







































 

_____________________________________________________________________________________________________ 
 
*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. “Endotyping Cellular 
and Humoral Immunoreactivity Against Allium Spices and Sulfites Preservatives in Allergic Patients: A Retrospective Study”. 
Asian Journal of Immunology 7 (1):185-200. https://journalaji.com/index.php/AJI/article/view/143. 

 
 

Asian Journal of Immunology 
 
Volume 7, Issue 1, Page 185-200, 2024; Article no.AJI.125235 
 

 
 

 

 

Endotyping Cellular and Humoral 
Immunoreactivity against Allium 

Spices and Sulfites Preservatives in 
Allergic Patients: A Retrospective 

Study 
 

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. The author CEO is responsible for the 
conceptualization, data curation, formal analysis, literature review, and writing the original draft. 
Authors DGP, APMT, CSM, JLSS, and RPSL performed 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/v7i1143  
 

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/125235  

 

 
 

Received: 21/08/2024 
Accepted: 23/10/2024 
Published: 28/10/2024 

 

Original Research Article 

https://doi.org/10.9734/aji/2024/v7i1143
https://www.sdiarticle5.com/review-history/125235


 
 
 
 

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ABSTRACT 
 

Background: Spices from the Allium genus season food worldwide, and their widespread use may 
produce allergic reactions by contact or ingestion. However, the Non–IgE-mediated 
immunoreactivity against these allergens has not yet been adequately investigated. 
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 two 
Allium spices (garlic and onion) and a sulfite derivative in patients with Non–IgE-mediated allergic 
phenotypes. 
Methodology: Two cohorts of allergic patients suspected of Non–IgE-mediated hypersensitivity 
against Allium spices and sulfite derivatives were investigated with the help of TTP or LAIT, 
simultaneously against extracts of garlic, onion, and sodium bisulfite. The results of the semi-
quantitative serum TTP against 1 mg/mL garlic extract, 1 mg/mL onion extract, and 1 mg/mL 
sodium bisulfite were distributed in ranges through cascade distribution charts. The results of the 
Leukocyte Adherence Inhibition (LAI) percentage promoted by the ex vivo challenges against 1 
mg/mL garlic extract, 1 mg/mL onion extract, and 1 mg/mL sodium bisulfite were distributed in 
ranges through cascade distribution charts. The statistical differences inside these cohorts were 
calculated.  
Results: Paired t-test indicated a non-significant difference between garlic TTP and onion TTP (p-
value = 0.761); a non-significant difference between sulfites TTP and garlic TTP (p-value = 0.112); 
a non-significant difference between sulfites TTP and onion TTP (p-value = 0.058); a non-
significant difference between garlic TIAL and onion TIAL (p-value = 0.942); a significant difference 
between sulfites TIAL and garlic TIAL (p-value < 0.001) and a significant difference between 
sulfites TIAL and onion TIAL (p-value < 0.001). 
Conclusion: Results support that the TTP and LAIT performed with sodium bisulfite, garlic, and 
onion extracts may discriminate diverse degrees of humoral and cellular immunoreactivity in 
patients suffering from diversified allergic phenotypes. 

 

 
Keywords: Allium sativum, Allium cepa; endotype; garlic; hypersensitivity; leukocyte adherence 

inhibition test; Non–IgE-mediated immunoreactivity; onion; precipitins; sulfite derivatives. 
 

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

1. INTRODUCTION  
 
Allium is a genus of monocotyledonous plants, 
characterized by their odorous volatile sulfur-
containing compounds (most derived from 
cysteine sulfoxides) with more than eight 
hundred species, among which quite a few are 
edible and cultivated as lore for culinary 
purposes or therapeutic indications, such as 
several kinds of garlic (e.g., Allium sativum L.), 
onions (e.g., Allium cepa L.), chives (e.g.,           
Allium schoenoprasum) and leeks (e.g., Allium 
porrum L.), among others [1]. Allium spices              
are reported to have been used as food                    
and as medicines in the ancient histories of 
Egypt, China, Japan, India, Greece, and         
Rome [2].  
 

The first reported clinical case of allergy to an 
Allium species was described in 1950 by 
Edelstein, who treated a food-pack worker 
presenting occupational dermatitis after skin 
contact with garlic and "severe cardiospasm" 
immediately after ingestion [3]. Shortly after that 
(1952), the Canadian Medical Association 
published a report of a cook who was also 
diagnosed with an occupational allergy to onion 
and garlic [4]. Finally (1954), the "classic aspects 
of onion and garlic dermatitis in housewives" was 
published by the American College of Allergists 
[5]. Hypersensitivities to Allium spices have been 
reported to produce contact dermatitis, gingivitis, 
stomatitis, allergic rhinitis, allergic conjunctivitis, 
and asthma [6,7]. In Spain, the reported 
prevalence of documented hypersensitivity 
against garlic and/or onions in a cohort of allergic 
patients submitted to routine skin allergic tests 
was almost 3% [8]. In Saudi Arabia, from a 
cohort of 108 patients with clinical suspicion of 
food allergy, 15 (13.8%) had garlic and onion-
specific IgE antibodies in their sera [9]. 
 



 
 
 
 

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Skin tests with spices gradually became routine 
to diagnose cooks with hand dermatitis, and the 
early attempts to identify the sensitizer revealed 
that it was soluble in water, ethanol, and acetone 
[10]. In the last decade of the 20th century, 
doctors became progressively aware of other 
clinical manifestations that could be caused by 
hypersensitivity to components of the Allium 
genus: several cases of bronchial asthma, 
rhinitis, conjunctivitis, and dermatitis were 
attributed to onion allergy, demonstrated by 
bronchial provocation tests, nasal provocation 
tests, and skin tests with onion extracts that 
cross-reacted with garlic and leek extracts; and 
in some cases, detection of specific IgE against 
onion and garlic was possible by Phadezyme® 
and CAP System RAST FEIA® [11]. Allergic 
rhinitis elicited by Allium allergens was also 
demonstrated by anterior rhinomanometry [12]. 
Susceptible patients may also develop urticaria, 
angioedema, and anaphylactic reactions, 
especially to young garlic (an unripe and 
underdeveloped bulb) and raw onions [13,14]. An 
experience done with volunteers and patients 
with gastroesophageal reflux submitted to an 
esophageal pH probe who had ingested              
onions did not increase any reflux variables 
measured in asymptomatic volunteers. However, 
the ingestion of onions significantly increased 
reflux episodes in symptomatic subjects 
compared with asymptomatic subjects after 
onion ingestion and when compared with meals 
with no-onion [15]. 
 
Even before the reports of Allium allergic 
reactions, the sulfur compounds of                       
Allium species were already studied, particularly 
the diallyl disulfide, an organic molecule                   
with formula C6H10S2 [16]. Diallyl disulfide                     
is one of the principal components of the distilled 
oil of garlic and other Allium genus                       
plants, produced during the decomposition                
of allicin released after crushing [17,18]. 
Identified as an active component due to its 
larvicidal activity, the diallyl disulfide was                  
also suspected of producing allergic reactions 
and was employed to perform diagnostic               
allergic skin tests (with the help of a 1%             
solution) [19-21]. Non-IgE-mediated allergic 
contact dermatitis is one of the most              
common symptoms of Allium allergy, mainly                
in cooks who handle the spices, despite the               
use of gloves permeable to diallyl disulfide               
[22]. Occasionally, when used as a naturopathic 
remedy by topical application, garlic has                     
also been linked to allergic contact dermatitis 
[23].  

With the help of Immunoblotting, in 2001, Asero 
et al. identified IgE-binding to 15-kDa and 43-kDa 
onion proteins; after preabsorption of the 
patient's serum with peach Lipid Transfer Protein 
(LTP), the IgE-binding to the 15-KDa protein 
disappeared [24]. Later (2007), Enrico et al., by 
immunoblotting an onion extract with the serum 
of a patient with onion allergy and anti-IgE, 
revealed a 12-kDa IgE-binding protein band that 
was inhibited by onion extract and the peach 
allergen rPru p 3 (a marker allergen for LTP) 
[25,26]. With the help of mass spectrometry, a 
Taiwan group marked the IgE-binding of a 56-
kDa protein as a major allergen for patients with 
garlic allergy, identified as the alliin lyase 
(E.C.4.4.1.4), the enzyme responsible for the 
lysis of alliin into the biologically active allicin 
molecule upon crushing of a garlic clove [27]. 
Italian investigators also reported cellular 
responses, and they identified specific B cell 
proliferation in onion-allergic patients' plasma 
incubated with onion extracts (but not in healthy 
controls) [28]. Cellular techniques using cell 
sorting, EliSpot, flow cytometry, and confocal 
microscopy have recently boosted a crescent 
interest in studying the interaction of LTP 
allergens with immune cells, particularly with 
group 2 innate lymphoid antigen-presenting cells, 
promoting Th2 cell responses involved in allergy 
pathogenesis [29].  
 
Besides diallyl disulfide, Allium species also 
produce other organic sulfides such as diallyl 
trisulfide, diallyl sulfide, dipropyl disulfide, 
dipropyl trisulfide, 1-propenyl propyl disulfide, 
allyl methyl disulfide, and dimethyl disulfide [30]. 
In organic chemistry, "sulfide" or "thioether" 
refers to the linkage "C–S–C". In inorganic 
chemistry, "sulfides" are corrosive ionic salts 
composed of the anion S2−, while "sulfites" (or 
chiefly British "sulphites") are salts composed of 
the sulfur dioxide (SO2

− ) which is found in 
aqueous solution as free SO2, HSO3

− and/or 
SO3

2−. In biological fluids, the sulfite ions are 
usually combined with carbonyl compounds such 
as acetaldehydes, proteins, sugars, and others 
[31]. The solution's free sulfite ions associate and 
dissociate in equilibria with carbonyl compounds 
according to the solution's pH at diverse 
equilibrium constants (pKs) [32]. 
 
Allium species' characteristic flavor and 
pungency depend on the content of "sulfates" 
(the sulfur tetraoxide SO4

-2) in the soil the plant 
grows [33]. In Naples, it was reported a particular 
association of pizza makers' hand dermatitis with 
positive skin allergy tests done with diallyl 



 
 
 
 

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disulfide and ammonium persulfate, a flour-
strengthening salt with formula (NH4)2+(S2O8)2- 
[34].  
 
There seems to be some hypersensitivity cross-
reactivity among the organic sulfides in Allium 
spices and the inorganic sulfites used as 
antioxidants and preservatives in industrialized 
foods, cosmetics, and medicines [35]. Sulfites 
are the group of inorganic compounds elected by 
the American Contact Dermatitis Society as the 
"Allergen of the Year for 2024" [36]. 
 
Sulfites were anciently aggregated into the 
human diet employing Saccharomyces 
cerevisiae fermentation, which reduces sulfate 
into sulfites and sulfides [37,38].  
 
Nowadays, sulfites are almost universally added 
to industrialized foods and beverages to control 
non-enzymic browning, enzymic browning, 
oxidation, and microorganisms growing [39]. 
Several sulfite salts are qualified to be added to 
industrial food, such as sulfur dioxide (E 220; 
SO2), sodium sulfite (E 221; Na2SO3), sodium 
bisulfite (E 222; NaHSO3), sodium metabisulfite 
(E 223; Na2S2O5), potassium metabisulfite (E 
224; K2S2O5), calcium sulfite (E 226; CaSO3), 
calcium bisulfite [E 227; Ca(HSO3)2] and 
potassium bisulfite (E 228; KHSO3) [40]. 
 
Since the seventies, sulfur dioxide has been 
known as an atmospheric pollutant associated 
with the rapid development of laryngeal and 
bronchial spasms, excessive bronchial 
secretions, and pulmonary edema in sensitive 
patients, either inhaled or ingested [41]. 
Anaphylaxis after ingestion of sodium bisulfite 
was also reported [42]. Besides respiratory 
symptoms and anaphylaxis, sulfite 
hypersensitivities are associated with nausea, 
stomach cramps, diarrhea, urticaria, 
angioedema, dermatitis, diaphoresis, tingling 
sensations, flushing, and loss of consciousness 
[43]. Sulfites are associated with headaches in 
sensitive individuals [44]. Sulfites are among the 
main suspects of allergies associated with 
alcoholic beverages [45]. Several prescription 
medicines incorporate sulfites as antioxidants, 
such as topical ophthalmic medications; inhaled 
bronchodilators (salbutamol, racepinephrine); 
beta-adrenoceptor agonists (isoprenaline, 
epinephrine); local anesthetics; injectable 
corticosteroids; injectable antibiotics; injectable 
antiarrhythmics; injectable analgesics; antishock 
agents, including Aramine®, Intropin® and 

Levophed®; and solutions for parenteral nutrition 
and dialysis [46-52]. 
 
The resources to diagnose sulfite hypersensitivity 
are in vivo and ex vivo provocation tests [53]. We 
routinely employ the Leukocyte Adherence 
Inhibition Test (LAIT) and the Tube Titration of 
Precipitins (TTP) in our facilities as a triage to 
evaluate Non-IgE-mediated immunoreactivity 
against suspected allergens before the 
performance of more exhaustive in vivo 
provocation tests [54-60]. To evaluate the 
potential of the LAIT and TTP to endotyping 
Non–IgE-mediated cellular and humoral 
immunoreactivity against garlic, onion, and 
sodium bisulfite, we retrospectively compiled the 
electronic medical charts of patients with Non–
IgE-mediated allergic rhinitis, allergic pharyngitis, 
allergic laryngitis, allergic bronchitis, allergic 
sinusitis, allergic migraine, atopic dermatitis, 
and/or urticaria who were investigated 
simultaneously for immunoreactivity against 
these three allergens by one of these assays.  
 
The present study is a proof of concept that 
hypothesizes that the LAIT and the TTP may 
differentiate diverse endotypes and degrees of 
immunoreactivity against Allium species and 
sodium bisulfite among patients suffering from 
common allergic phenotypes. As the tests were 
performed simultaneously with the same venous 
sample with the three allergens, it was possible 
to calculate a paired t-test to discriminate cross-
reactivity between them.  
 

2. MATERIALS AND METHODS  
 

2.1 Subjects 
 

After receiving Institutional Review Board 
approval from the Instituto Alergoimuno de 
Americana (Brazil; 07/2024), we reviewed the 
electronic chart of 9,450 outpatients who 
attended our facility from January 2018 to 
September 2024.  
 
A cohort of 100 consecutive outside patients 
(TTP cohort) had been simultaneously submitted 
to TTP with garlic extract, onion extract, and 
sodium bisulfite for presenting Non–IgE-
mediated allergic rhinitis, allergic pharyngitis, 
allergic laryngitis, allergic bronchitis, allergic 
sinusitis, allergic migraine, atopic dermatitis, 
and/or urticaria. This cohort counted 26 males; 
mean age 39.7 years; SD 16.9 years; range 5 to 
79 years; median 39.5 years; modes = 43 



 
 
 
 

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(appeared seven times); geometric mean = 35.2 
years.  
 
A cohort of 100 consecutive outside patients 
(LAIT cohort) had been simultaneously submitted 
to TIAL with garlic extract, onion extract, and 
sodium bisulfite for presenting Non–IgE-
mediated allergic rhinitis, allergic pharyngitis, 
allergic laryngitis, allergic bronchitis, allergic 
sinusitis, allergic migraine, atopic dermatitis, 
and/or urticaria. This cohort counted 33 males; 
mean age 42.9 years; SD 21.3 years; range 2 to 
91 years; median 43 years; modes = 43 and 48 
years (each appeared four times); geometric 
mean = 35.1 years.  
 
This study did not include patients under 
biological and/or systemic anti-inflammatory 
therapy. These procedures were offered to 
patients with clinical suspicion of Allium spices 
hypersensitivity who demonstrated a non-
reactive or inconclusive skin test against sodium 
bisulfite, garlic, and onion extracts [61]. 
 

2.2 Garlic Extract 
 
The peeled garlic was crushed, homogenized, 
and then left for 48 hours in Coca's solution at 
4°C for protein extraction before centrifugation 
and separation of the water-soluble fraction from 
solid particles and oily fraction [62]. The protein 
quantification of the allergen extracts was done 
according to Bradford's protein-dye binding 
methodology [63]. 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 garlic extract solution was used 
to perform the allergic skin tests, TTP, and LAIT. 
All relevant and mandatory laboratory health and 
safety measures have been complied with during 
the experiments.  
 

2.3 Onion Extract  
 
The onion extract solution was prepared using a 
similar technique employed for the garlic extract. 
 

2.4 Sodium Bisulfite Solution 
 
The sodium bisulfite powder (IUPAC ID: Sodium 
hydrogen sulfite; formula NaHSO3) was acquired 
from Bianquimica™ and diluted with distilled 
water at 1 mg/mL to perform the allergic skin 
tests, TTP, and LAIT.  

2.5 LAIT: Ex vivo Investigation: Leukocyte 
Adherence Inhibition Test 

 
2.5.1 LAIT: Procedure for allergen ex vivo 

challenging  
 
We performed the LAIT as previously described 
[64-73]. Shortly, each donor's fresh plasma was 
divided into two parts and used in paralleled ex 
vivo challenging tests with the Allium or onion 
extracts and the unchallenged plasma (added 
with antigen dilution solution as a control). We 
collected the 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). 
 
2.5.2 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 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.  
 
2.5.3 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. 



 
 
 
 

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2.6 TTP: In vitro Investigation: Tube 
Titration of Precipitins 

 
As previously reported, the semi-quantitative 
TTP against the aluminum solution was 
performed in a transparent vitreous tube array 
[74-76]. 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 antigen (1 mg/mL) 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 [77]. 
 

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 TTP for the garlic extract showed a 
distribution concentrated on the higher dilutions 

(Fig. 1). There was no negative result. The mean 
was estimated at 1:247; the median was 1:128; 
the standard deviation was estimated at 1:180; 
the mode was 1:512 (appeared nine times).  
 
The TTP for the onion extract showed a 
distribution concentrated on the higher dilutions 
(Fig. 2). There was no negative result. The mean 
was estimated at 1:254; the median was 1:256; 
the standard deviation was estimated at 1:178; 
the mode was 1:512 (appeared 29 times).  
 
The TTP for the sodium bisulfite solution showed 
a distribution concentrated on the higher dilutions 
(Fig. 3). There was no negative result. The mean 
was estimated at 1:207; the median was 1:158; 
the standard deviation was estimated at 1:176; 
the mode was 1:128 (appeared 29 times).  
 
The LAIT for the garlic extract showed a wide 
distribution range of results. Most results were 
concentrated in the more immunoreactive 
groups. There were five negative results. The LAI 
ranged from 0% to 89%. The mean was 51.3%; 
the median was 57.5%; the standard deviation 
was 24.0%; the mode was 0% (appeared five 
times). The cascade distribution demonstrates a 
wide range of LAI results (Fig. 4). 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 garlic allergens in a 
Non–IgE-mediated hypersensitivity condition in 
these patients. 

 

 
 
Fig. 1. Cascade distribution chart of the Tube Titration of Precipitins (x-axis %) resulting from 

the garlic extract against the serum of the TTP cohort of 100 tests/subjects (y-axis) 



 
 
 
 

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Fig. 2. Cascade distribution chart of the Tube Titration of Precipitins (x-axis %) resulting from 

the onion extract against the serum of the TTP cohort of 100 tests/subjects (y-axis) 
 

 
 

Fig. 3. Cascade distribution chart of the Tube Titration of Precipitins (x-axis %) resulting from 
the sodium bisulfite extract against the serum of the TTP cohort of 100 tests/subjects (y-axis) 

 

The LAIT for onion extract showed a wide 
distribution range of results. Most results were 
concentrated in the more immunoreactive 
groups. There were three negative results. The 
LAI ranged from 0% to 89%. The mean was 
52%; the median was 51.2%; the standard 
deviation was 24.9%; the mode was 62% 
(appeared seven times). The cascade 
distribution demonstrates a wide range of LAI 
results (Fig. 5). 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 onion allergens in a Non–IgE-
mediated hypersensitivity condition. 

The LAIT for sodium bisulfite showed a wide 
distribution range of results. Most results were 
concentrated in the less immunoreactive groups. 
There were 27 negative results. The LAI ranged 
from 0% to 88%. The mean was 27.5%; the 
median was 25.5%; the standard deviation was 
25.1%; the mode was 0% (appeared 27 times). 
The cascade distribution demonstrates a wide 
range of LAI results (Fig. 6). Most patients 
showed low or moderate immunoreactivity during 
the ex vivo challenge test. At the same time, a 
few displayed strong immunoreactivity, which 
could reflect the participation of sulfites in a Non–
IgE-mediated hypersensitivity condition in these 
patients. 



 
 
 
 

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The paired t-test indicated a non-significant, 
minimal difference between garlic TTP and onion 
TTP; p-value = 0.761. 
 
The paired t-test indicated a non-significant, 
minimal difference between sulfites TTP and 
garlic TTP; p-value = 0.112. 
 
The paired t-test indicated a non-significant, 
minimal difference between sulfites TTP and 
onion TTP; p-value = 0.058. 
 

The paired t-test results indicated a non-
significant minimal difference between garlic 
TIAL and onion TIAL; p-value = 0.942. 
 
The paired t-test indicated a significantly large 
difference between sulfites TIAL and garlic 
TIAL; p-value < 0.001. 
 
The paired t-test results indicated a significantly 
large difference between sulfites TIAL and onion 
TIAL; p-value < 0.001. 

 
 

Fig. 4. Cascade distribution chart of the range groups of Leukocyte Adherence Inhibition (LAI) 
results (x-axis %) of the ex vivo challenge against garlic 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) 
 

 
 
Fig. 5. Cascade distribution chart of the range groups of Leukocyte Adherence Inhibition (LAI) 

results (x-axis %) of ex vivo challenge against onion 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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193 

 

 
 
Fig. 6. Cascade distribution chart of the range groups of Leukocyte Adherence Inhibition (LAI) 

results (x-axis %) of ex vivo challenge with sodium bisulfite solution 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) 
  

4. DISCUSSION 
 
Soon after the discovery of penicillin (along with 
the antibiogram) in 1928, the empirical 
bactericidal properties of Allium spices were 
demonstrated to be "penicillin-like" and proved 
effective against Gram-positive bacteria, first in 
animals (1931) and later in humans (1945) 
[78,79]. Allium extracts also proved effective 
against fungi, especially their organosulfur 
derivatives [80,81].  
 
The early biochemical studies performed with the 
Allium species were motivated by the alliinase 
enzymatic ability to hydrolyze S-alkyl-substituted 
cysteine sulfoxide derivatives to the 
corresponding alkyl alkane thiosulfinates, 
ammonia, and pyruvic acid [82]. Allium species 
are a natural source of bioactive compounds, 
including more than 180 active secondary 
metabolites [83]. Several Allium bioactive 
compounds have been studied for their 
supposed anticancer, anti-diabetic, anti-
inflammatory, antimicrobial, immunomodulatory, 
and cardioprotective properties [84]. Allium 
spices were studied in several murine models of 
inflammatory diseases with promisor results [85]. 
Murine experiments demonstrated that onion 
bulb extract inhibited the house dust mite-
induced phosphorylation of EGFR, ERK1/2, and 
AKT pathways, inhibiting airway cellular influx, 
perivascular and peribronchial inflammation, 

goblet cell hyper/metaplasia, and ex vivo 
eosinophil chemotaxis [86,87]. 
 
Consequently, it is natural to hypothesize that 
such intense biological activity is probably 
associated with a great potential to promote 
immunoreactivity and/or hypersensitization, 
interfering with metabolic pathways, being 
proposed even for treating allergic diseases [88]. 
Therefore, when treating a patient with the 
Precision Medicine approach, diagnosing or 
endotyping an eventual allergy, hypersensitivity, 
or immunoreactivity against any naturopathic 
therapeutics or nutraceutical food, elective for 
long-term treatments is remarkably advisable for 
the prescription of eviction measures and 
tolerogenic strategies aimed to mitigate diseases 
symptoms phenotypes [89]. The four classical 
hypersensitivity mechanisms described by Gell 
and Coombs have been amplified to seven types 
with several subtypes, increasing the complexity 
of the required investigation of the 
hypersensitivity mechanisms responsible for the 
disease phenotypes [90,91]. Endotyping the 
hypersensitivity mechanisms against sulfites may 
also help distinguish superimposable phenotypes 
produced by common pathways such as allergic 
migraines and sinusitis [92].  
 
The retrospective compilation of our data showed 
a large distribution of results when we 
ascertained the results of TTP and TIAL to 



 
 
 
 

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explore humoral and cellular immunoreactivity 
against two Allium spices and a sulfite derivative. 
These immunoassays do not identify the exact 
mechanisms responsible for the clinical 
condition. Instead, they provide clues about 
sensitization and immunoreactivity distributed 
into an extensive spectral range between 
immune tolerance and symptomatic 
hypersensitivity. 
 
The semi-quantitative research and titration of 
precipitins is an essential laboratory exam upon 
which the fundamentals of Immunology were 
constructed [93]. Precipitating antibodies suggest 
a remarkable immune humoral response against 
the tested antigens [94]. 
 
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 
allergen [95]. The LAIT did not indicate which 
pathways produce the resulting final 
phenomenon (leukocyte adherence inhibition) 
[96-99].  
 
As indicators of a previous immune response, 
LAIT and TTP are suitable techniques to  
quantify an exposome measurement and               
their correspondent immune response, as 
proposed by the exposome-wide association 
study [100]. 
 
The comparative results obtained from the TIAL 
cohort demonstrated a more significative 
immunoreactivity from the tests performed with 
garlic extract and onion extract than the results 
obtained with the sodium bisulfite. This finding 
states that sulfites are not the only allergen 
responsible for cellular immunoreactivity against 
the Allium species. On the contrary, several 
protein allergens in Allium spices play a critical 
role in immunoreactivity against these species. 
This means that if a patient presents cellular 
immunoreactivity against Allium species, he/she 
will not necessarily present immunoreactivity 
against sulfites; however, reciprocation is not 
probable. 
 
This preliminary retrospective survey 
demonstrated extensive results from the TTP 
and the ex vivo challenge test monitored by LAIT 
against two Allium species in two cohorts of 
patients with various allergic symptoms. 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. Every patient was 
simultaneously tested with several chemical            
and biological allergens, demonstrating            
positive results for some of them. Our results 
suggest that reactive allergic patients may  
impair their symptoms by an additional 
immunoreactivity against Allium spices and 
sulfite derivatives. 
 

5. LIMITATIONS 
   
This study is a retrospective analysis of data 
collected over six years and nine months. There 
was no protocol research, and the subject's data 
were limited to the essentials available on our 
electronic sheets. Therefore, we could not 
establish a cross-comparison between positive 
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 (CEO) based 
on a clinical suspicion led purely by the 
anamnesis and physical examination. The study 
lost many of these patients to follow-up, so 
assuring the relationship between the 
immunoassays' results and the patient's clinical 
outcome is not possible yet. Unfortunately, it was 
impossible to compare the two procedures 
because they were taken from different groups of 
patients. 
 

6. CONCLUSION 
 
Our preliminary results show that the LAIT and 
TTP may differentiate diverse degrees of 
immunoreactivity against Allium spices in 
patients clinically diagnosed with Non–IgE-
mediated allergies. This methodology may be 
easily incorporated into specialized centers since 
the technologies to perform TIAL and TTP are 
inexpensive and can be performed with minimum 
laboratory equipment. However, the technique 
depends on trained biomedical personnel 
performing artisanal laboratory procedures. As 
preliminary results, the propaedeutic meaning of 
these results and the possibility of interferents 
must be yet established [101]. 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 immunoreactivity of patients 



 
 
 
 

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suspected of symptomatic hypersensitivity 
against Allium spices and sulfite derivatives 
[102]. 
  

CONSENT 
 
As a retrospective survey of results recorded                 
in cognito, consent was given collectively by              
the institution's ethics committee following              
the principles of the Declaration of Helsinki  
[103]. 
 

ETHICAL APPROVALS 
 
The authors have collected and preserved 
written ethical approval as per international 
standards. 
 

DISCLAIMER (ARTIFICIAL INTELLIGENCE) 
 
Authors hereby declare that NO generative             
AI technologies such as Large Language  
Models (such as ChatGPT, COPILOT, and 
similar) and text-to-image generators have         
been used during the writing or editing of 
manuscripts.  
 

ACKNOWLEDGEMENTS 
 
The Instituto Alergoimuno de Americana funded 
this work. We want to thank our new laboratory 
technician, Alessandra Vieira de Oliveira, for 
participating in the exams.  

 
COMPETING INTERESTS 
 
Authors have declared that no competing 
interests exist. 

 
REFERENCES 
 
1. Block E. Garlic and other Alliums: The Lore 

and the Science. The Royal Society of 
Chemistry; 2009. 

2. Rivlin RS. Historical perspective on the use 
of garlic. J Nutr. 2001;131(3s):951s-4s. 

3. Edelstein AJ. Dermatitis caused by garlic. 
Arch Dermatol Syphilol. 1950;61(1):111. 

4. Burgess JF. Occupational dermatitis due to 
onion and garlic. Can Med Assoc J. 
1952;66(3):275. 

5. Burks JW. Classic aspects of onion and 
garlic dermatitis in housewives. Ann 
Allergy. 1954;12(5):592-6. 

6. Cantisani C, Visconti B, Paolino G, 
Frascani F, Tofani S, Fazia G, et al. 

Unusual food allergy: Alioidea allergic 
reactions overview. Recent Patents on 
Inflamm & Allergy Drug Discovery. 
2014;8(3):178-184. 

7. Cantisani C, Mattozzi C, Richetta AG, 
Melis L, Frascani F, Calvieri S. Aliolidea 
allergic contact stomatitis. Eur J Dermatol. 
2011;21(6):991-2. 

8. Armentia A, Martín-Armentia S, Pineda F, 
Martín-Armentia B, Castro M, Fernández 
S, et al. Allergic hypersensitivity to garlic 
and onion in children and adults. Allergol 
Immunopathol (Madr). 2020;48(3):232-
236. 

9. Almogren A, Shakoor Z, Adam MH. Garlic 
and onion sensitization among Saudi 
patients screened for food allergy: A 
hospital based study. African Health 
Sciences. 2013;13(3):689-93. 

10. Bleumink E, Doeglas HM, Klokke AH, 
Nater JP. Allergic contact dermatitis to 
garlic. Br J Dermatol. 1972;87(1):6-9. 

11. Valdivieso R, Subiza J, Varela-Losada S, 
Subiza JL, Narganes MJ, Martinez-Cocera 
C, et al. Bronchial asthma, 
rhinoconjunctivitis, and contact dermatitis 
caused by onion. J Allergy Clin Immunol. 
1994;94(5):928-30. 

12. Jiménez-Timon A, Rodríguez-Trabado A, 
Hernández-Arbeiza FJ, Porcel-Carreño, S, 
Rodríguez- Martín E, Agustín-Herrero, J, 
et al. Rinomanometría anterior como teste 
diagnóstico en alergia ocupacional por 
liliáceas. Allergol Immunopathol (Madr). 
2002;30(5):295-299. 

13. Pérez-Pimiento AJ, Moneo I, Santaolalla 
M, de-Paz S, Fernández-Parra B, 
Domínguez-Lázaro AR. Anaphylactic 
reaction to young garlic. Allergy. 
1999;54(6):626-9. 

14. Arena A, Cislaghi C, Falagiani P. 
Anaphylactic reaction to the ingestion of 
raw onion. A case report. Allergol 
Immunopathol (Madr). 2000;28(5):287-9. 

15. Allen ML, Mellow MH, Robinson MG, Orr 
WC. The effect of raw onions on acid 
reflux and reflux symptoms. Am J 
Gastroenterol. 1990;85(4):377-80. 

16. National Center for Biotechnology. 
Information on Diallyl disulfide. Retrieved 
June 16, 2024. 

Available:https://pubchem.ncbi.nlm.nih.gov
/compound/Diallyl-disulfide 

17. Challenger F, Greenwood D. Sulphur 
compounds of Allium; detection of n-
propylthiol in the onion; the fission and 



 
 
 
 

Olivier et al.; Asian J. Immunol., vol. 7, no. 1, pp. 185-200, 2024; Article no.AJI.125235 
 
 

 
196 

 

methylation of diallyl disulphide in cultures 
of Scopulariopsis brevicaulis. Biochem J. 
1948;43(1):ix. 

18. Challenger F, Greenwood D. Sulphur 
compounds of the genus Allium; detection 
of n-prophylthiol in the onion; the fission 
and methylation of diallyl disulphide in 
cultures of Scopulariopsis brevicaulis. 
Biochem J. 1949;44(1):87-91. 

19. Amonkar SV, Banerji A. Isolation                   
and characterization of larvicidal principle 
of garlic. Science. 1971;174(4016):1343- 
4. 

20. Ekeowa-Anderson AL, Shergill B, 
Goldsmith P. Allergic contact cheilitis to 
garlic. Contact Dermatitis. 2007;56(3):174-
5. 

21. Hubbard VG, Goldsmith P. Garlic-fingered 
chefs. Contact Dermatitis. 2005;52(3):165-
6. 

22. Moyle M, Frowen K, Nixon R. Use of 
gloves in protection from diallyl disulphide 
allergy. Australas J Dermatol. 2004; 
45(4):223-225. 

23. Esfahani A, Chamlin SL. Garlic Dermatitis 
on the Neck of an Infant Treated for Nasal 
Congestion. Pediatr Dermatol. 2017;34(4): 
e212-e213. 

24. Asero R, Mistrello G, Roncarolo D, Amato 
S. A case of onion allergy. J Allergy Clin 
Immunol. 2001;108(2):309-310. 

25. Enrique E, Malek T, De Mateo JA, et al. 
Involvement of lipid transfer protein in 
onion allergy. Ann Allergy Asthma 
Immunol. 2007;98(2):202. 

26. Mothes-Luksch N, Raith M, Stingl G, 
Focke-Tejkl M, Razzazi-Fazeli E, 
Zieglmayer R. et al. Pru p 3, a marker 
allergen for lipid transfer protein 
sensitization also in Central Europe. 
Allergy. 2017;72(9):1415-1418. 

27. Kao SH, Hsu CH, Su SN, Hor WT, Chang 
TW, Chow LP. Identification and 
immunologic characterization of an 
allergen, alliin lyase, from garlic (Allium 
sativum). J Allergy Clin Immunol. 2004; 
113(1):161-168. 

28. Albanesi M, Pasculli C, Giliberti L, et al. 
Immunological characterization of onion 
(Allium cepa) allergy. Postepy 
Dermatologii i Alergologii. 2019;36(1):98-
103. 

29. Palomares F, Pérez-Sánchez N, Nieto N, 
et al. Group 2 innate lymphoid cells are 
key in lipid transfer protein allergy 

pathogenesis. Front Immunol. 
2024;15:1385101. 

30. Bastaki SMA, Ojha S, Kalasz H, Adeghate 
E. Chemical constituents and medicinal 
properties of Allium species. Mol Cell 
Biochem. 2021;476(12):4301-4321. 

31. Jiang X. Sulfur Chemistry. Springer 
International Publishing; 2019. 

32. Burroughs LF, Sparks AH. Sulphite-
binding power of wines and ciders. I. 
Equilibrium constants for the dissociation 
of carbonyl bisulphite compounds. J Sci 
Food Agric. 1973;24(2):187-198. 

33. Behairy AG, Mahmoud AR, Shafeek MR, 
Aisha H. Ali AH, MM H. Growth, yield and 
bulb quality of Onion plants (Allium cepa 
L.) as affected by foliar and soil application 
of potassium. Middle East Journal of 
Agriculture Research. 2015;4(1):60-66. 

34. Lembo S, Lembo C, Patruno C, Balato A, 
Balato N, Ayala F. Pizza makers' contact 
dermatitis. Dermatitis: contact, atopic, 
occupational, drug. 2014;25(4):191-194. 

35. Schlarbaum JP, Kimyon RS, Liou YL, 
Voller LM, Seyfer SJ, Warshaw EM. Hold 
the spice: Allergy to garlic and sulfites-
Possible relevance in a patient with 
cheilitis granulomatosa. Contact 
Dermatitis. 2019;81(5):397-398. 

36. Ekstein SF, Warshaw EM. Sulfites: 
Allergen of the Year 2024. Dermatitis. 
2023;35(1):6-12. 

37. Lester MR. Sulfite sensitivity: significance 
in human health. J Am Coll Nutr. 1995; 
14(3):229-232. 

38. De Guidi I, Serre C, Noble J, Ortiz-Julien 
A, Blondin B, Legras JL. QTL mapping 
reveals novel genes and mechanisms 
underlying variations in H2S production 
during alcoholic fermentation in 
Saccharomyces cerevisiae. FEMS Yeast 
Res. 2024;24:foad050.  

DOI: 10.1093/femsyr/foad050 

39. Roberts AC, McWeeny DJ. The uses                 
of sulphur dioxide in the food industry.                 
Int J Food Sci Technol. 1972;7(3):221- 
238. 

40. EFSA Panel on Food Additives. Nutrient 
Sources added to F. Scientific Opinion            
on the re-evaluation of sulfur dioxide                
(E 220), sodium sulfite (E 221), sodium 
bisulfite (E 222), sodium metabisulfite                
(E 223), potassium metabisulfite (E 224), 
calcium sulfite (E 226), calcium bisulfite           
(E 227) and potassium bisulfite (E 228) as 



 
 
 
 

Olivier et al.; Asian J. Immunol., vol. 7, no. 1, pp. 185-200, 2024; Article no.AJI.125235 
 
 

 
197 

 

food additives. EFSA Journal. 2016;14(4): 
4438. 

41. Kochen J. Sulfur dioxide, a respiratory 
tract irritant, even if ingested. Pediatrics. 
1973;52(1):145-146. 

42. Prenner BM, Stevens JJ. Anaphylaxis after 
ingestion of sodium bisulfite. Ann Allergy. 
1976;37(3):180-182. 

43. Gunnison AF, Jacobsen DW. Sulfite 
hypersensitivity. A critical review. CRC 
critical reviews in toxicology. 1987; 
17(3):185-214. 

44. Silva M, Gama J, Pinto N, et al. Sulfite 
concentration and the occurrence of 
headache in young adults: a prospective 
study. Eur J Clin Nutr. 2019;73(9):1316-
1322. 

45. Hinton AN, Goldminz AM. Alcohol-Related 
Dermatitis: A Review. Dermatitis: contact, 
atopic, occupational, drug. 2020;31(3):185-
190. 

46. Shaver RL, Warshaw EM. Contact 
allergens in prescription topical ophthalmic 
medications. Dermatitis: Contact, Atopic, 
Occupational, Drug. 2022;33(2):135-143. 

47. Yang WH, Purchase EC. Adverse 
reactions to sulfites. Cmaj. 1985;133(9): 
865-867, 880. 

48. Twarog FJ, Leung DY. Anaphylaxis to a 
component of isoetharine (sodium 
bisulfite). Jama. 1982;248(16):2030-2031. 

49. Koepke JW, Selner JC, Dunhill AL. 
Presence of sulfur dioxide in commonly 
used bronchodilator solutions. J Allergy 
Clin Immunol. 1983;72(5 Pt 1):504-508. 

50. Simon RA. Adverse reactions to drug 
additives. J Allergy Clin Immunol. 1984; 
74(4 Pt 2):623-630. 

51. Huang AS, Fraser WM. Are sulfite 
additives really safe? N Engl J Med. 1984; 
311(8):542. 

52. Schwartz HJ, Sher TH. Bisulfite sensitivity 
manifesting as allergy to local dental 
anesthesia. J Allergy Clin Immunol. 1985; 
75(4):525-527. 

53. Bush RK, Zoratti E, Taylor SL. Diagnosis 
of sulfite and aspirin sensitivity. Clin Rev 
Allergy. 1990;8(2-3):159-178. 

54. Kuratsuji T. Studies on leukocyte 
adherence inhibition test. Part II. Clinical 
applications of LAI test to detect delayed 
type hypersensitivity in infants and 
children. Keio J Med. 1981;30(2):65-69. 

55. Olivier CE, Pinto DG, Teixeira APM, 
Santana JLS, Santos RAPGS, Lima RPS, 

et al. Evaluating Non-IgE-mediated 
Allergens' Immunoreactivity in Patients 
with "Intrinsic" Persistent Rhinitis with Help 
of the Leukocyte Adherence Inhibition 
Test. Eur J Med Health Sci. 2023;5(1):17-
22. 

56. Olivier CE, Pinto DG, Teixeira APM, 
Santana JLS, Santos RAPGS, Lima RPS, 
et al. Evaluating Non-IgE-Mediated 
Allergens' Immunoreactivity in Patients 
Formerly Classified as "Intrinsic" 
Asthmatics with Help of the Leukocyte 
Adherence Inhibition Test. Eur J Clin Med. 
2023;4(2):1-7 

57. Olivier CE, Pinto DG, Teixeira APM, 
Santana JLS, Santos RAPGS, Lima RPS, 
et al. Contribution of the Leukocyte 
Adherence Inhibition Test to the Diagnosis 
of Innate Non–IgE-mediated 
Immunoreactivity against Alternaria 
alternata. Asian J Immunol 2023;6(1):243-
251. 

58. Olivier CE, Pinto DG, Teixeira APM, 
Santana JLS, Santos RAPG, Lima RPS. 
Contribution of the Leukocyte Adherence 
Inhibition Test to the Diagnosis of Innate 
Non–IgE-mediated Immunoreactivity 
against Saccharomyces cerevisiae. Asian 
J Immunol. 2023;6(1):234-241. 

59. Olivier CE, Pinto DG, Teixeira APM, 
Santana JLS, Santos RAPGS, Lima RPS, 
et al. Contribution of the Leukocyte 
Adherence Inhibition Test to the Diagnosis 
of Non–IgE-mediated Immunoreactivity 
against Candida albicans in Patients with 
Atopic Dermatitis. Asian J Immunol. 
2023;6(1):268-276. 

60. Olivier CE, Pinto DG, Teixeira APM, 
Santana JLS, Santos RAPGS, Lima RPS, 
et al. Contribution of the leukocyte 
adherence inhibition test in diagnosing 
Non–IgE-Mediated immunoreactivity 
against Aspergillus fumigatus in patients 
with allergic rhinitis and asthma. Asian J 
Immunol. 2024;7(1):12-20. 

61. Olivier CE, Argentão DGP, Santos RAPG, 
Silva MD, Lima RPS, Zollner RL. Skin 
scrape test: An inexpensive and painless 
skin test for recognition of immediate 
hypersensitivity in children and adults. The 
Open Allergy Journal. 2013;6:9-17. 

62. Coca AF. Studies in Specific 
Hypersensitiveness V. The preparation of 
fluid extracts and solutions for use in the 
diagnosis and treatment of the allergies 
with notes on the collection of pollens. The 



 
 
 
 

Olivier et al.; Asian J. Immunol., vol. 7, no. 1, pp. 185-200, 2024; Article no.AJI.125235 
 
 

 
198 

 

Journal of Immunology. 1922;7(2):163-
178. 

63. Bradford MM. A rapid and sensitive 
method for the quantitation of microgram 
quantities of protein utilizing the principle 
of protein-dye binding. Anal Biochem. 
1976;72:248-254. 

64. Olivier CE, Lima RPS, Pinto DG, Santos 
RAPG, Silva GKM, Lorena SLS, et al. In 
search of a tolerance-induction strategy for 
cow's milk allergies: Significant reduction 
of beta-lactoglobulin allergenicity via 
transglutaminase/cysteine polymerization. 
Clinics. 2012;67(10):1171-1179. 

65. Olivier CE, Santos RAPG, Lima RPS, 
Argentão DGP, Silva GKM, Silva MD. A 
novel utility for an old method: The 
leukocyte adherence inhibition test is an 
easy way to detect the immunoreactive 
interference of the collection tube 
anticoagulant on cellular immunoassays. 
Journal of Cell Adhesion. 2014;Article ID 
860427(http://dx.doi.org/10.1155/2014/860
427):1-6. 

66. Olivier CE, Pinto DG, Lima RPS, Silva MD, 
Santos RAPG, Teixeira APM, et al. 
Assessment of immunoreactivity against 
therapeutic options employing the 
leukocyte adherence inhibition test as a 
tool for precision medicine. Eur J Clin Med. 
2021;2(3):40-45. 

67. Olivier CE, Pinto DG, Santos RAPG, Lima 
RPS. Dextran's interference over the 
leukocyte adherence inhibition test. 
Academia Letter. 2021; Article (number): 
3792. 

68. Olivier CE, Pinto DG, Teixeira APM, 
Santana JLS, Santos RAPGS, Lima          
RPS. Immunoreactivity against 
Dermatophagoides pteronyssinus 
Assessed by the Leukocyte Adherence 
Inhibition Test in Patients with Intrinsic 
Atopic Dermatitis and Correlated "Intrinsic" 
Non–IgE-mediated Allergic Conditions. Eur 
J Clin Med. 2021;2(6):45-50. 

69. Olivier CE, Pinto DG, Teixeira APM, 
Santana JLS, Santos RAPGS, Lima RPS. 
Contribution of the leukocyte adherence 
inhibition test to the evaluation of cellular 
immunoreactivity against latex extracts for 
Non—IgE-Mediated latex-fruit-pollen 
syndrome in allergic candidates to 
exclusion diets and allergic 
desensitization. Eur J Clin Med. 2022; 
3(1):11-17. 

70. Olivier CE, Pinto DG, Teixeira APM, 
Santana JLS, Santos RAPGS, Lima RPS. 
Contribution of the leukocyte adherence 
inhibition test for the evaluation of 
immunoreactivity against gluten extracts in 
Non—IgE-mediated / non-autoimmune 
Gluten-Related Disorders. Eur J Clin Med. 
2022;3(2):1-7. 

71. Olivier CE, Pinto DG, Teixeira APM, 
Santana JLS, Santos RAPGS, Lima RPS. 
Leukocyte adherence inhibition test to the 
assessment of immunoreactivity against 
cow's milk proteins in Non—IgE-Mediated 
gastrointestinal food allergy. Eur J Clin 
Med. 2022;3(2):38-43. 

72. Olivier CE, Pinto DG, Teixeira APM, 
Santana JLS, Santos RAPG, Lima RPS. 
Contribution of the leukocyte adherence 
inhibition test to the diagnosis of 
immunoreactivity against cobalt. Asian J 
Immunol. 2023;6(1):174-184. 

73. Olivier CE, Pinto DG, Teixeira APM, 
Santana JLS, Santos RAPGS, Lima RPS. 
Exploring the role of leukocyte adherence 
inhibition test in assessing Non-IgE 
mediated immunoreactivity to benzoic acid 
in allergic patients. Asian J Immunol. 
2024;7(1):63-70. 

74. Olivier CE, Pinto DG, Teixeira APM, 
Santana JLS, Santos RAPGS, Lima RPS. 
Intrinsic atopic dermatitis: Titration of 
precipitins in the screening of food 
allergens for prescription of elimination 
diets and desensitization strategies. Eur J 
Clin Med. 2021;2(6):1-9. 

75. Olivier CE, Pinto DG, Teixeira APM, 
Santana JLS, Santos RAPGS, Lima RPS, 
et al.. Endotyping Non-IgE-Mediated 
Immunoreactivity to Dermatophagoides 
farinae: Implications for Allergic Patients. 
Asian J Immunol. 2024;7(1):90-99. 

76. Olivier CE, Pinto DG, Teixeira APM, 
Santana JLS, Santos RAPG, Lima RPS. 
Endotyping cellular and humoral 
immunoreactivity against aluminum in 
allergic patients: A retrospective study. 
Asian J Immunol. 2024;7(1):149-158. 

77. Williams CA, Chase MW. CHAPTER 13 - 
Precipitation Reactions. In: Reactions of 
Antibodies with Soluble Antigens. Vol 3. 
Academic Press. 1971;1-102. 

78. Gerberi D. Alexander Fleming: A second 
look. Journal of the Medical Library 
Association: JMLA. 2024;112(1):55-59. 

79. H. MW. Antibiotics in Onions and Garlic. 
Cal West Med. 1945;63(1):4-5. 



 
 
 
 

Olivier et al.; Asian J. Immunol., vol. 7, no. 1, pp. 185-200, 2024; Article no.AJI.125235 
 
 

 
199 

 

80. Lesnikov EP. Date on the fungicidal and 
fungistatic effects in vitro of fungicides of 
onion and garlic upon geotrichum. Biull 
Eksp Biol Med. 1947;24(7):70-72. 

81. Ledezma E, DeSousa L, Jorquera A, et al. 
Efficacy of ajoene, an organosulphur 
derived from garlic, in the short-term 
therapy of tinea pedis. Mycoses. 
1996;39(9-10):393-395. 

82. Schwimmer S, Mazelis M. Characterization 
of alliinase of Allium cepa (onion). Arch 
Biochem Biophysics. 1963;100(1):66-73. 

83. Ahmed SST, Fahim JR, Abdelmohsen UR. 
Chemical and biological studies on Allium 
sativum L. (1952-2020): A comprehensive 
review. J Advan Bioml Pharmac Sci. 
2022;5(1):1-22. 

84. El-Saadony MT, Saad AM, Korma SA,             
et al. Garlic bioactive substances and              
their therapeutic applications for improving 
human health: A comprehensive review. 
Front Immunol. 2024;15:1277074. 

85. Khajah MA, Orabi KY, Hawai S, Sary HG, 
El-Hashim AZ. Onion bulb extract reduces 
colitis severity in mice via modulation of 
colonic inflammatory pathways and the 
apoptotic machinery. J Ethnopharmacol. 
2019;241:112008. 

86. El-Hashim AZ, Khajah MA, Orabi KY, 
Balakrishnan S, Sary HG, Barakat              
AM. Treatment with onion bulb extract  
both prevents and reverses allergic 
inflammation in a murine model of     
asthma. Pharmac Biology. 2024;62(1):326-
340. 

87. El-Hashim AZ, Khajah MA, Orabi KY, 
Balakrishnan S, Sary HG, Abdelali AA. 
Onion bulb extract downregulates 
EGFR/ERK1/2/AKT signaling pathway and 
synergizes with steroids to inhibit allergic 
inflammation. Frontiers Pharmacol. 2020; 
11:551683. 

88. Li M, Yan YX, Yu QT, et al. Comparison of 
immunomodulatory effects of fresh garlic 
and black garlic polysaccharides on RAW 
264.7 macrophages. J Food Sci. 2017; 
82(3):765-771. 

89. Jutel M, Gajdanowicz P. Chapter 5 - 
Revised Disease Nomenclature Including 
Disease Endotypes. In: Agache I, Hellings 
P, eds. Implementing Precision Medicine  
in Best Practices of Chronic Airway 
Diseases. Academic Press. 2019;27-29. 

90. Gell PGH, Coombs RRA. Classification of 
allergic reactions responsible for clinical 
hypersensitivity and disease. In: Gell PGH, 
Coombs RRA, eds. Clinical Aspects of 

Immunology. 2nd ed. Oxford: Blackwell 
Scientific Publications. 1968;575-596. 

91. Jutel M, Agache I, Zemelka-Wiacek M, et 
al. Nomenclature of allergic diseases and 
hypersensitivity reactions: Adapted to 
modern needs: An EAACI position paper. 
Allergy. 2023;78(11):2851-2874. 

92. Olivier CE. Allergic sinusitis, allergic 
migraine, and sinus headaches. Online J 
Otolaryngol Rhinol. 
2024;7(1):OJOR.MS.ID.000652.  

DOI: 
000610.033552/OJOR.002024.000607.00
0652. 

93. Wells HG. Studies on the chemistry of 
anaphylaxis (III). Experiments with isolated 
proteins, especially those of the hen's egg. 
J Infect Dis. 1911;9:147-171. 

94. Gell PGH, Harington CR, Rivers RP. The 
antigenic function of simple chemical 
compounds; production of precipitins in 
rabbits. Brit J Exp Pathol. 1946;27(5):267-
286. 

95. Olivier CE, Lima RPdS, Pinto DG, Santos 
RAPGd. The plasma preincubation with 
papain before the assay suggests that a 
gell and coombs type II reaction is been 
demonstrated by the leukocyte adherence 
inhibition test. Biomedical Journal of 
Scientific & Technical Research. 
2021;36(3):28647-28655. 

96. Thomson DMP. Assessment of immune 
status by the leukocyte adherence 
inhibition test. New York: Academic Press; 
1982. 

97. Tong AW, Burger DR, Finke P, Barney C, 
Vandenbark AA, Vetto RM. Assessment of 
the mechanism of the leukocyte adherence 
inhibition test. Cancer Res. 1979;39(2 Pt 
2):597-603. 

98. Fink A, Heller L, Eliraz A, et al. Allergen-
specific leukocyte adherence inhibition 
(LAI) assay: Sensitivity, specificity and 
mechanism. Immunol Lett. 1987;16(1):65-
70. 

99. Halliday WJ, Maluish A, Miller S. Blocking 
and unblocking of cell-mediated anti-tumor 
immunity in mice, as detected by the 
leucocyte adherence inhibition test. Cell 
Immunol. 1974;10(3):467-475. 

100. Chung MK, House JS, Akhtari FS, et al. 
Decoding the exposome: data science 
methodologies and implications in 
exposome-wide association studies 
(ExWASs). Exposome. 2024;4(1): 
osae001. 



 
 
 
 

Olivier et al.; Asian J. Immunol., vol. 7, no. 1, pp. 185-200, 2024; Article no.AJI.125235 
 
 

 
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101. Anouar S, Hazim R, Brahim A. 
Interferences in immunological assays: 
Causes, detection, and prevention. Asian J 
Immunol. 2024;7(1):71-78. 

102. Chiarentin L, Gonçalves C, Augusto C, 
Miranda M, Cardoso C, Vitorino C. Drilling 
into "Quality by Design" approach for 

analytical methods. Crit Rev Anal Chem. 
2023;1-42. 

103. WMA. World Medical Association 
Declaration of Helsinki: Ethical            
principles for medical research involving 
human subjects. JAMA. 2013;310(20): 
2191-2194.

 
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