







































_____________________________________________________________________________________________________ 
 
*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, Nicole Sartoreto da Rocha, Jhéssica Letícia Santos Santana, and Regiane Patussi Santos Lima. 2025. 
“Endotyping Cellular and Humoral Immunoreactivity Against Pollen and Citrus Fruits in Patients With Non–IgE-Mediated 
Rhinoconjunctivitis”. Asian Journal of Immunology 8 (1):8-25. https://doi.org/10.9734/aji/2025/v8i1156. 

 
 

Asian Journal of Immunology 
 
Volume 8, Issue 1, Page 8-25, 2025; Article no.AJI.131451 
 

 
 

 

 

Endotyping Cellular and Humoral 
Immunoreactivity against Pollen and Citrus 

Fruits in Patients with Non–IgE-mediated 
Rhinoconjunctivitis 

 
Celso Eduardo Olivier a*, Daiana Guedes Pinto a,  

Ana Paula Monezzi Teixeira a, Cibele Silva Miguel a,  
Raquel Acácia Pereira Gonçalves Santos a,  

Nicole Sartoreto da Rocha b,  
 Jhéssica Letícia Santos Santana c  

and Regiane Patussi Santos Lima d  
 

a Instituto Alergoimuno de Americana, Brazil. 
b Faculdade de Americana, São Paulo, Brazil. 

c Instituto de Ensino e Pesquisa do Hospital de Amor de Barretos, Brazil. 
d Lavoisier laboratórios, São Paulo, Brazil. 

 
Authors' contributions  

 
The authors conducted this work in collaboration. The author CEO is responsible for the 

conceptualization, data curation, formal analysis, literature review, and writing the original draft. 
Authors DGP, APMT, CSM, NSR, JLSS and RPSL 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/v8i1156  
 

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/131451  

 
 

Received: 15/12/2024 
Published: 20/02/2025 

 
 
 
 

Original Research Article 

https://doi.org/10.9734/aji/2025/v8i1156
https://pr.sdiarticle5.com/review-history/131451


 
 
 
 

Olivier et al.; Asian J. Immunol., vol. 8, no. 1, pp. 8-25, 2025; Article no. AJI.131451 
 
 

 
9 
 

ABSTRACT 
 

Background: Allergies to citrus fruits are often associated with pollinosis due to cross-reactivity 
among pollen and food allergens (fruit-pollen syndrome) displayed in patients with several 
conditions, such as allergic rhinoconjunctivitis. 
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 
Citrus spices (orange and lemon) and a pollen extract in patients with non–IgE-mediated allergic 
rhinoconjunctivitis. 
Study Design: We retrospectively examined the medical charts of two cohorts of patients clinically 
diagnosed with non–IgE-mediated rhinoconjunctivitis with clinical suspicion of hypersensitivity 
against Citrus spices and pollen, who were investigated with the help of TTP or LAIT, 
simultaneously tested against individual extracts of orange, lemon and pollen. 
Methodology: The registered results of TTP and LAIT were distributed in ranges through a 
cascade distribution chart to outline the variability of the results. Dispersion graphs plotting the 
results of LAIT between the results of each pair of allergens were presented. The statistical 
significances were calculated.  
Results: The TTP for the pollen, orange, and lemon extracts showed a distribution concentrated 
on the higher dilutions, precluding an adequate differentiation among patients’ immunoreactivities. 
On the contrary, the LAIT results showed a wide distribution of results, demonstrating a better 
potential to differentiate patients and predict hypersensitivity. While the TTP results showed a slight 
correlation between the paired tests (Pearson’s correlation coefficient between r = 0.007 to 0.11), 
the LAIT results demonstrated a significant moderate correlation between the paired assays, 
projecting a better potential to predict cross-reactivity among the allergens (Pearson’s correlation 
coefficient between r = 0.43 to 0.56). 
Conclusion: Our preliminary results support that the TTP and LAIT performed with orange, lemon, 
and pollen extracts can potentially discriminate diverse degrees of humoral and cellular 
immunoreactivity in non–IgE-mediated allergic rhinoconjunctivitis patients. 
 

 

Keywords: Conjunctivitis; Hypersensitivity; Lemon; Leukocyte Adherence Inhibition Test; Orange; 
Pollen; Precipitins; Rhinitis. 

 

ABBREVIATIONS 
 

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

1. INTRODUCTION  
 

Citrus is a genus of wild and domesticated fruit 
plants in the Rutaceae family in which several 
cultivars are classified, such as oranges (e.g., 
Citrus sinensis), mandarins (e.g., Citrus 
reticulata), grapefruits (e.g., Citrus paradisi), 
pomelos (e.g., Citrus maxima), limes (e.g., Citrus 
latifolia), and lemons (e.g., Citrus limon) (Wu et 
al. 2018). 
 

A survey performed by the Good Housekeeping 
Institute pointed citrus fruits among the top ten 
foods perceived by food-allergic people as 
responsible for their symptoms (Sloan and 
Powers 1986).  
 

Allergies to citrus fruits are often associated with 
pollinosis due to a phenomenon of cross-

reactivity among pollen and food allergens (fruit-
pollen syndrome), displayed in patients with 
combined conditions such as allergic rhinitis, 
allergic conjunctivitis, allergic bronchitis, oral 
allergy syndrome, urticaria, angioedema, 
digestive symptoms and anaphylaxis (Iorio et al. 
2013, Li et al. 2022, Canonica et al. 2013, 
Armentia et al. 2016, Hasnain, Alqassim and Al-
Frayl 2017, Bartra et al. 2009). Nasal provocation 
with pollen extracts is an uncomplicated way to 
diagnose pollen hypersensitivity (Doyen et al. 
2018). Fruit-pollen syndrome is also frequently 
associated with latex allergy, sometimes called 
latex-pollen-fruit syndrome or latex-fruit-pollen 
syndrome (Čelakovská et al. 2024, Olivier et al. 
2022b). Pollen-derived products such as honey 
and royal jelly were described as triggers for 
anaphylaxis and urticaria in patients with 
pollinosis (Fuiano et al. 2006, Lombardi et al. 
1998). 

 
The Allergen Nomenclature Sub-Committee of 
the World Health Organization and International 
Union of Immunological Societies (WHO/IUIS) 



 
 
 
 

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has recognized so far four allergens weighting 
from 8 to 23 kDa, identified from the sweet 
orange (Citrus sinensis), according to their official 
nomenclature: Cit s 1 (Germin-like protein), Cit s 
2 (Profilin), Cit s 3 (Non-specific lipid-transfer 
protein type 1) and Cit s 7 (Gibberellin regulated 
protein) (Sub-Committee 2024c). The same Sub-
Committee listed one allergen from lemon (Citrus 
limon): Cit l 3, a 9.6 KDa non-specific lipid-
transfer protein type 1 (Sub-Committee 2024a), 
as well as one allergen from mandarin (Citrus 
reticulata): Cit r 3, a 9 KDa non-specific lipid-
transfer protein type 1 (Sub-Committee 2024b). 
 
The orange profilin, Cit s 2, has an amino acid 
sequence similar to pollen profilins, such as the 
birch Bet v 2 (73% identity) (López-Torrejón et al. 
2005). Profilins are plant pan-allergens 
responsible for cross-sensitization between 
pollen and plant-derived foods (Valenta et al. 
1992). Pollen and plant food profilin allergens 
show equivalent IgE and IgG reactivity, are 
quickly inactivated by gastric digestion, and are 
commonly involved in polysensitization of allergic 
patients (Sirvent et al. 2011). Profilin 
hypersensitivity is common in patients with cross-
reactivity to pollen and fruits such as oranges, 
pineapples, melons, watermelons, tomatoes, and 
bananas (Asero et al. 2003, Asero, Monsalve 
and Barber 2008).  
 
Gibberellin-regulated proteins (GRP) are a group 
of emergent allergens described in orange, 
Japanese apricots, sweet cherries, 
pomegranates, bell peppers, strawberries, and 
also in pollen from the Cupressaceae tree 
(cypress) family (Iizuka et al. 2022). GRP 
hypersensitivities are clinically associated with 
severe adverse reactions, such as a case of 
orange-induced anaphylaxis (Kàtcheff et al. 
2024, Özdemir 2024). Cystatin-like proteins 
found in freshly squeezed orange juice also 
produced angioedema, dysphonia, and dyspnea 
(Solórzano-Zepeda et al. 2021). Cystatins are 
enzymes playing several roles in 
microorganisms, plants, pollens, animals, and 
humans already considered autoallergens since 
they were implicated in autoallergies through 
cross-reaction IgE-mediated and T-cell mediated 
hypersensitivities (Rogers et al. 1993, Roesner et 
al. 2022). 
 
Citrus seeds also contain reaginic proteins that 
may produce allergic reactions when mixed with 
whole-fruit-crushed juices or when accidentally 
ingested, producing anaphylaxis, urticaria, and 
respiratory and digestive symptoms in patients 

who otherwise tolerate squeezed juices (Kayode 
et al. 2020, Glaspole et al. 2007, Wang 2008). 
Reactions to citrus seeds are related to 
hypersensitivity to citrin, an 11S globulin 
belonging to the cupin superfamily, which cross-
react with cashew and pistachio allergens 
(Konstantinou et al. 2023). Citrus-induced 
phytophotodermatitis is associated with the 
presence of coumarins and furocoumarins 
(psoralens, xanthotoxins, and bergaptens) in the 
peel or the juice of the fruit, involving cutaneous 
photosensitivity, phototoxicity, and/or 
photoallergy (Jiang et al. 2024, Belcadi, Oulad Ali 
and Senouci 2024). Sensory hypersensitivity 
(photophobia and osmophobia) in patients with 
migraine are also related to citrus fruits (Vitali-
Silva et al. 2024). 
 
 Non–IgE-mediated cellular immunoreactivity 
against food allergens had already been reported 
by our group with the help of the Leukocyte 
Adherence Inhibition Test (LAIT), as well as 
humoral immunoreactivity against food allergens 
with the help of Tube Titration of Precipitins 
(TTP) (Olivier et al. 2021d, Olivier et al. 2022a, 
Olivier et al. 2022c, Olivier et al. 2024c, Olivier et 
al. 2024b, Olivier et al. 2024a). We routinely 
employ the LAIT and the TTP in our facilities as a 
triage to evaluate non–Non–IgE-mediated 
immunoreactivity against suspected allergens 
before performing more exhaustive in vivo 
provocation tests (Kuratsuji 1981, Olivier et al. 
2023e, Olivier et al. 2023g, Olivier et al. 2023c, 
Olivier et al. 2023b, Olivier et al. 2023d, Olivier et 
al. 2024f). To evaluate the potential of the LAIT 
and TTP to endotyping Non–IgE-mediated 
cellular and humoral immunoreactivity against 
orange, lemon, and pollen extracts, we 
retrospectively compiled the electronic medical 
charts of patients diagnosed with non–IgE-
mediated rhinoconjunctivitis 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 LAIT and the TTP may 
differentiate diverse degrees of cellular and 
humoral immunoreactivity against orange, lemon, 
and pollen allergens among patients suffering 
from non–IgE-mediated rhinoconjunctivitis. As 
the tests were performed simultaneously with the 
same venous sample with the three allergens, it 
is possible to calculate two-sample paired t-tests 
between each pair of LAIT results (since they 
refer to the same quantitative variable), as well to 
present a dispersion graph between them to 



 
 
 
 

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11 

 

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; 01/2025), we reviewed the 
electronic chart of 9,980 outpatients who 
attended our facility from January 2018 to 
February 2025.  

 
A cohort of 100 consecutive outside patients 
(TTP cohort) had been simultaneously submitted 
to TTP with orange extract, lemon extract, and 
pollen extract for presenting non–IgE-mediated 
allergic rhinoconjunctivitis. This cohort counted 
28 males; mean age 37.8 years; SD 20.4 years; 
range 4 to 88 years; median 32.5 years; modes = 
29 (appeared seven times); geometric mean = 
29.4 years.  

 
A cohort of 100 consecutive outside patients 
(LAIT cohort) had been simultaneously submitted 
to TIAL with orange extract, lemon extract, and 
pollen extract for presenting Non–IgE-mediated 
allergic rhinoconjunctivitis. This cohort counted 
35 males; mean age 42.9 years; SD 18.7 years; 
range 8 to 76 years; median 38 years; modes = 9 
and 38 years (each appeared five times); 
geometric mean = 32.2 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 Citrus spices 
hypersensitivity who demonstrated a non-
reactive or inconclusive skin test against pollen, 
orange, and lemon extracts (Olivier et al. 2013). 

 
2.2 Extracts 
 
2.2.1 Orange extract 

 
The whole orange (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 the 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 orange extract solution 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 Lemon extract 
 
The lemon extract solution was prepared using a 
similar technique employed for the orange 
extract. 
 
2.2.3 Pollen extract 
 
The pollen’s protein extraction was performed as 
follows: in a beaker, 5g of dehydrated 
beekeeping pollen, acquired from a local 
provider, was added to the Coca-based extractor 
solution to cover the amount of pollen. The 
sample was crushed and then left for 48 hours at 
4 °C. The sample was centrifuged (4,500 rpm for 
10 min) and filtered. The protein concentration 
was estimated spectrophotometrically and diluted 
to 1 mg/mL in antigen dilution solution (NaCl 10g, 
KH2PO4 0.72g, Na3PO4 2.86g, methylparaben 
1g, propylparaben 0.5g, glycerin 400 mL, H2O 
600mL) and used to perform the LAIT and 
allergic skin 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, Olivier et al. 2022b, Olivier et al. 2022a, 
Olivier et al. 2022c, Olivier et al. 2023e, Olivier et 
al. 2023g, Olivier et al. 2023a, Olivier et al. 
2024e). Shortly, each donor's fresh plasma was 
divided into two parts and used in parallel ex vivo 
challenging tests with the orange or lemon 
extracts and the unchallenged plasma (added 
with antigen dilution solution as a control). We 
collected plasma with high leukocyte content 
(buffy coat) from the heparinized tube after one 
hour of sedimentation at 37 °C. Then, we 
distributed aliquots of 100 μL into Eppendorf 
tubes with (or without) the challenging extract 



 
 
 
 

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and kept them under agitation for 30 minutes 
(200 rpm at 37 °C). 

 
2.3.2 LAIT: Procedure for adherence assay  
 

After incubation, the plasma was allocated into a 
standard Neubauer hemocytometer counting 
chamber with a plain, non-metallic glass surface 
and left to stand for 2 hours at 37 °C in the 
humidified atmosphere of the covered water bath 
to allow leukocytes to adhere to the glass. Next, 
we counted the leukocytes, removed the 
coverslip, and washed the chamber by 
immersion in a beaker with phosphate buffer 
saline (PBS) at 37 °C. Then, we added a drop of 
PBS to the hemocytometer's chamber and 
allocated a clean coverslip over it. The remaining 
cells were counted in the same squares as 
previously examined.  

 
2.3.3 LAIT: Procedure for calculation  

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

 
2.4 TTP: In vitro Investigation: Tube 

Titration of Precipitins 
 
As previously reported, the semi-quantitative TTP 
against the aluminum 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 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 (Williams and 
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 orange extract showed a 
distribution concentrated on the higher dilutions 
(Fig. 1). There were no negative results. There 
were no positive results in the range from 1:1 to 
1:32 dilutions. The mean was estimated at 1:347; 
the median was 1:256; the standard deviation 
was estimated at 1:169; the mode was 1:512 
(appeared 49 times).  

 
The TTP for the lemon extract showed a 
distribution concentrated on the higher dilutions 
(Fig. 2). There were no negative results. There 
were no positive results in the range from 1:1 to 
1:32 dilutions. The mean was estimated at 1:358; 
the median was 1:256; the standard deviation 
was estimated at 1:157; the mode was 1:512 
(appeared 49 times).  

 
The TTP for the pollen extract showed a 
distribution concentrated on the higher dilutions 
(Fig. 3). There were no negative results. There 
were no positive results in the range from 1:1 to 
1:32 dilutions. The mean was estimated at 1:387; 
the median was 1:512; the standard deviation 
was estimated at 1:155; the mode was 1:512 
(appeared 59 times).  

 
The LAIT for the orange extract showed a wide 
distribution range of results (Fig. 4). Most results 
were concentrated in the more immunoreactive 
groups. There were five negative results. The LAI 
ranged from 0% to 98%. The mean was 62.6%; 
the median was 68.5%; the standard deviation 
was 26.9%; the mode was 0% (appeared five 
times). Some patients showed low or moderate 
immunoreactivity during the ex vivo challenge 
test. In contrast, others displayed strong 
immunoreactivity, which could reflect the 
participation of orange allergens in a Non–IgE-



 
 
 
 

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mediated hypersensitivity condition in these 
patients. 
 

The LAIT for lemon extract showed a wide 
distribution range of results (Fig. 5). Most were 
concentrated in the more immunoreactive 
groups. There were six negative results. The LAI 
ranged from 0% to 99%. The mean was 54%; the 
median was 56.5%; the standard deviation was 
27.7%; the mode was 0% (appeared six times). 
Some patients showed low or moderate 
immunoreactivity during the Ex vivo challenge 
test. In contrast, others displayed strong 
immunoreactivity, which could reflect the 
participation of lemon allergens in a Non–IgE-
mediated hypersensitivity condition. 
 

The LAIT for pollen extract showed a wide 
distribution range of LAI results (Fig. 6). There 
were three negative results. The LAI ranged from 
0% to 98%. The mean was 55.9%; the median 
was 60.5%; the standard deviation was 27.5%; 
and the mode was 79% (appeared four times). 
Some patients showed low or moderate 
immunoreactivity in response to the ex vivo 
challenge test. Most displayed strong 
immunoreactivity, which could reflect the 
participation of pollens in the non–IgE-mediated 
hypersensitivity of these patients. 
 

The paired-t test indicated a non-significant slight 
difference between the results of lemon TTP and 
orange TTP (p = .640). Pearson’s correlation 
indicated a non-significant small positive 
relationship between lemon TTP and orange TTP 
results: r(98) = .00798, p-value = .937. 

The paired-t test indicated a non-significant slight 
difference between pollen TTP and orange TTP 
results (p-value = .077). Pearson’s correlation 
indicated a non-significant small positive 
relationship between pollen TTP and orange TTP 
results: r(98) = .0761, p-value = .452. 
 
The paired-t test indicated a non-significant slight 
difference between pollen TTP and lemon TTP 
results (p-value = .172). Pearson’s correlation 
indicated a non-significant small positive 
relationship between pollen and lemon TTP 
results: r(98) = .114, p-value = .259. 
 
The paired t-test indicated a significant difference 
between orange and lemon LAIT results (p-
value = 0.02741). However, Pearson’s 
correlation indicated a significantly moderate 
positive relationship between the orange                    
and lemon LAIT results: r(98) = .45, p-value < 
.001. 

 
The paired t-test indicated a significant difference 
between pollen and orange LAIT results (p-
value = 0.08241). However, Pearson’s 
correlation indicated a significant positive 
relationship between pollen and orange LAIT 
results: r(98) = .56, p-value < .00. 

 
The paired t-test indicated a significant difference 
between pollen and lemon LAIT results (p-
value =. 0.6322). However, Pearson’s correlation 
indicated a significant positive relationship 
between pollen and lemon LAIT results: r(98) = 
.43, p-value < .001. 

 

 
 

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

 

 
 
Fig. 4. Cascade distribution chart of the range groups of Leukocyte Adherence Inhibition (LAI) 

results (x-axis %) of the ex vivo challenge test against orange 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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Fig. 5. Cascade distribution chart of the range groups of Leukocyte Adherence Inhibition (LAI) 
results (x-axis %) of ex vivo ex vivo challenge test against lemon 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. 6. Cascade distribution chart of the range groups of Leukocyte Adherence Inhibition (LAI) 
results (x-axis %) of ex vivo challenge test against pollen 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) 
 

4. DISCUSSION 
 

Charles Blackley reported the first diagnosis of 
pollinosis when conceiving the skin provocation 
test to associate the respiratory symptoms of his 
patients to pollen inhalation (Blackley 1873). 
 

Pollinosis is preferably referred to as pollen 
allergy or, by extension, allergic 
rhinoconjunctivitis due to the frequent association 
of nasal and conjunctival symptoms. Except                 
for desertic or icy regions, pollinosis is a disease 

that occurs worldwide (Peternel et al. 2007, 
Ferreiro, Dopazo and Aira 2002, Xu et al. 2000, 
Nakamura 1996, Tilandyová et al. 1989, 
Wüthrich et al. 1986, Noferi, Ferrante and              
Testa 1965, Negrini and Belloni 1963, Bean                
and Glaser 1960). Pollinosis in Brazil has 
regional characteristics dependent on local                 
flora and regional crops (Oliveira et al. 2020, 
Rosario 1990, Taketomi et al. 2006, Taketomi et 
al. 2005). Our facility borders a city called 
“Limeira” due to its excellent production of lime 



 
 
 
 

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16 

 

oranges, from where several of our patients 
come. 
 

The two major phenotypes related to allergic 
rhinoconjunctivitis are “seasonal” and “perennial.” 
Perennial allergic rhinoconjunctivitis is usually 
related to house dust mites, and seasonal 
allergic rhinoconjunctivitis is usually related to 
pollinosis (at least in regions with well-defined 
climatic stations) (Lluch-Bernal et al. 2024). 
Atmospheric pollen concentration is a standard 
parameter regularly measured by air controller 

agencies and is strongly related to pollinosis 
symptoms (Sheng et al. 2022). Allergic 
rhinoconjunctivitis is a prototype model of 
disease that may be classically produced by IgE-
mediated hypersensitivity and non–IgE-mediated 
hypersensitivity (Olivier et al. 2023f, Yamana, 
Yamana and Uchio 2022). Longitudinal 
Clustering Analysis has recently characterized 
novel rhinoconjunctivitis phenotypes; however, 
several questions have not yet been answered 
(Togias et al. 2025). 

 

 
 

Fig. 7. Dispersion chart of the Leukocyte Adherence Inhibition (LAI) results of the ex vivo 
challenge test against lemon extract (x-axis %), plotted against the LAI results of the ex vivo 

challenge test against orange extract (y-axis %) 
 

 
 

Fig. 8. Dispersion chart of the Leukocyte Adherence Inhibition (LAI) results of the ex vivo 
challenge test against pollen extract (x-axis %), plotted against the LAI results of the ex vivo 

challenge test against orange extract (y-axis %) 



 
 
 
 

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17 

 

 
 

Fig. 9. Dispersion chart of the Leukocyte Adherence Inhibition (LAI) results against pollen 
extract (x-axis %), plotted against LAI results of the ex vivo challenge test against lemon 

extract (y-axis %) 
 
A well-studied non–IgE-mediated hypersensitivity 
endotype responsible for pollen-related allergic 
conjunctivitis and allergic blepharitis is the 
Macrophage Migration Inhibitory Factor, a 
cytokine responsible for eosinophil accumulation 
in the conjunctivas and eyelid dermis exposed to 
pollen (Nagata et al. 2015). Migration Inhibition 
Factors (MIFs) were the first lymphokines related 
to delayed hypersensitivity (Bloom and Bennett 
1966). MIFs are pluripotent cytokines essential in 
non–IgE-mediated allergic inflammation, 
recruiting reaginic cells, such as macrophages 
and eosinophils, to the inflammatory site (Das et 
al. 2011). MIFs are essential cytokines for T cell 
activation and sustainment of innate 
proinflammatory responses (Mitchell et al. 2002). 
The LAIT is an easy and affordable way to put in 
evidence the possible participation of leukocyte 
(or macrophage) inhibition cytokines in ex vivo 
challenge tests with allergens (Rocklin 1974, 
Dunn and Halliday 1980). 
 
Despite being considered a respiratory condition, 
proteins bearing correlated pollen epitopes are 
usually eaten through fruits and vegetables, 
producing cross-reacting allergic reactions 
(Andersen, Hall and Dragsted 2011, Worm et al. 
2014).  
 
Endotyping biomarkers of cellular and humoral 
immunoreactivity and cross-reactivity are 
essential to build better strategies to impersonate 
treatments for allergic patients (Agache and 
Akdis 2020). At the clinical set, diagnosis of IgE-
mediated hypersensitivity is an easy task, 

accomplished by anamnesis, skin tests, and the 
laboratory research of specific IgE; however, to 
diagnose non–IgE-mediated hypersensitivity, it is 
necessary to employ a multi-omics approach to 
differentiate the particularities of the variety of 
clinical phenotypes and immune endotypes 
responsible for allergic diseases (Macowan et al. 
2025, Yoon and Bunyavanich 2025, Khan 2016). 
The concept of immune dysregulation is evolving, 
and besides the major primary 
immunodeficiencies, there are secondary 
immunodeficiencies following inflammatory 
conditions raised by immune hypersensitivities, 
clinically known as allergies (Henrickson 2025). 
 
The semi-quantitative research and titration of 
precipitins is a pioneering laboratory exam upon 
which the fundamental bases of Immunology 
were constructed (Wells 1911). Precipitating 
antibodies suggest the presence of a humoral 
immune response against the tested antigens 
(Gell, Harington and Rivers 1946). Before the 
discovery of IgE, the research of precipitins 
against pollen and mold allergens was the 
leading way to realize in vitro diagnostic of 
immunoreactivity against these agents (Augustin 
and Hayward 1960, Augustin, Hayward and 
Longbottom 1960). 
 
Precipitins to pollen allergens are obtained after 
sensitization of guinea pigs with Phleum pratense 
and Dactylis glomerata pollens, producing 
antiserum reactive against these pollens and 
cross-reactive against Festuca pratensis and 
Cynodon dactylon (Augustin 1953).  



 
 
 
 

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18 

 

The LAIT is an ex vivo challenge test performed 
with a viable leukocyte buffy coat that can 
theoretically explore most known immune 
pathways as it allows the interaction of all 
immune-circulating participants with the allergens 
Several immune pathways can produce the final 
leukocyte adherence inhibition (Olivier et al. 
2021a). 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 orange, lemon, 
and pollen allergens among patients suffering 
from non–IgE-mediated rhinoconjunctivitis. 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 
distinguish some order of cross-reactivity 
between them.  
 
The retrospective compilation of our data showed 
a large distribution of results when we 
ascertained the results of TTP and TIAL to 
explore humoral and cellular immunoreactivity 
against two Citrus allergens and a pollen extract. 
These immunoassays did not precisely identify 
the mechanisms responsible for clinical 
conditions. Instead, they provide evidence about 
cellular and humoral immunoreactivity distributed 
into an extensive spectral range that may 
suggest immune tolerance or hypersensitivity. 
 
The TTP for the pollen, orange, and lemon 
extracts showed a distribution concentrated on 
the higher dilutions, precluding an adequate 
differentiation among patients’ 
immunoreactivities. Further studies performed 
with assays extended to higher dilutions are 
needed to achieve more reliable conclusions. On 
the contrary, the LAIT results showed a wide 
distribution of results, demonstrating a better 
potential to differentiate patients and predict 
hypersensitivity. While the TTP results showed a 
slight correlation between the paired tests, the 
LAIT results demonstrated a moderate 
correlation between the paired assays, projecting 
a better potential to predict cross-reactivity 
among the allergens. 
 

On the contrary, the LAIT results showed a wide 
distribution of results, demonstrating a better 
potential to differentiate patients and predict 
hypersensitivity. While the TTP results showed a 
slight correlation between the paired tests 
(Pearson’s correlation coefficient between r = 
0.007 to 0.11), the LAIT results demonstrated a 
significant moderate correlation between the 
paired assays, projecting a better potential to 

predict cross-reactivity among the allergens 
(Pearson’s correlation coefficient between r = 
0.43 to 0.56). 
 
This preliminary retrospective survey 
demonstrated extensive results from the TTP and 
the ex vivo challenge test monitored by LAIT 
against pollen and two Citrus species in two 
cohorts of non–IgE-mediated rhinoconjunctivitis 
patients. 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. Our results 
suggest that pollen-allergic patients may impair 
their symptoms by additional cross-
immunoreactivity against Citrus allergens. 

 

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 
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 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 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 pollen, orange, and 
lemon extracts in patients clinically diagnosed 
with non–IgE-mediated allergic 
rhinoconjunctivitis. TIAL and TTP are 
inexpensive, can be performed with minimum 
laboratory equipment, and can be incorporated 
into strategies to address health disparities in 



 
 
 
 

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19 

 

respiratory and food allergies. As a preliminary 
report, the propaedeutic meaning of the 
presented results and the possibility of 
interferents must be yet established. 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 citrus fruits 
and pollen allergens. 
 

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. 

 

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. 
 

ETHICAL APPROVALS 
 

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

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 
 
The authors have declared that no competing 
interests exist. 

 
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