Original article The basophil activation test in the diagnosis of allergy: technical issues and critical factors Up to 7.5% of the general population is reported to have experienced systemic anaphylactic reactions after hyme- noptera stings (1). Personal history, skin testing and detection of allergen-specific immunoglobulin E (IgE) serum antibodies are the mainstays of the diagnostic procedure in cases of hymenoptera venom hypersensitiv- ity, but are only of limited value in many instances. The basophil activation test (BAT) is now generally accepted as an additional and reliable diagnostic tool. Compared with the determination of IgE in serum, BATs offer the additional advantage of being able to demonstrate functional responses as positive test results will occur only after successful cross-linking, but not by monovalent binding as seen in IgE assays. The first CD63-based protocol for allergy diagnosis was developed by Sainte-Laudy and Sabbah in the mid- 1990s and the first English description of this application was published in 1996 (2). In 2000, excellent sensitivity and specificity in the diagnosis of hymenoptera allergy were described (3) and subsequently confirmed in several studies (4–8). The first CD203c-based protocol for hymenoptera allergy was published in 2001 (9); these data were recently validated by another research group (7). Currently, BAT with CD63 is the best clinically validated test (10), whereas BAT based on CD203c still requires further investigation. Generally, the potentials and pitfalls of BAT are still a point at issue. Whether BAT should be performed only by experienced and validated laboratories is a debated question (11, 12). However, pitfalls are scarcely reported in the published literature; most reports are based on personal communication and small numbers of patients (13). Furthermore, BAT protocols are generally not standardized; diverse protocols are prone to yield differ- ent results. Flow cytometric quantification of activated basophils can be used either on whole blood or on basophils separated by buffy coat centrifugation or sedimentation over dextran. Nevertheless, there is a clear Background: The basophil activation test (BAT) is a widely validated and reli- able tool especially for the diagnosis of hymenoptera venom allergy. Neverthe- less, several pitfalls have to be considered and outcomes may differ because of diverse in-house protocols and commercially available kits. We aimed to identify the factors that may influence results of the CD63-based BAT. Methods: Basophil responses to monoclonal anti-IgE (clone E124.2.8) and bee and wasp venom were determined by BAT based on CD63. The effect of stim- ulating factors such as, IL-3, cytochalasin B and prewarming of the samples was investigated. Furthermore, we compared two different flow cytometer systems and evaluated the influence of storage time, different staining protocols and anti- allergic drugs on the test results. Results: Interleukin-3 enhanced the reactivity of basophils at 300 pM, but not at 75 and 150 pM. Prewarming of samples and reagents did not affect basophil reactivity. CD63 expression assayed after storage time of up to 48 h showed that basophil reactivity already started to decline after 4 h. Basophils stained with HLA-DR-PC5 and CD123-PE antibodies gated as HLA-DR neg /CD123 pos cells showed the highest reactivity. No effect on test outcomes was observed at therapeutic doses of dimetindene and desloratadine. Finally, slight differences in the percentage of activated basophils, depending on the cytometer system used, were found. Conclusion: Basophil activation test should be performed as early as possible after taking the blood sample, preferably within 4 h. In contrast to the skin test, BAT can be performed in patients undergoing treatment with antihistamines. For reasons of multiple influencing factors, BAT should be performed only at validated laboratories. G. J. Sturm 1 , B. Kranzelbinder 1 , E. M. Sturm 2 , A. Heinemann 2 , A. Groselj-Strele 3 , W. Aberer 1 1 Division of Environmental Dermatology and Venerology, Department of Dermatology, Medical University of Graz; 2 Institute of Experimental and Clinical Pharmacology, Medical University of Graz; 3 Center for Medical Research, Division of Biostatistics, Medical University of Graz, Graz, Austria Key words: allergy; BAT; CD63; flow cytometry; technical issues. Dr Gunter J. Sturm Division of Environmental Dermatology and Venerology Department of Dermatology Medical University of Graz Auenbruggerplatz 8 A-8036 Graz Austria Accepted for publication 23 January 2009 Allergy 2009: 64: 1319–1326 Ó 2009 John Wiley & Sons A/S DOI: 10.1111/j.1398-9995.2009.02004.x 1319