly without developing any symptoms, the probability
of having FA is negligible regardless of the allergy test results;
such patients should in fact not be tested as a false-positive result
could be confusing for the patient and lead to unnecessary food
avoidance. The clinical history provides information that enable
the clinician to establish a pretest probability of FA that will be
taken into account to determine the probability of clinical allergy
for a given allergy test result, that is, the post-test probability.
91
This reasoning is best described using nomograms that use
likelihood ratios to calculate the post-test probability based on a
given pretest probability. Likelihood ratios have the advantage of
not depending on the prevalence of the disease in the population,
as opposed to PPV, and can be calculated from the sensitivity
and specificity of the test.
91-93
Different factors can modulate
pretest probabilities and likelihood ratios, for instance, the pre-
vious allergic reaction(s), the dietary history, age, ethnicity,
TABLE V. Factors modulating the interpretation of allergy test results
Factors identified in the clinical history Effect on the probability of clinical allergy for a given specific IgE level
Reported immediate allergic reaction
to the specific food
A history of reacting to the tested food supports the clinical relevance of detected IgE.
(Younger) Age Lower levels of allergen-specific IgE have increased clinical relevance in young children.
(Black) Ethnicity Black race is associated with higher levels of allergen-specific IgE with decreased
clinical relevance.
Atopic eczema Polyclonal IgE response can be non-allergen-specific and thus decrease clinical relevance
of a given specific IgE level.
Concomitant inhalant allergies Pollen sensitization can cause false-positive results of specific IgE to plant food extracts.
Atopic population Positive predictive value of a given specific IgE level increases with the increase in
the prevalence of the disease in the population.
Geographical location Variable Clinical relevance of IgE to extracts and patterns of sensitization to allergen components
can vary with inhalant allergen exposure typical of certain geographical locations.
These factors affect the pretest probability and therefore influence the resulting post-tes t probability.
J ALLERGY CLIN IMMUNOL PRACT
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SANTOS AND BROUGH 243
concomitant atopic diseases, geographical location, and the
clinical setting. This is best studied for sIgE testing.
The clinical relevance of a given allergen sIgE result can vary
depending on the age of the patient, with lower levels of sIgE
having increased clinical relevance in younger patients.
94
Ninety-
five percentage PPV cutoffs have been established for children
<2 years at lower levels of food sIgE compared with cutoffs for
older children.
14,95
Diagnostic decision levels may be affected by the patients’
ethnicity. Black race is associated with a higher prevalence of
sensitization to foods
96
and a higher level of total IgE compared
with Caucasians
97
despite lower prevalence of FA.
96
This
discrepancy suggests that patients of black ethnicity may have
more clinically irrelevant IgE and therefore higher diagnostic
cutoffs. Indeed, the 95% cutoffs defined in the United
Kingdom
7
for peanut sIgE and Ara h 2 sIgE provided lower
PPVs in South African peanut-sensitized patients
98
; the optimal
cutoffs to diagnose PA in this population were 15 kU/L for
peanut sIgE and 8 kU/L for Ara h 2 sIgE, which had 80% and
93% PPV, respectively.
Concomitant atopic diseases can also modulate the clinical
relevance of a given allergy test result. Patients with atopic
eczema tend to have a polyclonal IgE response to allergens that
often lacks clinical expression. This underscores the importance
of a judicious selection of allergens to be tested. Grabenhenrich
et al
62
showed that for a given component-sIgE level, a high
total IgE (>500 kU/L) significantly reduced the probability of
clinical peanut or hazelnut allergy, respectively, particularly at
low levels of Ara h 2 sIgE or Cor a 14 sIgE. In patients with
birch or grass pollen allergy, high levels of sIgE to plant foods,
such as peanut or hazelnut, may have a low probability of a
systemic allergic reaction. These are the cases where deter-
mining sIgE to individual allergens that are involved in cross-
reactivity (eg, Ara h 8 and Cor a 1) can be helpful in dis-
tinguishing real FA from sensitization secondary to pollen al-
lergy, which can cause PFAS but usually not systemic allergic
reactions.
Geographical location is another factor that may influence the
clinical relevance of a given sIgE level. A study by Vereda et al
99
illustrates this nicely for PA. In Northern and Central Europe,
sensitization to birch pollen leads to high prevalence of sensiti-
zation to Ara h 8, the Bet v 1-homolog, which typically causes
oral allergic symptoms. In Spain, exposure to birch pollen and
sensitization to Ara h 8 are rare and peanut allergic patients are
often sensitized to Ara h 9 (nsLTP), probably as a consequence of
primary sensitization to peach LTP. In the United States and in
the United Kingdom, the most common pattern of sensitization
in peanut allergic patients is the combination of IgE to Ara h 1,
Ara h 2, and Ara h 3, although other patterns may be found in
individual patients.
63
Finally, the clinical setting influences the predictive value of
sIgE levels, with increasing likelihood of clinical allergy going
from the general population to secondary care and then to
specialist centers. In studies performed in the general population,
the prevalence of sensitization to foods such as cow’s milk and
egg was much lower than in a population recruited from
specialist centers. For example, approximately 8% and 3%-4% of
children in National Health and Nutrition Examination Survey
2005-2006
100
and 78% and 89% of children in Consortium of
Food Allergy Research (COFAR)
101
were sensitized to cow’s
milk and egg, respectively, although this is probably an extreme
example as a positive SPT to cow’s milk and/or egg was one of
the inclusion criteria in COFAR, and therefore it is a highly
selected population.
Factors influencing the decision of performing an
oral food challenge
The main reason to perform an OFC is to identify the food
that caused the allergic reaction for the initial diagnosis and for
monitoring resolution of FA. Other reasons for an OFC include
assessing the status of tolerance to cross-reactive foods (eg, tree
nuts in PA or peanut in egg allergy) and expanding the diet in
foods not yet introduced but with positive allergy tests. This
TABLE VI. Factors influencing the decision to perform an oral food challenge (OFC)
Factors Effect on the decision to perform an OFC
History of an allergic reaction A previous history of a reaction to the specific food increases the chance of reacting during the OFC.
Recent exposure to the food A recent allergic reaction or the consumption of age-appropriate amount of the food precludes the OFC.
(Low) specific IgE levels Current low level of food-specific IgE and >50% decline within the last year indicate lower likelihood
of a positive OFC.
Importance of the food The importance of the food to the child’s diet and social life and her or his willingness to eat the food
regularly in the case of a negative challenge favor performing an OFC.
Resources available The resources available may limit the number of OFCs offered to patients.
Patient preferences Variable Patient may wish to undergo an OFC or not and her or his preferences need to be taken into account.
The decision to perform an OFC is made when the probability of a systemic reaction is sufficient for thereto be concern and low enoughthat the OFC is likely to be passed. The arrows
indicate the effect on the decision to perform an OFC: the arrow pointing up means weighing pro and the arrow pointing down means weighing con performinganOFC.
J ALLERGY CLIN IMMUNOL PRACT
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244 SANTOS AND BROUGH
occurs more frequently because of the increased use of antici-
patory testing and has important resource implications.
102
Many factors affect the decision as to whether to perform an
OFC (Table VI). The most important considerations are pro-
vided by the clinical history; the previous reaction history and
recent exposure to the food in question may avert the need
(already consuming) or lead to deferment (recent reaction, poorly
controlled asthma) of an OFC. SPT and sIgE testing will also
affect this decision; in a patient being assessed for the initial
diagnosis of FA, a recent convincing history of an allergic reac-
tion to an identified food, concomitant SPT 3 mm and/or
sIgE to the whole allergen 0.35 kU/L may be sufficient to
confirm the diagnosis without the need for further testing or an
OFC. In cases where the history and SPT/sIgE testing do not
provide a clear answer, further testing with sIgE to components
or the BAT may be warranted, before deciding to perform an
OFC.
When monitoring a patient for resolution of FA, it is generally
recommended that children with a 50% chance of experiencing a
negative challenge be good candidates for an OFC.
103
A pre-
dictive 50% negative cutoff of 2 kU/L has been identified for the
resolution of egg and cow’s milk allergies.
104
The rate of decline
of IgE to cow’s milk and egg has also been shown to predict
resolution; a 50% decrease in respective sIgE over 12 months is
associated with a 52% probability of tolerance to egg and 31%
probability of tolerance to cow’s milk.
105
Baseline sIgE and SPT
wheal size and severity of eczema also affect the rate of resolution
and this has been incorporated into a practical computerized
algorithm by Wood et al
106
for CMA. In the case of peanut, sIgE
2 kU/L and 5 kU/L have been shown to give a 50% pre-
diction of a negative peanut challenge in children with and
without a history of peanut reaction, respectively.
107
A systematic
review by Peters et al
108
in 2013 provides further details on sIgE
and SPT cutoffs to predict the resolution of cow’s milk, egg, and
peanut allergies.
When considering performing an OFC it is vital that the pa-
tient or parents of the child undergoing the OFC understand the
rationale for this and the importance of introducing the food into
the diet after a negative challenge. Several studies have shown that
18% to 32% of patients do not introduce the food after passing an
OFC.
109-111
This is of concern as the recurrence of FA (partic-
ularly peanut) has been shown to occur if the food continues
to be avoided after the OFC or is consumed in very small
quantities.
112-114
This would suggest that the immune system
needs ongoing exposure to maintain tolerance; however, this
conflicts with the fact that children develop tolerance whilst
avoiding a food. Nonetheless, if the food is not important to the
patients and they are not planning to introduce it, then it may be
better not to proceed with the OFC. Dietetic advice to prepare
recipes that the child will accept and suggestions for foods for
mixing can avert failed OFCs; dietitians can also advise on ways to
introduce the food. Another important consideration is to avert
failed OFCs due to the patient or family not being prepared for
the OFC due to uncontrolled asthma, continued antihistamine
use, and inability or refusal to complete the OFC. Clear verbal
and written information before the OFC is therefore essential.
66
Severity
Identifying patients at high risk of a severe reaction to foods is
important for the management of patients diagnosed with FA.
Previous studies have shown contradictory results about the
utility of food-specific IgE levels in assessing severity of
FA.
8,115,116
sIgE to certain allergen components, such as Ara h 2
in peanut, has been associated with more severe reactions than
sIgE to whole peanut or other single allergens, which is
corroborated by in vitro studies of basophil activation and
mediator release assays where Ara h 2 and Ara h 6 have been
shown to be the most potent elicitors of effector cell response.
25
On the contrary, sIgE to Ara h 8 is associated with PFAS. Higher
reactivity on the BAT to food allergens has been shown to be
associated with greater severity of allergic reactions during an
OFC.
117-119
In a peptide microarray, a broader IgE epitope di-
versity is associated with more severe reactions and with a greater
degree of basophil activation and degranulation after allergen
stimulation.
120,121
The above data need to be applied with caution to the
assessment of individual patients. For example, patients with
raised Ara h 2 sIgE do not necessarily have severe PA and can
actually pass a peanut OFC
122
; 10% of patients with PFAS can
have systemic reactions and 1% to 2% experience anaphy-
laxis.
123,124
The risk assessment of allergic patients depends on
factors other than mere individual players of IgE-mediated food-
induced allergic reactions (such as single allergens or epitopes,
IgE, or basophils) and requires a holistic clinical evaluation of the
patient.
CONCLUSIONS
In vitro allergy tests are useful in diagnosing IgE-mediated FA
and support the decision of whether an OFC is necessary to
reach an accurate diagnosis. Validated cutoffs are reliable when
applied to a similar patient population to the one where they
were developed. Patient-specific factors can modulate the prob-
ability of clinical allergy of a given sIgE result. IgE to allergen
components can provide more precise information about IgE
specificity. The BAT assesses the function of IgE in its ability to
mediate allergen-induced effector cell activation. Further research
is needed to improve our understanding about how the infor-
mation of various tests can be combined for optimal diagnostic
accuracy to reduce the need to perform OFCs to a minimum.
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