Hymenoptera venom allergy constitutes an important health problem and correct diagnosis is a prerequisite for effective and potentially life-saving management, i.e. specific venom immunotherapy (VIT). Currently, diagnosis of venom anaphylaxis generally relies upon an evocative history corroborated by positive venom skin tests (VST) and venom-specific IgE. Although each method can provide useful information, both have in se limitations (1–7). Sting challenge tests have been thoroughly described (8), but several practical and mainly ethical issues, as well as the observation that a single negative sting challenge does not definitely indicate absence of hypersensitivity certainly limits its application. In fact, as deliberate sting challenges carry a considerably risk for anaphylaxis, the technique is not recommended as a diagnostic instrument in untreated patients (9–11). We have demonstrated venom-induced lymphocyte proliferation to have a sensitivity of 72% in wasp venom anaphylaxis (12). Therefore, supplementary in vitro tests to diagnose venom anaphylaxis are of tremendous interest.
Upon encounter of specific allergen that bridges FcεRI-bound IgE, basophils not only synthesize and secrete bioactive mediators but also up-regulate the expression of certain activation markers (e.g. CD63 and CD203c) that can be quantified by flow cytometry. The value of flow cytometry-assisted allergy diagnosis has been reported in allergies to pollen, house dust mite, foods, drugs, and Hevea latex (for review see Refs.13 and14). The technique also proved reliable to diagnose hymenoptera venom anaphylaxis (15–19), with a sensitivity and specificity for wasp venom between 85 and 92% and 80 and 83.3%, respectively (18, 19). Nevertheless, as recently pointed out by the EAACI Interest Group on Insect Venom Hypersensitivity (7), additional larger and comprehensive studies remain necessary to confirm these results. Therefore, the primary aim of this study was to compare the diagnostic performance of the basophil activation test (BAT) in IgE-mediated wasp venom allergy with established tools such as skin tests and venom-specific IgE.
In spite of the high efficiency of VIT, an effective instrument to monitor successful VIT is still lacking, and an important unanswered question relates to the optimal duration of maintenance immunotherapy. Both, venom-specific IgE and VST fail to reflect induction of clinical tolerance and seem of little predictive value for future (deliberate) sting reactions VIT (20–26). As the technique closely resembles the in vivo pathway leading to symptoms, the second purpose of this study was to evaluate whether the BAT could constitute an in vitro and thus safe instrument to monitor successful VIT. Therefore in a subgroup of patients, the test was repeated at different predefined time points during treatment. Finally, a separate cross-sectional analysis was performed to evaluate the contribution of the BAT in the decision of stopping VIT safely.
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The only effective and potentially life-saving treatment of wasp venom anaphylaxis is specific VIT. Currently diagnosis of wasp venom anaphylaxis rests upon an evocative history along with the demonstration of wasp venom-specific IgE antibodies in the skin or serum (for a recent review of the EAACI Interest Group on Insect Venom Hypersensitivity on the diagnosis of venom allergy the reader is referred elsewhere (7)). Our data confirm that VST and quantification of venom-specific IgE can be considered as quite performant diagnostic tools for wasp venom anaphylaxis. However, as already addressed in the introductory paragraph, none of the tests demonstrated absolute diagnostic reliability and even when performed in a complementary way did not identify all patients at risk for subsequent sting anaphylaxis.
Our dose-finding experiments demonstrate that wasp venom-induced basophilic activation, assessed flow cytometrically through the coexpression of IgE and the activation marker CD63, is not restricted to a single concentration of the allergen but rather covers different venom stimulation concentrations with a kind of plateau of maximal cell stimulation. These findings are in line with prior observations (15–19). Most importantly, the observation that the BAT discriminates between patients and controls for different allergen concentrations spanning several log scales allows determination of an optimal diagnostic stimulation concentration for further validation of the technique. Given the potentially life-threatening course of wasp venom anaphylaxis, evaluation of the diagnostic performance of the BAT was performed with a venom stimulation concentration (10 μg/mL) that proved most discriminative between patients and controls. According to a two-graph ROC-generated threshold of 26% above spontaneous CD63 expression, the BAT confirmed to be a reliable diagnostic instrument in wasp venom allergy and demonstrated an overall sensitivity of 83.8 and 100% specificity. Obviously, these figures include the patients who demonstrated nonresponsiveness to positive control stimulation with anti-IgE and for whom, in the absence of venom-induced CD63 expression, the BAT cannot be considered as a diagnostic tool. Often, those individuals whose results should be considered as “false-negatives” in specificity calculation are not reported in the literature. Obviously, this is not correct.
Conversely, the BAT allowed correct identification of the culprit venom in two patients with a compelling history of wasp-venom anaphylaxis that presented with urticaria, angio-edema, and respiratory symptoms, but who demonstrated a negative skin test and venom-specific IgE result. Similar findings on the BAT to constitute a potential diagnostic tool in patients with negative IgE and skin test results were recently observed by Kosnik et al. (30).
Despite the high efficiency of VIT, the mechanisms and biomarkers associated with clinical efficacy remain elusive (for review see Ref.31). Studies monitoring conversion of venom-specific IgE and skin test responses have repeatedly demonstrated these tests to be of poor predictive value and to fail to reflect induction of clinical tolerance (20–26). In our cross-sectional analysis, only 18% of the patients demonstrated negative wasp venom IgE after 3 years of treatment. This is in line with the data of van Halteren et al. (22) who found IgE negativation in approximately one-tenth of their patients. On the other hand, anaphylaxis due to sting challenges has been reported in patients with venom-specific IgE or skin test conversion (21, 23–26). Therefore, current practice is to offer VIT for a finite period of 3–5 years, irrespective the persistence of a positive skin test or IgE result (32–35).
Alternatively, a variety of studies have demonstrated in vitro desensitization of basophils during IgE-mediated stimulation of the cells. From these data, different mechanisms that might lead to loss of patient's responsiveness during semirush immunotherapy have been postulated (36–40). As in vitro stimulation of basophils closely resembles the in vivo pathway finally leading to symptoms, we speculated that suppression of venom-induced CD63 expression during treatment might be an in vitro correlate of clinical tolerance and be useful to monitor successful VIT. Therefore, the BAT was repeated at the end of a 5-day build-up course and after 6 months of maintenance treatment, at both time-points clinical protection has already been demonstrated by a well-tolerated sting challenge (18, 41, 42) and mediator release by basophils to some extent to be inhibited (43, 44). At the end of 5 days of semirush immunotherapy there was no significant change in basophilic expression of CD63, yet the patients tolerated successive escalating exposure to venom without difficulty. A possible explanation for this apparent paradox could be expression of CD63 not necessarily to reflect final behavior of the cell, i.e. mediator release, but merely to represent a distinct point in the signaling pathway or a different functional endpoint (45). In other words, the semirush course inhibits degranulation but not fusion of the intracellular granules with the outer basophil membrane.
Alternatively, after 6 months of maintenance treatment a decrease in venom-induced basophilic activation was observed. However, as compared to pretreatment values, inhibition of basophil activation was restricted to the submaximal stimulation experiments (0.01 μg/mL venom) and was not demonstrable when the cells were stimulated maximally (0.1 and 10 μg/mL), suggesting 6 months of immunotherapy to induce an up to 10-fold decrease of basophil responsiveness rather than a complete inhibition of the cells. This observation parallels the findings of Mothes et al. (46) who demonstrated inhibition of allergen-induced release of histamine by therapy-induced blocking IgG antibodies to be restricted to those experiments in which the basophils were stimulated submaximally. Moreover, our findings might explain the failure of Erdmann et al. (18) to demonstrate suppression of basophilic activation after 6 months of VIT. Revision of the dose-finding curves from this study reveals that the time-course of the BAT during VIT was assessed with a stimulation concentration resulting in maximal cell response.
To evaluate whether the BAT could guide the decision when to discontinue VIT safely, a separate cross-sectional comparison with 30 patients (8 being re-stung without reaction) treated for 3 years was performed. At this time-point, as compared to pretreatment values of systemic reactors, significant inhibition of basophil activation was demonstrable for stimulation with 0.01 and also for 0.1 μg/mL venom. Although one could argue longitudinal analysis over 3 years to provide more robust data, we believe our cross-sectional investigation in matched patients to be highly suggestive for basophil responsiveness further to decrease with time, with more and more of the patients becoming negative during prolonged maintenance therapy. This frequency of reduced and absent basophilic responsiveness is consistent with the trend to diminished skin test sensitivity demonstrated by others (24). In this study these authors demonstrated VIT administrated for more than 5 years to induce an at least 10-fold decline in skin test sensitivity in over 95% and complete negativation of skin test responses in approximately 60–70% of the patients, respectively.
Taken together, from our data it emerges flow cytometry constitute a reliable tool to diagnose wasp venom anaphylaxis. Furthermore, we confirmed the technique to provide the physician with an instrument to take the sting out of difficult cases with inconsistent IgE and skin test results that hinder correct identification of the culprit (19, 30, 47).
Finally, for the first time, it is demonstrated that the technique might offer an in vitro tool to monitor successful VIT. However, it should be acknowledged that responsiveness in our assay might not really represent a basophil property, but rather mirror less IgE that is cross-linked on the cell membrane because of decreased specific IgE or induction of blocking IgG antibodies.