FULLTEXT01

International Journal of
Molecular Sciences
Review
Asthma and Allergy: Unravelling a Tangled Relationship with a
Focus on New Biomarkers and Treatment
Pablo Rodriguez del Rio 1, * , Andrew H. Liu 2,3,4 , Magnus P. Borres 5,6 , Eva Södergren 5 , Fabio Iachetti 5
and Thomas B. Casale 7
1 Department of Allergy, University Children’s Hospital Nino Jesús, 28040 Madrid, Spain
2 Breathing Institute, Section of Pediatric Pulmonary & Sleep Medicine, Children’s Hospital Colorado,
Aurora, CO 80045, USA; [email protected]
3 Department of Pediatrics, University of Colorado School of Medicine, Aurora, CO 80045, USA
4 Department of Pediatrics, National Jewish Health, Denver, CO 80217, USA
5 Thermo Fisher Scientific, 75123 Uppsala, Sweden; [email protected] (M.P.B.);
[email protected] (E.S.); [email protected] (F.I.)
6 Department of Women’s and Children’s Health, Uppsala University, 75123 Uppsala, Sweden
7 Morsani College of Medicine, University of South Florida, Tampa, FL 33612, USA; [email protected]
* Correspondence: [email protected]
Abstract: Asthma is a major driver of health care costs across ages. Despite widely disseminated
asthma-treatment guidelines and a growing variety of effective therapeutic options, most patients still
experience symptoms and/or refractoriness to standard of care treatments. As a result, most patients
undergo a further intensification of therapy to optimize symptom control with a subsequent increased
risk of side effects. Raising awareness about the relevance of evaluating aeroallergen sensitizations in
asthmatic patients is a key step in better informing clinical practice while new molecular tools, such as
the component resolved diagnosis, may be of help in refining the relationship between sensitization
Citation: Rodriguez del Rio, P.; Liu, and therapeutic recommendations. In addition, patient care should benefit from reliable, easy-to-
A.H.; Borres, M.P.; Södergren, E.; measure and clinically accessible biomarkers that are able to predict outcome and disease monitoring.
Iachetti, F.; Casale, T.B. Asthma and
To attain a personalized asthma management and to guide adequate treatment decisions, it is of
Allergy: Unravelling a Tangled
paramount importance to expand clinicians’ knowledge about the tangled relationship between
Relationship with a Focus on New
asthma and allergy from a molecular perspective. Our review explores the relevance of allergen
Biomarkers and Treatment. Int. J.
testing along the asthma patient’s journey, with a special focus on recurrent wheezing children. Here,
Mol. Sci. 2022, 23, 3881. https://
doi.org/10.3390/ijms23073881
we also discuss the unresolved issues regarding currently available biomarkers and summarize the
evidence supporting the eosinophil-derived neurotoxin as promising biomarker.
Academic Editors: Blanca Cárdaba
and Jose Antonio Cañas Mañas
Keywords: asthma; allergy; molecular diagnosis; biomarkers; EDN; treatment
Received: 28 February 2022
Accepted: 21 March 2022
Published: 31 March 2022
1. Introduction
Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims in Asthma is a major driver of health care costs for all ages and the most common chronic
published maps and institutional affil- disease for children and adolescents [1,2]. The prevalence of asthma has risen steadily over
iations. the past decades, currently reaching about 300 million people worldwide and potentially
involving a further 100 million by 2025 [3,4].
Despite the availability of both widely disseminated asthma-treatment guidelines
and an ever-evolving variety of effective therapeutic options, most patients with asthma
Copyright: © 2022 by the authors. continue to experience symptoms, with one in two adults reporting very poorly controlled
Licensee MDPI, Basel, Switzerland. asthma [5]. Similarly, the prevalence of inadequately controlled asthma in the pediatric
This article is an open access article population is quite high, with 30–40% of all severe exacerbations occurring in children and
distributed under the terms and
adolescents. [6]. It has been well documented that a significant proportion of patients might
conditions of the Creative Commons
be refractory to standard of care treatments, resulting in further intensification of therapy
Attribution (CC BY) license (https://
to optimize symptom control, thus remaining symptomatic despite maximal therapy and
creativecommons.org/licenses/by/
subsequent increased risk of side effects [7].
4.0/).
Int. J. Mol. Sci. 2022, 23, 3881. https://doi.org/10.3390/ijms23073881 https://www.mdpi.com/journal/ijms

Int. J. Mol. Sci. 2022, 23, 3881 2 of 21
To date, implementation of existing guidelines has been inadequate, particularly in

primary care setting [8], and due to several barriers, there is limited emphasis on identifying
triggers or allergens, jeopardizing efforts to effectively improve asthma control and patients’
burden [9]. Such a management approach seems to not fully acknowledge that the allergies
are frequent triggers of asthma exacerbations [10] and up to 80% of childhood asthma and
more than 50% of adult asthma cases may have an allergic component [11]. To date, in
the USA, allergy evaluation has been discussed in about 33% of primary care office visits
for asthma with allergy testing being only documented in 2% of cases of asthma over the
course of a year [12].
Considering a precision medicine approach, to improve asthma patient care is paramount
to bridge the observable characteristics (phenotypes) with the mechanisms driving the
disease (endotypes) using biomarkers. While an endotype-driven treatment still needs to
face multiple challenges before its implementation in daily clinical practice [13], asthma
endotype classifications combined with specific biomarkers may hold great potential for
new therapeutic modalities and better treatment efficacy [14]. Although specific, sensitive,
and reliable (point-of-care) biomarkers would be critical for selecting the proper treatment
for a given patient, current biomarkers appear to be good indicators of T2 endotypes but
not strong predictors of response to targeted treatments [15] and most of them require
further validation [16].
In this narrative review, we explore the tangled relationship between asthma and
allergy from a molecular perspective, and the relevance of allergen sensitization and testing
in asthma diagnosis and therapy selection, with a special focus on recurrent wheezing
children. Here, we also discuss the unresolved issues regarding current biomarkers in
clinical practice and summarize the evidence supporting eosinophil-derived neurotoxin
(EDN) as promising biomarker.
2. Pursuing an Optimized Asthma Care: The Relevance of the Molecular Approach

Asthma is recognized as a disease with significant heterogeneity in clinical features
(phenotypes), disease severity, pattern of underlying disease mechanisms and respon-
siveness to specific treatments (e.g., responder/non-responder, corticosteroid sensitive
or resistant). Thus, precision medicine strategies are needed to better tailor therapy on
a patient’s clinical and immunological profile [17]. Accordingly, classifying asthma into
distinct endotypes in a laboratory- and clinical-evidence-based manner contributes to
personalized precision medicine by unravelling disease mechanisms. To date, asthma
has been classically associated with type 2 inflammation, characterized by high levels of
immunoglobin E (IgE), eosinophils, fractional exhaled nitric oxide (FeNO), and cytokines
frequently found in allergic responses including interleukin 4, 5, 13 and 9 (IL-4, IL-5, IL-13,
IL-9). However, none of these biomarkers have proven effective in differentiating responses
between specific drugs that target type 2 inflammation. Moreover, between 10 and 33%
of subjects with asthma are not associated with allergy (non-allergic asthma) and exhibit
non-type 2 inflammation (non-T2 or T2-low endotype) with a prevalence of neutrophils or
a paucigranulocytic pattern. Therefore, there remains an unmet clinical need in the study
of the mechanisms and biomarkers for both T2-high and T2-low endotypes as concerns
their ability to predict response to targeted therapy [18].
It has been recently proposed that the molecular allergology approach to allergic
asthma may contribute to a better understanding of disease mechanisms, a precise diagno-
sis through the description of the molecular allergen sensitization profile, as well as to an
optimal selection of responders to the targeted treatment, either with allergen immunother-
apy (AIT), or with biologicals [19]. Importantly, it has been recently recognized that the
heterogeneity of asthma can be mostly ascribed to the complex interactions between the
host and the environment, including aeroallergens [20]. To this end, defining the allergen
sensitization of a patient with asthma at the molecular level by measuring specific IgE to
purified natural or recombinant allergens can improve diagnostic accuracy and improve
asthma phenotyping [21]. Allergen components have been available for testing in the clinic
Int. J. Mol. Sci. 2022, 23, 3881 3 of 21
for almost two decades. The different allergenic proteins in some pollens and perennial
allergens (e.g., dust mite) were characterized early. Pet-derived allergens are the third
leading cause of respiratory allergies, after mites and pollens, and a significant number of
new findings are changing the understanding of this allergy. Of note, the prevalence of
sensitization to dander from various animals appears to be increasing worldwide, with 1 in
5 adults being sensitized to cats. More recently, the allergenic proteins for cats and dogs
have been characterized with three important lipocalins (Canis familiaris 4, 6 and Felis
domesticus 7) being made available for testing as late as last year [22,23].
Furthermore, molecular-based diagnostics has a direct effect on the strategy of choos-
ing which allergens should be used in AIT [24]. Of note, it is recognized that molecular
diagnosis allows for a personalized AIT thus sparing patients the burden of multiple treat-
ments or unnecessary lifestyle modifications involved in allergen avoidance [25]. In this
scenario, the component resolved diagnosis (CRD) stands as a promising starting point as it
allows to precisely identify the number and type of recognized molecules in the individual
patient that are clinically relevant.
Among the innovative molecular approaches to allergic asthma, epigenetics is gaining
significant interest by virtue of its role in immune cell differentiation and plasticity [26] and
given the observation that airway epithelium pathobiology in asthma is regulated by epige-
netic mechanisms [27]. Importantly, epigenetic modifications can mediate the effects of the
environment on the development of or protection from allergic diseases [26]. Accordingly,
DNA methylation changes might thus be used as molecular biomarkers to quantify the
different allergy enhancing or protective exposures [28]. Epigenetic mechanisms, including
methylation, can also contribute to childhood asthma; therefore, identifying DNA methyla-
tion profiles in asthmatic patients can inform disease pathogenesis. Of note, recent analyses
from the MeDALL (European Mechanisms of the Development of Allergy) consortium
reported a significant association between reduced whole blood DNA methylation at both
21 and 14 CpG sites and childhood allergy, thus providing novel insights into the shared
molecular mechanisms underlying asthma, rhinitis, and eczema [29,30].
3. The Relevance of Allergen Testing in Asthma Management

3.1. Diagnosis
It is increasingly recognized that evaluating aeroallergen sensitization in asthma pa-
tients is a key step to improve patients’ care [31]. In line with this, the National Institute of
Health Asthma Outcomes Task Force recommends the assessment of aeroallergen sensitiza-
tion as a core biomarker for classification of asthma [32] while recommendations from the
GA2 LEN/EAACI state that the clinical relevance of each allergen is more important than
the number of sensitizations itself [33]. Although the testing criteria and the timing of the
testing (either alongside or after a diagnosis of asthma has been made) may vary among
guidelines, integrating aeroallergen evaluation into asthma management is of paramount
importance to optimize the asthma patient journey from diagnosis to treatment.
Different types of aeroallergens and specific sensitization profiles are associated with
a different pattern of clinical symptoms and different levels of severity. It has been docu-
mented that there is a direct relationship between the degree of allergen sensitization, mea-
sured as serum specific IgE, and the likelihood of expression of asthma symptoms [34,35].
This association with allergen-specific IgE titers is especially marked in children, where
elevated specific IgE supports a T2-high profile. Of note, when associated with childhood
symptoms of atopy and asthma, positive IgE testing aids in diagnosing early-onset allergic
asthma [36].
Testing for molecular aeroallergen sensitization can be of help to identify individuals
who are sensitized to minor allergens or to cross-reactive allergens as well as to confirm two
or more coexisting sensitizations (polysensitization). Most patients with allergic asthma
seen by specialists are polysensitized, and many of them are also poly-allergic (sensitiza-
tions with clinical relevance) because polysensitization does not necessarily mean that all
sensitivities are clinically significant. To date, while in Europe allergen immunotherapy
Int. J. Mol. Sci. 2022, 23, 3881 4 of 21
practice recommends that 1 or 2 of the most important allergens for the patient are to be
included in the same extract, in the United States, mixtures including many or most of the
sensitizing allergens are commonly administered [25].
Aeroallergen sensitization testing enhances the ability to predict asthma development,
drug response, and risk for future asthma exacerbations in children. In addition, among
children and adolescents, the allergy testing may help identify common comorbid con-
ditions such as allergic rhinitis, which when treated appropriately, can improve asthma
control [37]. Within the pediatric population, preschool children with repeated wheeze
have been recently included among the patients’ profiles who may benefit the most from
aeroallergen sensitization evaluation [31]. As recently proposed by Casale et al., several
patients’ profiles should undergo aeroallergen testing: persistent asthmatics, patients who
need oral corticosteroids (OCS) or inhaled corticosteroids (ICS), patients who seek to get
advice on the presence of pet at home or regular pet contact or to better understand their
condition, and patients who may be eligible to receive AIT or biologicals [31].
Several allergen component tests are now available for clinicians to use in every-
day practice [38]. Allergen-specific IgE tests utilizing individual allergenic molecules
are regarded as a more precise and informative option, particularly in polysensitized pa-
tients compared to those tests based on whole allergenic extracts. A growing spectrum of
molecules, representing single allergens of clinical relevance, have been identified, char-
acterized, and produced for commercial in vitro assays. CRD can be of special help in
unveiling co-sensitization and/or cross-sensitization of closely related or widely different
allergen sources [39,40]. Furthermore, CRD can be of special interest when the prescription
of AIT needs to be accomplished in areas with high frequency of sensitization to “minor
allergens” [41]. In addition, this is relevant when one should identify whether the sensiti-
zation is primary (species-specific) or a result of cross-reactivity to proteins with similar
protein structures. In this regard, CRD is gaining greater recognition for pet allergy diag-
nostics [42]. To date, the frequency of co-sensitization with cat and dog may be explained
by shared proteins between the two species e.g., lipocalins, or serum albumins (Table 1).
Table 1. Major and minor dog and cat allergens. Can f, canis familiaris; Equ c, equus caballus; Fel d,
felis domesticus; IgE, immunoglobin E. Elaborated from data in [37,42–45].
Molecular Sensitization
Allergen Family Biological Function Cross-Reactivity Sensitizer Role
Allergen Rate (%) *
DOG, CANIS FAMILIARIS
Can f 1 shares 62%
aminoacid identity
Transporter for small
with Fel d 7. The
hydrophobic
sensitization during
Can f 1 Lipocalin molecules, such as 50–76 Fel d 7
childhood has been
lipids and
shown to be predictive
steroid hormones
marker of dog allergy
in adolescence
Limited
hydrophobic
patient-dependent
Can f 2 Lipocalin molecules, such as 22–35 Fel d 1
cross-reactivity with
lipids and
Fel d 4
steroid hormones
Regulation of colloid Serum albumins may
osmotic pressure, play a significant role
transporter for a as cross-reacting
Fel d 2, other
Can f 3 Serum albumin multitude of 25–59 allergens in
serum albumins
metabolites, individuals sensitized
nutrients, drugs, and to dander of multiple
other molecules animal species
Int. J. Mol. Sci. 2022, 23, 3881 5 of 21
Table 1. Cont.
Allergen Rate (%) *
Sensitization to Can f 4
hydrophobic
has been found in up
Can f 4 Lipocalin molecules, such as 35–59 Equ c 1
to 46% of patients
lipids and
allergic to dog.
steroid hormones
The Can f 5 amino acid
sequence shows no
significant similarity
to any known animal
Prostate-specific
dander or urinary
antigen of
Can f 5 Kallikrein Arginine-esterase 71 § allergen. Therefore,
human
monosensitization to
seminal plasma
Can f 5 could be a
highly specific marker
for allergy to
male dogs.
Can f 6 and
Transporter for small homologous allergens
hydrophobic may contribute to
Can f 6 Lipocalin molecules, such as 23–61 Fel d 4, Equ c 1 multisensitization and
lipids and symptoms in
steroid hormones individuals allergic
to mammals
CAT, FELIS DOMESTICUS
Fel d 1 is the most
important allergen in
cat allergy, shown to
Its production is react with IgE from
Fel d 1 Secretoglobin related to 60–100 Can f 2 90% of cat-sensitized
sexual hormones individuals, and to
account for up to 90%
of IgE reactivity to
cat dander
Regulation of colloid Serum albumins may
osmotic pressure, play a significant role
transporter for a as cross-reacting
Fel d 2 Serum albumin multitude of 14–54 Can f 3 allergens in
metabolites, individuals sensitized
nutrients, drugs, and to dander of multiple
other molecules animal species.
60–90% of cat allergic
patients showed IgE
Member of the binding to rec. Fel d 3
Fel d 3 # Cystatin cysteine protease 10 in plaque
inhibitor family immunoassay; 10%
showed IgE binding in
radioallergosorbent test.
Transporter for small Fel d 4 binds IgE at
hydrophobic relatively high
Fel d 4 Lipocalin molecules, such as 61–63 Can f 6, Equ c 1 frequency in
lipids and cat-sensitive
steroid hormones individuals
Int. J. Mol. Sci. 2022, 23, 3881 6 of 21
Table 1. Cont.
Allergen Rate (%) *
Transporter for small Fel d 7 has high
hydrophobic potential to crossreact
Fel d 7 Lipocalin molecules, such as 38 Can f 1 with Can f 1, with
lipids and which shares 62%
steroid hormones amino acid identity.
With an IgE-binding
It has a high
frequency of only 19%
degree of
Latherin-like among individuals
Fel d 8 # 19–20 homology to
protein allergic to cat, it is not
horse Equ c 4
considered a major
and Equ c 5
cat allergen.
* Sensitization rate in the general population. Sensitization frequencies are variable in different geographic regions.
The reported percentage range is the average reported in the literature as extrapolated from the references cited in
the Table legend. Allergens are defined as major when are recognized by 50% or more of the sensitized population
and minor when are recognized by less than 50% of the sensitized population. § in Spanish population. # Not yet
available in the commercial IgE immunoassays.
Four dog allergens (e.g., Can f 1, Can f 2, Can f 4, and Can f 6) and two cat allergens
(Fel d 4 and Fel d 7) are in the lipocalins family of proteins [42]. A recent study carried
out in 294 children and adults with suspected asthma and a positive skin prick test to
cat and dog showed that allergen components can unveil the molecular basis of animal
polysensitization and may be of help in both identifying primary sensitizers and explaining
how individual IgE patterns of expression may correlate with previous pet ownership [46].
3.2. Therapy Selection

Asthma management entails allergen avoidance: therefore, identifying allergen sensi-
tization is crucial to both foster education on allergen avoidance and guide appropriate
exposure control [47]. Patients with asthma undergoing allergy testing are more likely to
adopt preventive measures (including an asthma plan, trigger avoidance, and medication
adherence), thus experiencing fewer days with allergy symptoms than their counterparts
who had not been tested [48]. These outcomes were supported by a study of adults with
moderately severe asthma, who had an individualized plan, including environmental
control based on the results of allergy testing [49]. Although avoidance interventions at the
population level for the main allergens are not supported by robust evidence of efficacy and
are still somewhat controversial, knowing which specific allergen is triggering the allergic
asthma can be of help in preventing the appearance or worsening of bronchial symptoms
in sensitized selected individuals [50]. Importantly, it is key not only to employ appropriate
avoidance measures, but also to effectively sustain these interventions over time to attain a
long-lasting symptom improvement.
As suggested by Global Initiative for Asthma (GINA) guidelines, most asthma pa-
tients react to multiple triggers that are ubiquitous in the environment, thus making their
avoidance very burdensome for the patient. Inhalant allergens, including indoor (molds,
animal dander, and house dust mites) and outdoor ones such as pollens, appear to be the
most important for children and adults with asthma. However, medications to maintain
good asthma control have a key role because patients appear less affected by environmental
factors when their asthma is well controlled [10].
Allergen testing can predict response to corticosteroids in both children and adults.
Certain patterns of inhaled allergen sensitization in early life may help identify children
at high risk for severe exacerbations and those who are likely to respond well to ICS [51].
Similarly, a recent study in the adult population with asthma and sensitization to airborne
allergens found that among sensitized subjects, the decline in forced expiratory volume in
Int. J. Mol. Sci. 2022, 23, 3881 7 of 21
1 second (FEV1 ) was lower for long-term ICS users (1.3–8 and >8 years), compared with
nonusers and short-term users [52].
Identifying sensitizations in allergic rhinitis subjects may also help guide AIT for po-
tential asthma prevention. Therefore, positive specific IgE is considered a useful biomarker
for AIT candidate selection in the context of a clear history of symptoms on exposure to
the relevant allergen [25]. To date, AIT stands as the only treatment approach able to alter
the natural course of allergic respiratory diseases by decreasing frequency and severity of
symptoms and progression of rhinitis to asthma [25,53]. As AIT works through induction
of allergen-specific tolerance, its effectiveness relies on the inclusion of relevant allergens in
adequate concentrations to achieve tolerance. As a result, omission of relevant allergens or
the use of irrelevant allergens may reduce efficacy of immunotherapy.
Polysensitization develops over time and has clinical relevance for treatment decisions.
In many patients originally classified as polysensitized based on SPTs to whole extracts,
molecular diagnosis could identify clear sensitizations to major allergens with clinical
relevance to be considered for AIT [54]. A detailed molecular diagnosis will add value when
determining whether AIT is appropriate for a given patient and, if so, which allergen(s)
should be administered. A study performed on 141 patients with pollen-induced AR [55]
showed that molecular diagnosis would change the allergen composition in AIT. The
patients were tested with SPTs and the ISAC® microarray-based panel of allergens, and
prescriptions before and after the knowledge gained with the input of ISAC were compared.
In 1 out of 2 cases, the allergen composition of AIT was changed due to the molecular
diagnosis results.
Finally, one must bear in mind that that even negative results for aerollergen sensi-
tization can be meaningful to personalize asthma care, as patients will be spared from
taking drugs that will not be effective and from the inconvenience of avoiding triggers to
which they are not sensitized [31]. Last but not the least, negative test results may prompt
a search for other causes of the observed symptoms, thereby improving asthma phenotype
definition and a personalized therapeutic approach.
4. Biomarker Hunting: From Current Challenges to Promising Candidates

Asthma had been tackled for a long time with a “one size fits all” management, al-
though it encompasses a wide range of phenotypes differing in severity and natural history.
Therefore, elucidation of these phenotypes and identification of biomarkers with which
to recognize them and guide appropriate treatment remain a priority. Biomarkers stand
as measurable indicators, bridging an underlying pathway to a phenotype or endotype of
a disease, and are of great value to both predict treatment response and monitor disease
progression. Therefore, an ideal biomarker should be sensitive, specific, able to provide
positive and negative predictive values, while being simple to measure and cost-effective.
Currently available biomarkers allow one to dichotomize individuals to either T2-high
or non-T2-high groups, thus establishing criteria for therapy selection in routine practice.
Of note, some biomarkers may offer highest yields when applied in the background of
certain clinical characteristics (e.g., more reversible airway disease, age at disease onset).
Therefore, a combination of different biomarkers, or a biomarker panel, may be more
suitable than one to refine selection of biological therapies. Exhaled NO, blood eosinophils,
serum IgE, and periostin are well-studied and established biomarkers for T2-high asthma
endotype [36].
FeNO measurement can be a valuable tool as it predominantly signifies IL-4 and IL-13
activity, with FeNO concentrations greater than 50 ppb (>35 ppb in children) unveiling
an eosinophilic airway inflammation and ICS responsiveness [56]. In conjunction with
peripheral eosinophilia, an elevated FeNO is a risk factor for airway hyperreactivity and
uncontrolled asthma [57]. However, despite being a reasonable indicator of T2-driven
asthma, there are no specific guidelines on how FeNO can guide therapy with biologics and
FeNo may be affected by relevant confounders including smoking, dietary nitrate intake,
and virus infections. Nevertheless, a recent post hoc analysis of the Liberty Asthma Quest
Int. J. Mol. Sci. 2022, 23, 3881 8 of 21
study evaluated the additional value of baseline FeNO, adjusted for baseline eosinophil
level and other clinical characteristics, as a predictor of response in dupilumab-treated
patients with uncontrolled, moderate-to-severe asthma [58].
By virtue of IgE’s role as a key mediator in the development and maintenance of aller-
gic inflammation and of a documented association between allergic asthma and elevated
total IgE [11], serum total IgE has been proposed as a predisposing factor for allergic asthma
and often employed for epidemiological analyses. However, elevated levels of serum total
IgE are not only found in patients with allergic asthma but also with other conditions
including parasitic infestations, inflammatory diseases, and some primary immunodefi-
ciency diseases [59]. Accordingly, total levels of IgE should be carefully interpreted and
not considered as an indication for the presence of allergic diseases. In terms of diagnos-
tic and therapeutic relevance, a recent study proposed serum-free IgE rather than total
IgE as biomarker of type 2 asthma in adults [60] and suggested that it could be used to
determine therapeutic response. Given the emerging availability of biologicals targeting
type 2 cytokines, it is important to assess total IgE ability to assist clinicians in choosing
a biological therapy among the approved options. As suggested by Sanchez et al., the
available evidence indicates that total IgE, as well as the blood eosinophil count and FeNO,
are not enough to select one therapy over another [61]. The presence of specific IgE antibod-
ies to environmental allergens proves sensitization and is associated with allergic asthma.
However, it has been suggested that the interpretation of skin prick tests and specific IgE
to whole allergen extracts relies on arbitrary cutoffs, which do not distinguish between
clinically relevant and non-relevant sensitizations [11]. In addition, while positive serum
specific IgE levels and symptoms upon exposure to the sensitizing allergen are indicated as
inclusion criteria for starting AIT, specific IgE levels solely were found a poor predictor of
AIT indication. Overall, the clinical relevance of total serum IgE and specific serum IgE as
biomarker for therapy selection requires further studies.
Although its expression in bronchial tissue is not associated with asthma severity,
periostin has been shown to be a biomarker of persistent eosinophilic airway inflammation
despite corticosteroid use [62]. The potential use of serum periostin levels is the assessment
of greater response to anti-T2-based therapies. Of note, periostin disadvantages encompass
the presence of several periostin splice variants, that complicate its detection, and the
uncertainty regarding its use as potential biomarker in children, since baseline periostin
levels are higher in children given the ongoing bone growth during childhood.
The measurement of eosinophilia in peripheral blood cells (either by absolute count
(BEC) or by percent differential of total leukocytes) has been investigated as a potential sur-
rogate marker of bronchial and/or lung inflammation. Higher eosinophil counts identify
patients with more severe disease and poorer outcomes, patients for whom biologic thera-
pies targeting allergic and/or eosinophilic pathways are recommended. To date, the recent
European Academy of Allergy and Clinical Immunology (EAACI) guideline on the use of
biologicals in severe asthma suggested that the higher the blood eosinophils, the higher the
expected impact of benralizumab, dupilumab, and mepolizumab in reducing severe asthma
exacerbations as well as the higher improvement in asthma control in patients treated with
benralizumab and reslizumab. In contrast, the effect of omalizumab on exacerbations did
not depend on blood eosinophils [63–65], although a greater response in patients treated
with omalizumab, when peripheral blood eosinophils were ≥300 cells·µL−1 , was also
reported [66].
Although blood eosinophil numbers can easily and quantitatively be determined in
any hospital laboratory, standardization of the appropriate cut-off of clinically relevant
eosinophilia and the need for single or multiple measurements in different settings are
needed prior to the application in clinical practice. In addition, some confounding factors
should be acknowledged for blood eosinophils, including circadian variation, parasites (e.g.,
helminthiases, schistosomiasis, filariases), and treatment with systemic corticosteroids [15].
For example, a diurnal variability of BECs has been reported, with peak counts recorded
soon after midnight and lowest counts at noon. Regarding the blood eosinophil counts
Int. J. Mol. Sci. 2022, 23, 3881 9 of 21
variability, a recent clinical trial conducted in patients with severe uncontrolled asthma
receiving standard maintenance treatment showed that most patients shifted to a different
BEC group from their baseline group at some point during the study and such behavior
was most marked in patients receiving long-term OCS. Therefore, the need for several
measurements of BEC may be particularly relevant for patients receiving OCSs [67–69].
Biomarkers’ clinical value also relies on their ability to both predict treatment response
and monitor disease progression. While circulating blood eosinophil count of ≥300/µL
may be helpful to identify a T2 immune biology to initiate therapy with an anti-T2 mAb, it
has very limited value to monitor response [70,71]. Overall, peripheral eosinophil count is
commonly obtained due to ease of performance and clinical accessibility. However, one
should keep in mind the caveats in its use and the potential limitations as a biomarker for
phenotyping refractory asthma or selecting therapy in mild asthma.
Overall, more differentiating, noninvasive, simply measurable, reliable biomarkers
with well-defined cutoff values and documentation on their stability/behavior over time
are urgently needed. Research focusing on eosinophil-derived molecular products is now
emerging, which highlights more potential biomarkers for allergic diseases [15].
Eosinophil granular proteins are a useful eosinophilic activation marker in asthmatic
patients and include the eosinophil peroxidase (EPO), eosinophil cationic protein (ECP)
and EDN [72]. Earlier studies reported that in patients with asthma serum levels of EPO
negatively correlated with FEV1 and positively with the number of eosinophils in peripheral
blood [73]. Of note, it can be measured in saliva and is documented correlating with sputum
eosinophils. In addition, in asthmatic children, EPO levels were lower compared to healthy
controls and positively correlated with total IgE levels [74].
ECP has been proposed as a marker for eosinophilic disease and quantified in biolog-
ical fluids including serum, bronchoalveolar lavage, and nasal secretions. Elevated ECP
levels are found in allergic asthma [75] and, among asthmatics, were associated with high
neutrophil count [76]. ECP levels are affected by age, smoking, circadian rhythm, and
seasonal variation, although only smoking appears to be of clinical significance [77]. Serum
ECP was significantly higher in children with symptomatic asthma than in asymptomatic
patients [78]. In children, ECP may provide complementary value when used together with
lung function test and FeNO. A recent study suggested that plasma ECP concentrations
may be a useful marker of type 2 inflammation in children and may help identify those
children at highest risk for recurrent exacerbations who could benefit from corticosteroid
treatment [79].
EDN, also known as eosinophil protein X, is a granular protein released from activated
eosinophils [72]. Patients with severe asthma and uncontrolled asthmatics exhibit higher
serum EDN level which showed a good positive correlation to total eosinophil count
(TEC) [80], thus suggesting the use of EDN as biomarker of asthma severity in adults. A
recent study carried out in adults from the Epidemiological Study on the Genetics and
Environment of Asthma revealed that EDN could be a potential biomarker to monitor
asthma evolution in adults as its levels are associated with different asthma expression
patterns in adults [71]. It has been also reported that higher serum EDN levels could be
found in children at the acute phase than at the stable phase of asthma, and in contrast to
TEC, the serum EDN level can be predictive of the severity of asthma [81]. In the pediatric
population, EDN may hold great promise in distinguishing persistent wheezing children
from children presenting wheezing triggered by respiratory tract infections [82] and in
aiding in the diagnosis of school age childhood asthma [83] as well as to monitor response to
montelukast or budesonide in preschool children with asthma [84]. Given the limited ability
of BEC to monitor treatment response to biologicals, the observed significant correlation
between reduced serum EDN level from baseline and lung function improvement after
omalizumab, benralizumab and reslizumab [85–87] may help monitoring the treatment
response to IgE- and IL-5 targeted therapies.
Finally, from a clinical standpoint, the finding that EDN levels can be measured in
multiple specimen types, such as sputum, serum, urine, bronchoalveolar lavage fluid, and
Int. J. Mol. Sci. 2022, 23, 3881 10 of 21
nasal lavage fluid [88–92] and, in contrast to BEC, are less affected by circadian rhythm or
gender differences [93] expand its potential usefulness in routine practice. Table 2 illustrates
the relative pros and cos of available Type 2 asthma biomarkers.
Table 2. Comparison between the advantages and disadvantages of the most employed type 2
asthma biomarkers and those of ECP and EDN. AIT, allergen immunotherapy; ECP, eosinophil
cationic protein; EDN, eosinophil-derived neurotoxin; FeNO, fractional exhaled nitric oxide; ICS,
inhaled corticosteroid; IgE, immunoglobin E; IL, interleukin; mAb, monoclonal antibody. Elaborated
from data in [15,56–58,60,62,67–69,75–79,81–83,94].
Biomarker Usefulness Advantages Disadvantages

It can serve as prognostic
biomarker and to predict Easy to realize in the clinical
The optimal cut-off has yet to
responsiveness to setting, requires minimal
be established and its levels
corticosteroid therapy in patient effort, could be
may be elevated due to
asthmatic patients with type 2 collected across the age
co-existing conditions such as
inflammation. Baseline value spectrum, and it is
parasitic infestations or
Blood eosinophil count can be used to predict the cost-effective. Circulating
decreased due to concomitant
clinical efficacy of blood eosinophil count of
medications such as oral
mepolizumab, reslizumab), ≥300/µL may be helpful to
corticosteroids. An additional
anti-IL5 receptor antibody identify a T2 immune biology
confounding factor is the
(benralizumab) and anti-IL4 to initiate therapy with
diurnal variability.
receptor an anti-T2 mAb
antibody (dupilumab).
The stability of serum
The presence of several
periostin over disease
periostin splice variants
The potential use of serum progression in adults with
complicates its detection. The
periostin levels is the asthma (without seasonal
uncertainty regarding its use
Periostin assessment of greater effect) and in children
as potential biomarker in
response to between 4 and 11 years of age
children since baseline
anti-T2-based therapies supports its use as a
periostin levels are higher
biomarker for type
in children
2-high asthma.
The main advantages of
specific IgE measurements
over the skin prick test are
that virtually all available
The presence of several allergens can be tested, and
The interpretation of skin
periostin splice variants the results are not influenced
prick tests and specific IgE to
complicates its detection. The by antihistamines or eczema.
whole allergen extracts relies
uncertainty regarding its use Specific IgE tests are slightly
Serum-specific IgE on arbitrary cutoffs, which do
as potential biomarker in more useful to confirm or
not distinguish between
children since baseline reject the suspicion of specific
pathologic and non-clinically
periostin levels are higher sensitisation to a certain
relevant sensitizations.
in children allergen. Assessment of
sensitization at the molecular
level can play a crucial role
before prescribing AIT for the
right selection of components.
The role of FeNO may be It can be a valuable tool as it
additive as a biomarker in predominantly signifies IL-4
FeNo may be affected by
relation to asthma morbidity. and IL-13 activity. FeNO can
relevant confounders
In conjunction with peripheral be used to adjust ICS dose or
FeNO including smoking, dietary
eosinophilia, an elevated as a marker of adherence to
nitrate intake and
FeNO is a risk factor for treatment. It may be a
virus infections.
airway hyperreactivity and predictor of response
uncontrolled asthma. to dupilumab
Int. J. Mol. Sci. 2022, 23, 3881 11 of 21
Table 2. Cont.
Biomarker Usefulness Advantages Disadvantages

In children, ECP may provide ECP levels are affected by age,
complementary value when smoking, circadian rhythm,
used together with lung and seasonal variation,
function test and FeNO. although only smoking
Plasma ECP concentrations appears to be of clinical
may be a useful marker of It can be measured in serum, significance. Measurement of
ECP type 2 inflammation in bronchoalveolar lavage and ECP has shown both time and
children and may help nasal secretions. temperature dependency
identify those children at during serum sampling.
highest risk for recurrent There is no internationally
exacerbations who could established cut-off for serum
benefit from ECP for use in the diagnosis
corticosteroid treatment. of asthma.
It is easy to obtain from
multiple specimen types (e.g.,
It holds great promise in serum, urine, sputum,
distinguishing wheezing bronchoalveolar lavage fluid,
children from children with and nasal lavage fluid) and is
wheezing triggered by not affected by circadian Additional studies are needed
respiratory tract infections, in rhythm, smoking or gender to validate the benefits of
aiding in the diagnosis of differences. Compared to ECP, serum EDN for predicting
EDN school age childhood asthma EDN is significantly less long-term clinical outcomes
as well as to monitor response charged, making it easier to and selecting right biologics
to montelukast or budesonide work with in routine setting. for right patients with
in preschool children with The quantification of serum severe asthma.
asthma. It can be used as EDN is not influenced by the
biomarker to monitor asthma type of storage tube used. It is
evolution in adults. stable at room temperature or
for up to one year when
frozen at −20 ◦ C or −80 ◦ C.
5. The Wheezing Child: A Paradigm to Optimize Asthma Control in Adult Life

The global prevalence of current wheeze in children and adolescents was estimated
to be 14.1% and 11.7% and projected to increase by 0.06% and 0.13% annually, respec-
tively [95,96]. Wheezing is common among preschool children and infants; of note, young
children with recurrent wheezing encompass a heterogeneous group with different geno-
types and phenotypes that lead to different outcomes [97]. Overall, recurrent wheezing
is associated with a two-fold increase in outpatient physician consultation or even the
emergency department, an up to 5-fold greater risk to be admitted to hospital [98], missed
days at school, activity limitation, and sleep disturbances [99]. Several phenotypes of
preschool wheeze have been proposed to identify individuals at risk for persistent asthma
at school age and display a temporal pattern of symptoms (Table 3).
Table 3. Wheezing child phenotypes. IgE, immunoglobin E; NA, not available. Elaborated from data
in [100–105].
Phenotype Clinical Profile

• Onset before the age of 3 years
Transient early wheeze • Resolves by the age of 6 years without persistent
lung function impairment.
• Onset after 3 years of age and persists in childhood
• Linked to atopy, reduced lung function and
Late-onset wheeze
bronchial hyperresponsiveness.
• Higher likelihood of asthma in adolescence
Int. J. Mol. Sci. 2022, 23, 3881 12 of 21
Table 3. Cont.
Phenotype Clinical Profile

• Starts early life
• Associated with atopy, high IgE levels, early
Persistent wheeze allergen sensitization and diminished lung
function by school age.
• Higher likelihood of asthma in adolescence
• Onset between 6 and 54 months of age
• Not associated with airborne allergen sensitization
Prolonged early wheeze
• Weakly associated with higher airway
responsiveness and impaired lung function.
• Onset between 18 and 42 months
• Linked with persisting symptoms, atopy, poor lung
Intermediate-onset wheeze
function and at more risk of developing asthma
in childhood.
• Frequent exacerbations, hospitalizations, and
unscheduled visits, reduced lung function,
Persistent troublesome wheeze bronchial hyperreactive airways, greater inhalant
allergen sensitization in comparison with
other phenotypes.
Using the latent class analysis (LCA) technique, Fitzpatrick et al. identified four
phenotypes of recurrent wheezing in preschool children based on type-2 inflammatory
features including BEC, atopic eczema, aeroallergen, and food sensitization and/or pet
exposures [51]. These phenotypes are distinguishable with regards to exacerbation risk,
with inhaled allergen sensitization patterns being important risk factors, and, importantly,
predict favorable response to daily ICS treatment to prevent exacerbations. Thus, certain
patterns of inhaled allergen sensitization in early life can help identify children at high risk
for severe exacerbations and those who are likely to respond well to ICS [51]. Of note, ICS
are recommended as first choice of controller treatment in all preschool recurrent wheezing
children irrespective of phenotype, but they are particularly beneficial in terms of fewer
exacerbations in atopic children [106].
Genes and environmental factors such as respiratory viruses, tobacco smoke expo-
sure, and inhaled allergens can modify the phenotypes of early childhood wheezing [107].
Furthermore, early exposures, including that to older siblings, pets, farm animals, and
house-dust endotoxin, seem to influence the risk for persistent asthma [108,109]. To date,
children suffering from allergic asthma, especially those with a persistent moderate and
severe phenotype, were more often sensitized to all the three major dust mite allergens
(Der p1, Der p2, Der p23) [110]. Of note, Der p 23 sensitization has been recently described
as being associated with increased asthma risk [111]. Although many individuals later di-
agnosed with asthma exhibit their first symptoms during the preschool period, diagnosing
asthma in preschool children is challenging, resulting in undertreatment of young asthmatic
children and possibly overtreatment of transient wheezers [112]. Of note, transient early
wheezing and nonallergic wheezing generally subside by 4–6 years of age as the airways
enlarge in a growing child [31]. It is well documented that a lack of diagnostic criteria as
well as the incapability of pre-school age children to perform conventional lung function
tests, like exhaled nitric oxide or spirometry, hinder effective diagnosis and assessment in
children [113,114]. As diagnosing asthma in the pediatric population is still challenging in
clinical practice, defining the prognosis of preschool children requiring medical attention
for recurrent wheezing appears of great value to optimize asthma care.
The progression from recurrent wheezing to persistent asthma is variably predicted by
factors that emerged in cohort studies including the early sensitization to inhalant or food
allergens, atopy, family history of asthma male gender, peripheral blood eosinophilia, as
well as a history of wheezing with lower respiratory tract infections. The Asthma Predictive
Index (API), that includes recurrent wheeze (e.g., more than three wheezing episodes per
Int. J. Mol. Sci. 2022, 23, 3881 13 of 21
year) with risk factors such as parental asthma, atopic dermatitis, and allergen sensitization
in the child, may more effectively identify young persistent asthma risk [115]. Overall, API
has an appreciable likelihood ratio (~7.4) as it increases the probability of a prediction of
asthma by 2–7 times [106,116]. Children with a positive API were found 7 times more likely
to have active asthma at school age [117]. A modified version of API, modified API (mAPI),
allows clinicians to identify children from birth to age 4 at high risk of developing asthma
as well as to predict children response to ICS [118,119]. Importantly, mAPI acknowledges
the contribution of allergic sensitization to at least one aeroallergen as a major criterion.
The identification of sensitization patterns based on individual allergens and linking
these to disease morbidity for asthma in young children could have relevant clinical implica-
tions. Data from German MAS birth cohort study showed that schoolchildren sensitized to
perennial allergens with high exposure early in life are more prone to develop an impaired
lung function at school age than children without sensitization or sensitized to indoor
allergens but with a low exposure [120]. Therefore, diagnosing specific inhalant allergen
sensitizations in at-risk children can aid in designing allergen-avoidance strategies [121]
as well as in guiding exposure mitigation and AIT choice in asthma prevention if allergic
rhinitis is present. Allergen sensitization also negatively affected asthma outcomes in
children with poorly controlled asthma and sensitization as they were more likely to use
oral corticosteroids than their counterparts without allergen sensitization [122].
Furry animals, such as dogs and cats, represent important allergen sources [42]. It
has been reported that sensitization to dog, cat, and horse throughout childhood was
significantly associated with asthma at age 7 years [123]. In a population-based study
with 259-animal sensitized, most were sensitized to two or more dog allergens, and co-
sensitization to Can f 5 [prostate (i.e., male) specific allergen] and Can f 1/2 conferred
the greatest risk for asthma [124]. In line with this, a recent study in children with mild
to severe asthma showed that sensitization to a greater number of dog components may
identify children whose asthma severity is driven by dog exposure and may benefit from
targeted interventions such as exposure mitigation and immunotherapy [125]. To this end,
molecular-based allergy diagnostics such as CRD provides clinicians with opportunities to
better characterize children with polysensitization [126]. Nevertheless, one should keep
in mind that a disagreement currently exists between asthma guidelines on the routine
use of allergy testing in the diagnostic work-up of a child with persistent asthma [127].
Collectively, healthcare providers dealing with a preschool child with recurrent wheeze
may rely on several tools, such as mAPI, aeroallergen testing, and CRD, to effectively
predict the risk of persistent asthma in later childhood and adulthood. Armed with this
predictive information, personalized education and successful implementation of allergen
avoidance and appropriate therapies can help to reduce the substantial burden associated
with more severe, persistent, and exacerbation-prone disease. From a clinical standpoint, a
recurrent wheezing child may be a useful paradigm to optimize asthma care in children at
high risk of developing persistent, life-long asthma.
6. Conclusions
Asthma affects both children and adults and displays high morbidity. As asthma
should be regarded as a syndrome, because of its phenotypic and endotypic heterogeneity,
therapeutic approaches can be most effective if tailored to selected molecular targets
in the dysregulated pathways of that given patient [128]. Despite growing therapeutic
armamentarium, asthma control remains largely inadequate, and rates of healthcare use are
high in both pediatric and adult populations, thus posing a substantial burden on societies.
It has been suggested that raising awareness about the relevance of evaluating aeroallergen
sensitizations in asthmatic patients is a key step in improving asthma/allergy care and
informing clinical practice [31]. To date, mounting evidence is supporting the relevance of
aeroallergen testing across the asthma patient journey (Figure 1).
in the dysregulated pathways of that given patient [128]. Despite growing therapeutic ar-
mamentarium, asthma control remains largely inadequate, and rates of healthcare use are
high in both pediatric and adult populations, thus posing a substantial burden on socie-
ties. It has been suggested that raising awareness about the relevance of evaluating aeroal-
lergen sensitizations in asthmatic patients is a key step in improving asthma/allergy care
Int. J. Mol. Sci. 2022, 23, 3881 14 of 21
and informing clinical practice [31]. To date, mounting evidence is supporting the rele-
vance of aeroallergen testing across the asthma patient journey (Figure 1).
Figure 1. Relevance of allergen testing across asthma patient journey. AIT, allergen immunotherapy.
Nevertheless, allergic sensitization should be regarded as a quantifiable rather than

dichotomous trait, e.g., we can use the titer of allergen-specific sIgE antibodies and/or the
number of sensitizations to the major allergen components of an exposure (e.g., dog) [129].
Physicians are currently provided with tools to better describe sensitization such as CRD
that measures sIgE response to many major and minor allergens. This approach aids in
refining the relationship between sensitization, clinical outcomes, and asthma progres-
sion [130]. CRD stands as a useful tool to better characterize children with polysensitization,
to offer guidance in pet allergy treatment recommendations, including exposure reme-
diation and AIT prescription [42,126] based on major components present in allergenic
extracts used in AIT and the patient’s profile. The information retrieved through molecular
allergy diagnostics opens novel avenues to advance asthma care while raising concerns
about the correct timing for its application [131]. To implement the CRD approach in the
diagnostic and management algorithms for asthma, appropriate interpretation tools should
be developed to facilitate its use [129].
To unravel asthma molecular networks and to link phenotype with endotypes, great
efforts have been placed in the search of biomarkers endowed with several features such as
ease of performance, reliability, clinical accessibility, and the ability to predict outcome and
disease monitoring [15]. Despite drawbacks of currently employed biomarkers, therapy
selection still relies on them, as also documented in international guidelines and opinion
papers [14,40,53,63–65]. Total IgE, FeNO, and BEC are the main drivers for biologicals
prescription in patients with moderate to severe T2 high asthma with established cut off
values for likelihood of treatment response.
Int. J. Mol. Sci. 2022, 23, 3881 15 of 21
Furthermore, we are still lacking more effective biomarkers for therapy selection in
patients with overlapping phenotypes or in those for whom the presence of risk factors,
such as smoking, or of concomitant therapies (e.g., systemic corticosteroids), which strongly
hinders the reliability of some biomarker assessments. In this challenging scenario, EDN
is an analytically attractive biomarker that can be reliably quantified in multiple speci-
mens [93] and is stable during long-term storage. Of note, EDN may hold promise in
distinguishing wheezing children from children with respiratory tract infections [82] and
in aiding in the diagnosis of school age childhood asthma [83].
Future studies addressing the prognosis of preschool recurrent wheezing children
appear of great value to optimize asthma care. It has been well established that diagnosis
asthma in preschool children remains an unsolved issue. To date, preschool children with
recurrent wheezing display several phenotypes (reflecting different endotypes and differing
for clinical manifestations, natural history, response to therapy and inflammatory mecha-
nisms) and may also switch between phenotypes or progress into another. Furthermore,
not all the observed phenotypes are precursors of or manifestations of childhood asthma
and, when it is the case, may be differentially responsive to asthma-targeted therapies.
To this end, it has been advised to distinguish children based on allergen sensitization,
evidence of eosinophilia and evidence of neutrophilia and/or infection [98]. In line with
this, efforts should also be directed towards a better characterization of children with
polysensitization and a routine implementation of allergen testing in at-risk children to
design allergen-avoidance strategies as well as to guide AIT choice in asthma prevention.
Another area deserving further investigation is the search of biomarkers for nonallergic
type2-low children as the type-2 low inflammatory endotype is still poorly characterized in
the pediatric population. Finally, it is important to recognize that asthma phenotypes are
not static and can change with time in individual patients. Therefore, continued assessment
of patients unresponsive to therapies requires reassessment of relevant biomarkers.
Author Contributions: Conceptualization and methodology, P.R.d.R., A.H.L. and T.B.C.; writing—
original draft preparation, P.R.d.R., A.H.L. and T.B.C.; writing—review and editing, P.R.d.R., A.H.L.,
T.B.C., M.P.B., E.S. and F.I. All authors have read and agreed to the published version of the manuscript.
Funding: Medical writing support has been funded by Thermo Fisher Scientific (Uppsala, Sweden).
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Not applicable.
Acknowledgments: Medical writing support was provided by Chiara Degirolamo, of ContentEdNet
(Rome, Italy), which was in accordance with Good Publication Practice (GPP3) guidelines.
Conflicts of Interest: P.R.d.R. is a consultant for Thermo Fisher Scientific; has received personal
lecturing fees from ALK Abello, GSK, FAES, Miravo, Novartis, LETI Pharma, Allergy Therapeutics,
and Aimmune Therapeutics. A.H.L. is a Consultant for Phadia ThermoFisher, Avillion, Revenio,
and LabCorp; received non-monetary research support from Phadia Thermofisher and Propeller
Health/ResMed; and research funding from Avillion. All funds paid to University of Colorado. T.B.C.
reports consulting fees from Thermo Fisher Scientific, Genentech, Novartis, outside the submitted
work. M.P.B., E.S. and F.I. are employed by Thermo Fisher Scientific, Uppsala, Sweden.
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International Journal of

Int. J. Mol. Sci. 2022, 23, 3881. https://doi.org/10.3390/ijms23073881 https://www.mdpi.com/journal/ijms

To date, implementation of existing guidelines has been inadequate, particularly in

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Biomarker Usefulness Advantages Disadvantages

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Nevertheless, allergic sensitization should be regarded as a quantifiable rather than

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