Medizinische Universität Graz Austria/Österreich - Forschungsportal - Medical University of Graz
Gewählte Publikation:
Sturm, G.
The basophil activation test in diagnostic practice of hymenoptera venom allergy
[ Dissertation ] Medical University of Graz; 2011. pp. 103
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- Autor*innen der Med Uni Graz:
- Betreuer*innen:
- Aberer Werner
- Heinemann Akos
- Strunk Dirk
- Altmetrics:
- Abstract:
- The basophil activation test (BAT) is a widely validated and reliable tool especially for the diagnosis of hymenoptera venom allergy. Nevertheless, several pitfalls have to be considered and outcomes may differ due to diverse in-house protocols and commercially available kits. In the first step, we aimed to identify factors that may influence results of the CD63-based BAT. The effect of stimulating factors such as IL-3, cytochalasin B and pre-warming of the samples was investigated. Additionally, 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. IL-3 enhanced the reactivity of basophils at 300 pM, but not at 75 pM and 150 pM. Pre-warming of samples and reagents did not affect basophil reactivity. CD63 expression assayed after storage time of up to 48 hours showed that basophil reactivity already started to decline after 4 hours. Basophils stained with HLA-DR-PC5 and CD123-PE antibodies gated as HLA-DRneg/CD123pos cells showed the highest reactivity. No effect on test outcomes was observed at therapeutic doses of dimetindene and desloratadine. The second aim was to identify potent influencing factors of the CD203c-based BAT and to emphasize differences between CD63 and CD203c detection. The effects of IL-3 and degranulation enhancing substances were investigated and compared with CD63 up-regulation. Furthermore, the influence of different storage conditions and incubation times was evaluated and the impact of anti-allergic drugs on the test results was assessed. CD203c and CD63 expression was rapidly upregulated reaching a maximum after 20 to 30 min. Basophil CD203c up-regulation assayed after storage times up to 48 h declined already after 4h. IL-3 treatment increased CD203c and CD63 baseline levels and decreased basophil CD203c responses in a dose-dependent manner. Finally, therapeutic concentrations of dimetindene and desloratadine did not affect CD203c up-regulation. In the next step we evaluated the optimized BAT in patients with (clinically irrelevant) double sensitization to bee and wasp venom, a frequent problem in the diagnosis of hymenoptera venom allergy. Among 117 patients, double sensitization (DS) was observed in 63.7% by the Immulite, in 61.5% by the CAP, in 47.9% by the intradermal test (IDT), in 20.5% by the ADVIA, and in 17.1% by the BAT. In CAP double positive patients, western blot inhibition revealed cross-reactive carbohydrate determinant (CCD)-based DS in 50.8%, and the component resolved diagnosis (CRD) showed 41.7% of patients with true DS. BAT, CRD, and ADVIA showed the lowest rate of DS and were helpful in finding the culprit insect. However, the rate of DS was higher than expected by personal history, indicating that the matter of clinical relevance is still not solved even by novel tests. Clinically irrelevant sensitization is frequently observed. Therefore, we initiated the next study to prove if sensitized subjects without a history of systemic sting reactions (SSR) tolerate sting challenges with the respective insect. In addition, BAT and routine diagnostic tools were correlated with the outcome of sting challenges. In 94 subjects 131 sting challenges with bees and wasps were performed. As expected, only 5 of 94 (5.3%) subjects showed SSR after the sting. Large local reactions (LLR) occurred in 41 of 94 (43.6%) subjects. A telephone survey was conducted among 1,401 subjects to determine the prevalence of SSR and LLR with an accuracy of ±1% in the general pop ...