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.

Perceived high risk of COVID-19 vaccination: the revealing power of placebo.Beyens Michiel1,2; Toscano Alessandro1,2; Van Damme Pierre3; Dogné Jean-Michel4; Ebo Didier1,2,5*; Sabato Vito1,2,51 Department of Immunology, Allergology, Rheumatology and the Infla-Med Centre of Excellence, Faculty of Medicine and Health Sciences, University of Antwerp, Antwerp, Belgium2 Immunology, Allergology, Rheumatology, Antwerp University Hospital, Antwerp, Belgium3 Centre for the Evaluation of Vaccination and Vaccine & Infectious Disease Institute, University of Antwerp, Antwerp, Belgium.4 Department of Pharmacy, Namur Research Institute for LIfe Sciences, University of Namur, Namur, Belgium.5 Immunology and Allergology, AZ Jan Palfijn Gent, Ghent, Belgium

ABBREVIATIONSCA cannabis allergyCCDs cross-reactive carbohydrate determinantsCI confidence intervalCSA cannabis sativa allergic patientsnsLTP s nonspecific lipid transfer proteinsP+LTP- controls sensitized to pollen without nsLTP sensitizationP+LTP+ controls sensitized to pollen and nsLTPs(r) recombinantsIgE specific immunoglobulin EST skin teststIgE total immunoglobulin EKEYWORDSCannabis allergy, sIgE, total IgE, allergy diagnosis, sIgE-to-total IgE ratioTo the Editor,The most important “diagnostic test” for CA is a detailed history. However, a positive history is no absolute proof of CA, mainly because of physiological effects of cannabis i.e. (rhino)conjunctivitis presence and possibly because of incorrect interpretation or recollection of symptoms by the patients under the drug’s influence. Consequently, clinical suspicion of CA requires confirmatory testing. A cannabis challenge, being excluded for obvious ethical/legal reasons, documentation of CA generally starts with skin testing (ST) or specific IgE-quantification. However, in the absence of a standardized extract for ST, many will use prick-prick tests (e.g., with buds, leaves, seeds)1 2. Crude plant parts or extracts thereof, can contain both genuine and cross-reactive allergenic components. Consequently, positive results of crude extract ST and sIgE should always be interpreted cautiously, as it might merely reflect (cross-) sensitization instead of allergy.Here, we sought to investigate whether serological diagnosis of CA could benefit from an adjustment for tIgE. For this purpose, sIgE-to-tIgE ratios were calculated for sIgE hemp (FEIA, ImmunoCAP ThermoFisher Scientific), sIgE to recombinant (r) Can s 3 and rCan s 5 (Cytometric Bead Assay) as detailed elsewhere 3. A sIgE rCan s 3-to-rPru p 3 and sIgE rCan s 5-to-rBet v 1 ratio was also calculated. Negative sIgE values were excluded, as these cannot benefit from such an adjustment. Patients were selected from our previously published data3 4; cannabis sativa allergic patients (CSA), controls with a pollen but no nsLTP sensitization (P+LTP-) and controls with both pollen and nsLTP sensitizations (P+LTP+) were enrolled.Detailed demographic information is shown in table 1 of our previously published article3. As shown in figure 1, the distribution of the sIgE hemp-to-tIgE ratio and sIgE rCan s 5-to-tIgE differed significantly between controls and CSA. Using a cut-off of 0.02 for sIgE hemp-to-tIgE, it was shown that a specificity of 93% (95% confidence interval (CI), 85-98%) could be reached (table 1). A second, lower cut-off (0.005) can benefit most of the CSA population (sensitivity 77% (95% CI 67-85%)) and retains a relatively good specificity 79% (95% CI 68-87%).For sIgE rCan s 5-to-tIgE, a cut-off of 0.01 could possibly be beneficial. However, small group numbers make it difficult to estimate its true value. The ratio of sIgE rCan s 3-to-tIgE did not show added value (p=0.86).Comparing CSA patients with and without anaphylaxis; no added value was found to identify (a risk of) cannabis related anaphylaxis for any of the explored sIgE-to-tIgE ratios (sIgE hemp-to-tIgE->p=0.104; sIgE rCan s 3-to-tIgE->p=0.416; sIgE rCan s 5-to-tIgE->p=0.84, data not shown).Finally, ratios of similar protein families i.e. sIgE rCan s 3-to-sIgE rPru p 3 and sIgE rCan s 5-to-sIgE rBet v 1 were explored, showing no additional diagnostic value (see figure E1 in the online repository).These results indicate that sIgE-to-tIgE ratios might have a place in the diagnostic approach of CA. In the case of a definite history of cannabis related symptoms we recommend (figure E2) a sIgE hemp assay (sensitivity 82% (95% CI 74-89%) and specificity 32% (95% CI 20-45%)) 3. A negative result significantly reduces the chance of IgE-mediated CA. A positive result should be followed by a sIgE hemp-to-tIgE ratio as it notably increases test specificity. Where available, it is worthwhile using cannabis component resolved diagnostics as it was shown that over two-thirds of CSA who experienced anaphylaxis are Can s 3 sensitized 3. This study is bound by certain limitations; group numbers vary between the different analyses because of insufficient patients’ sera. Additionally, we could not explore sIgE rCan s 2-to-tIgE, sIgE rCan s 4-to-tIgE and sIgE rCan s 4-to-sIgE rBet v 2, because of insufficient group numbers. Finally, there is no guarantee that these results can be extrapolated to other CSA populations or different geographical regions. The results of these study are of a preliminary nature. Repetition of our methods in other, ideally larger populations are feasible to see whether these results can be confirmed and remain intact in different CA populations.FIGURES & TABLESFIGURE 1