The basophil activation test in wasp venom allergy: sensitivity, specificity and monitoring specific immunotherapy

BACKGROUND: As in vitro diagnosis of wasp venom sensitization by specific serum IgE has a sensitivity of only 60-80%, additional in vitro tests are desirable. Basophil activation is associated with the expression of CD63 and its measurement has been proposed as a novel in vitro test for immediate-type allergy. Furthermore, to date, no in vitro test exists to monitor successful specific immunotherapy (SIT) with wasp venom. Therefore, the potentially harmful sting challenge is still recommended. OBJECTIVE: We compared the CD63-based basophil activation test (BAT) in the diagnosis of wasp venom allergy with skin tests and measurement of specific IgE. Furthermore, we investigated whether BAT can predict the outcome of the sting challenge in patients on SIT. METHODS: Fifty patients with a systemic reaction caused by a wasp sting and 20 controls were studied. Intracutaneous tests were performed with wasp and bee venom in the suspected allergics. Specific IgE was determined by the CAP-FEIA method and basophil activation by flow cytometry upon double staining with anti-IgE/anti-CD63 mAb. Twenty-five patients were sting challenged 6 months after starting SIT and the BAT was repeated before challenge. RESULTS: Sensitivity of the intracutaneous tests, specific IgE and BAT was 100, 76, and 92%, respectively. Specificity of specific IgE and the BAT was 85 and 80%, respectively. The cut-off for a positive BAT was 15% CD63+ basophils. There was a positive correlation between IgE reactivity to wasp venom and the number of CD63+ basophils (r = 0.65). Although no patient had a systemic reaction upon sting challenge, in most subjects basophil activation did not decrease when compared with the BAT before SIT. CONCLUSIONS: Quantitation of basophil activation by CD63 expression is a valuable new in vitro method for diagnosis of allergy to hymenopteran venoms. The CD63-based BAT is a helpful tool for the complementation of routine diagnostic tests such as specific IgE as it increases sensitivity of in vitro detection of sensitization. However, this in vitro method does not offer an alternative to the sting challenge in monitoring successful SIT.     

Comparison of basophil activation tests using CD63 or CD203c expression in patients with insect venom allergy

BACKGROUND: Flow cytometric basophil activation tests have been developed as cellular tests for in vitro diagnosis of IgE-mediated reactions. Different activation markers (CD63 or CD203c) with distinct ways of regulation have been used after stimulation with various allergens. OBJECTIVE: It was the aim of the present study to compare basophil activation tests by measuring both CD63 and CD203c upregulation in patients with insect venom allergy. MATERIALS AND METHODS: 43 patients with a history of insect venom anaphylaxis were examined. A careful allergy history was taken, and skin tests and determination of specific IgE-antibodies were performed. Basophil activation tests (BAT) using CD63 or CD203c expression were done after stimulation with different concentrations of bee and wasp venom extracts. 25 healthy subjects with negative history of insect venom allergy were studied as controls. RESULTS: The CD203c protocol showed a slightly higher sensitivity than the CD63 protocol (97% vs. 89%) with regard to patients' history. The magnitude of basophil response was higher with CD203c in comparison to CD63 for both insect venoms. Specificity was 100% for the CD63 protocol and 89% for the CD203c protocol with regard to controls with negative history and negative RAST. CONCLUSION: These results support the reliability of basophil activation tests using either CD63 or CD203c as cellular tests in the in vitro diagnosis of patients with bee or wasp venom allergy with a slightly higher sensitivity for the CD203c protocol.     

A new basophil activation test using CD63 and CCR3 in allergy to antibiotics

Background Flow cytometric basophil activation tests (BAT) have been developed as cellular tests for in vitro diagnosis of IgE‐mediated reactions. Different markers and techniques have been used after stimulation with various allergens. Objective It was the aim of the present study to compare an established BAT (Flow‐CAST^®) with a newly developed basophil activation protocol using CD63 and CCR3 (Flow2 CAST^®) in patients with type‐I allergy to antibiotics. Materials and methods Twenty‐four patients with a history of type‐I allergy to antibiotics were examined. A careful allergy history was taken, and skin tests and determination of specific IgE antibodies were performed. Two different BAT using CD63 expression but different protocols were carried out after stimulation with different concentrations of antibiotics. Fifteen healthy subjects without a history of antibiotic allergy were studied as controls. Results The Flow2 CAST^® showed a higher sensitivity than the Flow‐CAST^® (55% vs. 53%) with regard to patients' history. Specificity was 80% both for the Flow2 CAST^® and for the Flow‐CAST^® with regard to controls with negative history and negative RAST. Conclusion These results show the value of two different BAT as cellular tests in the in vitro diagnosis of patients with antibiotic allergy with equal specificity and a slightly higher sensitivity for the Flow2 CAST^®. Cite this as: B. Eberlein, I. León Suárez, U. Darsow, F. Ruëff, H. Behrendt and J. Ring, Clinical & Experimental Allergy, 2010 (40) 411–418.     

Detection of allergen-induced basophil activation by expression of CD63 antigen using a tricolour flow cytometric method

In the field of allergy diagnosis, most in vitro functional tests are focused on basophils. Nevertheless, the very small number of circulating basophils limits these experiments and their clinical benefit remains controversial. As flow cytometry is a valuable tool for identifying cell populations, even at low concentrations, we developed a tricolour flow cytometric method for the study of allergen-induced basophil activation. Identification of cells was based both on CD45 expression and on the presence of IgE on the cell surface, since basophils express high-affinity receptors for IgE (Fc epsilon RI). Cell activation upon allergen challenge was assessed by the expression of CD63 antigen on the plasma membrane. Basophil isolation and activation (with the chemotactic peptide formyl-methionyl-leucyl-phenylalanine) were validated in 32 non-allergic patients. In 12 allergic patients, basophil stimulation by a relevant allergen was in most cases positive (10/12). Furthermore a concentration-dependent hook effect was observed. Of the allergic and non-allergic patients, none showed non-specific activation with an irrelevant allergen (specificity 100%). Overall, our preliminary results, even in a small population, suggest that this is a reliable and valuable method for the diagnosis of allergies complementing specific allergen IgE and skin test results. Obviously, additional clinical studies are needed to validate these first results.     

Monitoring of basophil activation using CD63 and CCR3 in allergy to muscle relaxant drugs

Allergic or pseudoallergic reactions that occur during anesthesia have been increasing for the last few years. To date, the diagnosis of allergy to muscle relaxants remains difficult. In this respect, we developed a flow cytometric method for the study of drug-induced basophil degranulation using CD63 and CCR3. Fifty patients who developed clinical features evocative of allergic reactions immediately after induction of anesthesia were included and classified into two groups. Group 1 (n = 39) comprised true allergic patients, who developed typical signs of shock associated to positive skin testing. Group 2 (n = 11) consisted of patients whose clinical history was not typical and skin testing was negative or nonconclusive. Seventeen control subjects were also studied in this report. We compared data from flow cytometry to skin tests, specific IgE, and histamine release results. Flow cytometry showed a sensitivity of 54%, while that of specific IgE was similar, at 62%. Interestingly, when considering the sensitivity of IgE + CD63 for diagnosis, we reached a sensitivity value of 80%. Of 15 negative results for specific IgE, we found 7 positive CD63 tests, while histamine release gave positive results in only 2 cases. Furthermore, the CD63 protocol showed good specificity (100%). We conclude that our flow cytometry protocol is a promising tool in allergy diagnosis since it is specific and complementary to specific IgE detection.     

CD63 expression on basophils as a tool for the diagnosis of pollen-associated food allergy: sensitivity and specificity

BACKGROUND: Basophil activation is associated with the expression of CD63. Because allergens can induce basophil activation by cross-linking specific IgE, increased CD63 expression has been proposed as a novel in vitro test for immediate type allergy. OBJECTIVE: We compared the CD63-based basophil activation test (BAT) in the diagnosis of allergy to carrot, celery and hazelnut with skin prick tests (SPT) and measurement of allergen-specific IgE. METHODS: Twenty-nine patients with a history of an oral allergy syndrome induced by carrot, celery or hazelnut (n = 20 for each allergen) and 20 controls were studied. SPT were performed with standardized and native carrot, celery and hazelnut extracts. Allergen-specific IgE was determined by the CAP FEIA method and basophil activation was determined by flow cytometry upon double staining with anti-IgE/anti-CD63 mAb. RESULTS: SPT with native carrot, celery and hazelnut showed sensitivities of 100%, 100% and 90%, and specificities of 80%, 80% and 90%. SPT with commercial extracts of the same allergens gave sensitivities of 85%, 80% and 85%, and specificities of 80%, 80% and 90%. Sensitivity of allergen-specific IgE and the BAT for carrot, celery and hazelnut was 80% vs. 85%, 70% vs. 85%, and 80% vs. 90%, with corresponding specificities of 80% vs. 85%, 80% vs. 80%, and 95% vs. 90%. The cut-off for a positive BAT was 10% CD63+ basophils. Moreover, there was a positive correlation between IgE reactivity and the number of CD63+ basophils for all food allergens (carrot: r = 0.69, celery: r = 0.67, hazelnut: r = 0.66). CONCLUSIONS: Quantification of basophil activation by CD63 expression is a valuable new in vitro method for diagnosis of immediate type food sensitization. Although double-blind placebo-controlled food challenges remain the gold standard, the CD63-based BAT may supplement routine diagnostic tests such as SPT or allergen-specific IgE in the future.     

Flow cytometric basophil activation test by detection of CD63 expression in patients with immediate‐type reactions to betalactam antibiotics

BACKGROUND: In this study, we used flow cytometry to determine the percentage of activated basophils that expressed the CD63 marker after in vitro stimulation by different betalactam antibiotics. The diagnostic reliability of the technique was assessed, as well as its correlation with specific IgE. METHODS: Fifty-eight patients with clinical allergy to betalactam antibiotics and presenting positive skin tests to at least one of the allergens (minor determinant mixture (MDM), benzylpenicilloyl-polylysine (PPL), penicillin, ampicillin, amoxicillin, cephalosporins) were tested. Thirty subjects non-allergic to betalactams were also studied as controls. The flow assay stimulation test (FAST) uses flow cytometry to determine the percentage of basophils that express CD63 as an activation marker after in vitro stimulation with allergen. Double labelling with monoclonal antibodies anti-CD63-PE and anti-IgE FITC was used. RESULTS: The allergic patients show a statistically greater number of activated basophils than the control subjects, after the incubation of cells with all the betalactams at various concentrations. The sensitivity of the technique is 50%, the specificity 93.3%, the likelihood ratio for a positive value 7.46 and the likelihood ratio for a negative value 0.54. In spite of having a greater sensitivity (37.9%) and specificity (86.7%) than CAP, differences between sensitivity and specificities of both techniques (CAP and FAST) do not reach statistical significance. CONCLUSION: The basophil activation test is a particularly useful technique in the diagnosis of patients with IgE-mediated allergy to betalactams and allows the identification of 50% of patients. Used in conjunction with CAP, it allows the identification of 65.5% of such patients.     

Marked improvement of the basophil activation test by detecting CD203c instead of CD63

BACKGROUND: The flow cytometric basophil activation test by detection of CD63 expression has been developed as an alternative method for in vitro diagnosis of IgE-mediated reactions to various allergens. Despite promising initial studies, the test remains disappointing in terms of sensitivity. CD203c has recently been demonstrated as a specific activation marker of basophils that is rapidly up-regulated after allergen challenge in sensitized patients. OBJECTIVE: The goal of the present study was to compare basophil activation tests by using either CD203c or CD63 in the diagnosis of immediate-type allergy to latex. METHODS: Twenty-seven patients (health care workers of our institution) who developed clinical features evocative of allergy after contact with latex were included and classified into two groups. Group 1 (n = 16) comprised true allergic patients who presented with typical signs of immediate allergic reaction associated with a positive skin test (prick test). Group 2 (n = 11) consisted of patients whose clinical history was not typical and had negative skin test. Twelve healthy subjects were also studied as controls. We compared the sensitivity of two triple-staining flow cytometric protocols measuring basophil activation after latex stimulation: CD45-IgE-CD63 and CD45-IgE-CD203c. RESULTS: The CD203c protocol showed a higher sensitivity than the CD63 protocol (75% vs. 50%). In comparison, latex-specific IgE sensitivity was found to be 69%. Furthermore, the magnitude of the basophil response was significantly higher with CD203c in comparison with CD63. Specificity was 100% for both protocols. CONCLUSION: Due to superior gating of basophils and a higher range of activation in response to allergen, the basophil activation test is markedly improved by use of CD203c instead of CD63.     

Position paper The sting challenge test in Hymenoptera venom allergy

Ruëff F, Przybilla B, Müller U, Mosbech H. The sting challenge test in Hymenoptera venom allergy. Position paper of the Subcommittee on Insect Venom Allergy of the European Academy of Allergology and Clinical Immunology. Allergy 1996: 51: 216–225. © Munksgaard 1996.     

Comparison of two basophil activation markers CD63 and CD203c in the diagnosis of amoxicillin allergy

Background To confirm allergy to β‐lactam (BL), a basophil activation test in flow cytometry based on CD63 up‐regulation was described. CD203c is a more recent basophil activation marker and up to day there is no consensus about which marker is the more sensitive one. CD203c has not yet been evaluated in the diagnosis of BL allergy. Objective The aim of the study was to compare the reliability of CD203c to CD63 for the diagnosis of amoxicillin (AX) allergy, which is nowadays the most frequent BL allergy. Methods Twenty‐seven patients with an immediate positive skin test (ST) to AX, 20 had had anaphylaxis with AX and 7 had urticaria and/or angioedema, were compared with 14 controls with no allergy to BL and to six patients with delayed positive ST to AX. Results In the anaphylaxis group, AX induced up‐regulation of CD203c in the basophils of 12 patients out of 20 (60%) and of CD63 in four patients (20%) (P<0.02). Two patients out of seven with urticaria or angioedema had a positive result with CD203c and CD63. In patients who had anaphylaxis, ampicillin (AMP) induced CD203c up‐regulation in eight out of 12 (67%) patients tested, and CD63 up‐regulation in 4 out of 12 (33%) (all patients who had anaphylaxis could not be tested with AMP). False‐positive results were observed with CD203c as well as CD63, and for 10 patients indeed this was confirmed by a negative drug provocation test. The origin of conflicting results between CD63 and CD203c might be at least the targeting of basophils based on anti‐IgE labelling. Among IgE⁺ gated cells, by means of CD33, a marker of monocytes, a contamination up to 50% by monocytes was detected. In contrast to CD63, CD203c is an activation marker specific of basophils with a basal low‐level expression in resting basophils. Thus, IgE and CD203c double targeting of basophils avoids the contamination by monocytes. Conclusion CD203c seems to be a more sensitive activation marker of basophils than CD63 for the diagnosis of amoxicillin allergy.     

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.
Conclusion:  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.     

The clinical utility of basophil activation testing in diagnosis and monitoring of allergic disease

The basophil activation test (BAT) has become a pervasive test for allergic response through the development of flow cytometry, discovery of activation markers such as CD63 and unique markers identifying basophil granulocytes. Basophil activation test measures basophil response to allergen cross‐linking IgE on between 150 and 2000 basophil granulocytes in <0.1 ml fresh blood. Dichotomous activation is assessed as the fraction of reacting basophils. In addition to clinical history, skin prick test, and specific IgE determination, BAT can be a part of the diagnostic evaluation of patients with food‐, insect venom‐, and drug allergy and chronic urticaria. It may be helpful in determining the clinically relevant allergen. Basophil sensitivity may be used to monitor patients on allergen immunotherapy, anti‐IgE treatment or in the natural resolution of allergy. Basophil activation test may use fewer resources and be more reproducible than challenge testing. As it is less stressful for the patient and avoids severe allergic reactions, BAT ought to precede challenge testing. An important next step is to standardize BAT and make it available in diagnostic laboratories. The nature of basophil activation as an ex vivo challenge makes it a multifaceted and promising tool for the allergist. In this EAACI task force position paper, we provide an overview of the practical and technical details as well as the clinical utility of BAT in diagnosis and management of allergic diseases.     

Validation of a two-color flow cytometric assay detecting in vitro basophil activation for the diagnosis of IgE-mediated natural rubber latex allergy

BACKGROUND: IgE-dependent triggering of basophils not only elicits the release of different mediators but also the up-regulation of certain markers, e.g. CD63, which can be detected by flow cytometry. We intended to investigate if flow cytometric analysis of basophil activation could be a valuable tool in the diagnosis of latex allergy, and to evaluate if the basophil activation test (BAT) could be helpful in determining the clinical significance of a positive latex IgE in individuals with negative history and negative latex skin test. Additionally we aimed to determine the role of cross-reactive carbohydrate determinants (CCDs) in causing positive latex IgE without apparent clinical significance. METHODS: Twelve healthy controls without a history of latex hypersensitivity with a negative latex IgE and skin test (group 1), 24 individuals without a history of latex hypersensitivity with a negative latex IgE and skin test but with other inhalant allergies (group 2), and 29 latex allergic patients with a compelling history of latex allergy with a positive latex IgE and prick test (group 3) were enrolled. The diagnostic performances of the BAT were further evaluated in 13 individuals with a history of latex allergy but with negative specific IgE and/or skin test (group 4). Twenty-four individuals with positive latex IgE without apparent clinical relevance, i.e. without history of latex hypersensitivity and negative latex skin tests, were also analyzed (group 5). The putative role of CCDs causing positive latex IgE results without apparent clinical significance was evaluated by quantification of IgE for bromelain. RESULTS: According, to the receiver operating characteristics(ROC)-generated threshold value of 17% between latex allergic patients and the pooled group of nonlatex allergic individuals, the sensitivity and specificity of the basophil activation test was 93.1% and 91.7%, respectively. In healthy controls, allergic patients without latex hypersensitivity and latex allergic patients the number of positive BATs was 0/12, 3/24 and 27/29, respectively. In the individuals with an evocative history of latex allergy but with negative latex IgE and/or skin test the BAT was positive in all 13 cases. Twenty of 24 individuals without apparent latex allergy but with positive latex IgE had a negative BAT. IgE for bromelain was positive in 1/19 sera from group 2, 1/24 sera from group 3, none of the 8 sera from group 4, but in 16/18 sera from group 5, respectively. CONCLUSION: Flow cytometric analysis of activated basophils seems a highly sensitive and specific tool for diagnosing latex allergy. In addition, the technique might help to determine the clinical relevance of positive IgE quantification in the absence of overt latex allergy. CCDs of natural rubber latex allergens were confirmed to mimic latex sensitization.     

Cross‐reactive carbohydrate determinants strongly affect the results of the basophil activation test in hymenoptera‐venom allergy

Background In hymenoptera‐venom allergy, sera of up to 60% of patients show in vitro reactivity to honeybee venom (HBV) and yellow jacket venom (YJV). This phenomenon is mainly caused by specific IgE (sIgE) against cross‐reactive carbohydrate determinants (CCD). Whether or not these antibodies can induce clinical symptoms is a longstanding debate. Objective The aim of this study was to investigate the biological activity of CCD‐sIgE and the suitability of the basophil activation test (BAT) in hymenoptera venom‐allergic patients having CCD‐sIgE. Methods The biological activity of CCD‐sIgE was analysed by application of native and CCD‐depleted YJV and HBV in BAT with the blood of 62 hymenoptera venom‐allergic patients and 16 non‐allergic controls. According to results of intracutaneous skin tests (IC) with YJV and HBV and the existence of CCD‐sIgE, patients were classified into six subgroups. Results In patients with mono‐positive IC and CCD‐sIgE, and thus double‐positive sIgE, BAT with native venoms was also double positive in up to 67% of the patients. In contrast, BAT with CCD‐depleted venoms was positive only with the IC‐positive venom. However, activation of basophils with the IC‐negative venom was significantly lower compared with the IC‐positive one. In IC mono‐positive patients without CCD‐sIgE, BAT was mono‐positive with the IC‐positive venom in the native and in the CCD‐depleted form. CCD‐positive patients with double‐positive IC were a heterogeneous group, with the majority of CCD‐positive patients also being double positive with the native forms of both venoms but mono‐positive with the CCD‐depleted ones. Conclusions In vitro BAT clearly demonstrates biological activity of CCD‐sIgE. However, because most of the patients showed a mono‐positive IC and activation of basophils with the IC‐negative venom was significantly lower compared with the IC‐positive one, the present data suggest that CCD‐sIgE is clinically irrelevant in these patients. Cite this as: M. Mertens, S. Amler, B. M. Moerschbacher and R. Brehler, Clinical & Experimental Allergy, 2010 (40) 1333–1345.     

Diclofenac induces basophil degranulation without increasing CD63 expression in sensitive patients

Diclofenac (Dc) induces an IgE-independent basophil (Ba) degranulation in susceptible individuals. CD63 Ba expression is utilized as an in vitro test for diagnosis of drug hypersensitivity. We tested the ability of Dc to induce CD63 Ba expression by flow cytometry (BAT) and Ba degranulation using light microscopy (HBDT) in patients sensitive to Dc. We studied 14 patients with diclofenac hypersensitivity, also two patients sensitive to Dermatophagoides pteronyssinus (Dp), and 12 normal controls. HBDT was performed by mononuclear cells toluidine blue staining. BAT determined CD63 expression in antiCD63/anti-IgE/anti-CD45-labelled whole blood. In each case, the percentage of activated Ba post-stimulation with 1 and 10 microg/ml Dc was determined. Positive controls included N-formyl-methionyl-leucyl-phenylalanine (fMLP) peptide-induced activation. IgE-mediated Ba activation was induced with a Dp allergenic extract. With Dc 1 microg/ml, mean HBDT in Dc-susceptible individuals was 33.62 +/- 18.35% and 8.49 +/- 4.79% in controls (P = 0.0001). Mean BAT was 2.04 +/- 1.68% and 1.93 +/- 1.40% in controls (P = 0.8). Ba preincubation with Dc did not affect fMLP-induced CD63 expression, neither in Dc-sensitive individuals (P = 0.8) (n = 4) nor in subjects without Dc hypersensitivity (P = 0.25) (n = 4). Ba from the two patients sensitive both to Dc and Dp responded to Dp but not to Dc by BAT: Dc, 1.99 +/- 0.78%; Dp: 60.87 +/- 9.28%; but showed degranulation by HBDT: Dc, 30.53 +/- 1.02%, Dp: 48.78 +/- 22.17%. Dc induces Ba degranulation in sensitive patients in a way that does not induce CD63 expression and is different from IgE-mediated and fMLP-mediated degranulation. Our results suggest that CD63 expression is not a reliable diagnostic method for diclofenac allergy.