







































_____________________________________________________________________________________________________ 
 

*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 Between Blomia Tropicalis and 
Farfantepenaeus Brasiliensis in Patients With Allergic Multimorbidity”. Asian Journal of Immunology 8 (1):163-78. 
https://doi.org/10.9734/aji/2025/v8i1169. 
 

Asian Journal of Immunology 
 
Volume 8, Issue 1, Page 163-178, 2025; Article no.AJI.139203 
 

 
 

 
 

Endotyping Cellular and Humoral 
Cross-Reactivity between Blomia 

tropicalis and Farfantepenaeus 
brasiliensis 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  
 

This work was carried out in collaboration among all authors. Author CEO conceptualized the study, 
did data curation, formal analysis, literature review, and wrote original draft of the manuscript. Authors 
DGP, APMT, CSM, NSR, JLSS and RPSL performed laboratory procedures. Author ESM cultured the 
Blomia tropicalis specimens and prepared their extract. Author RAPGS performed cutaneous tests. All 

authors read and approved the final manuscript 
 

Article Information 
 

DOI: https://doi.org/10.9734/aji/2025/v8i1169  
 

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/139203  

 

Received: 20/04/2025 
Published: 03/07/2025 

 

Original Research Article 

https://doi.org/10.9734/aji/2025/v8i1169
https://pr.sdiarticle5.com/review-history/139203


 
 
 
 

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164 

 

ABSTRACT 
 

Background: Panallergens, such as tropomyosin, produce cross-reactivity between indoor 
allergens and food allergens, including the house dust mite Blomia tropicalis and the Brazilian pink 
shrimp Farfantepenaeus brasiliensis, and are responsible for the clinical symptoms present in 
patients with allergic multimorbidity phenotypes. 
Aim: To evaluate the potential of the Tube Titration of Precipitins (TTP) and the Leukocyte 
Adherence Inhibition Test (LAIT) to discriminate and correlate cellular and humoral 
immunoreactivity against protein extracts of B. tropicalis and F. brasiliensis in patients with non–
IgE-mediated allergic multimorbidity phenotypes. 
Study Design: We examined retrospectively the medical charts of two cohorts of patients clinically 
diagnosed with non–IgE-mediated allergic multimorbidity phenotypes related to inhalation or 
contact with house dust and/or consumption of shrimps, who were concomitantly investigated for 
these allergens with the help of TTP or LAIT. 
Methodology: The registered results of the TTP and LAIT against protein extracts of B. tropicalis 
and F. brasiliensis were distributed in ranges through cascade distribution charts. The correlation 
between the paired assays was calculated using Pearson's methodology and demonstrated by 
dispersion graphs.  
Results: The TTP for the B. tropicalis extract showed a distribution concentrated on the higher 
dilutions. The mean was 1:353; the median was 1:256; the standard deviation was 1:163;                       
the mode was 1:512 (appeared 49 times). The TTP for the F. brasiliensis extract showed a 
distribution concentrated on the higher dilutions. The mean was 1:380; the median was 1:512;           
the standard deviation was 1:157; the mode was 1:512 (which appeared 57 times). The LAIT for 
the B. tropicalis extract showed a wide distribution range of results. The LAI ranged from 0% to 
100%. The mean was 49.8%; the median was 51.5%; the standard deviation was 30.0%; the mode 
was 0% (appeared eleven times). The LAIT for the F. brasiliensis extract showed a wide 
distribution range of results. The LAI ranged from 0% to 100%. The mean was 55%; the median 
was 56%; the standard deviation was 28.8%; the mode was 0% (appeared five times). There was 
no significant correlation between B. tropicalis and F. brasiliensis when analyzed by TTP or LAIT 
results. 
Conclusion: Our preliminary results suggest that the TTP and LAIT may reveal humoral and 
cellular immunoreactivity in patients with allergic multimorbidity phenotypes using protein extracts 
of B. tropicalis and F. brasiliensis. 

 

 
Keywords: Blomia tropicalis; Farfantepenaeus brasiliensis; hypersensitivity; house dust mite; 

leukocyte adherence inhibition test; Non–IgE-mediated immunoreactivity; shrimp; 
precipitins. 

 

ABBREVIATIONS 

 
LAI : Leukocyte Adherence Inhibition 

LAIT : Leukocyte Adherence Inhibition Test 

TTP : Tube Titration of Precipitins 
 

1. INTRODUCTION  
 

Blomia tropicalis is an arthropod (phylum) of the 
subphylum Chelicerata; class Arachnida; 
subclass Acari; superorder Acariformes; order 
Sarcoptiformes; suborder Astigmata; superfamily 
Glycyphagoidea; family Echimyopodidae 
(Schoch et al. 2020a). 
 

Farfantepenaeus brasiliensis (Penaeus 
brasiliensis) is an arthropod (phylum) of the 

subphylum Crustacea; class Malacostraca; order 
Decapoda; suborder Dendrobranchiata; 
superfamily Penaeoidea; family Penaeidae 
(Schoch  et al. 2020b). 
 
The first suspicion and evidence that house dust 
mites (HDM), from the genus Dermatophagoides, 
were causing respiratory allergies were 
publicized in the mid-1960s (Voorhorst et al., 
1964; Fain, 1966). The allergenicity of HDM from 
the Blomia genus and its cross-reactivity with 
other HDM species were studied using in vitro 
neutralization of skin-sensitizing antibodies, as 
described in Japan in the late 1960s  (Miyamoto 
et al. 1969). The species Blomia tropicalis was 
described in the early 1970s as a dust and 
storage mite found in tropical and subtropical 
regions (van Bronswijk et al., 1973). Soon, 



 
 
 
 

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several reports on the allergenicity of Blomia 
tropicalis in Western tropical countries were also 
published (Fernández-Caldas et al., 1990; 
Fernández-Caldas et al., 1993; Chew et al., 
1999; Baqueiro et al., 2006). 
 
Almost six decades after the discovery of their 
causality in respiratory allergies, several reports 
have indicated that food contamination with 
HDM, including Blomia tropicalis, also produces 
urticaria and anaphylaxis and is associated with 
hypersensitivity to non-steroidal anti-
inflammatory drugs (Sánchez-Borges et al., 
2013; Barrera-de-Pino, Murgas, and Miranda, 
2012). After that, it was noticed that the HDM, 
including Blomia tropicalis, also produced Atopic 
Dermatitis (Emran et al. 2019).  
 
The Allergen Nomenclature Sub-Committee of 
the World Health Organization and the 
International Union of Immunological Societies 
(WHO/IUIS) has recognized twenty-six allergens 
to date, ranging in molecular weight from 7 to 
110 kDa, which have been identified in Blomia 
tropicalis (Sub-Committee, 2025a; Chua et al., 
2007).  
 
Tropomyosin is a phylogenetically conserved 
heat-stable alpha-helical coiled-coil dimeric 
protein found in vertebrates and invertebrates 
that interacts with actin, producing muscle 
contraction (Bailey 1946) (Reese et al., 1999). 
Tropomyosin homologs are defined as the group 
10 HDM allergens, and Blo t 10 is the Blomia 
tropicalis tropomyosin, sharing amino acid 
sequence homology with several forms of 
tropomyosins that are involved in cross-reactivity 
with mites, cockroaches, shrimps, snails, oysters, 
crabs, lobsters, squids, and other invertebrates 
(Martínez et al. 2024, Papia et al., 2021). 
 
Tropomyosin is the major allergen of crustaceans 
from the genus Penaeus (Daul et al. 1994). 
Tropomyosin is considered a panallergen, a 
sensitizing protein acquired from different fonts, 
responsible for (apparently) unrelated clinical 
allergic reactions such as respiratory symptoms 
(elicited by inhalation of house dust mites) or 
systemic symptoms (elicited by the ingestion of 
edible invertebrates such as shrimps) (Wong et 
al., 2016, Yang et al. 2010). 
 
Farfantepenaeus brasiliensis (pink shrimp) is the 
most exploited shrimp species along the 
Brazilian Coast (Carvalho et al., 2019). Shrimp's 
tropomyosin is a 34- to 38-kDa heat-stable 
allergen that can provoke IgE-mediated 

immediate-type hypersensitivity reactions after 
ingestion (Shanti et al., 1993). Tropomyosin is 
considered the major allergen of shrimp and is 
usually classified as a group 1 allergen, following 
the group 1 allergens (Crac c 1, Exo m 1, Lit v 1, 
Met e 1, Pan b 1, Pen a 1, Pen i 1, Pen m 1) 
(Sub-Committee, 2025b). Nevertheless, 
uncharacterized, the Farfantepenaeus 
brasiliensis tropomyosin should receive the 
allergen nomenclature Far b 1). Besides the 
common major allergen, tropomyosin, several 
species-specific shrimp allergens may also 
participate in allergic reactions (Morgan et al. 
1989). 
 
Several symptoms have been attributed to 
shrimp allergy (urticaria, angioedema, chest 
tightness, shortness of breath, nausea, vomiting, 
diarrhea, fainting with documented hypotension, 
chills, fever, abdominal discomfort, abdominal 
pain, and finger stiffness) (Waring et al. 1985). 
 
Allergic Multimorbidity is defined as the presence 
of concomitant or consecutive allergic 
phenotypes that may be IgE-mediated, partly 
IgE-mediated, or non-IgE-mediated (Bousquet et 
al., 2015; Bousquet et al., 2025). Besides the 
IgE-mediated hypersensitivity mechanism, 
several types of hypersensitivity mechanisms are 
associated with allergic phenotypes (Jutel et al., 
2023). The humoral-dependent non–IgE-
mediated allergic phenotypes may be evaluated 
by the research of Precipitins (Augustin 1953; 
Augustin et al., 1960; Cunningham-Rundles et al. 
1978; Ferguson & Carswell 1972; Heiner et al., 
1962). The Tube Titration of Precipitins (TTP) is 
routinely used at our facilities to evaluate 
humoral immunoreactivity against suspected 
allergens as a triage test prior to the exhaustive 
in vivo provocation tests, which define 
hypersensitivities diseases (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 & Nilsson 
1979, Kuratsuji 1981, Thomson, 1982). The LAIT 
and the LMIT have also been classically used to 
differentiate non–IgE-mediated immunoreactivity 
against food allergens (Allardyce & Shearman 
1975, George & Vaughan 1962, Ashkenazi et al. 
1980, Butler et al. 1981, Papageorgiou et al. 
1983).  



 
 
 
 

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Non–IgE-mediated cellular immunoreactivity 
against food allergens had also been reported by 
our group using 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 LAIT and TTP to endotype 
cellular and humoral non–IgE-mediated 
immunoreactivity against allergens already 
proved to produce allergic symptoms by 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 
endotyping non–IgE-mediated cellular and 
humoral immunoreactivity against Blomia 
tropicalis and Farfantepenaeus brasiliensis 
extract, we retrospectively compiled the 
electronic medical charts of patients diagnosed 
with non–IgE-mediated Allergic Multimorbidity 
who were investigated for immunoreactivity 
simultaneously by one of these assays. 
 

The present study provides proof of concept that 
hypothesizes LAIT and the TTP may 
demonstrate cellular and/or humoral 
immunoreactivity against Blomia tropicalis and 
Farfantepenaeus brasiliensis proteins in patients 
suffering from non–IgE–mediated Allergic 
Multimorbidity. This paper is a retrospective 
study of the results of the immunoassays, so we 
do not have access to the nutritional background 
or the genetic constitution of the patients. 
However, we are planning to address these 
issues in further prospective studies. 
 

2. MATERIALS AND METHODS  
 

2.1 Subjects 
 

After receiving Institutional Review Board 
approval from the Instituto Alergoimuno de 
Americana (Brazil; 04/2025), 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 Blomia 
tropicalis or Farfantepenaeus brasiliensis 
extracts.  
 

A cohort of 100 consecutive outside patients 
(TTP cohort) had been submitted to TTP with 

Blomia tropicalis extract and Farfantepenaeus 
brasiliensis 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 42 males; mean age 41.2 years; SD 
18.4 years; range 10 to 82 years; median 39 
years; mode = 36 (appeared five times); 
geometric mean = 36.5 years.  
 

A cohort of 100 consecutive outside patients 
(LAIT cohort) had been submitted to TTP with 
Blomia tropicalis extract and shrimp 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 30 males; 
mean age 36.4 years; SD 23.5 years; range 2 to 
91 years; median 35 years; modes = 5 
(appeared five times); geometric mean = 26.4 
years.  
 

This study did not include patients under 
biological and/or systemic anti-inflammatory 
therapy. These procedures were offered to 
patients with allergen multimorbidity related to 
house dust and/or shrimp consumption who had 
indetectable specific IgE against these suspected 
agents and demonstrated non-reactive or 
inconclusive skin tests against Blomia tropicalis 
and Farfantepenaeus brasiliensis extracts 
(Olivier et al., 2013). 
 

2.2 Preparation of the Blomia tropicalis 
Extract 

 

2.2.1 Origen of the Blomia tropicalis strain 
 

The specimens of Blomia tropicalis were kindly 
provided by Prof. Dr. Jorge Martínez Quesada 
from the Faculty of Pharmacy at the University of 
the Basque Country, Spain. The culture medium 
was placed in 50 mL ThermoFisher® cell culture 
flasks (15 g per flask) in a sterile environment. 
These flasks were placed in an airtight container 
containing a saturated solution of distilled water 
and sodium chloride for at least 24 hours to 
maintain the humidity of the culture medium. 
They were brought to Brazil inside airtight bags 
containing cotton soaked in the saturated NaCl 
solution, thus maintaining humidity during the 
flight. 



 
 
 
 

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2.2.2. Preparation of the culture medium 
 

The culture and extraction of Blomia tropicalis 
proteins were performed at the BioAllergy 
laboratory (Álvaro de Carvalho, São Paulo, 
Brazil). The culture media (yeast extract and 
Tetramin®) were weighed on Thermomix's built-in 
scale (Vorwerk, TM6®), then crushed using the 
device's primary function for 30 seconds at 
maximum speed (10), and this step was 
repeated three times until a fine, homogenized 
powder was obtained. After the grinding step in 
the Thermomix®, all culture media were sieved 
through a 60-mesh sieve (250 μm opening) to 
ensure standardization and granulometry of the 
medium. The sieved culture medium was then 
weighed (10 g) and placed in Erlenmeyer flasks 
with a cotton plug and gauze to be transferred to 
a sterilization and drying oven (Ethik Technology, 
400D®), previously heated, where it remained for 
1 hour at 110ºC. After cooling, the contents of 
the Erlenmeyer flasks were transferred to 50 mL 
cell culture flasks using a previously sterilized 
plastic funnel, which was performed under 
unidirectional airflow. The flasks had been 
previously treated with ultraviolet light for 15 
minutes to prevent contamination by 
microorganisms and other mite species. The 50 
mL culture flasks containing only the culture 
medium were kept in airtight boxes with a 
saturated sodium chloride solution and distilled 
water for at least 48 hours to humidify them 
before inoculating the mites. The population peak 
(71,000 mites/g of culture) was reached after 90 
days of culture.  
 

2.2.3 Production of Blomia tropicalis 
allergenic extract 

 

The samples of whole culture (mites and culture 
medium) underwent the grinding process to 
improve the exposure of allergens to the 
extraction buffer. Next, they were subjected to 
the defatting process (using ethyl ether), which 
favored the loss of non-protein molecules. The 
material was sonicated to facilitate disruption. 
The sample was solubilized in Phosphate-
Buffered Saline (pH 7.2; Sodium Chloride (NaCl) 
– 81.9 g/L; Potassium Chloride (KCl) – 1.87 g/L; 
Dibasic Sodium Phosphate (Anhydrous 
Na2HPO4) – 14.19 g/L; Monobasic Potassium 
Phosphate (KH2PO4) - 2.38 g/L). The extraction 
was performed at a rate of 10 mL of buffer per 
gram of raw material (10%), using gentle 
magnetic stirring (1,000 rpm) for 4 hours at 4ºC. 
The material was centrifuged at 5,000 rpm 
(3,020g for 30 minutes, and the supernatant was 
preserved. The sediment was resuspended 

under the same conditions as in the first step and 
kept under stirring for 2 hours. The centrifugation 
was then repeated, and the supernatants were 
mixed. The final extract was dialyzed against the 
buffer to eliminate low-molecular-weight proteins 
(<5,000 Da) for 24 hours, with three buffer 
changes and a 1:50 dilution ratio. The process 
was continued by centrifugation at 10,000 rpm 
(12,100 g) for 30 minutes at 4 °C. Protein 
concentration was determined using the Bradford 
method (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. 
 

2.3 Shrimp Extract 
 

Farfantepenaeus brasiliensis was acquired from 
the local market and identified based on its 
morphological characteristics (Silveira et al., 
2022). The sample was grounded 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 allergen extract 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 



 
 
 
 

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168 

 

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 allocated a clean 
coverslip over it. 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 
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 
centrifugated 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 & 
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.  

 

 
 

Fig. 1. Cascade distribution chart of the tube titration of precipitins (x-axis %) resulting from 
the Blomia tropicalis 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 Farfantepenaeus brasiliensis extract against the serum of the TTP cohort of 100 

tests/subjects (y-axis) 
 

 
 

Fig. 3. Dispersion chart of the tube titration of precipitins results of the ex vivo challenge test 
against Blomia tropicalis extract (x-axis %), plotted against the Tube Titration of Precipitins 
results of the ex vivo challenge test against Farfantepenaeus brasiliensis extract (y-axis %) 

 

The TTP for the Blomia tropicalis extract showed 
a distribution concentrated on the higher dilutions 
(Fig 1). There was no negative result. The mean 
was estimated at 1:353; the median was 1:256; 
the standard deviation was estimated at 1:163; 
the mode was 1:512 (appeared 49 times). 
 
The TTP for the Farfantepenaeus brasiliensis 
extract showed a distribution concentrated on the 
higher dilutions (Fig 1). There was no negative 
result. The mean was estimated at 1:380; the 
median was 1:512; the standard deviation was 
estimated at 1:157; the mode was 1:512 (which 
appeared 57 times).  

Pearson's correlation indicated a non-significant, 
small positive relationship between TTP results 
for Blomia tropicalis and Farfantepenaeus 
brasiliensis; r(98) = 0.0629, p-value = 0.534 (see 
Fig. 3). 
 
The LAIT for the Blomia tropicalis extract showed 
a wide distribution range of results. The LAI 
ranged from 0% to 100%. The mean was 49.8%; 
the median was 51.5%; the standard deviation 
was 30.0%; the mode was 0% (appeared eleven 
times). The cascade distribution demonstrates a 
wide range of LAI results (Fig. 4). Some patients 
showed low or moderate immunoreactivity during 



 
 
 
 

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170 

 

the ex vivo challenge test. In contrast, others 
displayed strong immunoreactivity, which could 
reflect the participation of Blomia                          
tropicalis allergens in a non–IgE-mediated 
hypersensitivity condition in these patients (see 
Fig. 4) 
 
The LAIT for the Farfantepenaeus brasiliensis 
extract showed a wide distribution range of 
results. The LAI ranged from 0% to 100%. The 
mean was 55%; the median was 56%; the 
standard deviation was 28.8%; the mode was 0% 
(appeared five times). The cascade distribution 

demonstrates a wide range of LAI results (Fig. 
5). 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 shrimp allergens in a non–IgE-
mediated hypersensitivity condition in these 
patients (see Fig. 5). 
 
Pearson's correlation indicated a non-significant, 
small positive relationship between LAIT results 
for Blomia tropicalis and F. brasiliensis; r(98) = 
0.0713, p-value = 0.481 (see Fig. 6). 

 

 
 

Fig. 4. Cascade distribution chart of the range groups of leukocyte adherence inhibition (LAI) 
results (x-axis %) of the ex vivo challenge test against the Blomia tropicalis 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. 5. Cascade distribution chart of the range groups of leukocyte adherence inhibition (LAI) 
results (x-axis %) of the ex vivo challenge test against the Farfantepenaeus brasiliensis 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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171 

 

 
 

Fig. 6. Dispersion chart of the leukocyte adherence inhibition (LAI) results of the ex vivo 
challenge test against Blomia tropicalis extract (x-axis %), plotted against the LAI results of 

the ex vivo challenge test against Farfantepenaeus brasiliensis extract (y-axis %) 
 

4. DISCUSSION 
 

Polysensitization and cross-reactivity are related 
phenomena frequently observed in patients with 
allergic multimorbidity (Zhang et al., 2025; 
Čelakovská et al., 2024; Hasnain et al., 2017). 
When investigating the etiology of allergic 
reactions, it is advisable to maintain an open 
mind regarding cross-reactivity among 
panallergens, especially in polysensitized 
patients presenting with Allergic Multimorbidity 
(Miltner et al., 2024). The specificity and facility 
for researching precipitins transformed this 
immunoassay into a pioneering examination 
upon which the fundamentals of immunology 
were established (Hunter, 1905). Precipitins are 
yet used nowadays to monitor emerging 
diseases when more precise and sophisticated 
assays are inexistent or unavailable (Bellanger et 
al., 2022). Precipitating antibodies indicate a 
humoral immune response against the tested 
antigens (Gell, Harington, and Rivers, 1946). 
Before the discovery of IgE, the research of 
precipitins was the only viable way to 
demonstrate immunoreactivity against allergenic 
agents (Augustin & Hayward, 1960) (Wells, 
1911). Precipitin antibodies may belong to any 
Immunoglobulin class (except for IgG4), can 
mount immune complexes, and can participate in 
type II (antibody-dependent cell-mediated) and 
type III (immune complexes disease) Gell and 
Coombs hypersensitivity reactions, which may 
(theoretically) be demonstrated by the LAIT and 
the TTP assays, respectively (Gell & Coombs, 
1968).  

The LAIT is an ex vivo challenge test performed 
with live leukocytes, which allows the interaction 
of all immune-circulating cells with allergens. 
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 Blomia 
tropicalis and Farfantepenaeus brasiliensis 
extracts among patients suffering from non–IgE-
mediated Allergic Multimorbidity. As the tests 
were performed simultaneously using the same 
venous sample with both allergens, it was 
possible to calculate a correlation to                 
distinguish any order of cross-reactivity between 
them.  
 

The retrospective compilation of our data 
revealed a wide distribution of results when 
analyzing the LAIT results to explore cellular 
immunoreactivity. On the contrary, there was no 
such variability in the results of TTP, which 
demonstrated the presence of high titers of 
precipitin antibodies predominantly in the higher 
dilutions, suggesting the presence of humoral 
activity against these extracts in all selected 
patients, thereby limiting the analytical value of 
this immunoassay. Despite these immunoassays 
not precisely identifying the mechanisms 
responsible for the clinical condition, they provide 
general evidence about cellular and humoral 
immunoreactivity that may be involved in immune 



 
 
 
 

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tolerance, sensitization, and clinical 
hypersensitivity. 
 
This preliminary retrospective survey yielded 
diverse results from the TTP and the ex vivo 
challenge test, monitored by LAIT, against 
Blomia tropicalis and Farfantepenaeus 
brasiliensis extracts in two cohorts of patients 
diagnosed with non–IgE–mediated Allergic 
Multimorbidity. 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 patients 
with allergies to HDM may exacerbate their 
symptoms by experiencing additional cross-
immunoreactivity when consuming shrimp. 
 
Assessing diverse pathways of immunoreactivity 
and hypersensitivity through tools such as TIAL 
and TTP against the whole extract of suspected 
allergens, as well as suspected molecular 
allergens, is a multi-omics approach that 
evaluates both diagnosis and treatment 
effectiveness in allergic patients (Hubert et al., 
2023). 
 

5. LIMITATIONS 
 
This study is a retrospective analysis of data 
collected over a seven-year period 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 
were unable to 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, it was 
impossible to compare the two procedures                 
using paired statistics methodologies because 
they were derived from two distinct patient 
groups. 

6. CONCLUSION 
 
Our preliminary results suggest that the LAIT and 
TTP may distinguish between varying degrees of 
immunoreactivity against Blomia tropicalis and 
Farfantepenaeus brasiliensis extracts in patients 
clinically diagnosed with non–IgE–mediated 
Allergic Multimorbidity. Despite requiring trained 
personnel for execution, LAIT and TTP are 
inexpensive, can be performed with minimal 
laboratory equipment, and can be incorporated 
into strategies to address health disparities in 
diagnosing 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 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 aeroallergens 
and food 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 dangerous 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 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 LAIT may also 
contribute to streamlining biomedical research 
and improving tools, such as large language 
models, which clinicians often use as a decision 
support system to enhance diagnostic accuracy 
(Abers & Mathias, 2025). 
 

ETHICAL APPROVAL 
 

The authors have obtained and documented 
written ethical approval following international 
standards. As a retrospective survey of results 
recorded in cognito, consent was obtained 
collectively by the institution's ethics committee, 
following the principles outlined in the 
Declaration of Helsinki (World Medical 
Association, 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 they have no known 
competing financial interests or non-financial 
interests or personal relationships that could 
have appeared to influence the work reported in 
this paper. 
 

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