Diagnosis Of Hymenoptera Venom Allergy Research Articles

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

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.

Diagnosis of Hymenoptera Venom Allergy: State of the Art, Challenges, and Perspectives.

Hymenoptera venom allergy is the most common cause of anaphylaxis in adults and the second-most frequent in children. The proper diagnosis of this life-threatening allergy remains a challenge. This review focuses on the current knowledge regarding diagnostics of Hymenoptera venom allergy. The paper includes a brief description of the representatives of Hymenoptera order and the composition of their venoms. Then, diagnostic tests for allergy to Hymenoptera venom are described. Common diagnostic problems, especially double positivity in tests for IgE antibodies specific to honeybee and wasp venom, are also discussed. Special attention is paid to the search for new diagnostic capabilities using modern methodologies. Multidimensional molecular analysis offers an opportunity to characterize changes in body fluids associated with Hymenoptera venom allergy and yields a unique insight into the cell status. Despite recent developments in the diagnostics of Hymenoptera venom allergy, new testing methodologies are still needed to answer questions and doubts we have.

The role of component-resolved diagnosis in Hymenoptera venom allergy in clinical practice

Background: Hymenoptera venom allergy is an immunoglobulin (Ig) E mediated hypersensitivity reaction to Hymenoptera venoms. Obvious identification of the culprit insect that causes the clinical symptoms and, hence, the accurate selection of venom for curative treatment, is of great importance for the effectiveness and safety of venom immunotherapy. Objective: In this study, the contribution of component-resolved diagnostics (CRD) is evaluated in the diagnosis of Hymenoptera venom allergy. Method: Ninety-three patients from four different centers in Turkey were included in the study. Conventional tests, including prick and intradermal skin tests, with commercial venom extracts and serum specific IgE (sIgE) levels for whole venoms were performed. An sIgE analysis for venom allergen components, including rApi m 1, rApi m 2, rApi m 10, rVes v 1, rVes v 5, were evaluated by immunoblotting. Results: In conventional test results, 17 of 35 patients with bee venom allergy were positive to honey bee venom, whereas 18 patients were positive to bee and wasp venoms. In 28 of 35 patients with bee venom allergy, the diagnosis was confirmed with CRD. CRD revealed a sensitivity of 80% in patients with bee venom allergy. According to conventional tests, 7 of 24 patients with vespid venom allergy demonstrated sensitivity only to Vespula species, whereas 17 patients revealed double positivity. The total diagnostic sensitivity of Ves v 1 and Ves v 5 was calculated as 87.5%. Ten of 23 patients with a history of hypersensitivity to both venoms showed double sensitivity with CRD; one patient had cross-reactivity, one patient was found to be sensitive only to bee venom, and, eight patients were sensitive only to Vespula species. Eleven patients had an uncertain history in terms of the culprit insect type and six of them had double sensitivity in CRD. Conclusion: CRD seemed to be more helpful in diagnosing vespid venom allergy than bee venom allergy. It can also discriminate clinically significant sensitizations from irrelevant ones.

Current practices in diagnosis of Hymenoptera venom allergy in Poland.

Hymenoptera venom allergy (HVA) is associated with a high risk of anaphylaxis. Effective treatment of HVA patients requires allergologists' familiarity with the latest HVA recommendations. Evaluation of current practices in HVA diagnosis in Poland. A survey questionnaire was conducted in 32 HVA centres in Poland. The response rate was 97%. There were 1829 patients evaluated due to HVA in 2015. Sixty six percent (n = 21) of the centres used skin prick tests, out which 90% (n = 19) used 100 µg/ml of the venom extract as the highest concentration. All the centres performed intradermal tests (IDT) and serum specific IgE (sIgE), an initial diagnostic tool in 91% (n = 29). The highest venom concentration in IDT was 1 µg/ml in 75% (n = 24), 0.1 µg/ml in 16% (n = 5), 0.01 µg/ml in 3% (n = 1) and 10 µg/ml in 6% (n = 2). Baseline serum tryptase was assessed in 84% of the centres (n = 27), out of which 53% (n = 17) tested all their patients, whereas 31% (n = 10) checked only those with life-threatening reactions. In case of negative IDT/sIgE, 59% of the centres (n = 19) performed components evaluation, while 19% (n = 6) did the basophil activation test. In case of no identification of the culprit insect and sensitization to both venoms, VIT employed venom with higher sIgE. Most allergology centres in Poland follow HVA guidelines. We identified two inaccuracies in their HVA management including non-adequate venom concentration in IDT and a false belief in correspondence between sIgE concentration and severity of allergic reactions.

Predictors of severe anaphylaxis in Hymenoptera venom allergy: The importance of absence of urticaria and angioedema

BackgroundSevere anaphylaxis (SA) in Hymenoptera venom allergy has been associated with a number of risk factors. However, the effect of several of those risk factors on the severity of anaphylaxis is poorly defined. ObjectiveTo evaluate risk factors for SA in Hymenoptera venom allergy. MethodsWe evaluated data from 500 patients who were referred to our department for the diagnosis of Hymenoptera venom allergy during a period of 11 years to identify risk factors for SA. ResultsSix significant risk factors for SA were identified (P < .05): short interval from sting to reaction, absence of urticaria or angioedema (U/A) during anaphylaxis, older age, male sex, elevation of baseline serum tryptase (BST) level, and diagnosis of systemic mastocytosis. Moreover, elevation in BST level was significantly associated with the absence of U/A and older age. No association could be established between SA and comorbidities, concurrent cardiovascular medication, or the severity of the systemic reaction during the initiation of venom immunotherapy. ConclusionApart from BST and older age, male sex, short interval from sting to reaction, and absence of U/A are also risk factors for SA. The association between elevated BST level and SA was largely confined to those who had an absence of U/A after field sting, possibly because of the higher risk of concurrent systemic mastocytosis. Patients with an SA after a field sting do not have an elevated risk of systemic reactions during the initiation of venom immunotherapy compared with patients with mild anaphylaxis; therefore, additional preventive measures are not necessary.

Stinging Insect Allergens.

Hymenoptera venom allergy is one of the common causes of anaphylaxis. However, when physicians make the diagnosis of Hymenoptera venom allergy, the history of being stung is not always consistent with the results of venom-specific IgE. With the development of component-resolved diagnosis, it is possible to accurately localize an allergic reaction to certain sensitized proteins. This paper reviewed the studies that have addressed the identified allergenicity and cross-reactivity of Hymenoptera venom allergens accepted by the WHO/IUIS Nomenclature Sub-committee, the componentresolved diagnosis of Hymenoptera venom allergy and its predictive values for the efficacy and safety of venom immunotherapy. Also special attention was paid to the spread of Hymenoptera venom allergy in Asian countries.

Features of diagnosis of hymenoptera venom allergy

The article describes the experience and results of research in the field of allergy to insects, in particular to venom of Hymenoptera. The aspects of diagnostic algorithms of cases of hypersensitivity to insect stings are also presented and the results of research on this issue are described.

Hymenoptera venom allergy in outdoor workers: Occupational exposure, clinical features and effects of allergen immunotherapy

ABSTRACTObjectives. To describe (i) the clinical characteristics of workers, exposed to hymenoptera stings, with an ascertained diagnosis of Hymenoptera Venom Allergy (HVA), (ii) the specific role of occupational exposure, (iii) the effect of Venom Immunotherapy (VIT) in reducing the severity of allergic episodes in workers exposed to repeated stings of hymenoptera, and (iv) the management of the occupational consequences caused by allergic reactions due to hymenoptera stings.Methods. Between 2000 and 2013 an observational study, including patients referred to the regional reference hospital of Liguria, Italy, with an ascertained diagnosis of HVA and treated with VIT, was performed. A structured questionnaire was administered to all patients to investigate the occupational features of allergic reactions. These were graded according to standard systems in patients at the first episode, and after re-stings, during VIT.Results. One-hundred and 8four out of the 202 patients referred had a complete data set. In 32 (17.4%) patients, the allergic reaction occurred during work activities performed outdoor. Of these, 31.2% previously stung by hymenoptera at work, and receiving VIT, were re-stung during occupational activity. The grades of reaction developed under VIT treatment resulted clinically less severe than of those occurred at the first sting (p-value = 0.031).Conclusion. Our findings confirmed the clinical relevance of HVA, and described its occupational features in outdoor workers with sensitization, stressing the importance of an early identification and proper management of the professional categories recognized at high risk of hymenoptera stings. The Occupational Physician should be supported by other specialists to recommend appropriate diagnostic procedures and the prescription of VIT, which resulted an effective treatment for the prevention of episodes of severe reactions in workers with a proven HVA.

Utility of laboratory testing for the diagnosis of Hymenoptera venom allergy.

A diagnosis of Hymenoptera venom allergy is based on clinical history and the results of skin tests and/or laboratory methods. To analyze the utility of available laboratory tests in diagnosing Hymenoptera venom allergy. Ninety-five patients with Hymenoptera venom allergy with a history of bee (35) or wasp (60) anaphylactic sting reaction and positive skin test with bee or wasp venom were included in this analysis. Specific immunoglobulin E (to bee venom extract, wasp venom extract, available recombinant molecules, and a basophil activation test with venom extracts were assessed in all the patients. Test sensitivity and specificity were calculated by using standard threshold values; then, receiver operating characteristic curve analysis was performed to compute optimal threshold values. Also, statistical analysis of the utility of different combinations of laboratory tests was performed. The optimal threshold values were revealed to be the following: 1.0 kIU/L for bee venom extract (sensitivity, 97.14%; specificity, 100%), 0.35 kIU/L for rApi m 1 (sensitivity, 68.57%; specificity, 100%), 1.22 kIU/L for wasp venom extract (sensitivity, 88.33%; specificity, 95.45%), 0.7 kIU/L for rVes v 5 (sensitivity, 86.67%; specificity, 95.45%), 1.0 kIU/L for rVes v 1 (sensitivity, 56.67%; specificity, 95.45%), 6.5% for basophil activation test with bee venom extract (sensitivity, 80%; specificity, 95.45%), and 4.5% for basophil activation test with wasp venom extract (sensitivity, 91.53%; specificity, 95.45%). The best test combinations were found to be the following: bee venom extract plus rApi m 1 (sensitivity, 97.14%; specificity, 95.45%) in bee and either wasp venom extract plus rVes v 5, or rVes v 5 plus rVes v 1 (both sensitivity, 98.33%; specificity, 95.45%) in patients with wasp venom allergy. Our analysis confirmed that currently used laboratory tests represent effective tools in diagnosing Hymenoptera venom allergy. Moreover, our probabilistic approach offered another way to interpret concrete values of laboratory test results and opened possible direction on how to optimize the laboratory diagnostic procedure.

Clinical Features and the Diagnostic Value of Component Allergen-Specific IgE in Hymenoptera Venom Allergy

PurposeAlthough patient history is vital for the diagnosis of hymenoptera venom allergy, specific IgE detection is also important to identify the culprit insect and monitor the effect of immunotherapy. We evaluated the diagnostic value of serum-specific IgE detection of hymenoptera venom component allergens and documented changes in allergen-specific IgE after immunotherapy.MethodsFifty-six hymenoptera venom allergy patients receiving venom immunotherapy were recruited from Ajou University Hospital, Korea. The clinical manifestations of the patients were noted, and serum-specific IgE detection was performed, using conventional venom extracts as well as component allergens. Data were analyzed retrospectively.ResultsA total of 35 (62.5%) patients were male, and 33 (73.3%) patients were atopic. The mean patient age was 44.9±13.8 years. Localized reactions occurred in 23.2% of patients, and systemic reactions occurred in 76.8%. The most common clinical manifestations included skin involvement, such as urticaria and angioedema, and respiratory involvement. Yellow jackets were the most frequent culprit insect, followed by yellow hornets, white-faced hornets, honeybees, and paper wasps, as determined at the time of diagnosis. Double sensitization to both Apidae and Vespidae species was detected in 70.9% of patients. The positive predictive values (PPV) of rVes v 5-specific and rPol d 5-specific IgE detection were 85.7% and 87.5%, respectively, which correlated well with conventional venom extract-specific IgE detection (r=0.762 and r=0.757, respectively). In contrast, the PPV of rApi m 1-specific IgE detection at the time of diagnosis was 34.8%. Three years of venom immunotherapy resulted in decreased venom-specific IgE, particularly IgE specific for Vespidae venom components.ConclusionsStings by yellow jackets and male sex may be risk factors for hymenoptera venom allergy in Korea. Vespidae component-specific IgE, but not Apidae component-specific IgE, had diagnostic and monitoring value in hymenoptera venom allergy comparable to that of conventional hymenoptera venom extract-specific IgE.

Inconsistent Results of Diagnostic Tools Hamper the Differentiation between Bee and Vespid Venom Allergy

BackgroundDouble sensitization (DS) to bee and vespid venom is frequently observed in the diagnosis of hymenoptera venom allergy, but clinically relevant DS is rare. Therefore it is sophisticated to choose the relevant venom for specific immunotherapy and overtreatment with both venoms may occur. We aimed to compare currently available routine diagnostic tests as well as experimental tests to identify the most accurate diagnostic tool.Methods117 patients with a history of a bee or vespid allergy were included in the study. Initially, IgE determination by the ImmunoCAP, by the Immulite, and by the ADVIA Centaur, as well as the intradermal test (IDT) and the basophil activation test (BAT) were performed. In 72 CAP double positive patients, individual IgE patterns were determined by western blot inhibition and component resolved diagnosis (CRD) with rApi m 1, nVes v 1, and nVes v 5.ResultsAmong 117 patients, DS was observed in 63.7% by the Immulite, in 61.5% by the CAP, in 47.9% by the IDT, in 20.5% by the ADVIA, and in 17.1% by the BAT. In CAP double positive patients, western blot inhibition revealed CCD-based DS in 50.8%, and the CRD showed 41.7% of patients with true DS. Generally, agreement between the tests was only fair and inconsistent results were common.ConclusionBAT, CRD, and ADVIA showed a low rate of DS. However, the rate of DS is higher than expected by personal history, indicating that the matter of clinical relevance is still not solved even by novel tests. Furthermore, the lack of agreement between these tests makes it difficult to distinguish between bee and vespid venom allergy. At present, no routinely employed test can be regarded as gold standard to find the clinically relevant sensitization.

Evaluation of Different Glycoforms of Honeybee Venom Major Allergen Phospholipase A2 (Api m 1) Produced in Insect Cells

Allergic reactions to hymenoptera stings are one of the major reasons for IgE-mediated anaphylaxis. However, proper diagnosis using venom extracts is severely affected by molecular cross-reactivity. In this study recombinant honeybee venom major allergen phospholipase A2 (Api m 1) was produced for the first time in insect cells. Using baculovirus infection of different insect cell lines allergen versions providing a varying degree of cross-reactive carbohydrate determinants as well as a non glycosylated variant could be obtained as secreted soluble proteins in high yields. The resulting molecules were analyzed for their glycosylation and proved to show advantageous properties regarding cross-reactivity in sIgE-based assays. Additionally, in contrast to the enzymatically active native protein the inactivated allergen did not induce IgE-independent effector cell activation. Thus, insect cell-derived recombinant Api m 1 with defined CCD phenotypes might provide further insights into hymenoptera venom IgE reactivities and contribute to an improved diagnosis of hymenoptera venom allergy.

Detection of IgE to recombinant Api m 1 and rVes v 5 is valuable but not sufficient to distinguish bee from wasp venom allergy

Detection of IgE to recombinant Api m 1 and rVes v 5 is valuable but not sufficient to distinguish bee from wasp venom allergy

P38 mitogen‐activated protein kinase signal transduction in the diagnosis and follow up of immunotherapy of wasp venom allergy

P38 mitogen-activated protein kinase (MAPK) is known to govern IgE-mediated basophil activation. Intracellular phosphorylated p38 MAPK (Pp38 MAPK) in IgE-activated basophils can be quantified flow cytometrically. To study whether Pp38 MAPK constitutes a potential novel read-out for flow-assisted diagnosis of hymenoptera venom allergy and to investigate whether this marker allows follow-up of successful venom immunotherapy (VIT). Fifty-two patients with documented wasp venom allergy and seven wasp-stung asymptomatic control individuals were enrolled. Wasp venom-induced basophil activation was analyzed flow cytometrically with anti-IgE, anti-CD63, and anti-Pp38 MAPK to assess their activation status before starting immunotherapy. To assess whether p38 MAPK constitutes a candidate marker for monitoring VIT, we repeated the basophil activation test (BAT) in 25 patients on the fifth day of a build-up immunotherapy. In addition, we investigated whether the Pp38 MAPK-based BAT could contribute in the decision of discontinuing VIT in a cross-sectional analysis in 13 patients receiving treatment for 3 years and 14 patients receiving treatment for 5 years. Patients exhibited a dose-dependent basophil activation with phosphorylation of p38 MAPK and upregulation of downstream CD63. In contrast, stung controls demonstrated a dose-dependent but "abrogated" signal transduction in basophils with less and shorter duration of the phosphorylation of p38 MAPK and without subsequent upregulation of CD63. When repeated after 5 days of VIT and when investigated cross-sectionally after 3 years or 5 years of maintenance therapy, no effect of VIT on the phosphorylation of p38 MAPK was demonstrable. This study discloses that not only basophils from patients, but also from the stung control individuals, respond to wasp venom stimulation with phosphorylation of p38 MAPK, although to a lesser extend. No clear effect of VIT on the phosphorylation of p38 MAPK was shown. Thus, although p38 MAPK provides an additional tool in the diagnosis of wasp venom allergy, it does not contribute to the decision whether to stop successful VIT. © 2010 International Clinical Cytometry Society.

Recombinant phospholipase A1 (Ves v 1) from yellow jacket venom for improved diagnosis of hymenoptera venom hypersensitivity

BackgroundHymenoptera venoms are known to cause life-threatening IgE-mediated anaphylactic reactions in allergic individuals. Proper diagnosis of hymenoptera venom allergy using venom extracts is severely affected by molecular cross-reactivities. Although non-glycosylated marker allergens would facilitate the identification of the culprit venom, the major allergen phospholipase A1 (Ves v 1) from yellow jacket venom (YJV) remained unavailable so far.MethodsExpression of Ves v 1 as wild type and enzymatically inactivated mutant and Ves v 5 in insect cells yielded soluble proteins that were purified via affinity chromatography. Functionality of the recombinant allergens was assessed by enzymatic and biophysical analyses as well as basophil activation tests. Diagnostic relevance was addressed by ELISA-based analyses of sera of YJV-sensitized patients.ResultsBoth major allergens Ves v 1 and Ves v 5 could be produced in insect cells in secreted soluble form. The recombinant proteins exhibited their particular biochemical and functional characteristics and were capable for activation of human basophils. Assessment of IgE reactivity of sera of YJV-sensitized and double-sensitized patients emphasised the relevance of Ves v 1 in hymenoptera venom allergy. In contrast to the use of singular molecules the combined use of both molecules enabled a reliable assignment of sensitisation to YJV for more than 90% of double-sensitised patients.ConclusionsThe recombinant availability of Ves v 1 from yellow jacket venom will contribute to a more detailed understanding of the molecular and allergological mechanisms of insect venoms and may provide a valuable tool for diagnostic and therapeutic approaches in hymenoptera venom allergy.

Basophil activation test is better but not good enough for the diagnosis of hymenoptera venom allergy: the problem of cross-reactive carbohydrate determinants

Cite this as: F. Altmann, Clinical & Experimental Allergy, 2010 (40) 1290–1292.

Sensibilisation ou allergie aux venins d’hyménoptères : comment faire la différence ?

The diagnosis of hymenoptera venom allergy is based on the clinical history and proof of sensitisation to the venom. Recommendations and consensus conferences have given priority to skin tests and, even more, to intradermal tests. Tests using blood cells can be used as an additional diagnostic step. At present, no test can reveal more than sensitisation to the venom. The situation becomes difficult when the clinical history is not clear, when the insect has not been identified or when the diagnostic tests reveal multiple sensitivities. The question then is between sensitivity to multiple hymenoptera or cross-reactions between the venoms. After having eliminated the role of carbohydrate determinants, which are most often the cause of cross-reactions, the clinician is likely to turn towards immunotherapy with several specific venoms in cases of severe clinical reactions. The prognosis of asymptomatic sensitisations still remains uncertain.

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

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. 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. 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. 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.

Diagnosis of Hymenoptera venom allergy

The purpose of diagnostic procedure is to classify a sting reaction by history, identify the underlying pathogenetic mechanism, and identify the offending insect. Diagnosis of Hymenoptera venom allergy thus forms the basis for the treatment. In the central and northern Europe vespid (mainly Vespula spp.) and honeybee stings are the most prevalent, whereas in the Mediterranean area stings from Polistes and Vespula are more frequent than honeybee stings; bumblebee stings are rare throughout Europe and more of an occupational hazard. Several major allergens, usually glycoproteins with a molecular weight of 10-50 kDa, have been identified in venoms of bees, vespids. and ants. The sequences and structures of the majority of venom allergens have been determined and several have been expressed in recombinant form. A particular problem in the field of cross-reactivity are specific immunoglobulin E (IgE) antibodies directed against carbohydrate epitopes, which may induce multiple positive test results (skin test, in vitro tests) of still unknown clinical significance. Venom hypersensitivity may be mediated by immunologic mechanisms (IgE-mediated or non-IgE-mediated venom allergy) but also by nonimmunologic mechanisms. Reactions to Hymenoptera stings are classified into normal local reactions, large local reactions, systemic toxic reactions, systemic anaphylactic reactions, and unusual reactions. For most venom-allergic patients an anaphylactic reaction after a sting is very traumatic event, resulting in an altered health-related quality of life. Risk factors influencing the outcome of an anaphylactic reaction include the time interval between stings, the number of stings, the severity of the preceding reaction, age, cardiovascular diseases and drug intake, insect type, elevated serum tryptase, and mastocytosis. Diagnostic tests should be carried out in all patients with a history of a systemic sting reaction to detect sensitization. They are not recommended in subjects with a history of large local reaction or no history of a systemic reaction. Testing comprises skin tests with Hymenoptera venoms and analysis of the serum for Hymenoptera venom-specific IgE. Stepwise skin testing with incremental venom concentrations is recommended. If diagnostic tests are negative they should be repeated several weeks later. Serum tryptase should be analyzed in patients with a history of a severe sting reaction.

The use of cellular in vitro assays in the diagnosis of hymenoptera venom allergy

RATIONALE: Immediate type allergies to hymenoptera venoms are currently diagnosed by clinical history, skin tests (ST) and determination of venom-specific IgE (IgE). Here we investigate the clinical utility of a sulfidoleukotriene release assay (CAST™-ELISA) and a basophil activation assay (expression of CD63 surface marker) by flow cytometry (Flow-CAST™).