







































 

_____________________________________________________________________________________________________ 
 
*Corresponding author: E-mail: celso@alergoimuno.med.br; 
 
Cite as: Olivier, Celso Eduardo, Daiana Guedes Pinto, Ana Paula Monezzi Teixeira, Jhéssica Letícia Santos Santana, Raquel 
Acácia Pereira Gonçalves Santos, and Regiane Patussi Santos Lima. 2024. “Endotyping Cellular and Humoral 
Immunoreactivity Against Aluminum in Allergic Patients: A Retrospective Study”. Asian Journal of Immunology 7 (1):149-58. 
https://journalaji.com/index.php/AJI/article/view/139. 

 
 

Asian Journal of Immunology 
 
Volume 7, Issue 1, Page 149-158, 2024; Article no.AJI.122945 
 

 
 

 

 

Endotyping Cellular and Humoral 
Immunoreactivity against Aluminum  
in Allergic Patients: A Retrospective 

Study 
 

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

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

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. All authors read and approved the final 
manuscript. 

 

Article Information 
 

DOI: https://doi.org/10.9734/aji/2024/v7i1139  
 

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/122945  

 
 
 

Received: 09/07/2024 
Accepted: 11/09/2024 
Published: 14/09/2024 

 
 

ABSTRACT 
 

Background: Several publications report that aluminum is responsible for hypersensitivity 
reactions. There is no standardized lab exam that can endotype (determine the pathophysiology 
responsible for the phenotype) hypersensitivity to aluminum. 

Original Research Article 

https://doi.org/10.9734/aji/2024/v7i1139
https://www.sdiarticle5.com/review-history/122945


 
 
 
 

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150 

 

Aim: To evaluate the potential of the Tube Titration of Precipitins (TTP) and the Leukocyte 
Adherence Inhibition Test (LAIT) to endotype humoral and cellular immunoreactivity against 
aluminum in patients clinically diagnosed with allergic contact dermatitis, intrinsic atopic dermatitis, 
and/or non–IgE-mediated urticaria. 
Study Design: We retrospectively examined the medical charts of two cohorts of 100 patients 
each; range 6 to 87 years; diagnosed with allergic phenotipes, who were investigated with the help 
of TTP (first cohort) or ex vivo challenge tests monitored by LAIT (second cohort) against 
aluminum.  
Methodology: The registered results of TTP against 1 mg/mL aluminum solution were distributed 
in ranges through a cascade distribution chart to outline the variability of the results inside the first 
cohort. The registered results of the Leukocyte Adherence Inhibition (LAI) percentage promoted by 
the ex vivo challenges with 1 mg/mL aluminum solution were distributed in ranges through a 
cascade distribution chart to outline the variability of results inside the second cohort.  
Results: Most TTP results concentrated on higher dilutions. The mean was estimated at 1:301; the 
median was 1:256; the standard deviation was estimated at 1:204; the mode was 1:512 (appeared 
46 times). The LAI ranged from 0% to 88%. The mean was 52%; the median was 49%; the 
standard deviation was 23.9%; the mode was 60% (appeared five times). The cascade graphs 
demonstrate a wide range of distribution of TTP and LAI results. 
Conclusion: Our preliminary results support that the TTP and LAIT performed with 1 mg/mL 
aluminum solution may discriminate diverse humoral and cellular immunoreactivity degrees in 
patients suffering from allergic contact dermatitis, intrinsic atopic dermatitis, and/or non–IgE-
mediated urticaria. 
 

 
Keywords: Aluminum; endotype; hypersensitivity; leukocyte adherence inhibition; leukocyte 

adherence inhibition test; phenotype; precipitins; precision medicine. 
 

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

1. INTRODUCTION  
 

Aluminum (or aluminium) is a post-transition 
metal with the symbol Al, atomic number 13, and 
a relative atomic mass of 26.98, which has a 
great affinity to oxygen [1]. The American 
Contact Dermatitis Society elected aluminum as 
the "Allergen of the Year 2022" [2]. Aluminum 
may be present naturally in jewelry, piercings, 
cosmetics, tubes of toothpaste, medicines, tattoo 
inks, foods (at shallow levels), or due to using 
aluminum cooking utensils and food additives [3]. 
Some foods, such as potatoes, spinach, and 
some teas, may contain naturally higher levels of 
aluminum [4]. Remarkably, the mean 
concentration of aluminum in soy-based infant 
formulas is remarkably higher than in milk-based 
and corn-based infant formulas [5]. The sixty-
seventh meeting of the Joint FAO/WHO Expert 
Committee on Food Additives held in Rome in 
2006 established the provisional tolerable weekly 
aluminum intake as 1 mg/kg body weight [6]. 
Aluminum in its metal presentation (E 173) and 
aluminum salts are bactericides frequently used 

as preservatives for foods and cosmetics [7]. 
Aluminum sulfates (E 520 to E 523) are mainly 
used in the food industry as thickeners and 
emulsifiers [8]. Aluminum-containing additives 
are also used for specific purposes: A) as raising 
agent in baking powder, such as aluminum 
sodium sulfate (E 521) and sodium aluminum 
phosphate-acidic (E 541); B) as firming agents 
during the processing of jellyfish and pickles 
such as aluminum potassium sulfate (E 522); C) 
as emulsifiers in processed cheese, such as 
sodium aluminum phosphate-basic (E 541); D) 
as anti-caking agents in powder mixes for 
beverage mixers and non-dairy creams such as 
sodium aluminosilicate (E 554); E) as anti-caking 
agents for table salt and vanilla powder such as 
calcium aluminum silicate (E 556);  F) as carriers 
for pigments (titanium dioxide and iron oxides) 
such as the potassium aluminum silicate (E 555) 
and aluminum powder (E 173) in decorating 
sugar-coated flour confectionery candy coatings, 
also known as lakes [9-14]. 
 
Aseptic aluminum-containing plastic packages 
and cartons prevent oxidation and light damage 
to perishable food [15]. Elemental aluminum and 
its salts (such as sulfate, phosphate, hydroxide, 
and silicate) have a wide variety of uses, such as 
food additives, cooking utensils, food packaging 
(beverage cans and foil), water treatment, 



 
 
 
 

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cosmetics (sunscreens), toiletry products 
(antiperspirants, tubes of toothpaste and sun 
blockers), antiacids with aluminum hydroxide, 
anti-diarrheic with kaolin (hydrated aluminum 
silicate or E 559), producing contact dermatitis, 
granuloma, and urticaria in sensitized people [16-
19]. Pool water flocculants are made with 
aluminum salts and may produce occupational 
allergies [20]. Aluminum-containing compounds 
have been employed as adjuvants in vaccines to 
amplify antigen-specific Th2 responses [21]. 
There is a positive dose-response relationship 
between plasmatic aluminum concentrations and 
cognitive impairment among workers with 
occupational exposure to aluminum [22]. 
Aluminum nanoparticles are associated with 
memory impairment and hippocampal 
inflammation induced by microglial activation, 
following upregulation of the inflammatory 
cytokine IL-1β (interleukin-1β) in mice [23]. 
Current lab models to study Alzheimer's disease 
are established through intraperitoneal injection 
of aluminum chloride (AlCl3) in rats or aluminum 
nanoparticles in mice [24,25]. Diagnosis of 
delayed hypersensitivity to aluminum is usually 
clinically made in patients with contact dermatitis 
with the help of cutaneous path tests             
performed with aluminum salts dispersed in 
petrolatum [26]. However, systemic aluminum 
immunoreactivity has already been documented 
by systemic immunoassay markers, such as 
eosinophilia [27].  
 
The clinical suspicion of immediate and delayed 
hypersensitivity against aluminum is usually 
confirmed through skin allergy tests and patch 
tests, respectively. Until now, no widespread lab 
exam has been able to document specific 
immunoreactivity against aluminum to help with 
clinical diagnosis. 
 
The Leukocyte Adherence Inhibition Test (LAIT) 
and the Tube Titration of Precipitins (TTP) are 
performed in our facilities as triage tests for 
suspected allergens prescribed before the 
performance of more exhaustive in vivo 
provocation tests [28-34].  
 
The present study hypothesizes that the LAIT 
and the TTP may differentiate endotypes and 
degrees of immunoreactivity against aluminum 
among patients suffering from common allergic 
phenotypes. To evaluate the potential of the LAIT 
and the TTP to discriminate humoral and cellular 
immunoreactivity against aluminum, we 

retrospectively compiled the electronic medical 
charts of patients clinically diagnosed with 
allergic contact dermatitis, intrinsic atopic 
dermatitis, and/or non–IgE-mediated urticaria 
who were investigated with these procedures.  

 

2. MATERIALS AND METHODS  
 

2.1 Subjects 
 
After receiving Institutional Review Board 
approval from the Instituto Alergoimuno de 
Americana (Brazil; 06/2024), we proceeded with 
the electronic chart review of 9,200 outpatients 
who attended our facility from January 2018 to 
August 2024.  
 
The first cohort of 100 outside patients had been 
submitted to TTP with 1 mg/mL of aluminum 
solution for presenting allergic contact dermatitis, 
intrinsic atopic dermatitis, and/or non–IgE-
mediated urticaria. This cohort counted 24 males 
and 76 females; mean age 38.4 years; SD 20.4 
years; range 6 to 87 years; median 36.5 years; 
mode = 46 (appeared five times); geometric 
mean = 32 years.  
 
The second cohort of 100 outside patients had 
been submitted to an ex vivo allergen challenge 
test with aluminum solution 1mg/mL monitored 
with LAIT for presenting allergic contact 
dermatitis, intrinsic atopic dermatitis, and/or non–
IgE-mediated urticaria. This cohort counted 28 
males and 72 females; mean age 40.3 years; SD 
17.5 years; range 8 to 75 years; median 42 
years; mode = 42 (appeared six times); 
geometric mean = 35.2 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 aluminum 
hypersensitivity who demonstrated a non-
reactive or inconclusive skin test against 
aluminum 1 mg/mL solution [35]. The cascade 
graphs were mounted using the functionalities of 
the spreadsheet editor Microsoft Excell® 
software. 
 

2.2 Aluminum Solution  
 

The aluminum solution was prepared with 
powdered acetate aluminum diluted with distilled 
water at 1 mg/mL to perform the allergic skin 
tests, TTP, and LAIT.  

 

 



 
 
 
 

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2.3 Ex vivo Investigation: Leukocyte 
Adherence Inhibition Test 

 
2.3.1 Procedure for allergen ex vivo 

challenging  
 
We performed the LAIT as previously described 
[29,30,36-45]. Shortly, each donor's fresh plasma 
was divided into two parts and used in paralleled 
ex vivo challenging tests with aluminum acetate 
solution 1 mg/mL and the unchallenged plasma 
assay. 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 kept under agitation for 30 
minutes (200 rpm at 37 °C) with aluminum 
solution (10μL of a solution with 1mg/mL) or 
without aluminum solution (when used as 
control). 

 
2.3.2 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.  

 
2.3.3 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. 

2.3.4 In vitro Investigation: Tube Titration of 
Precipitins (TTP) 

 

As previously reported, the semi-quantitative 
TTP against the aluminum solution was 
performed in a transparent vitreous tube [46]. 
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. The allergen extracts were 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 [47]. 
 

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 showed a wide 
distribution range of TTP results. There was one 
negative result. Most positive results 
concentrated on the higher dilutions (Fig. 1). The 
mean was estimated at 1:301; the median was 
1:256; the standard deviation was estimated at 
1:204; the mode was 1:512 (appeared 46 times). 
All Sia tests were negative.  
 

The LAI ranged from 0% to 88%. The mean was 
52%; the median was 49%; the standard 
deviation was 23.9%; the mode was 60% 
(appeared five times). The cascade distribution 
graph demonstrates a wide range of distribution 
of LAI results (Fig. 2). Three patients ignored the 
presence of the allergen on the plasma and 
presented no inhibition of leukocyte adherence 
(LAI = 0%) after contact with the aluminum 
solution. Some patients showed low or moderate 
immunoreactivity during the ex vivo challenge 
test, while most displayed strong 
immunoreactivity, suggesting aluminum's 
participation in the hypersensitivity condition. 
 

4. DISCUSSION 
 

Aluminum was one of the first inorganic 
adjuvants envisaged to boost the immune 



 
 
 
 

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response of infectious vaccines and 
subcutaneous allergen immunotherapies [48]. 
The first description of using aluminum salts to 
boost immune response was published in 1924 
when used as an adjuvant to tetanus and 
diphtheria toxoids [49]. Further (1938), aluminum 
salts were presumed as adjuvants to 
subcutaneous allergen immunotherapies for their 
depot effect, increasing the permanence of the 
immunotherapeutic agent on the site of the 
injection, allowing more time for the innate 
immune system to process the antigens 
(adsorptive adjuvant) [50]. Later (1985), it was 
established that the presence of aluminum salts 
increased the uptake of allergens by antigen-
presenting cells, stimulating antigen-induced T-
cell proliferation [51]. Aluminum-containing 
adjuvants also activate innate immune response 
by stimulating dendritic cells, inducing CD4+ T 
cell differentiation [52]. Aluminum acts as a 
vaccine adjuvant by producing cellular necrosis 
that releases inflammatory cytokines (such as IL-
33), stimulating innate and adaptive immunity 
[53]. It is a corollary to think that ingestion, 
contact, and parenteral administration of this 
metal may produce residual asymptomatic or 
even symptomatic conditions in a society 
"drowned" in aluminum through food additives, 
cosmetic preservatives, medicines, vaccine 

adjuvants, and others [54]. Aluminum 
hypersensitivity might represent just the iceberg's 
tip of an organism saturated by its effects, 
alerting the patient's conscious mind to avoid 
contact and ingestion. 
 
Personalized medicine is an approach dedicated 
to diagnosing the endotypes responsible for 
disease phenotypes. [55]. While phenotypes are 
defined by the visible clinical manifestations of 
the conditions (some of these induced by 
multiple underlying mechanisms), an endotype is 
a subtype of a given phenotype defined by its 
pathophysiological mechanism [56]. The 
classical four hypersensitivity mechanisms 
described by Gell and Coombs in the sixties are 
now amplified to seven types with several 
subtypes, implying an increased responsibility of 
the clinical caretakers to investigate the 
mechanisms responsible for the disease 
phenotypes in order to recommend better 
strategies to avoid the allergens and to prescribe 
tailored treatments for these infirmities [57,58]. 
Endotyping the hypersensitivity mechanisms may 
also help distinguish superimposable phenotypes 
presenting similar symptoms that may hamper 
establishing a precise diagnosis [59]. To detect 
immune responses against aluminum, we 
spreadsheet a retrospective compilation of data

 

 
 

Fig. 1. Cascade distribution chart of the Tube Titration of Precipitins (TTP on the x-axis) 
resulting from the aluminum solution against the serum of a cohort of 100 tests/subjects  

(y-axis) 



 
 
 
 

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154 

 

 
 

Fig. 2. Cascade distribution chart of the range groups of Leukocyte Adherence Inhibition (LAI) 
results (x-axis %) of ex vivo aluminum solution monitored by the Leukocyte Adherence 

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

 
produced at our facilities by TTP and TIAL, 
exploring humoral and cellular 
immunoreactivities against aluminum. 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 and skin 
tests are inconclusive or unfeasible due to the 
patient's skin conditions. 
 
These assays do not identify the exact immune 
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. TTP and LAIT must be 
interpreted as triage immune markers of the 
humoral and cellular responses after contact with 
a specific antigen, configuring themselves as 
techniques to quantify an exposome 
measurement, as proposed by the exposome-
wide association study [60].  
 
This preliminary retrospective survey 
demonstrated extensive results from the TTP 
and the ex vivo challenge test monitored by LAIT 
against aluminum in two cohorts of patients with 

various allergic symptoms. None of our patients 
presented an exclusive reaction to aluminum. 
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 environmental or 
ingested aluminum. 

 

5. CONCLUSION 
 
Our preliminary results show that the TTP and 
LAIT may differentiate diverse degrees of 
immunoreactivity against aluminum in patients 
clinically diagnosed with non–IgE-mediated 
cutaneous allergies. This methodology can 
provide a socioeconomic impact since the 
methodologies to perform TTP and LAIT are 
inexpensive and can be performed in a single lab 
attached to the facilities with minimum laboratory 
equipment. However, the propaedeutic meaning 
of these results and the possibility of interferents 
must be better established [61]. More studies 
focused on the quality-by-design approach with 
prospective larger double-blind cohorts need to 
evaluate the potential contribution of TTP and 
LAIT for endotyping immunoreactivity of patients 



 
 
 
 

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suspected of symptomatic hypersensitivity 
against aluminum and other similar food 
processing additives [62,63]. 
 

6. LIMITATIONS 
 
This study is a retrospective analysis of data 
collected over six years. 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 
physician's point of view who indicated the exam 
(CEO) based on a clinical suspicion led purely by 
the anamnesis, physical examination, routine lab 
exams, and allergic skin tests. 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 
impossible. 
 

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.  

 

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

ACKNOWLEDGEMENTS 
 
The Instituto Alergoimuno de Americana funded 
this work. We want to thank laboratory technician 
Alessandra Vieira de Oliveira for her help with 
the exams. 
 

COMPETING INTERESTS 
 

Authors have declared that no competing 
interests exist. 
 

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