Test di attivazione dei basofili (BAT): quale utilità nella diagnostica dell'allergia a veleno di imenotteri?

Abstract

BACKGROUND: The diagnosis of Hymenoptera venom allergy (HVA) relies on detailed anamnesis, skin tests and determination of specific IgE (sIgE) with extractive and molecular allergens of Apidae and Vespidae. In many patients, however, multiple venoms sIgE positivity is detected and it is not always possible to clearly determine the primary sensitization or whether there is true double sensitization, and therefore to prescribe a proper venom-specific immunotherapy (VIT). In these cases, the in vitro sIgE inhibition test can add valuable information. However, this test requires extensive experience and has some limitations in case of low sIgE values and in case of high levels of sIgE anti-cross reactive carbohydrate determinants (CCD). Recently, the use of basophil activation test (BAT) has been recommended in order to identify the primary sensitization in subjects in which molecular testing and/or sIgE inhibition have been inconclusive. Aim of the study was to evaluate the usefulness of BAT in a selected cohort of subjects who experienced allergic reactions following hymenoptera sting, history suggestive of Vespid sting and inconclusive sIgE assay.METHODS: Nineteen subjects with allergic reactions following hymenoptera sting (17 with systemic reactions and two with extensive local reactions) were studied. Of those, six patients showed multiple sIgE positivity for Vespidae and positivity sIgE for Apis mellifera (Apis m) and anti-CCD (5/6) too; six patients complained reactions after hymenoptera sting but with negativity of Apis m and Vespidae sIgE (≤0.10 kU/L), and seven patients showed very low sIgE levels (0.11-0.84 kU/L) for extractive and/or molecular Vespidae allergens. In all subjects, the following were performed: the sIgE assay for Vespula sp, Polistes dominulus (Polistes d), Vespa crabro, Apis m, Ves v 1, Ves v 5 and Pol d 5 (ImmunoCAP 1000, Thermofisher Scientific), and BAT in which extractive venoms of Vespula sp (BAG2-I3) and Polistes d (BAG2-I77), (Bühlmann Laboratories AG), were used for basophil stimulation. The response, measured as percentage of activated basophils (CD63+) at the different venom concentrations, was evaluated by cytofluorimetric method with FACSCanto™ II (Becton Dickinson). The sIgE inhibition assay was performed by overnight incubation at 4 °C of serum with venom extract in solution (Anallergo), at decreasing concentrations of 300, 30, 3 and 0.3 µg/mL, followed by sIgE assay.RESULTS: BAT detected the primary sensitizer in 6/19 (32%) subjects including one with negative sIgE and three with very low sIgE, while sIgE assay identified the primary sensitizer in 5/19 (26%) including four concordant with BAT. BAT in 6/19 (32%) cases allowed diagnosis of dual sensitization, of which half (3/6) confirmed by inhibition test, five concordant with sIgE assay by molecular technique, and one with negative sIgE. In one of seven cases of dual sensitization evidenced by molecular testing, BAT was decisive and in one case negative. In 2/7 (29%) patients with negative sIgE for molecular allergens, BAT was positive for Vespula sp (in one of these, similar activation was obtained for Vespula sp and Polistes d at different venom concentrations).CONCLUSIONS: This study proved that BAT is useful in confirming true double sensitizations and slightly more sensitive than molecular testing in identifying single sensitization to Vespidae venom, both in cases with negative sIgE (≤ 0.10 kU/l), and in subjects with multiple positivities for Vespidae, even where anti-CCD antibodies are present. These data, therefore, confirm the importance of combining multiple laboratory methods for precision diagnosis of HVA and more accurate choice of VIT.