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1.
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. 相似文献
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Yasnowsky KM Dreskin SC Efaw B Schoen D Vedanthan PK Alam R Harbeck RJ 《The Journal of allergy and clinical immunology》2006,117(6):1430-1434
BACKGROUND: Approximately 40% of patients with chronic idiopathic urticaria have antibodies to the alpha subunit of the high-affinity IgE receptor. CD203c is a basophil activation marker known to be upregulated by cross-linking of the FcepsilonRIalpha receptor and may serve as a useful marker to identify these patients. OBJECTIVE: The primary objective was to assess the affect of sera from patients with chronic idiopathic urticaria on basophil CD203c expression. Secondary objectives were to correlate CD203c expression with basophil histamine release and size of the autologous serum skin test and to determine whether the mechanism is mediated by an IgG antibody. METHODS: Sera were obtained from patients with chronic idiopathic urticaria and positive autologous serum skin test or negative autologous serum skin test and normal controls. Sera were incubated with donor whole blood. Activated basophils from whole blood were identified by flow cytometry on the basis of the presence of CD203c on high-expressing IgE positive cells. RESULTS: Incubation of donor basophils with sera from patients with chronic idiopathic urticaria and positive autologous serum skin test demonstrated significant upregulation of CD203c. IgG depletion of representative sera from patients with chronic idiopathic urticaria resulted in significant decrease in CD203c expression on donor basophils. CD203c expression correlated with basophil histamine release and the size of the autologous serum skin test. CONCLUSION: Sera from patients with chronic idiopathic urticaria and positive autologous serum skin test significantly upregulate basophil CD203c and correlate with basophil histamine release. CLINICAL IMPLICATIONS: This article describes an activation marker on basophils whose expression is increased by sera from patients with chronic idiopathic urticaria. 相似文献
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BACKGROUND: Endogenous histamine-releasing factors (HRFs) are involved in 30-60% of patients with chronic urticaria (CU). Evidence for their existence comes from in vivo studies of autoreactivity with the autologous serum skin test (ASST), in vitro immunoassays demonstrating autoantibodies against the immunoglobulin E (IgE) or the high affinity IgE receptor (FcepsilonRI) and serum-induced histamine release (HR) from basophils and mast cells. We have examined the correlation between the ASST and a new basophil histamine-releasing assay (the HR-Urtikaria test) in a group of well-characterized CU patients and subsequently determined the frequency of HR-Urticaria-positive sera from a larger population of CU patients. SUBJECTS: Group 1 consisted of 28 patients with CU (16 were ASST-positive) 20 patients with atopic dermatitis, 24 patients with allergy to birch and nine healthy controls. Group 2 consisted of 873 unselected CU patients. METHODS: White blood cells containing 1-2% basophils from a healthy nonatopic donor were incubated with patients sera in the presence of interleukin (IL)-3. Histamine was measured by the glass fibre method. RESULTS: Using the ASST as the true outcome, the HR-Urticaria test showed a sensitivity and specificity of 75% in group 1 using a cut-off value for HR of >16.5%. None of the controls was positive in the HR-Urticaria test. In group 2, we found no difference in the frequency of positives between male (34.6%, n = 254) and female adults (35.1%, n = 576) but twice as many females as males were tested. CONCLUSIONS: Our studies have shown that the HR-Urticaria test has a good sensitivity and specificity for endogenous HRFs demonstrated by the ASST in patients with CU and that about one-third of unselected patients with CU have a positive result. 相似文献
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In a limited number of severe chronic idiopathic urticaria (CIU) patients, low-dose cyclosporin A (CsA) treatment was found to be effective. This open study aimed to extend this clinical observation and determine the safety of treatment with CsA. In addition, it aimed to determine the prevalence and characteristics of the autologous serum skin test (AST) in such patients, and whether this test is affected by CsA treatment. Thirty-five patients who suffered from severe CIU (score 3), and who were followed for 6 months (using a clinical urticaria-severity score [range 0–3]) were divided into three groups: 19/35 were treated for 3 months with low-dose CsA, and thereafter followed for an additional 3 months; 6/35 dropped out of protocol treatment; and 10/35 untreated patients (followed for the same period) served as a disease controls. In the treated group, no side-effects were observed, and by the end of treatment, 13/19 (68%) patients were in full remission (score 0) and the remainder scored 1. In contrast, the 10 CsA-untreated patients scored 3 for the whole follow-up period of 6 months. Positive AST was found in 14/35 (40%) of patients, whereas none were detected in 20 healthy control subjects. AST neither correlated with disease activity nor predicted response to treatment. This uncontrolled study shows that low-dose CsA is effective in treating CIU patients, and can be given safely for 3 months. However, CIU patients requiring initially high doses of glucocorticosteroids and with a long clinical history are less amenable to CsA treatment. 相似文献
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Flow cytometric analysis of basophil numbers in chronic urticaria: basopenia is related to serum histamine releasing activity 总被引:1,自引:0,他引:1
C. E. H. GRATTAN D. WALPOLE D. M. FRANCIS† N. NllMI† G. DOOTSON‡ S. EDLER M. F. CORBETT§ R. M. BARR† 《Clinical and experimental allergy》1997,27(12):1417-1424
Background and Objective Peripheral blood basophils are reduced in some chronic urticaria patients when counted with granule stains. Approximately 30% of patients with severe chronic urticaria have functional autoantibodies which release histamine from healthy donor basophils in vitro but the relationship between basophil numbers in vivo and serum histamine releasing activity has not been studied. Objective To determine the relationship between basophil numbers and serum basophil histamine releasing activity and to assess whether basophils are present, but undetectable, in peripheral blood with granule stains by using a new Row cytometric method based on surface immunophenotype. Methods Basophils were counted manually by a chamber method using a granule stain and by flow cytometry using dual staining with anti-IgE and anti-FceRI in 25 chronic idiopathic urticaria patients and 25 healthy controls. Serum histamine releasing activity was assessed on healthy donor basophils in vitro and by the weal response to autologous serum skin testing in vivo (patients only). Results Basophils were significantly reduced in chronic urticaria by manual counting and How cytometry. A subgroup of seven patients with in vitro histamine releasing activity showed a marked reduction or absence of basophils by both methods. There were no obvious distinguishing clinical characteristics between these patients and the others; six of them showed positive autologous serum skin-test responses. On comparing the two methods, the manual basophil counts were generally lower than flow cytometric counts. Agreement over the full range of values was not strong and therefore counts obtained by the two methods are not directly interchangeable. Conclusions Basopenia in chronic idiopathic urticaria is associated with serum basophil bistamine releasing activity in a subgroup of patients. The lack of granule and surface immunophenotype staining suggests a reduction in numbers rather than an inability to detect circulating degranulated cells by conventional counting methods. 相似文献
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BACKGROUND: The presence of anti-FcepsilonRI and anti-IgE autoantibodies in a subset of patients with chronic urticaria suggests their aetiopathogenetic role. In clinical practice, the presence of these antibodies is usually considered when the autologous serum skin test (ASST) is positive. AIMS: To evaluate if the positive ASST follows up the activity of chronic urticaria. METHODS: Autologous serum skin test and thyroid autoantibody detection were performed in 82 patients with chronic urticaria and repeated 1 year later, when the vast majority of patients were symptom-free. Twenty patients with Hashimoto thyroiditis (HT), who had never suffered from urticaria, represented the control group. RESULTS: At the start of the study, the prevalence of positive ASST was 46.6%. The association of HT-urticaria was 29.3%. ASST was positive in 62 and 39% of patients with and without HT, respectively (P > 0.05 ns). One year later, 28 of 34 patients with a positive ASST were symptom-free, but 50% of them were positive for ASST. The ASST was positive in 86.7 and 8% of patients with and without HT, respectively (P < 0.001). In the control group, ASST was always negative. CONCLUSIONS: The co-existence of autoimmune thyroiditis with chronic urticaria seems to induce a significant difference in the persistence of a positive ASST. Consistent with previous reports, a positive ASST correlates with disease exacerbation in chronic urticaria patients without thyroiditis. In patients with thyroiditis and urticaria, positive ASST persists even after the urticaria has disappeared, thus questioning whether a positive ASST to be a surrogate marker of the functional role of anti-FcepsilonRI and anti-IgE autoantibodies. 相似文献
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Marked improvement of the basophil activation test by detecting CD203c instead of CD63 总被引:6,自引:0,他引:6
R. Boumiza G. Monneret M.-F. Forissier† J. Savoye† M.-C. Gutowski W. S. Powell‡ J. Bienvenu 《Clinical and experimental allergy》2003,33(2):259-265
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. 相似文献
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Comparison of two basophil activation markers CD63 and CD203c in the diagnosis of amoxicillin allergy 总被引:1,自引:0,他引:1
N. Abuaf H. Rostane B. Rajoely H. Gaouar J. E. Autegarden F. Leynadier R. Girot 《Clinical and experimental allergy》2008,38(6):921-928
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. 相似文献
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K. Vasagar B. M. Vonakis L. M. Gober A. Viksman S. P. Gibbons Jr. S. S. Saini 《Clinical and experimental allergy》2006,36(6):770-776
BACKGROUND: Approximately 40% of chronic idiopathic urticaria (CIU) subjects have autoantibodies to either FcepsilonRIalpha or IgE. The effect of such autoantibodies on circulating basophil activation status is unknown. OBJECTIVE: The expression of cell surface activation markers on basophils from CIU, non-allergic, and allergic subjects were compared. Further, the relationship between marker expression and serum factors reported in CIU, such as histamine-releasing activity (HRA) and immunoreactivity to FcepsilonRIalpha were examined. METHODS: Peripheral blood was obtained from CIU, allergic, and non-allergic donors and fractionated by density gradients. Enriched basophils (1-12%) were analysed by flow cytometry for expression of activation markers including CD63, CD69, and CD203c. Dilutions of serum (5-50%) were analysed for HRA on basophils from a normal donor. Serum was tested for immunoreactivity by western blotting to a standard cell lysate prepared from an RBL-SX38 cell line transfected with human FcepsilonRIalpha. RESULTS: CIU subjects (n=9) and allergic subjects (n=8) exhibited enhanced expression of CD63 and CD69, as compared with non-allergic subjects (n=7); however, no difference was seen among groups for CD203c expression. Five CIU and two non-allergic subjects had evidence of significant serum HRA (>20%), whereas two CIU, two allergic, and three non-allergic subjects had evidence of serum immunoreactivity to FcepsilonRIalpha. Serum HRA and serum immunoreactivity to FcepsilonRIalpha were not associated with enhanced surface marker expression. CONCLUSION: Basophil activation marker expression is increased in CIU subjects and is not associated with serum factors. In addition, serum HRA and FcepsilonRIalpha immunoreactivity are not unique to CIU, or related to enhanced circulating basophil marker expression. 相似文献
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Comparison of basophil activation tests using CD63 or CD203c expression in patients with insect venom allergy 总被引:2,自引:0,他引:2
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. 相似文献
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Background Chronic idiopathic urticaria (CIU) is a distressing skin condition involving recurrent itchy hives lasting 6 weeks or longer. The mechanism involves mast cell and basophil degranulation, which releases inflammatory mediators including histamine. In our clinical practice, we have observed that the onset of CIU is often preceded by a major life event. Objective To investigate the role of the hormones of the hypothalamic–pituitary–adrenal (HPA) axis in the link between psychological stress and CIU. Methods Thirty people with CIU and 30 normal controls were recruited. A flow cytometric CD63 expression assay was used to quantify basophil activation, and serum cortisol concentrations were measured as an indication of stress. Results Both corticotrophin releasing factor (CRF) and adrenocorticotrophic hormone (ACTH) were shown to activate basophils. There was no significant difference between numbers of CIU patients and normal controls responding to CFR, ACTH or cortisol. However, the responses in the CIU patients were stronger than those in normal controls. There was also a trend towards higher serum cortisol concentrations in CIU patients. The basophil response to CRF and ACTH correlated with the serum cortisol concentration in normal controls, but not in CIU patients. Conclusions Although our data have not supported the hypothesis that stress makes a major contribution to CIU, the heightened basophil response to CFR and ACTH and higher levels of serum cortisol do suggest a derangement of the HPA axis in CIU. 相似文献
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B. Eberlein I. León Suárez U. Darsow F. Ruëff H. Behrendt J. Ring 《Clinical and experimental allergy》2010,40(3):411-418
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. 相似文献
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Nicole Schoepke Riccardo Asero Andr Ellrich Marta Ferrer Ana Gimenez‐Arnau Clive E. H. Grattan Thilo Jakob George N. Konstantinou Ulrike Raap Per Stahl Skov Petra Staubach Arno Kromminga Ke Zhang Carsten Bindslev‐Jensen Alvaro Daschner Tamar Kinaciyan Edward F. Knol Michael Makris Nadine Marrouche Peter Schmid‐Grendelmeier Gordon Sussman Elias Toubi Martin K. Church Marcus Maurer 《Allergy》2019,74(12):2427-2436
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Flow cytometric basophil activation test by detection of CD63 expression in patients with immediate‐type reactions to betalactam antibiotics 总被引:3,自引:0,他引:3
M. L. Sanz P. M. Gamboa I. Antépara C. Uasuf L. Vila C. GarciaAvilés M. Chazot A. L. De Weck 《Clinical and experimental allergy》2002,32(2):277-286
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. 相似文献
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Enza DAuria Mara De Amici Amelia Licari Silvia Caimmi Cecilia Mantegazza GianVincenzo Zuccotti Gianluigi Marseglia 《Immunobiology》2019,224(1):30-33
Introduction
Chronic spontaneous urticaria (CSU) is characterized by recurrent itchy wheals, angioedema or both, that persist for longer than six weeks. In children, up to 40% of chronic spontaneous urticaria is due to mast cells and basophils-activating autoantibodies, mostly directed against the IgE high-affinity receptor subunit (FcεRI). Indirect basophil activation test (BAT) has been proposed in the diagnosis of autoimmune urticaria.Materials and methods
Sera from sixteen patients, aged from 2 to 15 yrs, with CSU were evaluated through indirect BAT by flow cytometry using a commercial kit (Flow CAST®, BUHLMANN Laboratories, Schonenbuch, Switzerland) according to the manufacturer’s instructions.Results
Indirect BAT test gave a much better diagnosis in our cohort than the gold standard ASST. Six children (37.5%) showed a positive indirect BAT while we could perform ASST in only 3 patients with just one patients showing a positive ASST. The specificity of BAT positive results was confirmed by the absence of significant difference between the BAT results obtained from negative controls vs negative sera of the patient (p?=?0.65) on the basophil donors, indicating that the serum is not activating basophil per se.Conclusions
This pilot study suggests the utility of BAT to identify the subtype of autoimmune CSU in children in clinical practice. 相似文献20.
M. L. Sanz G. Sánchez P. M. Gamboa L. Vila C. Uasuf M. Chazot I. Diéguez A. L. De Weck 《Clinical and experimental allergy》2001,31(7):1007-1013
BACKGROUND: In this study, we determined by flow cytometry the percentage of basophils activated after in vitro stimulation by allergens and expressing the CD63 marker. The diagnostic reliability of the technique was assessed as well as its correlation with other in vitro diagnostic parameters. METHODS: Fifty-three patients suffering from asthma and/or allergic rhinitis following sensitization to Dermatophagoides pteronyssinus and 51 patients sensitized to Lolium perenne were investigated. Twenty-four atopic patients not sensitive to these allergens and 38 healthy subjects were also selected as controls. The basophil activation test determines the percentage of basophils which express CD63 as an activation marker, by means of flow cytometry, after in vitro stimulation with allergen, using double labelling with monoclonal antibody anti-CD63-PE and anti-IgE-FITC. RESULTS: No differences in basal values (non-activated control) were found between sensitized patients, atopic controls and healthy controls. On the other hand, sensitized patients showed a significantly higher percentage of activated basophils after stimulation by allergens in vitro than both control groups (P < 0.001). We found a significant correlation between skin tests and basophil activation tests (r = 0.72, P < 0.001). We also found a positive and significant correlation between basophil activation tests and histamine release tests (r = 0.80, P < 0.001), allergen-specific sulphidoleukotriene production (r = 0.7, P < 0.001) and the occurrence of serum allergen-specific IgE (r = 0.71, P < 0.001). CONCLUSION: The basophil activation test is a highly reliable technique in the diagnosis of allergy to inhalant allergens. The sensitivity of the basophil activation test was 93.3%, and its specificity 98.4%, when using a cut-off point of 15% activated basophils as positive result. 相似文献