REAL-LIFE INSIGHTS AND CLINICAL CHARACTERISTICS OF HEREDITARY ALPHA TRYPTASEMIA IN ANAPHYLACTIC PATIENTSAuthors: Loli-Ausejo D1,2,4*, Gonzalez-Matamala MF1,2*, Martí D3, Ruano-Zaragoza M1,2,4, Mir-Ihara P1,2,4, Solis-Ynga K1,2, Lara R3, Bartra J1,2,4, Pascal M2,3,4**, Muñoz-Cano R 1,2,4**1. Department of Allergy, Hospital Clinic Barcelona, Universitat de Barcelona (UB), Barcelona, Spain.2. Clinical and Experimental Respiratory Immunoallergy (IRCE), Institut d’Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Barcelona, Spain.3. Department of Immunology, Hospital Clínic Barcelona, Universitat de Barcelona (UB), Barcelona, Spain.4. Redes de Investigación Cooperativa Orientadas a Resultados en Salud (RICORS), Red de Enfermedades Inflamatorias (REI), Instituto de Salud Carlos III (ISCIII), Madrid, Spain.* Loli-Ausejo D and Gonzalez-Matamala MF contributed equally to this work** Pascal M and Muñoz-Cano R contributed equally to this workCorrespondance : rmunoz@clinic.cat

Does cypress pollen exposure influence sensitisation to Pru p 7 as a major allergen in peach allergy? To the editor:Gibberellin-regulated protein (GRP) was recently identified as an allergen in pollen-fruit syndrome1. Similar to peach Lipid Transfer protein (LTP) Pru p 3, peach GRP (Pru p 7) has been associated with severe allergic reactions2. In addition, Pru p 7 has been proposed as a possible major allergen in peach, in areas with high cypress pollen counts3. However, the relevance of this allergen in an area with high prevalence of Pru p 3 sensitization and high cypress pollen exposure has not been fully established.A descriptive, observational study was conducted in Hospital Clínic, Barcelona between January 2021 and August 2023. Patients diagnosed of peach allergy based on clinical history and positive skin prick test to peach were included. Clinical data as age, gender, severity of reactions (classified by oFASS-3 scoring4), other offending plant foods, and presence of cofactors were also collected. Total IgE and serum specific IgE (sIgE) to Pru p 3, Pru p 7 and peach were tested (ImmunoCAP®, ThermoFisher Scientific). In addition, the presence of both cypress pollen sensitization and pollen count data from our area were assessed. (Detailed methods-Supplementary Material).A total of 140 patients were included; 123 (87.9%) were monosensitized to Pru p 3, 10 (7.1%) were sensitized to both (co-sensitized), and 7 (5.0%) were monosensitized to Pru p 7. Their main characteristics are summarized in Table 1. Patients monosensitized to Pru p 7 were all women, showed higher levels of sIgE Pru p 7 than co-sensitized patients (p=0.15), and lower peach sIgE values than those monosensitized to Pru p 3 (p=0.001) or co-sensitized (p=0.03). Moreover, two of these patients had negative sIgE to peach.Pru p 7 monosensitized patients had similar clinical features. Almost all (6/7) presented anaphylactic reactions, and.cofactors were frequently involved (5/7), exercise being the most common (4/7). Rosaceae and citrus fruits were the most frequent culprit foods; other offending foods were apple, fig, halzenut and pear (table 2). All these subjects were also sensitized to cypress pollen.The cypress pollen count (Annual Pollen Integral, or sum of the mean daily pollen concentrations along the year) in our area (Barcelona-Bellaterra) during the study period were 17.530 pollen*day/m3 in year 2021, 8.122 pollen*day/m3 in year 2022 and 8.320 pollen*day/m3 in year 2023.Pru p 7 has recently been reported as a peach major allergen in southern France, a region geographically close to ours (north-east Spain)3. In this study, out of the five analyzed regions in France, the most represented has a cypress annual pollen count of 18,782 pollen*day/m3, comparable to our area. Interestingly, despite the similarity of the cypress pollen exposure, geographical proximity, and climatic resemblance, we have observed a different peach sensitization profile that confirms the role of LTP as a major allergen and GRP as a minor allergen in our area. This difference suggests that other factors apart from cypress pollen exposure may influence Pru p 7 sensitisation.Concerning the severity of reactions, Pru p 7 monosensitized patients exhibited more severe reactions compared to Pru p 3 and Pru p 3+Pru p 7 groups (Table 1). Conversely, the presence of cofactors was consistent across all groups.Our study observed that peach-specific IgE is a poor biomarker of Pru p 7 sensitisation as over 50% of Pru p 7 monosensitized patients had negative or very low peach-specific IgE levels. For this reason and to avoid false negative results, we recommend testing Pru p 7 in all patients with suspected peach allergy and those with citric fruit-related reactions.Finally, our results suggest that others plant foods different from those previously described 5 could be triggering reactions in GRP allergic patients. Future studies are needed for a better understanding of the clinical pattern of GRP food allergy and the potential cross reactivity of this protein among plant foods not taxonomical related, to define a Gibberellin-regulated protein syndrome that would present very similar characteristics, but not identical, to LTP syndrome.REFERENCESTuppo L, Alessandri C, Pomponi D, et al. Peamaclein–a new peach allergenic protein: similarities, differences and misleading features compared to Pru p 3. Clin Exp Allergy. 2013;43(1):128-140. doi:10.1111/cea.12028.Inomata N, Okazaki F, Moriyama T, et al. Identification of peamaclein as a marker allergen related to systemic reactions in peach allergy. Ann Allergy Asthma Immunol. 2014;112(2):175-177.e3. doi:10.1016/j.anai.2013.11.003.Klingebiel C, Chantran Y, Arif-Lusson R, et al. Pru p 7 sensitization is a predominant cause of severe, cypress pollen-associated peach allergy. Clin Exp Allergy. 2019;49(4):526-536. doi:10.1111/cea.13345Fernández-Rivas M, Gómez García I, Gonzalo-Fernández A, et al. Development and validation of the food allergy severity score. Allergy. 2022 May;77(5):1545-1558. doi: 10.1111/all.15165. Epub 2021 Nov 12. PMID: 34716996; PMCID: PMC9298738Takei M, Nin C, Iizuka T, et al. Capsicum Allergy: Involvement of Cap a 7, a New Clinically Relevant Gibberellin-Regulated Protein Cross-Reactive With Cry j 7, the Gibberellin-Regulated Protein From Japanese Cedar Pollen. Allergy Asthma Immunol Res. 2022;14(3):328-338. doi:10.4168/aair.2022.14.3.328FIGURESTable 1 . Demographic and clinical characteristics of patients according to their sensitization to Pru p 3 and Pru p 7.

Background: Recent studies indicated that fish-allergic patients may safely consume certain fish species. Multiplex IgE testing facilitates the identification of species tolerated by individual patients. Methods: Sera were collected from 263 fish-allergic patients from Austria, China, Denmark, Luxembourg, Norway and Spain. Specific (s) IgE to parvalbumins (PVs) from 10 fish species along with IgE to 7 raw and 6 heated fish extracts was quantified using a research version of the ALEX 2 assay. IgE-signatures of individual patients and patient groups were analyzed using SPSS and R. Results: sIgE to alpha-PV from ray, a cartilaginous fish, was not detected in 78% of the patients while up to 41% of the patients, depending on their country of origin, tested negative for at least one beta-PV. sIgE values were highest for mackerel and tuna PVs (>10 kUA/L) and significantly lower for cod (4.9 kUA/L) and sole PVs (2.55 kUA/L). 17% of the patients, although negative for PVs, tested positive for the respective fish extracts. Based on the absence of IgE to PVs and extracts, up to 21% of the patients were identified as potentially tolerating one or more bony fish. Up to 90% of the patients tested negative for ray. The probability of negativity to one fish based on negativity to others was calculated. Negativity to tuna and mackerel emerged as a good marker of negativity to additional bony fish. Conclusion: Measuring sIgE to PVs and extracts from evolutionary distant fish species indicates bony and cartilaginous fish species for tolerance-confirming food challenges.

Background: As the use of multiplex specific IgE (sIgE) detection methods becomes increasingly widespread, proper comparative validation assessments of emerging new platforms are vital. The objective of this study was to assess the clinical and technical performance of the ALEX platform (MacroArray Diagnostics), in comparison to the ImmunoCAP ISAC 112 microarray and the ImmunoCAP singleplex method (ThermoFisher Scientific) in the diagnosis of pollen (cypress, grass, olive), dust mite Dermatophagoides pteronyssinus, Alternaria alternata, fruit (apple, peach) and nut (walnut, hazelnut and peanut) allergy. Methods: We enrolled 153 allergic patients and 16 non-atopic controls. sIgE assays were conducted using ISAC112, ALEX version 2 (ALEX2), and ImmunoCAP for whole extracts and major components. Technical validation of ALEX2 was performed by measuring repeatability and inter-assay, inter-batch and inter-lab reproducibility. Results: When measured globally (detection by one or more allergen components), ALEX2 showed adequate sensitivity and specificity for most of the allergens studied, comparable in general to that of ISAC112 (except for olive pollen and walnut) and similar to that of ImmunoCAP whole extract measurements. Component-by-component analysis showed comparable results for all techniques, except for Ole e 1 and Jug r 3 in both ISAC112 and ImmunoCAP comparisons, and Alt a 1, when compared with ISAC112. Continuous sIgE levels correlate with sIgE by ImmunoCAP. Good reproducibility and repeatability were observed for ALEX2. Conclusions: ALEX2 shows sound technical performance, and adequate diagnostic capacity, comparable in general to that of ISAC112 and ImmunoCAP for some aeroallergens and plant-food allergies in Mediterranean patients.

Epidemiological studies have explored the relationship between allergic diseases and cancer risk or prognosis in AllergoOncology. Some studies suggest an inverse association, but uncertainties remain, including in IgE-mediated diseases and glioma. Allergic disease stems from a Th2-biased immune response to allergens in predisposed atopic individuals. Allergic disorders vary in phenotype, genotype, and endotype, affecting their pathophysiology. Beyond clinical manifestation and commonly used clinical markers, there is ongoing research to identify novel biomarkers for allergy diagnosis, monitoring, severity assessment, and treatment. Gliomas, the most common and diverse brain tumours, have in parallel undergone changes in classification over time, with specific molecular biomarkers defining glioma subtypes. Gliomas exhibit a complex tumour-immune interphase and distinct immune microenvironment features. Immunotherapy and targeted therapy hold promise for primary brain tumour treatment, but require more specific and effective approaches. Animal studies indicate allergic airway inflammation may delay glioma progression. This collaborative European Academy of Allergy and Clinical Immunology (EAACI) and European Association of Neuro-Oncology (EANO) Position Paper summarizes recent advances and emerging biomarkers for refined allergy and adult-type diffuse glioma classification to inform future epidemiological and clinical studies. Future research is needed to enhance our understanding of immune-glioma interactions to ultimately improve patient prognosis and survival.

A consensus protocol for the Basophil Activation Test for multicenter collaboration and External Quality AssuranceAuthors: Pascal, M# 1, Edelman SM#2, Nopp, A#3, Möbs, C4, Geilenkeuser, WJ5, Knol, EF6, Ebo, DG7, Mertens C7, Shamji, MH8, Santos, AF9,10, Patil, S11, Eberlein, B*12, Mayorga, C*13, Hoffmann HJ14*Affiliations1 Immunology Department, Centre de Diagnòstic Biomèdic, Hospital Clínic de Barcelona, Barcelona, Spain; Institut d’Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Universitat de Barcelona, Spain.2 Skin and Allergy Hospital, Helsinki University Central Hospital, Helsinki, Finland, present address Aimmune Therapeutics, Finland3 Department of Clinical Science and Education, Karolinska Institutet, Södersjukhuset, and Sachs´ Children and Youth Hospital, Södersjukhuset, Stockholm, Sweden4 Department of Dermatology and Allergology, Philipps-Universität Marburg, Marburg, Germany5 Reference Institute for Bioanalytics, Bonn, Germany6 Center of Translational Immunology and Dermatology/Allergology, University Medical Center Utrecht, Utrecht, The Netherlands.7 Faculty of Medicine and Health Sciences, Department of Immunology-Allergology- Rheumatology, University of Antwerp, Antwerp, Belgium8 National Heart and Lung Institute, Imperial College London, UK and NIHR Imperial Biomedical Research Centre, UK9 Department of Women and Children’s Health (Pediatric Allergy) & Peter Gorer Department of Immunobiology, Faculty of Life Sciences and Medicine, King’s College London, London, United Kingdom10 Children’s Allergy Service, Evelina London Children’s Hospital, Guy’s and St Thomas’ Hospital, London, United Kingdom11 Division of Allergy and Immunology, Departments of Medicine and Pediatrics, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts, United States12 Department of Dermatology and Allergy Biederstein, School of Medicine, Technical University Munich, Munich, Germany13 Allergy Clinical Unit, Hospital Regional Universitario de Málaga and Allergy Research Group, Instituto de Investigación Biomédica de Málaga-IBIMA-BIONAND, Malaga, Spain;14 Department of Clinical Medicine, Aarhus University, Department of Respiratory Diseases and Allergy, Aarhus University Hospital, Denmark# shared first authors, * shared senior authorsCOIM Pascal, SM Edelman, A Nopp, C Möbs, EF Knol, SU Patil and C Mayorga have no conflict of interest regarding this work. B Eberlein received methodological and technical support from the company BUEHLMANN Laboratories AG (Schönenbuch, Switzerland) outside the submitted work. Dr Hoffmann reports grant from the Innovation Fund of Denmark, outside the submitted work. Dr Shamji reports grants awarded to institution from the Immune Tolerance Network, UK Medical Research Council, Allergy Therapeuitics, LETI Laboratories, Revolo biotherapeutics and Angany Inc. He has received consulting fees from Bristol Meyers Squibb and lecture fees from Allergy Therapeutics and LETI laboratories, all outside the submitted work. Dr. Santos reports grants from Medical Research Council (MR/M008517/1; MC/PC/18052; MR/T032081/1), Food Allergy Research and Education (FARE), the NIH, Asthma UK (AUK-BC-2015-01), the Immune Tolerance Network/National Institute of Allergy and Infectious Diseases (NIAID, NIH) and the NIHR through the Biomedical Research Centre (BRC) award to Guy’s and St Thomas’ NHS Foundation Trust, during the conduct of the study; speaker or consultancy fees from Thermo Scientific, Nutricia, Infomed, Novartis, Allergy Therapeutics, IgGenix, Stallergenes, Buhlmann, as well as research support from Buhlmann and Thermo Fisher Scientific through a collaboration agreement with King’s College London, outside the submitted work. Dr Geilenkeuser is an employee of Referenizinstitut für Bioanalytik, DE that provided logistic assistance and reagent support for the study.To the editorThe basophil activation test (BAT) has significant potential as a diagnostic tool to better phenotype and manage patients with IgE-mediated allergies, so that only a small proportion of patients need to be challenged. Sample, reagent, laboratory procedure, analysis protocols, and population characteristics can influence BAT performance (1,2). Regulatory approval and clinical implementation require extensive standardization of laboratory protocols, cytometer settings, and results interpretation (3). European national authorities require External Quality Assurance (EQA) of the performance of modern diagnostic laboratories by agencies independent of test suppliers to meet ISO 15189:2012, 15189:2013 and 9001:2015.Based on an online survey among 59 responding European laboratories performing BAT in 2017 (4,5) (Online Supplement; Results of the online survey), a Task Force was launched in 2018 to create the basis for a BAT-EQA. Round Robins (RR) were organized with seven shipments of 2 donors each to 7-10 European centers with overnight courier service from Bonn, DE. To minimize variation, prior to shipment, blood basophils were activated with 1 ul FcεRI antibody/ml of blood and stabilized with 0.2 mL Transfix (Cytomark, UK) per mL of blood to stabilize activated basophils up to 48 hours for staining (6). Fresh blood was included for stimulation and staining at the participating laboratory sites.We met after the third shipment to reach consensus on a protocol for BAT (Online Supplement; Proposed SOP for in house BAT). The threshold set on an unstimulated control sample was determined empirically on an independent data set as equal or greater than 2.5% with ROC curves based on data from patients with hypersensitivity to amoxicillin and patients with peanut allergy, (Online supplement, tables S1 and S2). This proposal did not find universal consensus among the authors.Data analysis started with identification of the relevant region in a scatter plot, followed by identification of basophils with the relevant markers, for instance, using low SSC and CD193 only or CD193 and CD123. Finally, the threshold was set at 2.5% of CD63 expression on resting basophils (Figure 1A). >5% CD63+basophils above that threshold in an activated sample was considered a positive response. This setting was used to obtain the percentage of CD63+ cells in centrally preactivated and locally activated blood samples; however, it was not adopted in all labs. Data from participating labs analyzed with their proprietary and the above standardized analysis compared well (online supplement, figure S4).The first two RR were used to establish coherence between participating laboratories. Data from RR3–RR7 were comparable. The standard deviation of activation measured at all participating centers was 16.8% in preactivated blood (Figure 1B) compared with 49.2% for samples activated and analyzed locally, illustrating the utility of using preactivated blood for EQA. Shipment to Málaga took 48h, and local activation of blood basophils was consistently suboptimal, consistent with a preliminary round robin from 2012, where the clinical outcome was robust up to 24 h. Centrally activated basophils performed as well in Málaga as in other centers.EQA for BAT is critical to facilitate routine implementation of this assay in the field of in vitro allergy diagnostics. The variability of the responses to our survey highlighted the importance and need for multicenter validation. Full validation and standardization of the BAT protocol and analysis is essential and possible for setting the grounds for controlled multicenter research studies as well as EQA. The BAT-EQA Task Force provides a standard operating protocol (Online supplement; Proposed SOP for in house BAT) and reference materials for the test to standardize and enhance the accuracy of BAT for both clinical and research collaborations and EQA.