







































_____________________________________________________________________________________________________ 
 
*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, Raquel Acácia Pereira Gonçalves Santos, and 
Everton Salgado Monteiro. 2025. “Endotyping Cellular and Humoral Cross-Reactivity Among Canine, Feline, and Swine 
Allergens in Patients With Allergic Multimorbidity”. Asian Journal of Immunology 8 (1):142-62. 
https://doi.org/10.9734/aji/2025/v8i1168. 

 

Asian Journal of Immunology 
 
Volume 8, Issue 1, Page 142-162, 2025; Article no.AJI.138781 
 

 
 

 

 

Endotyping Cellular and Humoral 
Cross-reactivity among Canine, Feline, 

and Swine Allergens in Patients with 
Allergic Multimorbidity 

 
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, 
Raquel Acácia Pereira Gonçalves Santos a 

and Everton Salgado Monteiro e 

 
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. 
e Faculty of Medicine, São Paulo University, Brazil. 

 
Authors’ contributions 

 
The authors conducted this work in collaboration with each other. The author CEO is responsible for 

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

extracted the proteins from the fur of cats and dogs. Author RAPGS performed cutaneous tests. All 
authors read and approved of the final manuscript. 

 
Article Information 

 
DOI: https://doi.org/10.9734/aji/2025/v8i1168  

 
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/138781  

 
  

https://doi.org/10.9734/aji/2025/v8i1168
https://pr.sdiarticle5.com/review-history/138781


 
 
 
 

Olivier et al.; Asian J. Immunol., vol. 8, no. 1, pp. 142-162, 2025; Article no.AJI.138781 
 
 

 
143 

 

Received: 16/06/2025 
Published: 03/07/2025 

 
 

ABSTRACT 
 

Background: The cat-pork syndrome and the cross-reactivity between cat dander and dog dander 
are particular situations associated with the allergic multimorbidity phenotypes. The perception of 
this cross-reactivity must benefit the allergist dealing with these conditions. 
Aim: To evaluate the capacity of the Tube Titration of Precipitins (TTP) and the Leukocyte 
Adherence Inhibition Test (LAIT) to discriminate cellular and humoral immunoreactivity against cat 
fur and dog fur extracts as well as pork meat extract in patients with non–IgE-mediated 
multimorbidity phenotypes. 
Study Design: We examined retrospectively the medical charts of two cohorts of patients clinically 
diagnosed with non–IgE-mediated multimorbidity allergic phenotypes related to inhalation or contact 
with cat fur, dog fur, and/or consumption of pork meat, who were investigated with the help of TTP 
or LAIT. 
Methodology: The correlation between the paired assays was calculated using Pearson's 
methodology and demonstrated by dispersion graphs.  
Results: Pearson's correlation indicated a significant positive relationship between the cat                            
fur and dog fur extracts in LAIT results: r (98) = 0.395, p-value < 0.001. The paired-t test                   
indicated a significant difference between the TTP results of cat fur and pork meat extracts                     
(p-value = 0.009). Pearson correlation analysis indicated a non-significant, negative                    
relationship between the TTP results of cat fur and pork meat (p-value = 0.644). The paired t-test 
indicated a significant difference between the TTP results of dog fur and pork meat extracts                   
(p < 0.001). 
Conclusion: The preliminary results suggest that the TTP and LAIT may discriminate between 
diverse humoral and cellular immunoreactivity levels in patients with various allergic phenotypes, as 
observed in cat and dog fur and pork meat extracts. A significant association was found between 
the immunoreactivity of cat fur and dog fur. There was no clear association between pork meat and 
fur immunoreactivity. 
 

 
Keywords:  Endotype; hypersensitivity; cat fur; dog fur; leukocyte adherence inhibition test; non–ige-

mediated immunoreactivity; pork meat; precipitins. 
 

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

1. INTRODUCTION 
 
Hypersensitivity conditions, such as allergic 
rhinoconjunctivitis and allergic bronchitis caused 
by sensitization to allergens from furry pets, 
affect more than 10% of the worldwide 
population, producing a deleterious impact on 
patients' quality of life (Chan and Leung 2018; 
van Hage et al. 2023; Konradsen et al. 2015).  
 
Allergic Multimorbidity (defined in patients with 
concomitant or consecutive allergic phenotypes) 
is the subject study for the "Mechanisms of the 
Development of ALLergy" (MeDALL) project to 

understand the links between multimorbidity and 
polysensitization in allergic phenotypes that may 
be IgE-mediated, partly IgE-mediated, or non-
IgE-mediated (Bousquet et al. 2015; Bousquet et 
al. 2025).  
 
Allergic Multimorbidity is a concept associated 
with the "Atopic March", a pattern of allergic 
disease development recognized in children with 
sequential and concomitant sensitization to food 
allergens and inhalant allergens (Hahn and 
Bacharier 2005; Kulig et al. 1999). 
 
Allergic sensitization may happen at any age. In 
general, no clinical symptoms are detectable at 
birth; however, the earlier sensitization is usually 
directed against food proteins (hen's egg and 
cow's milk, occurring via the mother's milk), while 
sensitization to environmental allergens and 
other food allergens (such as soy and wheat 

Original Research Article 



 
 
 
 

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144 

 

allergens) are increasingly progressing after the 
first birthday (Wahn 2000). 
 
Initial signs of allergic disease are atopic 
dermatitis and food allergies, which typically 
have their most significant incidence peaks 
during the first two years of life. Nearly half of 
children with atopic dermatitis develop symptoms 
within the first six months of life, and 
approximately 85% of individuals with eczema 
have symptoms onset by the age of five years 
(Kay et al. 1994). 
 
Allergic multimorbidity represents a group of 
conditions that deleteriously impair the quality of 
life and usually involves polysensitization in 
which cat and dog dander may be particularly 
associated (Li et al. 2025). Cross-reactivities 
among related and unrelated allergens are 
standard features in allergic patients for whom 
allergen immunotherapy represents the 
appropriate treatment, necessitating the 
identification of responsible allergens (Liang et 
al.; 2024; Alvarez-Cuesta et al.; 2007; Patel et 
al.; 2013; Varney et al.; 1997; Worm; et al. 2013). 
Some murine models have been developed to 
study cross-reactivity among multiple food 
allergens (Musa et al. 2024). Cross-reactivity 
between furry animals and animal-derived food 
allergens is under review (Gromek et al., 2025). 
 
The Allergen Nomenclature Sub-Committee of 
the World Health Organization and International 
Union of Immunological Societies (WHO/IUIS) 
has recognized so far eight allergens weighing 
from 11 to 1,000 KDa, identified from the 
domestic cat (Felis domesticus) named after Fel 
d 1 (uteroglobin) to Fel d 8 (latherin-like protein) 
(Sub-Committee 2025b). The same Sub-
Committee has recognized eight allergens so far, 
weighing from 14 to 29 kDa, identified in the 
domestic dog (Canis familiaris), named after Can 
f 1 (Lipocalin) to Can f 8 (Cystatin) (Sub-
Committee, 2025a). The Allergen Nomenclature 
Sub-Committee of the World Health Organization 
and the International Union of Immunological 
Societies (WHO/IUIS) has recognized one 
allergen from the domestic pig (Sus scrofa) to 
date: a serum albumin of 60 kDa, named Sus s 1 
(Sub-Committee, 2025c). 
 
Cross-reactivity between cat fur and dog fur is a 
particular situation, as dog extracts contain a Fel 
d 1-like allergen that is cross-reactive to Fel d 1 
(Hellu et al., 2024). Molecular mimicry between 
unrelated mammalian proteins from different 
animal species has been associated with 

unexpected cross-reactive allergic reactions, 
particularly between proteins from cat dander 
and pork meat (Kile et al., 2023). The first reports 
of a direct cross-reactivity between cat dander 
and pork meat came from France in 1994, 
manifested as urticaria associated with 
abdominal symptoms, and were named the "cat-
pork syndrome" (also referred to as pork-cat 
syndrome) (Drouet et al. 1994; Drouet; et al 
1994a; Drouet et al. 1994). The first case report 
was accompanied by an extensive investigation 
performed with skin tests, specific IgE 
quantification, electrophoresis, Western Blot, and 
chromatography that confirmed a crossed 
reaction against a common epitope from pork 
meat and cat extract, a protein with a molecular 
weight of 67 kDa (Sabbah et al. 1994a; Sabbah 
et al. 1994b). After this initial description, several 
cases in France were further reported, 
associating the syndrome with cross-reactivity 
against similar allergens, such as dog dander, 
boar meat, and heparin (Couturier et al. 1999; 
Drouet et al. 2001; Drouet; et al. 1997; Drouet 
and Sabbah 1996). 
 
The first description of the so-called cat-pork 
syndrome in the United States of America was 
published in 2013, reporting eight patients with 
elevated specific IgE against cat dander, dog 
dander, and pork meat allergens. The more 
illustrative cases presented abdominal cramping, 
nausea, itching, and hives after ingestion of pork 
(Posthumus et al. 2013). In 2014, Spanish 
physicians also described a case of occupational 
asthma in a patient sensitized to cat dander, dog 
dander, and pork meat (Alvarez-Perea et al., 
2014). Soon, in 2015, the cat-pork syndrome 
became just one more example of cross-
reactivity among aeroallergens and food 
allergens (Popescu 2015). 
 
Further, in 2019, Japanese investigators reported 
a case of early childhood onset pork-cat 
syndrome associated with dog sensitization. A 6-
year-old girl presented with recurrent episodes of 
urticaria after consumption of pork meat with 
specific IgE (≥ 50 UI/mL) against cat dander, dog 
dander, pork meat, Sus s 1, Fel d 2, Can f 1, Can 
f 2, and Can f 3. Western blotting analysis 
demonstrated specific IgE activity against a 67-
kDa protein in pork meat and cat dander extract. 
Cross-reactivity between these two proteins was 
confirmed by an inhibition test (Yamada et al. 
2019). More recently, Component Resolved 
Diagnosis associated Fel d 2 and Sus s 1 as the 
main molecular allergens involved in cross-
reactivity (Barradas Lopes et al. 2022). 



 
 
 
 

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The Alpha-gal syndrome is another condition that 
warrants careful diagnosis, as it may present 
cross-reactive allergens in cat and dog dander, 
as well as pork meat (Commins and Platts-Mills, 
2013). First put in evidence in 2009, the alpha-
gal syndrome is a cross-reactive allergic 
multimorbidity condition elicited by IgE and non-
IgE antibodies against the galactose-α-1,3-
galactose (α-gal), a carbohydrate moiety 
commonly expressed on non-primate 
mammalian proteins (such as beef, pork, and 
lamb), and in the cat IgA (Commins et al. 2009). 
Allergy against α-gal is usually triggered by tick 
bites (Van Nunen et al. 2009). 
 
Besides the classic Gell and Coombs IgE-
mediated type I hypersensitivity mechanism, 
several types and subtypes of non-IgE-mediated 
hypersensitivity mechanisms are being studied, 
associated with various allergic phenotypes 
(Jutel et al., 2023). 
 
Humoral immunoreactivity against food allergens 
and aeroallergens has been traditionally 
evaluated through research on precipitins 
(Augustin, 1953; Augustin, et al. 1960; 
Cunningham-Rundles et al., 1978; Ferguson and 
Carswell, 1972; Heiner, et al. 1962). 

 
We also routinely employ the Tube Research of 
Precipitins (TTP) in our facilities as a triage to 
evaluate humoral non–IgE-mediated 
immunoreactivity against suspected allergens 
before performing more exhaustive in vivo 
provocation tests (Olivier et al. 2023e; Olivier et 
al. 2021e; Olivier et al. 2021d; Olivier et al. 
2024e; Olivier et al. 2024c). 

 
The Leukocyte Adherence Inhibition Test (LAIT) 
and its similar assay, the Leukocyte Migration 
Inhibition Test (LMIT), have traditionally been 
used to differentiate non–IgE-mediated 
immunoreactivity against microorganisms and 
aeroallergens (Fink et al. 1987; Kallen and 
Nilsson 1979; Kuratsuji 1981; Thomson 1982). 
The LAIT and the LMIT have also been used to 
differentiate non–IgE-mediated immunoreactivity 
against food allergens (Allardyce and Shearman 
1975; George and Vaughan 1962; Ashkenazi et 
al. 1980; Butler et al. 1981; Papageorgiou et al. 
1983). 

 
Non–IgE-mediated cellular immunoreactivity 
against food allergens had also been reported by 
our group with the help of the LAIT (Olivier et al. 
2022b, Olivier et al. 2022a, Olivier et al. 2022c, 
Olivier et al. 2023a). Non–IgE-mediated cellular 

immunoreactivity against aeroallergens and 
microorganisms had also been reported by our 
group with the help of the LAIT (Olivier et al. 
2023d, Olivier et al. 2023f, Olivier et al. 2023b, 
Olivier et al. 2023c, Olivier et al. 2024f). We also 
routinely employ the Tube Research of 
Precipitins (TTP) in our facilities as a triage to 
evaluate non–IgE-mediated immunoreactivity 
against suspected allergens before performing 
more exhaustive in vivo provocation tests (Olivier 
et al. 2024b, Olivier et al. 2024d, Olivier et al. 
2024g, Olivier et al. 2025). 
 
To evaluate the potential of the LAIT and TTP to 
endotype non–IgE–mediated cellular and 
humoral immunoreactivity against cat dander, 
dog dander, and pork meat extract, we 
retrospectively compiled the electronic medical 
charts of patients diagnosed with non–IgE–
mediated allergic multimorbidity who were 
investigated for immunoreactivity simultaneously 
using one of these assays. 
 
The present study serves as a proof-of-concept, 
hypothesizing that LAIT and the TTP may 
demonstrate a correlation between cellular 
and/or humoral immunoreactivity against cat 
dander, dog dander, and pork meat proteins in 
patients suffering from non–IgE–mediated 
Allergic Multimorbidity. 
 

2. MATERIALS AND METHODS 
 

2.1 Subjects 
 

After receiving Institutional Review Board 
approval from the Instituto Alergoimuno de 
Americana (Brazil; 03/2025), we reviewed the 
electronic chart of 10,500 outpatients who 
attended our facility from January 2018 to June 
2025 selecting patients diagnosed with allergic 
multimorbidity who were evaluated 
simultaneously with LAIT or TTP against cat 
dander, dog dander and pork meat extracts. 
 
A cohort of 100 consecutive outside patients 
(TTP cohort) had been submitted to TTP with cat 
dander, dog dander, and pork meat extract for 
presenting Non–IgE-mediated Allergic 
Multimorbidity as defined by the concomitant or 
consecutive presence of at least two allergic 
phenotypes (allergic rhinoconjunctivitis, allergic 
bronchitis, atopic dermatitis, urticaria, 
gastrointestinal hypersensitivity, and/or 
anaphylaxis). This cohort counted 39 males; 
mean age 32.2 years; SD 20.2 years; range 3 to 
88 years; median 31 years; mode = 7 (appeared 
six times); geometric mean = 24.6 years. 



 
 
 
 

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A cohort of 100 consecutive outside patients 
(LAIT cohort) had been submitted to LAIT with 
cat dander, dog dander, and pork meat extract 
for presenting non–IgE-mediated Allergic 
Multimorbidity as defined by the concomitant or 
consecutive presence of at least two allergic 
phenotypes (allergic rhinoconjunctivitis, allergic 
bronchitis, atopic dermatitis, urticaria, 
gastrointestinal hypersensitivity, and/or 
anaphylaxis). This cohort counted 29 males; 
mean age 30.3 years; SD 17.2 years; range 5 to 
86 years; median 30 years; mode = 32 
(appeared five times); geometric mean = 24.9 
years. 
 
Inclusion criteria: these procedures were offered 
to patients with allergic multimorbidity and had an 
undetectable specific IgE and had demonstrated 
a non-reactive or inconclusive skin test against 
cat fur, dog fur, and pork meat extracts (Olivier et 
al. 2013a). 
 
Exclusion criteria: this study excluded patients 
receiving biological and/or systemic anti-
inflammatory therapy.  
 

2.2 Pork Meat Extract 
 
Pork meat (shank) acquired from the local 
market 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 an 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 in amber, opaque glass vials. The pork 
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. 
 

2.3 Dog and Cat Fur Extracts 
 
The dog and cat fur's protein extraction was 
performed as follows: the material was furnished 
by a veterinarian who took special care not to 
mix cat fur with dog fur collected from several 
animals. The fur was treated with acetone to 

remove the fat. After this, the acetone was 
removed from the sample using the autoclave. 
The sample was ground for 48 hours at 4 °C with 
a Coca-based extractor solution added to cover 
the amount of antigen. 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 to perform allergic skin 
tests, TTP, and LAIT. 
 

2.4 LAIT: Ex vivo Investigation: 
Leukocyte Adherence Inhibition Test 

 
2.4.1 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. 2021a, Olivier et al. 2021b, Olivier et al. 
2021c). Shortly, each donor's fresh plasma was 
divided into two parts and used in parallel ex vivo 
challenging tests with the three allergen extracts 
(cat fur, dog fur, and pork meat) 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 and kept them 
under agitation for 30 minutes (200 rpm at 37 
°C). 
 
2.4.2 LAIT: Procedure for adherence assay 
 
 After incubation, the challenged 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 a humidified atmosphere of the 
covered water bath, allowing leukocytes to 
adhere to the glass. Next, we counted the 
leukocytes, removed the coverslip, and washed 
the chamber by immersing it in a beaker 
containing phosphate-buffered saline (PBS) at 37 
°C. Then, we added a drop of PBS to the 
hemocytometer's chamber and covered it with a 
clean coverslip. The remaining cells were 
counted in the same squares as previously 
examined.  
 
2.4.3 LAIT: Procedure for calculation 
 
The percentage of Leukocyte Adherence (LA) of 
each assay was estimated as: (the number of 
leukocytes observed on the hemocytometry 



 
 
 
 

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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 
Leukocyte Adherence (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 the 
unchallenged control plasma, multiplied by 100 
(%). To further calculate the Leukocyte 
Adherence Inhibition (LAI), we subtracted the 
LAR from 100 (%). We utilized the LAI results for 
the cascade distribution chart and the statistical 
calculations, both of which were performed using 
the Microsoft Excel statistical package. 
 

2.5 TTP: In vitro Investigation: Tube 
Titration of Precipitins 

 
As previously reported, the semi-quantitative 
TTP was performed in a transparent vitreous 
tube array (Olivier et al. 2024a). Shortly, the 
patient's blood was collected in a clot-activator 
collecting tube. After separation, the serum was 
centrifuged at 2,000 rpm for 10 minutes. Each 
allergen extract was allocated in sets of eleven 
glass tubes at progressively diluted serum 
concentrations. The progressive dilutions were 
combined with separated aliquots of 15 μL of the 
allergen extract 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, 
performed with 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, no research protocol 
was in place; therefore, we report the incidental 
immune investigation as documented in the 
digital medical charts. 
 
The TTP for the cat fur extract showed a 
distribution concentrated on the higher dilutions 
(Fig. 1). There was no negative result. The mean 
was estimated at 1:352; the median was 1:512; 
the standard deviation was estimated at 1:170; 
the mode was 1:512 (appeared 51 times); the 
geometric mean was estimated at 1:296 (see 
Fig. 1). 

The TTP for the dog fur extract showed a 
distribution concentrated on the higher dilutions 
(Fig. 2). There was no negative result. The mean 
was estimated at 1:318; the median was                 
1:256; the standard deviation was estimated at 
1:176; the mode was 1:512 (appeared 42 times); 
the geometric mean was estimated at 256 (see 
Fig. 2). 
 
The TTP for the pork meat extract showed a 
distribution concentrated on the higher dilutions 
(Fig. 3). There was no negative result. The mean 
was estimated at 1:413; the median was 1:512; 
the standard deviation was estimated at 1:144; 
the mode was 1:512 (appeared 67 times); the 
geometric mean was estimated at 1:377 (see 
Fig. 3). 
 
The LAIT for the cat fur extract showed a wide 
distribution range of results. The LAI ranged from 
0% to 100%. The mean was 56.2%; the median 
was 57.5%; the standard deviation was 27.0%; 
the mode was 68% (appeared six times). The 
cascade distribution demonstrates a wide range 
of LAI results. Most patients exhibited strong 
immunoreactivity, which could reflect the 
participation of cat fur allergens in a Non–IgE-
mediated hypersensitivity condition in these 
patients (see Fig. 4). 
 
The LAIT for the dog fur extract showed a wide 
distribution range of results. The LAI ranged from 
0% to 100%. The mean was 56.1%; the median 
was 60.5%; the standard deviation was 29.1%; 
the mode was 0% (appeared seven times). The 
cascade distribution demonstrates a wide range 
of LAI results. Most patients exhibited strong 
immunoreactivity, which may reflect the 
involvement of dog fur allergens in a Non–IgE-
mediated hypersensitivity condition in these 
patients (see Fig. 5). 
 
The LAIT for the pork meat extract showed a 
wide distribution range of results. The LAI ranged 
from 0% to 100%. The mean was 46.8%; the 
median was 48%; the standard deviation was 
27.6%; the mode was 0%                                  
(appeared six times). The cascade distribution 
demonstrates a wide range of LAI results. Most 
patients showed low or moderate 
immunoreactivity during the ex vivo challenge 
test. In contrast, others displayed strong 
immunoreactivity, which could reflect the 
involvement of pork meat allergens in a Non–
IgE-mediated hypersensitivity condition in these 
patients (see Fig. 6). 



 
 
 
 

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Fig. 1. Cascade distribution chart of the tube titration of precipitins (x-axis %) resulting from the cat dander extract against the serum of the TTP 
cohort of 100 tests/subjects (y-axis). The graph shows a concentration of results in higher titrations 



 
 
 
 

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The paired t-test indicated a non-significant, 
slight difference between the TTP results of cat 
fur and dog fur (p-value = 0.165). Pearson's 
correlation indicated a non-significant small 
positive relationship between TTP results of cat 
fur and dog fur; r(98) = 0.0337; p-value = 0.739 
(see Fig. 07). 
 

The paired t-test indicated a significant small 
difference between the TTP results of cat fur and 
pork meat (p-value = 0.009). Pearson correlation 
indicated a non-significant, minimal negative 
relationship between TTP results of cat fur and 
pork meat; r(98) = 0.0468; p-value = 0.644 (see 
Fig. 08). 
 

The paired t-test indicated a significant small 
difference between the TTP results of dog fur 
and pork meat (p < 0.001). Pearson's correlation 
indicated a significant small negative relationship 
between TTP results between dog fur and pork 
meat; r(98) = 0.248; p-value = 0.013)                        
(see Fig. 09). 

The paired t-test indicated no significant 
difference between cat fur and dog fur LAIT 
results (p-value = 0.9949). Pearson's correlation 
indicated a significantly moderate positive 
relationship between the cat fur and dog fur LAIT 
results: r(98) = 0.395, p-value < 0.001 (see Fig. 
10). 
 

The paired t-test indicated a significant small 
difference between cat fur and pork meat LAIT 
results (p-value = 0.009082). Pearson's 
correlation indicated a non-significant, small 
positive relationship between cat fur and pork 
meat LAIT results: r(98) = 0.193, p-value = 0.054 
(see Fig. 11). 
 

The paired t-test indicated a significant small 
difference between dog fur and pork meat LAIT 
results (p-value = 0.01968). However, Pearson's 
correlation indicated a non-significant, minimal 
positive relationship between dog fur and pork 
meat LAIT results: r(98) =0.0519, p-value = 
0.608 (see Fig. 12) 

 

 
 

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

graph shows a concentration of results in higher titrations 
 

 
 

Fig. 3. Cascade distribution chart of the tube titration of precipitins (x-axis %) resulting from 
the pork meat extract against the serum of the TTP cohort of 100 tests/subjects (y-axis). The 

graph shows a concentration of results in higher titrations 



 
 
 
 

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150 

 

 
 

Fig. 4. Cascade distribution chart of the range groups of Leukocyte Adherence Inhibition (LAI) results (x-axis %) of the ex vivo challenge test 
against cat fur 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 graph shows a wide distribution of results 
 

 
 

Fig. 5. Cascade distribution chart of the range groups of Leukocyte Adherence Inhibition (LAI) results (x-axis %) of the ex vivo challenge test 
against dog fur 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 graph shows a wide distribution of results 



 
 
 
 

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Fig. 6. Cascade distribution chart of the range groups of Leukocyte Adherence Inhibition (LAI) results (x-axis %) of the ex vivo challenge test 
against pork meat 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 graph shows a wide distribution of results 



 
 
 
 

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Fig. 7. Dispersion chart of the Tube Titration of Precipitins (TTP) against cat fur extract (x-axis 

%), plotted against the TTP against dog fur extract (y-axis %). The paired t-test indicated a non-
significant, slight difference between the TTP results of cat fur and dog fur (p-value = 0.165). 

Pearson's correlation indicated a non-significant small positive relationship between TTP 
results of cat fur and dog fur; r(98) = 0.0337; p-value = 0.739 

 

 
 

Fig. 8. Dispersion chart of the Tube Titration of Precipitins (TTP) against cat fur (x-axis %), 
plotted against the TTP against pork meat extract (y-axis %). The paired t-test indicated a 

significant small difference between the TTP results of cat fur and pork meat (p-value = 0.009). 
Pearson correlation indicated a non-significant, minimal negative relationship between TTP 

results of cat fur and pork meat; r(98) = 0.0468; p-value = 0.644 
 



 
 
 
 

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Fig. 9. Dispersion chart of the Tube Titration of Precipitins (TTP) against dog fur extract (x-axis 
%), plotted against the TTP against pork meat extract (y-axis %). The paired t-test indicated a 

significant small difference between the TTP results of dog fur and pork meat (p < 0.001). 
Pearson's correlation indicated a significant small negative relationship between TTP results 

between dog fur and pork meat; r(98) = 0.248; p-value = 0.013) 
 

 
 

Fig. 10. Dispersion chart of the Leukocyte Adherence Inhibition (LAI) results of the ex vivo 
challenge test against cat fur extract (x-axis %), plotted against the LAI results of the ex vivo 

challenge test against dog fur extract (y-axis %). The paired t-test indicated no significant 
difference between cat fur and dog fur LAIT results (p-value = 0.9949). Pearson's correlation 
indicated a significantly moderate positive relationship between the cat fur and dog fur LAIT 

results: r(98) = 0.395, p-value < 0.001 



 
 
 
 

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Fig. 11. Dispersion chart of the Leukocyte Adherence Inhibition (LAI) results of the ex vivo 
challenge test against cat dander (x-axis %), plotted against the LAI results of the ex vivo 

challenge test against pork meat extract (y-axis %). The paired t-test indicated a significant 
small difference between cat fur and pork meat LAIT results (p-value = 0.009082). Pearson's 
correlation indicated a non-significant, small positive relationship between cat fur and pork 

meat LAIT results: r(98) = 0.193, p-value = 0.054 
 

 
 

Fig. 12. Dispersion chart of the Leukocyte Adherence Inhibition (LAI) results of the ex vivo 
challenge test against dog dander extract (x-axis %), plotted against the LAI results of the ex 

vivo challenge test against pork meat extract (y-axis %). The paired t-test indicated a 
significant small difference between dog fur and pork meat LAIT results (p-value = 0.01968). 

However, Pearson's correlation indicated a non-significant, minimal positive relationship 
between dog fur and pork meat LAIT results: r(98) =0.0519, p-value = 0.608 

 



 
 
 
 

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4. DISCUSSION 
 
Currently, efforts to understand the Innate 
Immune System's role in allergic diseases are 
increasing, accompanied by a growing arsenal of 
tools (Lee and Cohen, 2025). 
 
The correlation and distribution of simultaneous 
positive specific IgE against food allergens and 
inhalant allergens are usually weak; however, 
polysensitization and cross-reactivity are more 
the rule than the exception (Zhang et al., 2025; 
Čelakovská et al., 2024; Hasnain, et al. 2017). 
 
The treatment of monosensitized allergic patients 
through subcutaneous shots raised the paradigm 
of allergen-specific Immunotherapy (Olivier 
2017). However, despite the use of precision 
techniques such as the Component Resolved 
Diagnosis to identify allergens at a molecular 
level, several allergens (such as the major cat 
allergen Fel d 1) present great diversity 
(dissimilar substitutions in the protein sequence) 
among cat species reflecting the genetic 
evolution of Felidae (Cleveland et al. 2024). 
Therefore, it is common to see patients who 
report allergic symptoms elicited by some cats 
but not by others. The same happens concerning 
dogs. 
 
In the context of polysensitization, multimorbidity, 
and cross-sensitization, where several allergens 
appear to be clinically relevant, a more rational 
treatment strategy consists of the use of group-
specific multiallergen desensitization 
immunotherapy, which subcutaneous shots 
cannot administer due the extension of the local 
inflammatory response, but rather by the 
sublingual-swallow route, where the collateral 
effects are minimal (Olivier et al. 2013b, Khan 
2016). 
 
Assessing diverse ways of immunoreactivity and 
hypersensitivity against allergens responsible for 
clinical symptoms is a multi-omics approach to 
evaluate both the diagnosis and treatment of 
allergic patients (Czolk et al.,                                
2021).  
 
Alternative approaches, such as the LAIT and 
the TTP, are proposed not to pinpoint the 
molecular allergen responsible for the allergic 
phenotype but to obtain an overall view of the 
patient's immunoreactivity against the whole 
extract. However, executing these 
immunoassays with molecular allergens to 
demonstrate a specific reaction is also possible. 

We usually do not use molecular allergens purely 
due to a lack of resources. 
 
To contour the difficulties in diagnosing Non–IgE-
mediated hypersensitivity, some scientists have 
committed to evaluating the utility of the specific 
IgG in helping clinically diagnose their patients 
(Alkhateeb 2020). This is controversial, as IgG 
may function as both a hypersensitivity trigger 
and an allergen blocker, depending on the 
reaction of the other immune players (Atwah and 
Koshak, 2024). IgG antibodies can participate in 
type II (antibody-dependent cell-mediated) and 
type III (immune complex disease) Gell and 
Coombs hypersensitivity reactions, which may be 
theoretically reproduced by the LAIT and the TTP 
assays, respectively (Olivier et al., 2021a; Olivier 
et al., 2021d). 
 
The semi-quantitative titration of precipitins is a 
pioneering laboratory exam that laid the 
fundamental basis of immunology (Wells 1911). 
Precipitating antibodies indicate the presence of 
a humoral immune response against the tested 
antigens (Gell, Harington, and Rivers, 1946). 
Before the discovery of IgE, research on 
precipitins was the leading method for in vitro 
diagnosis of immunoreactivity against allergenic 
agents (Augustin and Hayward, 1960). 
 
The LAIT is an ex vivo challenge test performed 
with a viable leukocyte buffy coat, which can 
theoretically explore the most well-known 
immune pathways, as it allows the interaction of 
all immune-circulating participants with the 
allergens (Olivier et al., 2021a). Several immune 
pathways can inhibit leukocyte adherence (Tong 
et al., 1979; Halliday, et al. 1974). 
 
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 cat dander, 
dog dander, and pork meat allergens among 
patients suffering from non–IgE-mediated allergic 
multimorbidity. As the tests were performed 
simultaneously with the same venous sample 
with the three allergens, it was possible to 
calculate a correlation to distinguish some order 
of cross-reactivity between them. 
 
The retrospective compilation of our data 
revealed a wide distribution of results when we 
assessed the outcomes of TTP and LAIT to 
explore humoral and cellular immunoreactivity 
against the studied allergens. These 
immunoassays did not precisely identify the 



 
 
 
 

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mechanisms responsible for clinical conditions. 
Instead, they provide evidence about cellular and 
humoral immunoreactivity distributed across an 
extensive spectral range, which may suggest 
immune tolerance or hypersensitivity. 
 
This preliminary retrospective survey 
demonstrated extensive results from the TTP 
and the ex vivo challenge test monitored by LAIT 
against cat fu, dog fur, and pork meat in two 
cohorts of non–IgE-mediated allergic 
multimorbidity 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 
evaluated for several chemical and biological 
allergens, demonstrating positive results for 
some of them. Our results suggest that dog and 
cat dander allergy patients may experience 
additional symptoms when consuming pork meat 
and vice versa. 
 

5. LIMITATIONS 
 
This study is a retrospective analysis of data 
collected over seven years since our facility 
began employing laboratory immune assays. 
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 
the positive and negative controls to validate the 
results. The number of subjects is suitable for a 
preliminary study; however, future studies should 
be more comprehensive. The lack of a research 
protocol implies the possibility of bias introduced 
by the physician's point of view (CEO) based on 
a clinical suspicion driven solely by the 
anamnesis and physical examination. The study 
lost many of these patients to follow-up, so it is 
not yet possible to ensure the relationship 
between the immunoassay results and the 
patient's clinical outcome. Unfortunately, 
comparing the two procedures using paired t-
tests was impossible because they were 
obtained from distinct patient groups. 
 

6. CONCLUSION 
 
The preliminary results suggest that the TTP and 
LAIT, when applied to cat and dog fur extracts 
and pork meat extract, may discriminate between 
diverse humoral and cellular immunoreactivity 
levels in patients with various allergic 

phenotypes. A significant association was found 
between the immunoreactivity of cat fur and dog 
fur. The association between pork meat and fur 
immunoreactivity was not clear. LAIT and TTP 
are inexpensive, can be performed with minimum 
laboratory equipment, and can be incorporated 
into strategies to address health disparities in 
respiratory and food allergies (Anagnostou et al. 
2025). As a preliminary report, the propaedeutic 
significance of the presented results and the 
potential interfering factors must be further 
established (Anouar, et al. 2024). More studies 
focused on the quality-by-design approach with 
larger, prospective, double-blind cohorts are 
needed to evaluate the potential contribution of 
LAIT and TTP for endotyping cellular and 
humoral immunoreactivity in patients suspected 
of hypersensitivity to cat fur, dog fur, and pork 
meat allergens (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 patients from 
undergoing unnecessary, exhaustive, and 
potentially hazardous in vivo challenge tests. 
Exploring the humoral and the cellular arms of 
immune systems, the TTP and LAIT alone or 
combined may represent, in the near future, a 
tool for allergists to elaborate etiologic diagnosis 
for their patients, as well as determine the 
endotypes (mechanisms) of hypersensitivity, in 
order to choose more convenient and 
personalized therapies (Adly e. al., 2021). Adding 
data provided by TTP and LAIT may also 
contribute to streamlining biomedical research 
and improving tools, such as large language 
models, which clinicians often use as decision 
support systems to enhance diagnostic accuracy 
(Abers and Mathias, 2025). 
 

CONCENT 
 

It is not applicable. 
 

ETHICAL APPROVAL 
 
The authors have obtained and documented 
written ethical approval in accordance with 
international standards.As a retrospective 
compilation of results recorded in cognito, 
consent was obtained collectively by the 
institution's ethics committee, following the 
principles of the Declaration of Helsinki (WMA, 
2013). 



 
 
 
 

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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 assistance 
with the exams 
 

COMPETING INTERESTS 
 
Authors have declared that no competing 
interests exist. 
 

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