_____________________________________________________________________________________________________ *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) Olivier et al.; Asian J. Immunol., vol. 8, no. 1, pp. 8-25, 2025; Article no. AJI.131451 10 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 Olivier et al.; Asian J. Immunol., vol. 8, no. 1, pp. 8-25, 2025; Article no. AJI.131451 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 Olivier et al.; Asian J. Immunol., vol. 8, no. 1, pp. 8-25, 2025; Article no. AJI.131451 12 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- Olivier et al.; Asian J. Immunol., vol. 8, no. 1, pp. 8-25, 2025; Article no. AJI.131451 13 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) Olivier et al.; Asian J. Immunol., vol. 8, no. 1, pp. 8-25, 2025; Article no. AJI.131451 14 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) Olivier et al.; Asian J. Immunol., vol. 8, no. 1, pp. 8-25, 2025; Article no. AJI.131451 15 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 Olivier et al.; Asian J. Immunol., vol. 8, no. 1, pp. 8-25, 2025; Article no. AJI.131451 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 %) Olivier et al.; Asian J. Immunol., vol. 8, no. 1, pp. 8-25, 2025; Article no. AJI.131451 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). Olivier et al.; Asian J. Immunol., vol. 8, no. 1, pp. 8-25, 2025; Article no. AJI.131451 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 Olivier et al.; Asian J. Immunol., vol. 8, no. 1, pp. 8-25, 2025; Article no. AJI.131451 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. REFERENCES Agache, I., & Akdis, C. A. (2020). Endotypes of allergic diseases and asthma: An important step in building blocks for the future of precision medicine. Allergology International, 65(3): 243-252. Andersen, M. B., Hall, S., & Dragsted, L. O. (2011). Identification of European allergy patterns to the allergen families PR-10, LTP, and profilin from Rosaceae fruits. Clinical Reviews in Allergy and Immunollogy, 41(1): 4-19. Armentia, A., Pineda, F., Martin-Armentia, B., Ramos, C., Gil Martin, F. J., & Palacios, R. (2016). Endophthalmitis related to lemon allergy in a heroin addict. Allergologia et Immunopathologia, 44(5): 472-474. Asero, R., Mistrello, G., Roncarolo, D., Amato, S., Zanoni, D., Barocci, F., et al. (2003). Detection of clinical markers of sensitization to profilin in patients allergic to plant-derived foods. Journal of Allergy and Clinical Immunology, 112(2): 427-432. Asero, R., Monsalve, R., & Barber, D. (2008). Profilin sensitization detected in the office by skin prick test: a study of prevalence and clinical relevance of profilin as a plant food allergen. Clinical and Experimental Allergy, 38(6): 1033-1037. Augustin, R. (1953). Precipitins to grass pollen proteins. Nature, 172(4372): 307. Augustin, R., & Hayward, B. J. (1960). Human reagins to grass pollens and moulds: their purification and physico-chemical characterization. Immunology, 3(1): 45-73. Augustin, R., Hayward, B. J., & Longbottom, J. L. (1960). Isolation and physico-chemical characterization of reagins, blocking antibodies and precipitins to grass pollens. Acta Allergologica Supplementum (Copenhagen), 7: 31-37. Bartra, J., Sastre, J., del Cuvillo, A., Montoro, J., Jáuregui, I., Dávila, I., et al. (2009). From pollinosis to digestive allergy. Journal of Investigative Allergology and Clinical Immunology, 19 Suppl 1: 3-10. Olivier et al.; Asian J. Immunol., vol. 8, no. 1, pp. 8-25, 2025; Article no. AJI.131451 20 Bean, G. W., & Glaser, J. (1960). Incidence of subsequent ragweed pollinosis in patients with perennial bronchial asthma and/or perennial allergic rhinitis. Ann Allergy, 18: 1126-1129. Belcadi, J., Oulad Ali, S., & Senouci, K. (2024). Lime Dermatitis. Dermatitis, 35(5): 429- 430. Blackley, C. H. (1873). Experimental Researches on the Causes and Nature of Catarrhus Aestivus: Hay Fever or Hay Asthma - Available oline on http://www.archive.org/details/experimental res00blacgoog (Facsimile ed.): Oxford Historical Books. Bloom, B. R., & Bennett, B. (1966). Mechanism of a reaction in vitro associated with delayed-type hypersensitivity. Science, 153(3731): 80-82. Bradford, M. M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analls of Biochemistry, 72: 248-254. Canonica, G. W., Ansotegui, I. J., Pawankar, R., Schmid-Grendelmeier, P., van Hage, M., Baena-Cagnani, C. E., et al. (2013). A WAO - ARIA - GA²LEN consensus document on molecular-based allergy diagnostics. World Allergy Organization Journal, 6(1): 1-17. Čelakovská, J., Čermákova, E., Andrýs, C., Boudkova, P., & Krejsek, J. (2024). Sensitization to latex and food allergens in atopic dermatitis patients according to ALEX2 Allergy Xplorer test. Molecular Immunology, 175: 89-102. Coca, A. F. (1922). Studies in Specific Hypersensitiveness V. The Preparation of Fluid Extracts and Solutions for Use in the Diagnosis and Treatment of the Allergies with Notes on the Collection of Pollens. The Journal of Immunology, 7(2): 163-178. Das, R., Moss, J. E., Robinson, E., Roberts, S., Levy, R., Mizue, Y., et al. (2011). Role of macrophage migration inhibitory factor in the Th2 immune response to epicutaneous sensitization. Journal of Clinical Immunollogy, 31(4): 666-680. Doyen, V., Braun, J. J., Lutz, C., Khayath, N., & de Blay, F. (2018). [The usefulness of nasal provocation tests for respiratory physicians]. Reviste de Maladies Respiratoires, 35(8): 788-795. Dunn, I. S., & Halliday, W. J. (1980). Interactions between T and B lymphocytes and macrophages in the production of leukocyte adherence inhibition factor. Cellular Immunology, 52(1): 48-61. Ferreiro, M., Dopazo, A., & Aira, M. J. (2002). Incidence of pollinosis in the city of A Coruña: correlation with aerobiological data. Journal of Investigative Allergology and Clinical Immunology, 12(2): 124-129. Fuiano, N., Incorvaia, C., Riario-Sforza, G. G., & Casino, G. (2006). Anaphylaxis to honey in pollinosis to mugwort: a case report. European Annals of Allergy and Clinical Immunollogy, 38(10): 364-365. Gell, P. G. H., Harington, C. R., & Rivers, R. P. (1946). The antigenic function of simple chemical compounds; production of precipitins in rabbits. British Journal of Experimental Pathology, 27(5): 267-286. Glaspole, I. N., de Leon, M. P., Rolland, J. M., & O'Hehir, R. E. (2007). Anaphylaxis to lemon soap: citrus seed and peanut allergen cross-reactivity. Annals of Allergy, Asthma & Immunology, 98(3): 286-289. Gosset-Student, W. S. (1908). The probable error of a mean. Biometrika, 6(1): 1-25. Hasnain, S. M., Alqassim, A., & Al-Frayl, A. (2017). Diagnosis of allergy: IgE mediated cross-reactions amongst selected asthma elicitors. Journal of Disease and Global Health, 10(4): 111-122. Henrickson, S. E. (2025). Evolution of the concept of immune dysregulation and current classification. Journal of Allergy and Clinical Immunology, 155(1): 89-91. Iizuka, T., Barre, A., Rougé, P., Charpin, D., Scala, E., Baudin, B., et al. (2022). Gibberellin-regulated proteins: Emergent allergens. 3. Frontiers in Allergy, 3:877553. Iorio, R. A., Del Duca, S., Calamelli, E., Pula, C., Lodolini, M., Scamardella, F., et al. (2013). Citrus Allergy from Pollen to Clinical Symptoms. PLOS ONE, 8(1): e53680. Jiang, S. Y., Wright, C. M., Hinton, A., Green, G., Moyer, A. R., Gerstbacher, et al. (2024). Lime-Induced Phytophotodermatitis: A Rash That Requires Explicit Questioning. Journal of Allergy and Clinical Immunollogy in Practice, 12(6): 1631-1632. Kàtcheff, S. C., Labrador-Horrillo, M., Bartolomé, B., & Garriga-Baraut, T. (2024). Allergy to Gibberellin-regulated proteins in an adolescent: A case of orange-induced anaphylaxis mediated by cofactors. Allergollogie et Immunopathollogie (Madrid), 52(2): 48-50. Olivier et al.; Asian J. Immunol., vol. 8, no. 1, pp. 8-25, 2025; Article no. AJI.131451 21 Kayode, O. S., Prado, N., Thursfield, D. J., Till, S. J., & Siew, L. Q. C. (2020). Lemon seed allergy: a case presentation. Allergy, Asthma & Clinical Immunology, 16(1): 32. Khan, M. M. (2016). Allergic Disease. In M. M. Khan (Ed.), Immunopharmacology: 197- 225. Cham: Springer International Publishing. Konstantinou, G. N., Baker, M. G., Yu, J., Ford, L. S., Bencharitiwong, R., Grishina, G., et al. (2023). Citrin: a novel food allergen in citrus seeds and citrus-derived pectin that shows cross-reactivity with cashew and pistachio. Annals of Allergy, Asthma and Immunology, 131(6): 759-765.e753. Kuratsuji, T. (1981). Studies on leukocyte adherence inhibition test. Part II. Clinical applications of LAI test to detect delayed type hypersensitivity in infants and children. Keio Journal of Medicine, 30(2): 65-69. Li, J. D., Gu, J. Q., Xu, Y. Y., Cui, L., Li, L. S., Wang, Z. X., et al. (2022). Serum IgE profiles in Chinese pollinosis patients with grass pollen sensitisation. World Allergy Organization Journal, 15(1): 100624. Lluch-Bernal, M., Pedrosa, M., Domínguez- Ortega, J., Colque-Bayona, M., Correa- Borit, J., Phillips-Anglés, E., et al. (2024). Sensitization to Quercus ilex Pollen Is Clinically Relevant in Patients With Seasonal Pollen Allergy. Journal of Investigative Allergology and Clinical Immunology, 34(5): 338-340. Lombardi, C., Senna, G. E., Gatti, B., Feligioni, M., Riva, G., Bonadonna, P., et al. (1998). Allergic reactions to honey and royal jelly and their relationship with sensitization to compositae. Allergol Immunopathol (Madr), 26(6): 288-290. López-Torrejón, G., Ibáñez, M. D., Ahrazem, O., Sánchez-Monge, R., Sastre, J., Lombardero, M., et al. (2005). Isolation, cloning and allergenic reactivity of natural profilin Cit s 2, a major orange allergen. Allergy, 60(11): 1424-1429. Macowan, M., Pattaroni, C., Bonner, K., Chatzis, R., Daunt, C., Gore, M., et al. (2025). Deep multiomic profiling reveals molecular signatures that underpin preschool wheeze and asthma. Journal of Allergy and Clinical Immunology, 155(1): 94-106. Mitchell, R. A., Liao, H., Chesney, J., Fingerle- Rowson, G., Baugh, J., David, J., et al. (2002). Macrophage migration inhibitory factor (MIF) sustains macrophage proinflammatory function by inhibiting p53: regulatory role in the innate immune response. Proceedings of the National Academy of Science USA, 99(1): 345-350. Nagata, Y., Yoshihisa, Y., Matsunaga, K., Rehman, M. U., Kitaichi, N., & Shimizu, T. (2015). Role of macrophage migration inhibitory factor (MIF) in pollen-induced allergic conjunctivitis and pollen dermatitis in mice. PLoS One, 10(2): e0115593. Nakamura, S. (1996). [Seven years study on the incidence of Japanese cedar pollinosis among university students and its transition while in college]. Arerugi, 45(4): 378-385. Negrini, A. C., & Belloni, L. (1963). [Considerations on the incidence of various pollens in the etiology of pollinosis in Liguria]. Pathologica, 55: 363-370. Noferi, A., Ferrante, E., & Testa, A. (1965). [Pollinosis in Naples: characteristics and incidence in our case statistics from 1958 to 1964]. Folia Allergol (Roma), 12(3): 166- 176. Oliveira, T. B., Persigo, A. L. K., Ferrazza, C. C., Ferreira, E. N. N., & Veiga, A. B. G. (2020). Prevalence of asthma, allergic rhinitis and pollinosis in a city of Brazil: A monitoring study. Allergologie et Immunopathologie, 48(6): 537-544. Olivier, C. E., Argentão, D. G. P., Santos, R. A. P. G., Silva, M. D., Lima, R. P. S., & Zollner, R. L. (2013). Skin scrape test: an inexpensive and painless skin test for recognition of immediate hypersensitivity in children and adults. The Open Allergy Journal, 6: 9-17. Olivier, C. E., Lima, R. P. d. S., Pinto, D. G., & Santos, R. A. P. G. d. (2021a). The Plasma Preincubation with Papain Before the Assay Suggests that a Gell and Coombs Type II Reaction is Been Demonstrated by the Leukocyte Adherence Inhibition Test. Biomedical Journal of Scientific & Technical Research, 36(3): 28647 - 28655. Olivier, C. E., Lima, R. P. S., Pinto, D. G., Santos, R. A. P. G., Silva, G. K. M., Lorena, et al. (2012). In search of a tolerance-induction strategy for cow's milk allergies: significant reduction of beta- lactoglobulin allergenicity via transglutaminase/cysteine polymerization. Clinics, 67(10): 1171-1179. Olivier, C. E., Pinto, D. G., Lima, R. P. S., Silva, M. D. d., Santos, R. A. P. G., Teixeira, A. P. M., et al. (2021b). Assessment of Olivier et al.; Asian J. Immunol., vol. 8, no. 1, pp. 8-25, 2025; Article no. AJI.131451 22 Immunoreactivity against Therapeutic Options Employing the Leukocyte Adherence Inhibition Test as a Tool for Precision Medicine. European Journal of Clinical Medicine, 2(3): 40-45. Olivier, C. E., Pinto, D. G., Santos, R. A. P. G., & Lima, R. P. S. (2021c). Dextran’s interference over the Leukocyte Adherence Inhibition Test. Academia Letter, Article (number): 3792. Olivier, C. E., Pinto, D. G., Teixeira, A. P. M., Miguel, C. S., Santos, R. A. P. G., Santana, J. L. S., et al. (2024a). Cellular and Humoral Immunoreactivity against Hen’s Egg White: Relevance in Allergic Patients. Asian Journal of Immunology, 7(1): 274-284. Olivier, C. E., Pinto, D. G., Teixeira, A. P. M., Miguel, C. S., Santos, R. A. P. G., Santana, J. L. S., et al. (2024b). Endotyping Cellular and Humoral Immunoreactivity against Allium spices and Sulfites preservatives in Allergic Patients. A Retrospective Study. Asian Journal of Immunology, 7(1). Olivier, C. E., Pinto, D. G., Teixeira, A. P. M., Santana, J. L. S., Santos, R. A. P. G., & Lima, R. P. S. (2021d). Self-imposed food restriction and oral food challenges are correlated with precipitin's accuracy in the diagnosis of non-IgE-mediated food- related adulthood acute episodes of urticaria. Journal of Allergy & Therapy, 12(8): 1-8. Olivier, C. E., Pinto, D. G., Teixeira, A. P. M., Santana, J. L. S., Santos, R. A. P. G., & Lima, R. P. S. (2023a). Contribution of the Leukocyte Adherence Inhibition Test to the Diagnosis of Immunoreactivity against Cobalt. Asian Journal of Immunology, 6(1): 174-184. Olivier, C. E., Pinto, D. G., Teixeira, A. P. M., Santana, J. L. S., Santos, R. A. P. G., & Lima, R. P. S. (2023b). Contribution of the Leukocyte Adherence Inhibition Test to the Diagnosis of Innate Non–IgE-mediated Immunoreactivity against Saccharomyces cerevisiae. Asian Journal of Immunology, 6(1): 234-241. Olivier, C. E., Pinto, D. G., Teixeira, A. P. M., Santana, J. L. S., Santos, R. A. P. G., & Lima, R. P. S. (2024c). Contribution of Leukocyte Adherence Inhibition Test in the Evaluation of Non–IgE-mediated Immunoreactivity against Peanut Proteins in Children and Adults with Atopic Dermatitis. Asian Journal of Immunology, 7(1): 55/-62. Olivier, C. E., Pinto, D. G., Teixeira, A. P. M., Santana, J. L. S., Santos, R. A. P. G., & Lima, R. P. S. (2024d). Endotyping Cellular and Humoral Immunoreactivity against Aluminum in Allergic Patients: A Retrospective Study. Asian Journal of Immunology, 7(1): 149-158. Olivier, C. E., Pinto, D. G., Teixeira, A. P. M., Santana, J. L. S., Santos, R. A. P. G., & Lima, R. P. S. (2024e). Exploring the Role of Leukocyte Adherence Inhibition Test in Assessing Non-IgE Mediated Immunoreactivity to Benzoic Acid in Allergic Patients. Asian Journal of Immunology, 7(1): 63-70. Olivier, C. E., Pinto, D. G., Teixeira, A. P. M., Santana, J. L. S., Santos, R. A. P. G., Lima, R. P. S., et al. (2023c). Contribution of the Leukocyte Adherence Inhibition Test to the Diagnosis of Innate Non–IgE-mediated Immunoreactivity against Alternaria alternata. Asian Journal of Immunology, 6(1): 243-251. Olivier, C. E., Pinto, D. G., Teixeira, A. P. M., Santana, J. L. S., Santos, R. A. P. G., Lima, R. P. S., et al. (2023d). Contribution of the Leukocyte Adherence Inhibition Test to the Diagnosis of Non–IgE-mediated Immunoreactivity against Candida albicans in Patients with Atopic Dermatitis. Asian Journal of Immunology, 6(1): 268-276. Olivier, C. E., Pinto, D. G., Teixeira, A. P. M., Santana, J. L. S., Santos, R. A. P. G., Lima, R. P. S., et al. (2023e). Evaluating Non-IgE-mediated Allergens’ Immunoreactivity in Patients with “Intrinsic” Persistent Rhinitis with Help of the Leukocyte Adherence Inhibition Test. European Journal of Medical and Health Sciences, 5(1): 17-22. Olivier, C. E., Pinto, D. G., Teixeira, A. P. M., Santana, J. L. S., Santos, R. A. P. G., Lima, R. P. S., et al. (2023f). Far Beyond the IgE: Insights into the Clinical Profile of Allergic Patients with Selective IgE Deficiency, Urticarial Vasculitis, Allergic Pharyngitis, and Perennial Allergic Conjunctivitis. , 7(1), 35–45. Asian Journal of Immunology, 7(1): 35-45. Olivier, C. E., Pinto, D. G., Teixeira, A. P. M., Santana, J. L. S., Santos, R. A. P. G., Lima, R. P. S., et al. (2024f). Contribution of the Leukocyte Adherence Inhibition Test Olivier et al.; Asian J. Immunol., vol. 8, no. 1, pp. 8-25, 2025; Article no. AJI.131451 23 in Diagnosing Non–IgE-Mediated Immunoreactivity against Aspergillus fumigatus in Patients with Allergic Rhinitis and Asthma. Asian Journal of Immunology, 7(1): 12-20. Olivier, C. E., Pinto, D. G., Teixeira, A. P. M., Santana, J. L. S., Santos, R. A. P. G., Lima, R. P. S., et al. (2024g). Endotyping Non-IgE-Mediated Immunoreactivity to Dermatophagoides farinae: Implications for Allergic Patients. Asian Journal of Immunology, 7(1): 90-99. Olivier, C. E., Pinto, D. G., Teixeira, A. P. M., Santana, J. L. S., Santos, R. A. P. G. S., & Lima, R. P. S. (2021e). Immunoreactivity against Dermatophagoides pteronyssinus Assessed by the Leukocyte Adherence Inhibition Test in Patients with Intrinsic Atopic Dermatitis and Correlated “Intrinsic” Non–IgE-mediated Allergic Conditions. European Journal of Clinical Medicine, 2(6): 45-50. Olivier, C. E., Pinto, D. G., Teixeira, A. P. M., Santana, J. L. S., Santos, R. A. P. G. S., & Lima, R. P. S. (2021f). Intrinsic Atopic Dermatitis: Titration of Precipitins in the Screening of Food Allergens for Prescription of Elimination Diets and Desensitization Strategies. European Journal of Clinical Medicine, 2(6): 1-9. Olivier, C. E., Pinto, D. G., Teixeira, A. P. M., Santana, J. L. S., Santos, R. A. P. G. S., & Lima, R. P. S. (2022a). Contribution of the Leukocyte Adherence Inhibition Test for the evaluation of immunoreactivity against gluten extracts in non—IgE-mediated / non-autoimmune Gluten-Related Disorders. European Journal of Clinical Medicine, 3(2): 1-7. Olivier, C. E., Pinto, D. G., Teixeira, A. P. M., Santana, J. L. S., Santos, R. A. P. G. S., & Lima, R. P. S. (2022b). Contribution of the Leukocyte Adherence Inhibition Test to the Evaluation of Cellular Immunoreactivity against Latex Extracts for Non—IgE-Mediated Latex-Fruit-Pollen Syndrome in Allergic Candidates to Exclusion Diets and Allergic Desensitization. European Journal of Clinical Medicine, 3(1): 11-17. Olivier, C. E., Pinto, D. G., Teixeira, A. P. M., Santana, J. L. S., Santos, R. A. P. G. S., & Lima, R. P. S. (2022c). Leukocyte Adherence Inhibition Test to the assessment of Immunoreactivity Against Cow’s Milk Proteins in Non—IgE-Mediated Gastrointestinal Food Allergy. European Journal of Clinical Medicine, 3(2): 38-43. Olivier, C. E., Pinto, D. G., Teixeira, A. P. M., Santana, J. L. S., Santos, R. A. P. G. S., Lima, R. P. S., et al. (2023g). Evaluating Non-IgE-Mediated Allergens’ Immunoreactivity in Patients Formerly Classified as “Intrinsic” Asthmatics with Help of the Leukocyte Adherence Inhibition Test. European Journal of Clinical Medicine, 4(2): 1-7. Olivier, C. E., Santos, R. A. P. G., Lima, R. P. S., Argentão, D. G. P., Silva, G. K. M., & Silva, M. D. (2014). A Novel Utility for an Old Method: The Leukocyte Adherence Inhibition Test Is an Easy Way to Detect the Immunoreactive Interference of the Collection Tube Anticoagulant on Cellular Immunoassays. Journal of Cell Adhesion, Article ID 860427(http://dx.doi.org/10.1155/2014/860 427): 1-6. Özdemir, Ö. (2024). Gibberellin-regulated protein allergy and orange-induced anaphylaxis. Allergollogie et Immunopathologie (Madrid), 52(5): 105-106. Peternel, R., Milanović, S. M., Hrga, I., Mileta, T., & Culig, J. (2007). Incidence of Betulaceae pollen and pollinosis in Zagreb, Croatia, 2002-2005. Annals of Agricultural and Environmental Medicine, 14(1): 87-91. Rocklin, R. E. (1974). Products of Activated Lymphocytes: Leukocyte Inhibitory Factor (LIF) Distinct from Migration Inhibitory Factor (MIF). Journal of Immunology, 112(4): 1461-1466. Roesner, L. M., Swiontek, K., Lentz, D., Begemann, G., Kienlin, P., Hentges, F., et al. (2022). Patients With Atopic Dermatitis Sensitized to Pet Dander Mount IgE and T- Cell Responses to Mammalian Cystatins, Including the Human Self-Protein. Journal of Investigative Allergology and Clinical Immunology, 32(5): 383-392. Rogers, B. L., Pollock, J., Klapper, D. G., & Griffith, I. J. (1993). Sequence of the proteinase-inhibitor cystatin homologue from the pollen of Ambrosia artemisiifolia (short ragweed). Gene, 133(2): 219-221. Rosario, N. A. (1990). Pollinosis in Brazil: changing concepts. Journal of Allergy and Clinical Immunology, 85(4): 819-820. Sheng, W., Liu, A., Peng, H., Wang, J., & Guan, L. (2022). A time-series analysis on generalized additive model for atmospheric pollen concentration and the number of Olivier et al.; Asian J. Immunol., vol. 8, no. 1, pp. 8-25, 2025; Article no. AJI.131451 24 visits of allergic conjunctivitis, Beijing, China. Environmental Science Pollution Research International, 29(40): 61522- 61533. Sirvent, S., Tordesillas, L., Villalba, M., Díaz- Perales, A., Cuesta-Herranz, J., Salcedo, G., et al. (2011). Pollen and plant food profilin allergens show equivalent IgE reactivity. Annals of Allergy, Asthma & Immunology, 106(5): 429-435. Sloan, A. E., & Powers, M. E. (1986). A perspective on popular perceptions of adverse reactions to foods. Journal of Allergy and Clinical Immunology, 78(1 Pt 2): 127-133. Solórzano-Zepeda, C., Pérez-Allegue, I., Pastor- Vargas, C., Bartolomé-Zavala, B., & González-de-Olano, D. (2021). Allergy to orange with cystatine-like protein as one of its allergens. Annals of Allergy, Asthma and Immunology, 127(2): 266-267. Sub-Committee, A. N. (2024a). The official list of allergens from Citrus limon, Vol. 2024: WHO/International Union of Immunological Societies. Sub-Committee, A. N. (2024b). The official list of allergens from Citrus reticulata, Vol. 2024: WHO/International Union of Immunological Societies. Sub-Committee, A. N. (2024c). The official list of allergens from Citrus sinensis, Vol. 2021: WHO/International Union of Immunological Societies. Taketomi, E. A., Sopelete, M. C., Moreira, P. F. S., & Vieira, F. A. M. (2006). Pollen allergic disease: pollens and its major allergens. Brazilian Journal of Otorhinolaryngology (English Edition), 72(4): 562-567. Taketomi, E. A., Sopelete, M. C., Moreira, P. F. S., Silva, D. A. O., Santos, C. M., Sá- Júnior, A., et al. (2005). Sensitization to Lolium multiflorum, a major Brazilian grass that causes pollinosis in patients living in Southern Brazil. Journal of Allergy and Clinical Immunology, 115(2): S125. Tilandyová, D., Uhlárová, M., Sámel, L., & Muchová, E. (1989). [Incidence of pollinosis in school-age children in urban and rural areas]. Ceskoslovenska Pediatrie, 44(8): 459-462. Togias, A., Gergen, P. J., Liu, A. H., Kim, H., Wood, R. A., O’Connor, G. T., et al. (2025). Rhinoconjunctivitis Symptoms in Children and Adolescents with Asthma: A Longitudinal Clustering Analysis. JACI, in press. Valenta, R., Duchene, M., Ebner, C., Valent, P., Sillaber, C., Deviller, P., et al. (1992). Profilins constitute a novel family of functional plant pan-allergens. Journal of Experimental Medicine, 175(2): 377-385. Vitali-Silva, A., Bello, V. A., Poli-Frederico, R. C., Oliveira, C. E. C., Reiche, E. M. V., Bossa, B. B., et al. (2024). Relationship between food triggers and sensory hypersensitivity in patients with migraine. Arquivos de Neuropsiquiatria, 82(11): 1-7. Wang, E. T. (2008). Anaphylaxis caused by tangerine seeds but not tangerine juice. Annals of Allergy, Asthma & Immunology, 101(5): 553-554. Wells, H. G. (1911). Studies on the chemistry of anaphylaxis (III). Experiments with isolated proteins, especially those of the hen's egg. Journal of Infectious Diseases, 9: 147-171. Williams, C. A., & Chase, M. W. (1971). CHAPTER 13 - Precipitation Reactions, Reactions of Antibodies with Soluble Antigens, Vol. 3: 1-102: Academic Press. . Worm, M., Jappe, U., Kleine-Tebbe, J., Schäfer, C., Reese, I., Saloga, J., et al. (2014). Food allergies resulting from immunological cross-reactivity with inhalant allergens: Guidelines from the German Society for Allergology and Clinical Immunology (DGAKI), the German Dermatology Society (DDG), the Association of German Allergologists (AeDA) and the Society for Pediatric Allergology and Environmental Medicine (GPA). Allergo Journal International, 23(1): 1-16. Wu, G. A., Terol, J., Ibanez, V., López-García, A., Pérez-Román, E., Borredá, C., et al. (2018). Genomics of the origin and evolution of Citrus. Nature, 554(7692): 311-316. Wüthrich, B., Schnyder, U. W., Henauer, S. A., & Heller, A. (1986). [Incidence of pollinosis in Switzerland. Results of a representative demographic survey with consideration of other allergic disorders]. Schweizeriche Medizinische Wochenschrift, 116(27-28): 909-917. Xu, L. J., Zhang, S. Y., Yang, Q., Cheng, L., Yin, M., & Miyoshi, A. (2000). [A preliminary study on the incidence of cedar pollinosis in district of Wuhan]. Lin Chuang Er Bi Yan Hou Ke Za Zhi, 14(11): 505-506. Olivier et al.; Asian J. Immunol., vol. 8, no. 1, pp. 8-25, 2025; Article no. AJI.131451 25 Yamana, Y., Yamana, S., & Uchio, E. (2022). Relationship among total tear IgE, specific serum IgE, and total serum IgE levels in patients with pollen-induced allergic conjunctivitis. Graefes Archives Clinical and Experimental Ophthalmology, 260(1): 281-287. Yoon, Y., & Bunyavanich, S. (2025). Multi-omic Approaches for Endotype Discovery in Allergy/Immunology. Journal of Allergy and Clinical Immunology. In press. 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