We read the excellent review by Ebo et al. with great interest. Over the past years, we have been working on technical aspects of the CD63-based basophil activation test (BAT) and evaluating this novel method in different fields of allergy diagnosis such as allergy to foods, drugs, Hymenoptera and natural rubber latex applying receiver–operating curve analysis [1, 2]. We strongly believe that there is a role for this promising technique of in vitro diagnosis and would like to comment on some aspects addressed in this review. To date mostly only natural allergen extracts have been applied to study allergen-induced CD63 expression. Hence, Ebo et al. suggest the benefit of applying recombinant allergens, for instance, as a basis for component-resolved allergy diagnosis should be evaluated in clinical studies. In a current study on birch pollen-associated allergy to foods we applied the recombinant allergens Bet v 1, Bet v 2, Api g 1, Dau c 1 and Mal d 1 in the CD63-based BAT using basophils from 30 food allergics with oral allergy syndrome induced by apple, carrot or celery and 20 controls. Results were compared with routine tests such as specific IgE (CAP method) and skin prick tests with native foods [3]. From this study, we draw several conclusions: (1) Application of recombinant allergens in the CD63-based BAT is feasible. (2) Sensitivity of the BAT for the three foods investigated ranged between 70% and 90%, exceeding sensitivity of specific IgE alone and increased overall sensitivity of in vitro tests. (3) In dose–response curves aiming at optimal allergen concentrations of the applied recombinant allergens a broad range of allergen concentrations (10−3–100 ng) induced CD63 expression in allergics without resulting in reduced sensitivity. (4) The BAT did not differentiate between clinically relevant or irrelevant sensitization. (5.) In apple allergy in particular, use of Mal d 1 in the BAT considerably improved in vitro sensitivity as compared with specific IgE against apple (80% vs. 55%). This is of importance for practical use since application of a few or even one single allergen concentration will eventually considerably ease the use of the BAT in daily clinical practice. Further on, application of recombinant allergens in the BAT may contribute to elucidate patterns of cross-reactivity, for instance, in food or latex allergy. We are presently evaluating the BAT with a set of recombinant latex allergens (Hev b 2–8) in latex allergics (health-care workers, patients with Spina bifida, food allergics). Our data indicate that in the BAT, application of recombinant latex allergens is feasible as well, however with very distinct ranges of varying allergen concentrations for each recombinant allergen, and a pattern of sensitization characteristic of the different subgroups of latex allergics with regard to source of sensitization. Interestingly, Ebo et al. demonstrated in a study on natural rubber latex allergy with natural allergen extracts that the BAT distinguished clinically relevant from irrelevant sensitization in several subjects [4]. Although we strongly support use of the BAT as a considerable progress of in vitro diagnostics we agree with Ebo et al. that quite a few open questions still remain and surprisingly to date several issues have not even been addressed at all. For instance, the problem of interference of prior drug intake such as glucocorticoids with the results of basophil activation as judged by CD63 expression or CD203c up-regulation has not been elucidated at all. In a small pilot study on the influence of oral administration of antihistamines we could not find a reduced rate of basophil activation in 10 birch pollen-sensitized patients after oral intake of 10 mg ceterizine as compared with basophil activation in the same subjects without intake of antihistamines (data unpublished). On the other hand with regard to some ‘technical aspects’ of this method (leucocyte suspensions vs. whole blood assays, pre-incubation with IL-3) some of these aspects might be of minor importance and in our eyes should not be overestimated. In another pilot study for technical evaluation of the CD63-based BAT, we demonstrated in 20 allergics (10 wasp and 10 birch pollen allergics) that the outcome of the BAT in an allergic subject (percentage of CD63+ basophils) was not influenced by the degree of basophil recovery within a broad range of 100–600 basophils. In other words, flow cytometric evaluation of a high quantity of basophils as compared with a low quantity does not improve the clinical and assay sensitivity, although at first sight this might be expected. With regard to the issue of pre-incubating basophils with IL-3 in a pilot study in 20 wasp allergics we established dose–response curves pre-incubating basophils with increasing amounts of IL-3 up to a final dose of 2 ng IL-3. Allergen-induced mean basophil activation was only increased by 5% by pre-incubating with IL-3 as compared with samples without pre-incubation with IL-3; however, priming with IL-3 may be helpful in drug allergy since drugs per se seem to induce a lower rate of basophil activation as compared with natural allergens in allergics [5]. The last point illustrates once more that at present not everyone should ‘go with the flow’ [6] but it is most desirable to establish multi-centre studies with uniform test protocols to tackle and answer these open questions and improve this promising in vitro method for daily clinical practice.
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Erdmann et al. (2004) studied this question.
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