The basophil activation test in the diagnosis of allergy: technical issues and critical factors.
BACKGROUND
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 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 stimulating 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.


CONCLUSIONS
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.
Sturm
G J
GJ
Department of Dermatology, Medical University of Graz, Austria.
Kranzelbinder
B
B
Sturm
E M
EM
Heinemann
A
A
Groselj-Strele
A
A
Aberer
W
W
D000818:Animals; D000888:Antibodies, Anti-Idiotypic / Q000494:pharmacology; D015703:Antigens, CD / Q000276:immunology*; D001491:Basophils / Q000187:drug effects / Q000276:immunology*; D001514:Bee Venoms / Q000494:pharmacology; D004115:Dimethindene / Q000494:pharmacology; D006634:Histamine H1 Antagonists / Q000494:pharmacology; D006801:Humans; D006927:Hymenoptera / Q000276:immunology*; D006967:Hypersensitivity / Q000175:diagnosis*; D007159:Immunologic Tests; D007377:Interleukin-3 / Q000494:pharmacology; D017336:Loratadine / Q000031:analogs & derivatives / Q000494:pharmacology; D010980:Platelet Membrane Glycoproteins / Q000276:immunology*; D060149:Tetraspanin 30; D014862:Wasp Venoms / Q000494:pharmacology
D000888:Antibodies, Anti-Idiotypic; D015703:Antigens, CD; D001514:Bee Venoms; C554586:CD63 protein, human; D006634:Histamine H1 Antagonists; D007377:Interleukin-3; D010980:Platelet Membrane Glycoproteins; D060149:Tetraspanin 30; D014862:Wasp Venoms; C026408:anti-IgE antibodies; D004115:Dimethindene; D017336:Loratadine; C121345:desloratadine