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.

The field of food allergy has seen tremendous change over the past 5-10 years with seminal studies redefining our approach to prevention and management and novel testing modalities in the horizon. Early introduction of allergenic foods is now recommended, challenging the previous paradigm of restrictive avoidance. The management of food allergy has shifted from a passive avoidance approach to active interventions that aim to provide protection from accidental exposures, decrease allergic reaction severity and improve the quality of life of food-allergic patients and their families. Additionally, novel diagnostic tools are making their way into the clinical practice with the goal to reduce the need for food challenges and assist physicians in the -- often complex -- diagnostic process. With all the new developments and available choices for diagnosis, prevention and therapy, shared decision-making has become a key part of the medical consultation, enabling patients to make the right choice for them, based on their values and preferences. Communication with patients has also become more complex over time, as patients are seeking advice online and through social media, but the information found online may be outdated, incorrect, or lacking in context. The role of the allergist has evolved to embrace all the above exciting developments and provide patients with the optimal care that fits their needs. In this review, we discuss recent developments, as well as the evolution of the field of food allergy in the next decade.

Adolescence is a critical stage of rapid biological, emotional and social change and development. Adolescents and young adults (AYA) with asthma and allergies need to develop the knowledge and skills to self-manage their health independently. Healthcare professionals (HCP), parents and their wider network play an essential role in supporting AYA in this process. Previous work showed significant limitations in transition care across Europe. In 2020, the first evidence-based guideline on effective transition for AYA with asthma and allergies was published by EAACI. We herein summarize practical resources to support this guideline’s implementation in clinical practice. For this purpose, multi-stakeholder Task Force members searched for resources in peer review journals and grey literature. These resources were included if relevant and of good quality, and were pragmatically rated for their evidence-basis and user friendliness. Resources identified covered a range of topics and targeted healthcare professionals, AYA, parents/carers, schools, workplace, and wider community. Most resources were in English, web-based and had limited evidence-basis. This position paper provides a valuable selection of practical resources for all stakeholders to support effective transitional care for AYA with asthma and allergies. Future research should focus on developing validated, patient-centred tools to further assist evidence-based transition care.

Progressive knowledge of allergenic structures resulted in a broad availability of allergenic molecules for diagnosis. Component resolved diagnosis allowed a better understanding of patient sensitization patterns, facilitating allergen immunotherapy decisions. In parallel to the discovery of allergenic molecules, there was a progressive development of a regulation framework that affected both in vitro diagnostics and Allergen Immunotherapy products. With a progressive understanding of underlying mechanisms associated to Allergen immunotherapy and an increasing experience of application of molecular diagnosis in daily life, we focus in analyzing the evidences of the value provided by molecular allergology in daily clinical practice, with a focus on Allergen Immunotherapy decissions.

The basophil activation test (BAT) is a functional assay that measures the degree of degranulation following stimulation with allergen or controls by flow cytometry and is directly correlated with histamine release. From the bell-shaped curve resulting from BAT in allergic patients, basophil reactivity (given by %CD63+ basophils) and basophil sensitivity (given by EC50 or similar) are the main outcomes of the test. BAT takes into account all characteristics of IgE and allergen and thus can be more specific than sensitization tests in the diagnosis of allergic disease. BAT reduces the need for in vivo procedures, such as intradermal tests and allergen challenges, which can cause allergic reactions of unpredictable severity. As it closely reflects the patients’ phenotype, it can potentially be used to monitor the natural resolution of food allergies and to predict and monitor clinical response to immunomodulatory treatments, such as allergen-specific immunotherapy and biologicals. Clinical application of BAT requires analytical validation, clinical validation, standardization of procedures and quality assurance to ensure reproducibility and reliability of results. Currently, efforts are ongoing to establish a platform that could be used by laboratories in Europe and in the USA for certification.

Groundbreaking Discoveries in ImmunologyTitle : IgE sialylation: unravelling a key anaphylactic mediatorAuthors : Beatriz Moyaa, Chiara Tontinib and Alexandra Santosc, d, e, fa. Allergy Service. Hospital Universitario 12 de Octubre, Madrid, Spainb. Lydia Becker Institute for Immunology and Inflammation, Faculty of Biology, Medicine and Health, University of Manchester, Manchester, UKc. Department of Women and Children’s Health (Paediatric Allergy), School of Life Course Sciences, Faculty of Life Sciences and Medicine, King’s College London, London, UKd. Peter Gorer Department of Immunobiology, School of Immunology and Microbial Sciences, King’s College London, London, UKe. Children’s Allergy Service, Guy’s and St Thomas’ Hospital, London, UKf. Asthma UK Centre of Allergic Mechanisms of Asthma, London, UKCorrespondence to : Beatriz Moya. Allergy Service. Hospital Universitario 12 de Octubre, Madrid, SpainEmail: [email protected]: Ig, Immunoglobulin; Fab, antigen-binding fragments; Fc, fragment crystallizable region; FcεRI, Fc epsilon receptor I; MC, mast cells.Word count: 637/1000

Adolescent and young adult (AYA) patients need additional support while they experience the challenges associated with their age. They need specific training to learn the knowledge and skills required to confidently self-manage their allergies and/or asthma. Transitional care is a complex process which should address the psychological, medical, educational and vocational needs of AYA in the developmentally appropriate way. The European Academy of Allergy and Clinical Immunology has developed a clinical practice guideline to provide evidence-based recommendations for healthcare professionals to support the transitional care of AYA with allergy and/or asthma. This guideline was developed by a multi-disciplinary working panel of experts and patient representatives based on two recent systematic reviews. It sets out a series of general recommendations on operating a clinical service for AYA, which include: (i) starting transition early (11-13 years), (ii) using a structured, multidisciplinary approach, (iii) ensuring AYA fully understand their condition and have resources they can access, (iv) active monitoring of adherence and (v) discussing any implications for further education and work. Specific allergy and asthma transition recommendations include (i) simplifying medication regimes and using reminders; (ii) focusing on areas where AYA are not confident and involving peers in training AYA patients; (iii) identifying and managing psychological and socioeconomic issues impacting disease control and quality of life; (iv) enrolling the family in assisting AYA to undertake self-management and (v) encouraging AYA to let their friends know about their allergies and asthma. These recommendations may need to be adapted to fit into national healthcare systems.