







































_____________________________________________________________________________________________________ 
 
*Corresponding author: E-mail: celso@alergoimuno.med.br; 
 
Cite as: Olivier, Celso Eduardo, Daiana Guedes Pinto, Ana Paula Monezzi Teixeira, Cibele Silva Miguel, Raquel Acácia 
Pereira Gonçalves Santos, Jhéssica Letícia Santos Santana, and Regiane Patussi Santos Lima. 2024. “Cellular and Humoral 
Immunoreactivity Against Hen’s Egg White: Relevance in Allergic Patients”. Asian Journal of Immunology 7 (1):274-84. 
https://doi.org/10.9734/aji/2024/v7i1150. 

 
 

Asian Journal of Immunology 
 
Volume 7, Issue 1, Page 274-284, 2024; Article no.AJI.127742 
 

 
 

 

 

Cellular and Humoral Immunoreactivity 
against Hen’s Egg White: Relevance in 

Allergic Patients 
 

Celso Eduardo Olivier a*, Daiana Guedes Pinto a,   
Ana Paula Monezzi Teixeira a, Cibele Silva Miguel a,  

Raquel Acácia Pereira Gonçalves Santos a,  
Jhéssica Letícia Santos Santana b  

and Regiane Patussi Santos Lima c 
 

a Instituto Alergoimuno de Americana, Brazil. 
b Instituto de Ensino e Pesquisa do Hospital de Amor de Barretos, Brazil. 

c Lavoisier Laboratórios, São Paulo, Brazil. 
 

Authors’ contributions  
 

This work was carried out in collaboration among all authors. Author CEO is responsible for the 
conceptualization, data curation, formal analysis, literature review, and wrote the original draft. 

Authors DGP, APMT, CSM, JLSS and RPSL performed the laboratory procedures. Author RAPGS 
performed cutaneous tests. All authors read and approved the final manuscript. 

 

Article Information 
 

DOI: https://doi.org/10.9734/aji/2024/v7i1150  
 

Open Peer Review History: 
This journal follows the Advanced Open Peer Review policy. Identity of the Reviewers, Editor(s) and additional Reviewers,  

peer review comments, different versions of the manuscript, comments of the editors, etc are available here: 
https://www.sdiarticle5.com/review-history/127742  

 

 

Received: 04/10/2024 
Accepted: 06/12/2024 
Published: 09/12/2024 

 
 

ABSTRACT 
 

Background: Hypersensitivity to chicken eggs is the second most common food allergy, presenting 
IgE-mediated and non-IgE-mediated hypersensitivity mechanisms. While IgE-mediated 
hypersensitivity is easily detected, non-IgE-mediated endotypes present challenging obstacles to 
diagnosis.  

Original Research Article 

https://doi.org/10.9734/aji/2024/v7i1150
https://www.sdiarticle5.com/review-history/127742


 
 
 
 

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275 

 

Aim: To evaluate the potential of the Tube Titration of Precipitins (TTP) and the Leukocyte 
Adherence Inhibition Test (LAIT) to discriminate cellular and humoral immunoreactivity against 
hen's egg white (albumen).  
Study Design: We retrospectively examined the medical charts of two cohorts of patients clinically 
diagnosed with diverse allergic phenotypes with clinical suspicion of non–IgE-mediated 
hypersensitivity against hen's egg white, who were investigated with the help of TTP or LAIT. 
Methodology: The registered results of the semi-quantitative serum TTP against 1 mg/mL albumen 
extract (TTP cohort) as well as the registered results of the Leukocyte Adherence Inhibition (LAI) 
percentage promoted by the ex vivo challenges against 1 mg/mL albumen extract (LAIT cohort) 
were distributed through a cascade distribution chart to outline the variability of the results. The 
statistical characteristics inside each cohort were calculated.  
Results: The cascade distribution for TTP with hen's egg extract showed a relatively uniform 
distribution. There were eight negative results. The mean was estimated at 1:156; the median was 
1:64; the standard deviation was estimated at 1:185; the mode was 1:512 (appeared eighteen 
times). The cascade distribution for LAIT with hen's egg extract showed a relatively uniform 
distribution. There were twenty and one negative results. The LAI ranged from 0% to 98%. The 
mean was 44.2%; the median was 48.5%; the standard deviation was 32.2%; the mode was 0% 
(appeared 21 times).   
Conclusion: Our preliminary results support that the TTP and LAIT performed with hen's albumen 
extract may discriminate diverse humoral and cellular immunoreactivity degrees in patients suffering 
from diversified allergic phenotypes. 
 

 
Keywords: Albumen; endotype; hen's egg white; hypersensitivity; leukocyte adherence inhibition test; 

non–ige-mediated immunoreactivity; phenotype; precipitins. 
 

ABBREVIATIONS 
 
LAI : Leukocyte Adherence Inhibition 
LAIT : Leukocyte Adherence Inhibition Test 
TTP : Tube Titration of Precipitins 
 

1. INTRODUCTION  
 

The domestic chicken Gallus domesticus 
resulted from hybridizing and domesticating 
several Asiatic wild Gallus species that started 
around eight thousand years ago [1]. The 
extensive creation of chicken for broilers or the 
production of eggs destined for human nutrition 
is nowadays one of the many contributors to the 
reconfiguration of the biosphere [2]. Hens are 
female adult chickens that lay eggs even if not 
fertilized. Eggs are organic vessels oviparous 
animals produce to incubate an embryo outside 
the animal's body [3]. After being laid, eggs may 
be a target of predators in nature for their high 
nutritional content [4]. Chicken eggs are 
constituted by a protective shell, a yolk (or 
vitellus), an egg white (or albumen), and an 
embryo (when fertilized) [5]. Egg white mainly 
consists of water (88%), protein (10.5%), 
carbohydrate (0.5%), ash (0.8%), and lipids 
(0.2%) [6]. The most abundant protein in 
albumen is ovalbumin (54%), a 
phosphoglycoprotein that converts to s-
ovalbumin (a less reactive form of albumin) 

during storage [7]. Ovotransferrin (also known as 
conalbumin, an iron-binding glycoprotein) is the 
second most abundant protein (12%), followed 
by ovomucoid (11%), a heat-resistant major 
allergen which functions as a trypsin inhibitor 
[8,9]. Other proteins were also quantified: 
ovomucin (3.5%), lysozyme (3.5%), ovoinhibitor 
(1.5%), ovoglycoprotein (1.0%), ovoflavoprotein 
(0.8%), ovomacroglobulin (0.5%), avidin (0.5%) 
and about more thirty proteins found in 
decrescent proportions [10,11]. 
                         
In addition to their nutritional values, albumen's 
proteins present several functional properties 
explored by the artisanal culinary and food 
industry [12]. Usually referred to in cosmetic 
ingredients as albumen, egg white proteins are 
used to enhance skin and hair properties, 
enhance skin tone, and reduce wrinkles. It is 
incorporated into face masks, cleansers, and 
skincare formulations [13]. 
 
Hypersensitivity to egg proteins is the second 
most common food allergy, with a self-report 
prevalence of up to 7% of the inquired population 



 
 
 
 

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[14]. Allergy to egg white proteins was one of the 
first thoroughly investigated reported cases of 
food allergy, successfully treated with 
desensitization in 1912 [15]. Patients may 
present cutaneous manifestations (urticaria, 
angioedema, atopic and contact dermatitis), 
gastrointestinal symptoms (allergic proctocolitis, 
eosinophilic esophagitis, food-protein induced 
enterocolitis syndrome), respiratory 
inflammations (rhinitis, bronchitis) and 
anaphylaxis [16,17].  
 
The Allergen Nomenclature Sub-Committee of 
the World Health Organization and International 
Union of Immunological Societies (WHO/IUIS) 
has recognized ten allergens related to Gallus 
domesticus, from which four are abundant in 
albumen according to their official nomenclature: 
Gal d 1 (ovomucoid), Gal d 2 (ovalbumin),                         
Gal d 3 (ovotransferrin) and Gal d 4 (lysozyme C) 
[18]. 
 
Bird-egg syndromes are symptomatic allergic 
cross-reactivities among egg proteins, poultry 
meat, and aeroallergens from feathers [19]. Egg 
white proteins may also function as 
aeroallergens, producing respiratory allergic 
phenotypes [20]. 
 
Non-IgE-mediated cellular immunoreactivity 
against food allergens had already been reported 
by our group with the help of the Leukocyte 
Adherence Inhibition Test (LAIT), as well as 
humoral immunoreactivity against other food 
allergens with the help of Tube Titration of 
Precipitins (TTP) [21-23]. These tools are 
evaluated as laboratory tools to endotype 
immunoreactivity conditions as a Personalized 
Medicine strategy [24]. 
 
We routinely employ the LAIT and the TTP in our 
facilities as a triage to evaluate non–IgE-
mediated immunoreactivity against suspected 
allergens before performing more exhaustive in 
vivo provocation tests [25-31]. To evaluate the 
potential of the LAIT and TTP to endotyping non–
IgE-mediated cellular and humoral 
immunoreactivity against hen's eggs albumen 
extracts, we retrospectively compiled the 
electronic medical charts of patients investigated 
by one of these assays. 
 
The present study is a proof-of-concept that 
hypothesizes that LAIT and the TTP may 
differentiate diverse degrees of cellular and 
humoral immunoreactivity against hen's egg 
white proteins in allergic patients.  

2. MATERIALS AND METHODS  
 

2.1 Subjects 
 

After receiving Institutional Review Board 
approval from the Instituto Alergoimuno de 
Americana (Brazil; 9/2024), we reviewed the 
electronic chart of 9,600 outpatients who 
attended our facility from January 2018 to 
November 2024. Among these we found 100 
patients submitted to TTP with hen's egg white 
and 100 patients submitted to LAIT with hen's 
egg white. 
 
A cohort of 100 consecutive outside patients 
(TTP cohort) had been submitted to TTP with 
hen's egg white for presenting in patients with 
non–IgE-mediated allergic phenotypes (urticaria, 
angioedema, atopic and contact dermatitis, 
allergic proctocolitis, eosinophilic esophagitis, 
food-protein induced enterocolitis, rhinitis, and/or 
bronchitis). This cohort counted 23 males; mean 
age 42.7 years; SD 20.3 years; range 4 to 92 
years; median 42.5 years; modes = 35 and 38 
years (each appeared five times).  
 
A cohort of 100 consecutive outside patients 
(LAIT cohort) had been submitted to TIAL with 
hen's egg white for presenting in patients with 
non–IgE-mediated allergic phenotypes (urticaria, 
angioedema, atopic and contact dermatitis, 
allergic proctocolitis, eosinophilic esophagitis, 
food-protein induced enterocolitis, rhinitis, and/or 
bronchitis). This cohort counted 27 males; mean 
age 39.9 years; SD 23.4 years; range 0 to 85 
years; median 41.5 years; mode = 1 (appeared 
five times).  
 
This study did not include patients under 
biological and/or systemic anti-inflammatory 
therapy. These procedures were offered to 
patients with clinical suspicion of hen's egg white 
hypersensitivity who demonstrated a non-
reactive or inconclusive skin test against hen's 
egg white extract, as well with total IgE inside 
reference levels, and non-detectable levels of 
specific IgE against any allergen, including hen’s 
egg white proteins [32,64]. 
 

2.2 Hen's Egg White Extract 
 
Hen's egg white was homogenized and then left 
for 48 hours in a Coca-based extractor solution 
(propylparaben 0.5g, methylparaben 1g, sorbitol 
30g, NaCl 5g, NaHCO3 2.5g, 1,000mL H2O) at 4 
°C for protein extraction before centrifugation 
and separation of the water-soluble fraction from 



 
 
 
 

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solid particles and oily fraction [33]. The protein 
quantification of the allergen extract was done 
according to Bradford's protein-dye binding 
methodology [34]. The solution was diluted in 
antigen dilution solution (NaCl 10g; KH2PO4 
0.72g; Na3PO4 2.86g; methylparaben 1g; 
propylparaben 0.5g; glycerin 400mL; H2O 
600mL) to an estimated protein concentration of 
1 mg/mL and stored at 4 °C into amber opaque 
glass vials. The hen's egg white extract solution 
was used for allergic skin tests, TTP, and LAIT. 
All relevant and mandatory laboratory health and 
safety measures have been complied with during 
the experiments.  
 

2.3 LAIT: Ex vivo Investigation: 
Leukocyte Adherence Inhibition 
Test 

 

2.3.1 LAIT: Procedure for allergen ex vivo 
challenging  

 
We performed LAIT as previously described [35-
41]. Shortly, each donor's fresh plasma was 
divided into two parts and used in parallel ex vivo 
challenging tests with the hen's egg white extract 
and the unchallenged plasma (added with 
antigen dilution solution as a control). We 
collected plasma with high leukocyte content 
(buffy coat) from the heparinized tube after one 
hour of sedimentation at 37 °C. Then, we 
distributed aliquots of 100 μL into Eppendorf 
tubes with (or without) the challenging extract 
and kept them under agitation for 30 minutes 
(200 rpm at 37°C). 
 
2.3.2 LAIT: Procedure for adherence assay  
 
 After incubation, the plasma was allocated into a 
standard Neubauer hemocytometer counting 
chamber with a plain, non-metallic glass surface 
and left to stand for 2 hours at 37°C in the 
humidified atmosphere of the covered water bath 
to allow leukocytes to adhere to the glass. Next, 
we counted the leukocytes, removed the 
coverslip, and washed the chamber by 
immersion in a beaker with phosphate buffer 
saline (PBS) at 37°C. Then, we added a drop of 
PBS to the hemocytometer's chamber and 
allocated a clean coverslip over it. The remaining 
cells were counted in the same squares as 
previously examined.  

 
2.3.3 LAIT: Procedure for calculation  
 
The percentage of Leukocyte Adherence (LA) of 
each assay was estimated as: (the number of 

leukocytes observed on the hemocytometry 
chamber after washing divided by the number of 
leukocytes observed on the hemocytometry 
chamber before washing) and multiplied by 100 
(%). The Leukocyte Adherence Ratio (LAR) was 
estimated based on the ratio between the LA 
from the antigen-specific challenged plasma and 
the LA from the unchallenged control plasma: 
LAR = LA of the challenged sample divided by 
LA of unchallenged control plasma multiplied by 
100 (%). To further calculate the Leukocyte 
Adherence Inhibition (LAI), we subtracted the 
LAR from 100 (%). We employed the LAI results 
for the cascade distribution chart and the 
statistics calculations, both performed with the 
help of the Microsoft Excel® statistical package. 
 

2.4 TTP: In vitro Investigation: Tube 
Titration of Precipitins 

 
As previously reported, the semi-quantitative 
titration of precipitins (TTP) against the allergen 
extract was performed in a transparent vitreous 
tube array [42-44]. Shortly, the patient's blood 
was collected in a clot-activator collecting tube. 
After separation, the serum was centrifugated at 
2,000 rpm for 10 minutes. Each allergen extract 
was allocated in sets of eleven glass tubes at 
progressive duplicated serum dilutions. The 
progressive dilutions were combined with the 15 
μL of the hen's egg extract with 250 μL of the 
patient's serum, progressively diluted into 
physiological saline solution (NaCl 0,9%) in the 
dilution ratios of 1:1; 1:2; 1:4; 1:8; 1:16; 1:32; 
1:64; 1:128; 1:256; and 1:512. One tube was a 
blank control done with the water and serum to 
observe occasional spontaneous precipitation 
(Sia Test). After 24 hours, the tubes were 
examined, and the titers (the highest dilution 
factor that yields a positive reading) were 
recorded [45]. 
 

3. RESULTS 
 
As a retrospective survey, there was no research 
protocol; therefore, we report the incidental 
immune investigation as registered in the digital 
medical charts.  
 

The cascade distribution graph (cascade 
distribution graph is the designation of the 
standard graph derived from the Excell® 

software) for the TTP cohort with hen's egg 
extract showed a relatively uniform distribution 
(Fig. 1). There were eight negative results. The 
mean was estimated at 1:156; the median was 
1:64; the standard deviation was estimated                     



 
 
 
 

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at 1:185; the mode was 1:512 (appeared 
eighteen times). Some patients showed low or 
moderate concentrations of precipitins against 
hen's egg white. In contrast, others showed                  
high concentrations of precipitins against the 
hen's egg white, which could reflect the 
participation of hen's egg white allergens in these 
patients' non–IgE-mediated hypersensitivity 
conditions. 
 
The cascade distribution for the LAIT cohort with 
hen's egg extract showed a relatively uniform 

distribution (Fig. 2). There were twenty and one 
negative results. The LAI ranged from 0% to 
98%. The mean was 44.2%; the median was 
48.5%; the standard deviation was 32.2%; the 
mode was 0% (appeared 21 times). Some 
patients showed low or moderate 
immunoreactivity during the ex vivo challenge 
test. In contrast, others displayed strong 
immunoreactivity, which could reflect the 
participation of hen's egg white allergens in these 
patients' non–IgE-mediated hypersensitivity 
conditions. 

 

 
 

Fig. 1. Cascade distribution chart of the tube titration of precipitins (x-axis %) resulting from 
the hen's egg white extract against the serum of the TTP cohort of 100 tests/subjects (y-axis). 

 

 
 

Fig. 2. Cascade distribution chart of the range groups of Leukocyte Adherence Inhibition (LAI) 
results (x-axis %) of the ex vivo challenge test against hen's egg white extract monitored by 

the Leukocyte Adherence Inhibition Test (LAIT), according to the respective number of 
outcomes over the LAIT cohort with 100 tests/subjects (y-axis) 



 
 
 
 

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4. DISCUSSION 
 
When scientists need an animal model of chronic 
allergy to egg proteins, they administer mice 
subcutaneous injections of 10 μg of ovalbumin 
with an adjuvant such as aluminum hydroxide 
[46].  
 
In 1931, Woodruff and Goodpasture reported the 
first technique employing chicken eggs to 
propagate viruses [47]. Nowadays, several 
immunization vaccines (such as for measles, 
mumps, rubella, influenza, yellow fever, Q fever, 
and rabies) are prepared from viruses 
propagated in eggs and present residual or 
significative contents of egg proteins, originating 
controversy about vaccination in patients with 
egg allergy [48-50]. Most concerns and safety 
evaluations about human parenteral egg 
components are always taken looking for 
immediate anaphylactic responses. However, no 
effective surveillance has ever been done about 
the possibility of worsening of the 
immunoreactivity after further immunogenic 
stimulus, or ever the risk presented by the 
parenteral administration of a food allergen 
protein to a non-yet-sensitized individual and the 
risk of developing further hypersensitization. 
 
Laboratory allergy diagnosis to hen's egg white 
proteins is yet far from ideal, and continuous 
research is being done to improve the laboratory 
tools by developing new techniques [51].  
 
LAIT and TTP performed with egg proteins seem 
to be promising tools for evaluating cellular and 
humoral responses after immunization with egg-
propagated virus vaccines to evaluate the risk of 
development of eventual hypersensitivity 
reactions. 
 
The primary strategy of personalized medicine is 
to use endotype biomarkers for cellular and 
humoral immunoreactivity to personalize 
treatments for allergic patients [52]. The semi-
quantitative titration of precipitins is a pioneering 
laboratory exam upon which the fundamental 
bases of Immunology were constructed [53]. 
Precipitating antibodies suggest the presence of 
a humoral immune response against the tested 
antigens [54]. Before the discovery of IgE, 
researching precipitins against food allergens 
was the primary way to realize in vitro diagnosis 
of humoral immunoreactivity [55,56].            
 
The LAIT is an ex vivo challenge test performed 
with a viable leukocyte buffy coat that can 

theoretically explore most known immune 
pathways as it allows the interaction of all 
immune-circulating participants with the allergens 
[57]. Several immune pathways can produce the 
final leukocyte adherence inhibition [58-61]. 
 
The present study is a proof-of-concept that 
hypothesizes that LAIT and the TTP may 
endotype or differentiate diverse degrees of 
cellular and humoral immunoreactivity against 
hen's egg albumen among patients suffering 
from several non–IgE-mediated allergic 
phenotypes (urticaria, angioedema, atopic and 
contact dermatitis, allergic proctocolitis, 
eosinophilic esophagitis, food-protein induced 
enterocolitis, rhinitis, and/or bronchitis. The 
retrospective compilation of our data showed a 
large distribution of results when we ascertained 
the results of TTP and TIAL to explore humoral 
and cellular immunoreactivity against the 
albumen extract. These immunoassays provide 
evidence about cellular and humoral 
immunoreactivity distributed into an extensive 
spectral range that may suggest immune 
tolerance or hypersensitivity. 
 
This preliminary retrospective survey 
demonstrated extensive results from the TTP, 
and the ex vivo challenge test monitored by LAIT 
against hen's eggs albumen in two cohorts of 
patients with diverse non–IgE-mediated allergic 
phenotypes. TTP and LAIT are complementary 
triage tests used at our facilities to select 
worthwhile antigens to proceed with more 
laborious in vivo provocation tests when the 
specific IgE is undetectable. None of our patients 
presented an exclusive reaction to these 
allergens. When we perform these assays, we 
perform them within a battery of several other 
allergens to choose the more reactive ones to 
prioritize the in vivo provocation tests. Every 
patient was simultaneously evaluated for 
immunoreactivity against several chemical and 
biological allergens, demonstrating positive 
results for some of them. Our results suggest 
that some of our patients may impair their allergic 
symptoms by additional immunoreactivity against 
hen's egg albumen allergens. 

 

5. LIMITATIONS 
   

This study is a retrospective analysis of data 
collected over six years and ten months. There 
was no protocol research, and the subject's data 
was limited to the essentials available on our 
electronic sheets. Therefore, we could not 
establish a cross-comparison between positive 



 
 
 
 

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and negative controls to validate the results. The 
number of subjects is appropriate for a 
preliminary study; however, future studies must 
be more comprehensive. The lack of a research 
protocol implies the possibility of a bias produced 
by the point of view of the physician who 
indicated the exam based on a clinical suspicion 
led by anamnesis, physical examination, 
undetectable specific IgE results, and non-
reactive or inconclusive skin tests. The purpose 
of TTP and LAIT was not to discriminate reactive 
patients from healthy people. This was done with 
in vivo provocation tests. These assays aim to 
prioritize allergens to be evaluated by further in 
vivo provocation tests. The study lost the follow-
up of many of these patients, making it 
impossible to establish a relationship between 
the immunoassays' results and the patient's 
clinical outcome. Unfortunately, it was not 
possible to compare the two procedures with 
paired tests because they were taken from 
distinct groups of patients. The methodology of 
TTP and LAIT are standardized in our laboratory, 
and there is no significant operator-dependent 
variability every time they are submitted to quality 
control, however, as any laboratory assay, the 
results depends of the sample and the immune 
conditions of the patient at the moment of the 
collection, what explain possible variations of the 
results along different samples took at different 
moments of life, what justify the statistical 
analyses of a great group pf patients. 
 

6. CONCLUSION 
 

Our preliminary results show that the LAIT and 
TTP may differentiate diverse degrees of 
immunoreactivity against hen's egg albumen in 
patients clinically diagnosed with diverse non–
IgE-mediated allergic phenotypes 
(rhinoconjunctivitis, rhinosinusitis, bronchitis, 
atopic dermatitis, and urticaria). This 
methodology may be easily incorporated into 
specialized centers since the technologies to 
perform TIAL and TTP are relatively inexpensive 
and can be performed with minimum laboratory 
equipment. However, the technique depends on 
trained biomedical personnel performing 
artisanal and time-consuming laboratory 
procedures. As preliminary results, the 
propaedeutic meaning of these results and the 
possibility of interferents must be yet established 
[62]. More studies focused on the quality-by-
design approach with prospective larger double-
blind cohorts need to evaluate the potential 
contribution of LAIT and TTP for endotyping 
cellular and humoral immunoreactivity in patients 

suspected of hypersensitivity against hen's egg 
white allergens [63]. 

  

7. FUTURE DIRECTIONS AND 
RECOMMENDATIONS FOR CLINICAL 
PRACTICE 

 
The primary intended use of in vitro or ex vivo 
allergen challenge tests is to spare the patients 
from being submitted to exhaustive and 
dangerous in vivo challenge tests. Exploring the 
humoral and the cellular arms of immune 
systems, the TTP and TIAL alone or combined 
may represent, in the near future, a tool for 
allergists to construct an etiologic diagnosis from 
their patients, as well as determine the 
endotypes (mechanisms) of hypersensitivity, in 
order to choose more convenient and 
personalized therapies for them. Future studies 
with prospective planning and control groups will 
be necessary to avoid the characteristic biases of 
retrospective studies. 

 
DISCLAIMER (ARTIFICIAL INTELLIGENCE) 

 
Author(s) hereby declare that NO generative AI 
technologies such as Large Language Models 
(ChatGPT, COPILOT, etc.) and text-to-image 
generators have been used during the writing or 
editing of this manuscript.  

 
CONSENT  
 
As per international standards or university 
standards, patient(s) written consent has been 
collected and preserved by the author(s). 

 
ETHICAL APPROVAL 
 
As per international standards or university 
standards written ethical approval has been 
collected and preserved by the author(s). 

 
ACKNOWLEDGEMENTS 
 
The Instituto Alergoimuno de Americana funded 
this work. We want to thank the laboratory 
technicians Alessandra Vieira de Oliveira and 
Luciana Sacilotto Carvalho for their help with the 
exams.  

 
COMPETING INTERESTS 
 
Authors have declared that no competing 
interests exist. 



 
 
 
 

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