







































_____________________________________________________________________________________________________ 
 
*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 Tartrazine in Allergic Patients: A Retrospective Study”. Asian Journal of Immunology 7 
(1):217-27. https://journalaji.com/index.php/AJI/article/view/146. 

 
 

Asian Journal of Immunology 
 
Volume 7, Issue 1, Page 217-227, 2024; Article no.AJI.126461 
 

 
 

 

 

Endotyping Cellular and Humoral 
Immunoreactivity against Tartrazine 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. Author CEO conceptualized the study, 
did data curation, formal analysis, literature review and wrote 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/v7i1146  
 

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/126461 

 
 
 

Received: 22/10/2024 
Accepted: 06/11/2024 
Published: 11/11/2024 

 

Original Research Article 

https://doi.org/10.9734/aji/2024/v7i1146
https://www.sdiarticle5.com/review-history/126461


 
 
 
 

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218 

 

ABSTRACT 
 

Background: Several publications report that tartrazine is responsible for IgE-mediated and non–
IgE-mediated hypersensitivity reactions. There is no standardized lab exam to endotype non–IgE-
mediated immunoreactivity against tartrazine besides in vivo provocation tests. 
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 
tartrazine in patients clinically diagnosed with non–IgE-mediated allergic phenotypes associated 
with various non-IgE-mediated allergic conditions. 
Study Design: We retrospectively examined the medical charts of two cohorts of patients 
diagnosed with the aforementioned allergic phenotypes with clinical suspicion of tartrazine 
hypersensitivity, who were investigated with the help of TTP (first cohort) or ex vivo challenge tests 
monitored by LAIT (second cohort) against tartrazine.  
Methodology: The registered results of the semi-quantitative serum TTP against 1 mg/mL 
tartrazine 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 tartrazine solution 
were distributed in ranges through a cascade distribution chart to outline the variability of results 
inside the second cohort. The statistical characteristics of these cohorts were calculated.  
Results: Most positive TTP results concentrated on the higher dilutions. The mean was estimated 
at 1:290; the standard deviation was estimated at 1:200. The LAI ranged from 0% to 88%. The 
mean was 35.4%; the standard deviation was 24,7%. The cascade distribution graph demonstrates 
that the LAIT distribution is mainly over the negative and weaker results. 
Conclusion: Our preliminary results support that the TTP and LAIT performed with 1 mg/mL 
tartrazine solution may discriminate diverse humoral and cellular immunoreactivity degrees in 
patients suffering from several non–IgE-mediated allergic conditions, encouraging further 
prospective studies and validation. 
 

 

Keywords: Asthma; rhinitis; endotype; hypersensitivity; leukocyte adherence inhibition test; 
precipitins; tartrazine; urticaria. 

 

ABBREVIATIONS 
 

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

1. INTRODUCTION  
 
Tartrazine is a tasteless synthetic yellow-lemon 
water-soluble aromatic mono-azo dye used as its 
sodium salt, potassium salt, calcium salt, and 
aluminum lake [1]. Azo dyes (organic compounds 
bearing the functional group R−N=N−R′ where R 
and R′ are aryl groups) such as tartrazine, sunset 
yellow, and carmoisine are additives aggregated 
to provide color to medicines and processed 
foods in products such as canned vegetables, 
chewing gum, frankfurters, macaroni, soft drinks, 
spaghetti, bread, butter, cheese, concentrated 
fruit juices, ice cream, jellies, tomato ketchup, 
jam, candies, pickles, and others, regardless any 
intrinsic nutrition value, preservative activity or 
health benefit [2,3]. 
 
Tartrazine may be used alone or associated with 
aluminum lakes or blue colorants to produce 

green shades [1,4]. Tartrazine is also known by 
the codes: E102, INS 102, CI 19140, FD&C 
Yellow 5, Yellow 5 Lake, Acid Yellow 23, Food 
Yellow 4, and trisodium 1-(4-sulfonatophenyl)-4-
(4-sulfonatophenylazo)-5-pyrazolone-3-
carboxylate [5].  
 

The European Food Safety Authority Panel 
calculated a theoretical maximum daily exposure 
to tartrazine of 8.1 mg/kg/day for adults and 13.1 
mg/kg/day for children, establishing an 
Acceptable Daily Intake (ADI) of 7.5 mg/kg/day 
[6]. However, these limits do not represent 
enough environmental safety since they can 
damage aquatic fauna such as the freshwater 
zebrafish (Danio rerio) [7]. 
 
Tartrazine has been known to cause allergic 
reactions since 1958 [8]. The extensive clinical 
spectrum of tartrazine hypersensitivity includes 
urticaria, angioedema, anaphylactic shock, 
asthmatic bronchitis, rhinitis, throat tickle, cough, 
vasculitis (purpura), and contact dermatitis       
[9-14]. A patient with textile contact dermatitis 
after using a yellow cloth was diagnosed with the 
help of a skin contact test with tartrazine [15].  



 
 
 
 

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The fact that tartrazine is a drug additive with no 
proper function besides coloring pills and 
solutions makes it one of the first aims to 
diagnose when facing allergic conditions elicited 
by colored medications such as antihistamines 
and steroids used to treat allergies and asthma 
[16]. 
  
Tartrazine belongs to a select group of aromatic 
substances (aspirin, salicylates, sulfites, 
benzoates, and azo dyes) known to produce 
non–IgE-mediated “allergic-like” dose-dependent 
symptoms such as urticaria and bronchospasm 
at remarkably similar molar doses in allergic 
patients submitted to progressive oral challenges 
[17-19]. These reactions appear to have a 
familiar incidence, presenting cross-reactivity 
among these substances [20].  
 
Tartrazine hypersensitivity has been associated 
with aspirin sensitivity; however, the mechanism 
is obscure since aspirin inhibits the 
cyclooxygenases responsible for producing 
prostaglandins, while tartrazine does not have 
this effect [21,22]. A multicenter study performed 
with patients with aspirin-induced asthma found a 
frequency of 2.6% of patients with tartrazine 
hypersensitivity [23].  
 
Due to tartrazine hypersensitivity, there is a 
worldwide concern to substitute tartrazine from 
medicines and industrialized food for natural 
colorants such as curcumin (E100), riboflavin 
(E101), beta-carotene (E160a), annatto (E160b), 
or other chemical dyes such as quinoline yellow 
(E104), or yellow iron oxide (E172) [24,25].  
 
Azo dyes are relatively small compounds that 
cannot interact alone with antibodies, so in 
hypersensitivity reactions, they function as 
haptens, usually linked to complex proteins, such 
as albumins [26]. Commercial anti-tartrazine 
polyclonal antibodies are usually produced for 
research purposes by conjugating tartrazine with 
ovalbumin or allophycocyanin [27]. Although one 
can find commercial lab kits to detect specific IgE 
against conjugated tartrazine, antibodies of the 
IgE class are unlikely to mediate tartrazine 
hypersensitivity [28].  
 
The primary laboratory marker of patients 
reacting to dyes is eosinophilia [29]. A 
radioimmunoassay inhibition assay was 
designed to detect anti-tartrazine IgD and IgE 
antibodies. However, patients diagnosed with 
tartrazine hypersensitivity could be distinguished 
from controls only by their specific IgD, not by 

their specific IgE antibodies against tartrazine 
[30].  
 

There is not yet any reliable routine lab exam to 
quantify immunoreactivity against tartrazine, and 
the clinical diagnosis of hypersensitivity to 
tartrazine is founded chiefly on in vivo tests 
based on oral or cutaneous provocations. 
 

Cellular immunoreactivity against tartrazine (and 
other food additives) has already been 
demonstrated by ex vivo challenging tests 
monitored by the granulocytic myeloperoxidase 
release reaction [31]. Ex vivo challenges 
employing leukocytes to detect immunoreactivity 
against tartrazine were also already monitored by 
sulfidoleukotriene production in allergic patients 
[32]. 
 

The direct effect of tartrazine on lymphocytes is 
suggested by cytotoxic experiments employing 
ex vivo experiments employing human 
lymphocytes [33]. Ex vivo studies in cultured 
human leukocytes demonstrated that tartrazine 
at 70 μg/mL induces DNA damage, suggesting a 
genotoxic potential [34].  
 

The Leukocyte Adherence Inhibition Test (LAIT) 
and the Tube Titration of Precipitins (TTP) are 
performed in our facilities as triage tests to 
identify immunoreactivity against suspected 
allergens executed before the performance of 
more exhaustive in vivo provocation tests [35-
41]. The present study hypothesizes that LAIT 
and TTP may differentiate endotypes and 
degrees of immunoreactivity against tartrazine 
among patients suffering from common allergic 
phenotypes.  
 

To evaluate the potential of the LAIT and the 
TTP to discriminate humoral and cellular 
immunoreactivity against tartrazine, we 
retrospectively compiled the electronic medical 
charts of patients clinically diagnosed with non–
IgE-mediated allergic phenotypes associated 
with chronic and/or recurrent conditions such as 
rhinitis, sinusitis, conjunctivitis, bronchitis, allergic 
contact dermatitis, intrinsic atopic dermatitis, 
urticaria systemic anaphylactic reactions and/or 
gastrointestinal disorders who were investigated 
with these procedures.  
 

2. MATERIALS AND METHODS  
 

2.1 Subjects 
 

After receiving Institutional Review Board 
approval from the Instituto Alergoimuno de 



 
 
 
 

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Americana (Brazil; 08/2024), we reviewed the 
electronic chart of 9,500 outpatients who 
attended our facility from January 2018 to 
October 2024.  
 
The first cohort (TTP cohort) consisted of                 
100 outside patients clinically diagnosed with 
non–IgE-mediated allergic phenotypes 
associated with chronic and/or recurrent 
conditions such as rhinitis, sinusitis, 
conjunctivitis, bronchitis, allergic contact 
dermatitis, intrinsic atopic dermatitis, urticaria 
systemic anaphylactic reactions and/or 
gastrointestinal disorders. These patients had 
been submitted to TTP with 1 mg/mL of 
tartrazine solution.  
 
The TTP cohort counted 23 males and 77 
females; mean age 35,5 years; SD 21.8 years; 
range 1 to 79 years; median 35 years; modes = 
9, 27, 42, 43, and 69 (each appeared four times); 
geometric mean = 25.3 years.  
 
The second cohort (LAIT cohort) consisted of 
100 outside patients clinically diagnosed with 
non–IgE-mediated allergic phenotypes 
associated with chronic and/or recurrent 
conditions such as rhinitis, sinusitis, 
conjunctivitis, bronchitis, allergic contact 
dermatitis, intrinsic atopic dermatitis, urticaria 
systemic anaphylactic reactions and/or 
gastrointestinal disorders. These patients had 
been submitted to an ex vivo allergen challenge 
test with tartrazine solution 1mg/mL monitored 
with LAIT.  
 
The LAIT cohort counted 36 males and 64 
females; mean age 40 years; SD 21.2 years; 
range 4 to 90 years; median 38 years; mode = 
22 (appeared four times); geometric mean = 33.2 
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 tartrazine 
hypersensitivity who demonstrated a non-
detectable specific IgE against tartrazine and a 
non-reactive or inconclusive skin test done with 
tartrazine 1 mg/mL solution [42]. 
 

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

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 
[43-52]. Shortly, each donor's fresh plasma was 
divided into two parts and used in parallel ex vivo 
challenging tests with tartrazine acetate solution 
1 mg/mL and the unchallenged plasma assay. 
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 kept under agitation for 30 minutes (200 
rpm at 37°C) with tartrazine solution (10μL of a 
solution with 1mg/mL) or without tartrazine 
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 (phosphate-
buffered saline) 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. 



 
 
 
 

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

 
As previously reported, the semi-quantitative 
TTP against the tartrazine solution was 
performed in a transparent vitreous tube               
array [53-55]. 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 [56]. 
 

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 
distribution range of TTP results. There was one 
negative result, while more positive results 
concentrated on the higher dilutions (Fig. 1). The 
mean was estimated at 1:290; the median was 
1:256; the standard deviation was estimated at 
1:200; the mode was 1:512 (appeared 42 times). 
All Sia tests were negative.  
 
The LAI ranged from 0% to 88%. The mean was 
35.4%; the median was 36.5%; the standard 
deviation was 24,7%; the mode was 0% 
(appeared nineteen times). The cascade 
distribution graph demonstrates distribution 
mostly over the negative and weaker results  
(Fig. 2). Nineteen patients (19%) ignored the 
allergen, presenting no inhibition of leukocyte 
adherence (LAI = 0%) after contact of the  
plasma with the tartrazine solution. Some 
patients showed low or moderate 
immunoreactivity during the ex vivo challenge 
test, while most displayed strong 
immunoreactivity, suggesting tartrazine's 
participation in the hypersensitivity condition. 

4. DISCUSSION 
 
To detect humoral and cellular 
immunoreactivities against tartrazine, we 
retrospectively compiled the data registered in an 
Excell® spreadsheet resulting from TTP and TIAL 
against tartrazine at our facilities. These triage 
tests are performed before the in vivo 
provocation tests when skin tests are unfeasible 
due to the patient’s skin conditions. 
 
The humoral and the cellular immunoreactivity 
profiles present divergent results. The humoral 
profile, represented by the TTP, presented the 
most results in the more diluted titrations, 
suggesting that most patients produced 
prominent levels of anti-tartrazine antibodies. 
However, the cellular profile, represented by the 
TIAL, presented most results in the negative or 
the weaker LAI results, suggesting a less 
determinant participation of the cellular 
immunoreactivity. The fact that the results were 
obtained from two cohorts and measured by 
different methodologies (quantitative and semi-
quantitative) did not allow us to perform a paired 
t-test analysis. 
 
The primary strategy of Personalized Medicine is 
to diagnose the endotypes responsible for the 
disease’s phenotypes [57]. The former limited 
capacity to diagnose hypersensitivity had created 
in physicians the common idea that diagnosing a 
single hypersensitivity would be enough to treat 
their patients, conferring them a “mono-
sensitization label”. As medical knowledge and 
resources advance, more diagnoses are being 
performed, making physicians aware that poli-
sensitization is more a rule than an exception. 
Panallergens responsible for cross-reactivity 
between allergens from diverse sources, such as 
tropomyosins or profilins, contribute to this 
awareness, allowing a more extensive 
comprehension of the allergic conditions [58,59]. 
Cross-reactivity among azo dyes and similar 
preservatives, such as the sulfides, a group of 
inorganic salts added to processed foods and 
also naturally found in Allium spices and 
fermented beverages, is a large field for studies 
that must be explored to understand 
hypersensitivity conditions better, endotyping 
their mechanisms, and tailor the treatment of 
allergic patients [60,61]. 
 
The great insight Gell and Coombs gave in the 
sixties resisted the test of time and now is 
amplified as a compass to understand the wide 
variety of hypersensitivity reactions that are still 



 
 
 
 

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far from being completely elucidated [62,63]. 
Endotyping underlying hypersensitivity 
mechanisms may help distinguish 
superimposable phenotypes presenting similar 
symptoms that may hamper establishing a 
precise diagnosis [64]. 
 
TTP and LAIT assays do not identify the exact 
immune mechanisms responsible for clinical 
hypersensitivity; instead, they are general 
immune markers of the humoral and cellular 
responses against allergens, quantifying an 
exposome measurement [65].  
 

This retrospective proof of concept analysis 
demonstrated several degrees of humoral and 
cellular immunoreactivity, as demonstrated by 
TTP and LAIT against tartrazine in two cohorts of 
patients with several allergic conditions. None of 
the patients presented an exclusive reaction to 
tartrazine. Every patient was simultaneously 
evaluated for other suspected allergens, 
demonstrating positive and negative results. Our 
results suggest that tartrazine can elicit humoral 
and cellular immunoreactivity in allergic patients, 
theoretically able to impair their allergic 
symptoms. 

 
 

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

(y-axis) 
 

 
 

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



 
 
 
 

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5. CONCLUSION 
 
In vitro humoral assays and ex vivo cellular 
challenges can detect immunoreactivity against 
potentially lethal allergens without posing any 
risk for the patient and are occasionally 
employed in daily routine when conventional 
allergy diagnostic procedures are not elucidative 
or contra-indicated [66]. Our preliminary results 
show that the TTP and LAIT may differentiate 
diverse degrees of immunoreactivity against 
tartrazine 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 [67]. 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 
suspected of symptomatic hypersensitivity 
against tartrazine and other similar food 
processing additives [68]. 
 

6. FUTURE DIRECTIONS AND 
RECOMMENDATIONS FOR CLINICAL 
PRACTICE 

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

7. LIMITATIONS 
   
This study is a proof-of-concept retrospective 
analysis of data collected over six 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 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. 
 

CONSENT AND 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 [69]. 
 

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.  
 

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

 
COMPETING INTERESTS 
 
Authors have declared that no competing 
interests exist. 
 

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