







































_____________________________________________________________________________________________________ 
 
*Corresponding author: E-mail: celso@alergoimuno.med.br; 
 
Cite as: Olivier, Celso Eduardo, Daiana Guedes Pinto, Ana Paula Monezzi Teixeira, Cibele Silva Miguel, Jhéssica Letícia 
Santos Santana, Regiane Patussi Santos Lima, Nicole Sartoreto Rocha, and Raquel Acácia Pereira Gonçalves Santos. 2025. 
“Immunoreactivity to Cocoa and Nickel in Atopic and Allergic Contact Dermatitis”. Asian Journal of Immunology 8 (1):35-49. 
https://doi.org/10.9734/aji/2025/v8i1158. 

 
 

Asian Journal of Immunology 
 
Volume 8, Issue 1, Page 35-49, 2025; Article no.AJI.133582 
 

 
 

 

 

Immunoreactivity to Cocoa and Nickel 
in Atopic and Allergic Contact 

Dermatitis 
 

Celso Eduardo Olivier a*, Daiana Guedes Pinto a,  

Ana Paula Monezzi Teixeira a, Cibele Silva Miguel a, 
Jhéssica Letícia Santos Santana b,  

Regiane Patussi Santos Lima c, Nicole Sartoreto Rocha d  

and Raquel Acácia Pereira Gonçalves Santos a 
 

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. 
d Faculdade de Americana, São Paulo, Brazil. 

 
Authors’ contributions  

 
This work was carried out in collaboration among all authors. The author CEO did conceptualization, 

data curation, formal analysis, literature review, and writing the original draft. Authors DGP, APMT, 
CSM, JLSS, RPSL and NSR performed laboratory procedures. Author RAPGS performed cutaneous 

tests. All authors read and approved of the final manuscript. 
 

Article Information 
 

DOI: https://doi.org/10.9734/aji/2025/v8i1158  
 

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://pr.sdiarticle5.com/review-history/133582  

 
 

Received: 01/02/2025 
Published: 05/04/2025 

 
  

Original Research Article 

https://doi.org/10.9734/aji/2025/v8i1158
https://pr.sdiarticle5.com/review-history/133582


 
 
 
 

Olivier et al.; Asian J. Immunol., vol. 8, no. 1, pp. 35-49, 2025; Article no.AJI.133582 
 
 

 
36 

 

ABSTRACT 
 

Background: Cocoa is one of the foods more consistently associated with high nickel content, a 
metal that functions as a hapten responsible for various non–IgE-mediated symptomatic 
hypersensitivities.  
Aim: 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 cocoa extract and a 
nickel solution, as well to verify some sort of cross-reactivity between them in patients suffering from 
non–IgE-mediated Atopic and Allergic Contact Dermatitis. 
Methodology: We examined the medical charts of two cohorts of patients clinically diagnosed with 
Intrinsic Atopic Dermatitis and Allergic Contact Dermatitis, who were simultaneously investigated 
with cocoa extract and nickel solution with the help of TTP or LAIT. The registered results of the 
semi-quantitative serum TTP against cocoa extract and nickel solution, as well as the registered 
results of the Leukocyte Adherence Inhibition (LAI) percentage promoted by the ex vivo challenges 
against a cocoa extract and a nickel solution, were distributed in ranges through a cascade 
distribution chart to outline the variability of the results inside the cohorts. 
Results: The mean for the TTP for the cocoa extract was estimated at 1:385; the median at 1:512; 
and the SD at 1:180. The mean or the TTP for the nickel was estimated at 1:309; the median was 
1:256; the SD was estimated at 1:199. The mean for for the Leukocyte Adherence Inhibition (LAI) 
the cocoa extract was 51.9%; the median was 56%; the SD was 26.3%. The mean for the LAI for 
nickel solution was 33%; the median was 33%; the SD was 26,6%. The Pearson correlation 
indicated that there is a significant medium positive relationship between TTP results between 
Cocoa (x-axis) and Nickel (y-axis), r(98) = .335; p < .001. The Pearson correlation indicated that 
there is a significant medium positive relationship between LAIT results between Cocoa (x-axis) and 
Nickel (y-axis), r(98) = .425; p < .001. 
Conclusion: The results demonstrated a more significant immunoreactivity from the tests 
performed with cocoa extract than obtained with the nickel solution. These findings state that cocoa 
possesses other allergens responsible for cellular immunoreactivity besides nickel. This means that 
if a patient presents cellular immunoreactivity against cocoa, he/she will not necessarily present 
immunoreactivity against nickel; however, reciprocation is less probable. 
 

 

Keywords: Cocoa, dermatitis, endotype; hypersensitivity; leukocyte adherence inhibition test; nickel; 
non–ige-mediated immunoreactivity; precipitins. 

 

ABBREVIATIONS 
 

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

1. INTRODUCTION  
 

Self-reported allergy to chocolate is a common 
complaint in the clinical practice of Allergology; 
however, IgE-mediated cocoa hypersensitivity is 
not a commonly demonstrable endotype 
responsible for the phenotypes described as 
cocoa allergies (Nin-Valencia et al. 2024; Sloan 
& Powers 1986). When thoroughly investigated, 
IgE-mediated reactions to chocolate are usually 
due to cow's milk, hen's egg, peanut, soybean, 
hazelnut, almond, and other ingredients and 
contaminants added to cocoa to make sweet 
chocolate (Lopes et al. 2019, Pilolli et al. 2024). 
“The Allergen Nomenclature Sub-Committee of 
the World Health Organization and International 
Union of Immunological Societies (WHO/IUIS) do 

not recognize any protein derived from 
Theobroma cacao that could officially be stated 
as a major (or a minor) IgE-mediated allergen. A 
candidate to be recognized as a significant IgE-
mediated cocoa allergen is the Pathogenesis-
Related protein PR10 (TcPR-10), a protein with 
homology to pollen and food allergens derived 
from the Birch (Betula verrucosa), the pear 
(Prunus persica), the apple (Malus domestica), 
the cherry (Prunus avium) and the carrot 
(Daucus carota)” (Menezes et al. 2012).  
 

However, cocoa is one of the foods more 
consistently associated with high nickel content, 
a metal that functions as a hapten responsible for 
various non–IgE-mediated symptomatic 
hypersensitivities associated with several toxic 
and allergic diseases (Cubadda et al. 2020). 
Nickel is the fifth most abundant metal in the 
earth's crust. It is found in soil, absorbed by 
cocoa roots, and transported upright to 
accumulate in cocoa beans (Mostafa et al., 
2024). 



 
 
 
 

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37 

 

Besides nickel, cocoa beans may present 
several toxic metals absorbed from the soil, 
posing human health risks (Frimpong et al., 
2024). Besides cocoa, nickel may be present in 
licorice, lettuce, soybeans, oatmeal, nuts, 
almonds, legumes, peanuts, oats, grains, whole 
wheat (and whole meal flours), and mainly in 
canned food with high nickel contents, liberated 
from a nickel-plated tin alloy (Sharma 2013, 
Abeck et al., 1993; Veien & Menné, 1990; Veien 
et al., 1985). 
 
Nickel is one of the leading causes of allergic 
contact dermatitis, which is diagnosed by patch 
tests (Isufi et al., 2025). Nickel is a hapten 
collected by dendritic cells through the Toll-like 
receptor 4 and presented to naïve T cells in 
regional lymph nodes (Schmidt et al., 2010, 
Roediger & Weninger, 2011). “Nickel 
Hypersensitivity may be responsible for Allergic 
Contact Dermatitis, Allergic Contact Mucositis, 
and Systemic Nickel Allergy Syndrome (Schäfer 
et al., 2001; Ahlström et al., 2019; Greco et al., 
2023; Ricciardi et al., 2014). Systemic Nickel 
Allergy is a term commonly used to describe 
clinical manifestations of the systemic 
provocation by haptens associated with atopic 
dermatitis, psoriasis, urticaria, angioedema, 
rhinitis, asthma, headache, chronic fatigue, post-
prandial dyspnea, cystitis, vulvovaginitis, acne, 
and iron deficiency anemia (Nijhawan et al., 
2009; Veien, 2011; Akiba et al., 2025). 
 
“Sometimes, the exposition through a systemic 
route of immunoreactive haptens may          
produce localized cutaneous hypersensitivity 
phenotypes such as dyshidrotic eczema 
(pompholyx), toxicoderma-like rash, chronic 
pruritus, maculopapular rash, vasculitis-like 
lesions, flexural dermatitis, papuloerythroderma-
like eruptions, and baboon syndrome” (Antico & 
Soana, 2015).  
 
Nickel hypersensitivity may be successfully 
treated with sublingual desensitization and a low-
nickel diet. (Filatova & Cherpak, 2020; Ricciardi 
et al., 2013; Morris, 1998). Nickel allergy can be 
diagnosed with cutaneous and oral challenge 
tests (Veien et al., 1987). Non–IgE-mediated 
symptomatic Nickel hypersensitivity may be 
differentiated with the help of the Leukocyte 
Adherence Inhibition Test (Olivier et al., 2023c). 
“Non–IgE-mediated cellular immunoreactivity 
against several food allergens had already been 
reported by our group with the help of the 
Leukocyte Adherence Inhibition Test (LAIT) 
(Olivier et al., 2021d; Olivier et al., 2022a; Olivier 

et al., 2022c; Olivier et al., 2024a). The humoral 
immunoreactivity against several food allergens 
with the help of the Tube Titration of Precipitins 
(TTP) was also evaluated” (Olivier et al., 2024b; 
Olivier et al., 2024c; Olivier et al., 2025). “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” (Kuratsuji, 1981; Olivier et 
al., 2023f; Olivier et al., 2023h; Olivier et al., 
2023d). To evaluate the potential of the                
LAIT and TTP to endotyping Non–IgE-mediated 
cellular and humoral immunoreactivity against 
cocoa extract and nickel solution, we 
retrospectively compiled the electronic medical 
charts of patients diagnosed with non–IgE-
mediated hypersensitivity who were investigated 
simultaneously for immunoreactivity against 
these two allergens by one of these assays 
(Olivier et al., 2023b; Olivier et al., 2023e; Olivier 
et al., 2024f). 
 

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 cocoa extract 
and nickel solution, as well to verify some cross-
reactivity between them through the calculation 
of a paired t-test to distinguish some order of 
cross-reactivity in patients suffering from non–
IgE-mediated clinically diagnosed allergies.  
 

“As the tests were performed simultaneously with 
the same venous sample for the two allergens, it 
is possible to calculate a paired t-test between 
LAIT results (since they refer to the same 
quantitative variable), as well as to present a 
dispersion graph between them to distinguish 
some order of correlation suggesting (or not) 
cross-reactivity” (Gosset-Student 1908).  
 

2. MATERIALS AND METHODS  
 

2.1 Subjects 
 

After receiving Institutional Review Board 
approval from the Instituto Alergoimuno de 
Americana (Brazil; 02/2025), we reviewed the 
electronic chart of 10.170 outpatients who 
attended our facility from January 2018 to March 
2025.  
 

A cohort of 100 consecutive outside patients 
(TTP cohort) had been simultaneously submitted 
to TTP with cocoa extract and nickel solution for 
presenting non–IgE-mediated intrinsic atopic 
dermatitis and contact dermatitis. This cohort 



 
 
 
 

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38 

 

counted 27 males; mean age 38.6 years; SD 
21.4 years; range 1 to 84 years; median 37.5 
years; modes = 4, 7, 27, 30, 32, 33, 41, 45, 46, 
50 years (each appeared 3 times); geometric 
mean = 29.9 years.  
 
A cohort of 100 consecutive outside patients 
(LAIT cohort) had been simultaneously submitted 
to TIAL with cocoa extract and nickel solution for 
presenting non–IgE-mediated intrinsic atopic 
dermatitis and contact dermatitis. This cohort 
counted 27 males; mean age 43.3 years; SD 
19.4 years; range 10 to 91 years; median 40 
years; mode = 35 years (appeared seven times); 
geometric mean = 38.7 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 cocoa and/or 
nickel hypersensitivity who demonstrated an 
undetectable specific IgE against cocoa and non-
reactive or inconclusive skin tests against cocoa 
extract and nickel solution (Olivier et al., 2013). 
 

2.2 Extracts 
 
2.2.1 Cocoa extract 
 
The whole cocoa (pulp, peel, and seeds) was 
crushed, 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 
solid particles and oily fraction (Coca, 1922). The 
protein quantification of the allergen extracts was 
done according to Bradford's protein-dye binding 
methodology (Bradford, 1976). 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 cocoa extract was used to 
perform allergic skin tests, TTP, and LAIT. All 
relevant and mandatory laboratory health and 
safety measures have been complied with during 
the experiments.  
 
2.2.2 Nickel solution 
 
The [NiSO4 (H2O)6] was acquired from Labcenter 
Campinas. The powder was weighed and diluted 
in a buffer solution [NaCl 10g; KH2PO4 0,72g; 
Na3PO4 2,86g; H2O 600mL] to achieve the final 

concentration of 1 mg/mL to be employed in the 
LAIT, TTP, and cutaneous tests. 

 
2.3 LAIT: Ex vivo Investigation: 

Leukocyte Adherence Inhibition Test 
 
2.3.1 LAIT: Procedure for allergen ex vivo 

challenging  

 
We performed the LAIT as previously described 
(Olivier et al., 2012, Olivier et al., 2014, Olivier et 
al., 2021b, Olivier et al., 2021c, Olivier et al., 
2021e). Shortly, each donor's fresh plasma was 
divided into two parts and used in parallel ex vivo 
challenging tests with the cocoa extract, the 
nickel solution, and the unchallenged plasma 
(added with antigen dilution solution as a control) 
(Olivier et al., 2022b, Olivier et al., 2022a, Olivier 
et al., 2022c, Olivier et al., 2023f, Olivier et al., 
2023h). 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) (Olivier et al., 2023a, Olivier 
et al., 2024e). 

 
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 



 
 
 
 

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39 

 

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 
TTP against the cocoa extract and the nickel 
solution was performed in a transparent vitreous 
tube array (Olivier et al. 2021f, Olivier et al. 
2024g, Olivier et al. 2024d, Olivier et al. 2024b, 
Olivier et al. 2024a). 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 
separated aliquots of 15 μL of the antigen (cocoa 
extract or nickel solution) 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 distilled 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 (Williams & Chase, 1971). 

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 cocoa extract showed a 
distribution concentrated on the higher dilutions 
(Fig. 1). There was one negative result. The 
mean was estimated at 1:385; the median was 
1:512; the standard deviation was estimated at 
1:180; the mode was 1:512 (appeared 65 times).  
 
The TTP for the nickel showed a distribution 
concentrated on the higher dilutions (Fig. 2). 
There was one negative result. The mean was 
estimated at 1:309; the median was 1:256; the 
standard deviation was estimated at 1:199; the 
mode was 1:512 (appeared 46 times).   
 
The LAIT for the cocoa extract showed a wide 
distribution range of results. Most results were 
concentrated in the higher immunoreactive 
groups. There were seven negative results. The 
LAI ranged from 0% to 98%. The mean was 
51.9%; the median was 56%; the standard 
deviation was 26.3%; the mode was 0% 
(appeared seven times). The cascade 
distribution demonstrates a wide range of LAI 
results (Fig. 3). Some patients showed low or 
moderate immunoreactivity during the ex vivo 
challenge test. In contrast, others displayed 
strong immunoreactivity, which supposedly could 
reflect the participation of cocoa allergens in the 
Non–IgE-mediated hypersensitivity condition of 
these patients. 

 

 
 

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



 
 
 
 

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40 

 

 
 

Fig. 2. Cascade distribution chart of the tube titration of precipitins (x-axis %) resulting from 
the nickel solution against the serum of the TTP cohort of 100 tests/subjects (y-axis). 

 

 
 

Fig. 3. Cascade distribution chart of the range groups of Leukocyte Adherence Inhibition (LAI) 
results (x-axis %) of the ex vivo challenge test against cocoa 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) 

 

The LAIT for nickel solution showed a wide 
distribution range of results. Most results were 
concentrated in the lower and moderate 
immunoreactive groups. There were eighteen 
negative results. The LAI ranged from 0% to 
89%. The mean was 33%; the median was 33%; 
the standard deviation was 26,6%; the mode was 
0% (appeared eighteen 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 nickel in the Non–IgE-          
mediated hypersensitivity condition of these 
patients. 
 



 
 
 
 

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41 

 

The Pearson correlation indicated that there is a 
significant medium positive relationship between 
TTP results between Cocoa (x-axis) and Nickel 
(y-axis), r(98) = .335; p < .001; see Fig. 5. 
 

The Pearson correlation indicated that there is a 
significant medium positive relationship between 
LAIT results between Cocoa (x-axis) and Nickel 
(y-axis), r(98) = .425; p < .001; see Fig. 6. 

 
 

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

 

 
 

Fig. 5. Dispersion chart of the Leukocyte Adherence Inhibition (LAI) results of the Tube 
Titration of Precipitins against cocoa extract (x-axis %), plotted against the LAI results of the 

Tube Titration of Precipitins against nickel solution (y-axis %). 
 



 
 
 
 

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42 

 

 
 

Fig. 6. Dispersion chart of the Leukocyte Adherence Inhibition (LAI) results of the ex vivo 
challenge test against cocoa extract (x-axis %), plotted against the LAI results of the ex vivo 

challenge test against nickel solution (y-axis %). 
 

4. DISCUSSION 
 
Cocoa polyphenols have several effects on 
innate inflammatory response and systemic and 
intestinal adaptive immune systems (Pérez-Cano 
et al., 2013). Animal studies have observed that 
polyphenols in cocoa extract prevent IgE 
synthesis and can suppress the development of 
Atopic Dermatitis by downregulating 
inflammatory markers, inflammatory chemokines, 
and cell infiltration into lesion areas, turning this 
food a potential therapy for patients with skin 
diseases; however, these preliminary data does 
not justify yet its empirical prescription for the 
treatment of allergic patients (Kang et al., 2017; 
Abril-Gil et al. ,2012). 
 
Despite the results demonstrating a positive 
correlation between cocoa and nickel 
immunoreactivity, the cocoa extract 
demonstrated significantly higher immune 
responses, suggesting that nickel is not the only 
active allergen in the cocoa extract. This infers 
that evaluating one allergen does not eliminate 
the need to evaluate the other. This observation 
also demonstrates that cocoa polyphenols do not 
directly suppress the immunoreactivity evaluated 
by the assays, leading to more studies about 
their alleged downregulation effects over 
inflammatory chemokines and immune cells, as 
reported in animal studies. 
 

Endotyping cellular and humoral 
immunoreactivity biomarkers against specific 
allergens, haptens, and their respective cross-
reactivities are tools to build effective strategies 
to personalize exclusion diets and 
desensitization treatments for allergic patients 
(Agache & Akdis, 2020; Khan, 2016). Due to the 
facility's ability to diagnose hypersensitivities 
associated with the IgE-mediated endotype, 
there is a tendentious bias among physicians to 
just "ignore" the non-IgE-mediated mechanisms 
(Zhang et al., 2024). To demonstrate a non–IgE-
mediated allergic endotype, sometimes it is 
necessary to employ a multi-omics approach to 
differentiate the variety of clinical phenotypes 
and immune endotypes (Macowan et al., 2025; 
Yoon & Bunyavanich, 2025).  
 
The substantial number of sources for nickel is a 
confounding factor when investigating clinical 
hypersensitivity against this hapten. Initially, it 
may appear to be a polysensitization picture 
since the patients react to a confounder number 
of unrelated sources. The patient may also 
present unrelated symptoms, leading to the 
diagnosis of several allergic phenotypes. Until 
the conclusion that all phenotypes are produced 
by only one endotype mechanism elicited by only 
one allergen, it is usually a long journey requiring 
complex dietary interventions (Mikajiri et al., 
2024).  
 



 
 
 
 

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As a hapten, even in the presence of an IgE-
mediated hypersensitivity, it is almost impossible 
to develop a specific IgE-antibody for a 
commercial immunoassay against the multitude 
of carrier-haptens conjugates that may be 
produced once nickel gains blood circulation and 
conjugates with albumin, nickeloplasmin, or 
another serum protein (Barceloux, 1999). The 
best strategy is to challenge the patients' living 
plasma (ex vivo) or fresh serum (in vitro) with 
nickel solution and quantify the resulting 
immunoreactivity. 
 
The correlation and the distribution of 
simultaneous positive specific IgE against food 
and inhalant allergens is usually weak. However, 
polysensitization is more of a rule than an 
exception (Zhang et al., 2025; Čelakovská et al., 
2024).  
 
The research of precipitins is the pioneering 
laboratory exam upon which the bases of 
Immunology were constructed (Wells, 1911). 
Precipitating antibodies testifies a humoral 
immune response against the tested antigens 
(Gell et al.,1946). Before the discovery of IgE, 
the research of precipitins was the leading way to 
realize in vitro diagnosis of immunoreactivity 
against allergens (Augustin & Hayward, 1960; 
Augustin et al.,1960a; Augustin, 1953).   
 
Precipitins against food proteins and circulating 
immune complexes are strongly associated with 
patients with selective IgA deficiency since the 
secretory IgA is one of the main participants of 
the mechanism of immune exclusion promoted 
into the intestinal lumen to prevent the absorption 
of undigested proteins to blood circulation 
(Cunningham-Rundles et al., 1978). Primary 
selective immune deficiencies (such as IgA or 
IgE) lead to immune dysregulation, a concept 
that is evolving as specific endotypes associated 
with secondary immunodeficiencies raised by the 
inflammatory conditions provided by the allergic 
reactions (Henrickson, 2025; Olivier et al., 
2023g; Sapartini et al., 2025). 
 
The LAIT was designed as an ex vivo challenge 
test performed with a viable leukocyte buffy coat 
exploring several immune pathways, resulting in 
a final result quantified as the allergen-specific 
leukocyte adherence inhibition (Olivier et al. 
2021a; Thomson, 1982; Tong et al., 1979; Fink 
et al., 1987; Halliday et al.,1974). 
 
The comparative results obtained from the TIAL 
cohort demonstrated a more significative 

immunoreactivity from the tests performed with 
cocoa extract than those obtained with the nickel 
solution. This finding states that cocoas possess 
more allergens responsible for cellular 
immunoreactivity besides nickel. This means that 
if a patient presents cellular immunoreactivity 
against cocoa, he/she will not necessarily 
present immunoreactivity against nickel; 
however, reciprocation is less probable. 
 
This preliminary retrospective survey 
demonstrated extensive results from the TTP 
and the ex vivo challenge test monitored by LAIT 
against cocoa extract and nickel solution in two 
cohorts of patients with Allergic Contact and 
Atopic Dermatitis. 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 for several chemical and biological 
allergens, demonstrating positive results for 
some of them.  

 

5. LIMITATIONS 
  

This study is a retrospective analysis of data 
collected over seven years. 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 
and negative controls to validate the results. The 
number of subjects is appropriate for preliminary 
study; however, future studies must be more 
comprehensive. The lack of a prospective 
research protocol implies the possibility of a bias 
produced by the physician's point of view, which 
indicated the exam was based on clinical 
suspicion led purely by anamnesis and physical 
examination. The study lost many of these 
patients to follow-up, so assuring the relationship 
between the immunoassay results and the 
patient's clinical outcome is not possible yet. The 
two procedures were not compared with paired 
tests because they were taken from distinct 
groups of patients. 
 

6. CONCLUSION 
 

Our preliminary results show that the LAIT and 
TTP may differentiate diverse degrees of 
immunoreactivity against cocoa extracts and 
nickel solution in patients clinically diagnosed 
with non–IgE-mediated atopic and allergic 



 
 
 
 

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44 

 

contact dermatitis. TIAL and TTP are 
inexpensive, can be performed with minimum 
laboratory equipment, and can be incorporated 
into strategies to address health disparities in 
managing allergies (Anagnostou et al., 2025). As 
a preliminary report, the propaedeutic meaning of 
the presented results and the possibility of 
interferents must be yet established (Anouar, et 
al., 2024). 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 nickel and nickel-
containing foods (Chiarentin et al., 2023). 

 

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 unnecessary, 
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. 
Adding data provided by TTP and TIAL may also 
contribute to streamlining biomedical research 
and improving tools such as Large Language 
Models, usually used by clinicians as a decision 
support system to enhance diagnostic accuracy 
(Abers & Mathias, 2025). 
 

CONSENT  
 

It is not applicable. 
 

ETHICAL APPROVALS 
 

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 (WMA, 
2013). 
 

DISCLAIMER (ARTIFICIAL INTELLIGENCE) 
 

Authors 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.  
 

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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