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Background: Correct diagnosis of immediate drug hypersensitivity reactions (IDHRs) can pose a significant challenge, mainly because of the absence of reliable in vitro tests, uncertainties associated with skin testing, and incomplete understanding of the underlying mechanisms., Aim: To summarize and hypothesize on the potential of basophil activation test (BAT) as a safe aid to explore the mechanistic endotypes of IDHR, to identify antibody recognition sites, and to monitor drug desensitization., Methods: A literature search was conducted using the keywords "allergy, basophil activation, CD63, CD203c, diagnosis, drugs, hypersensitivity, flow cytometry"; this was complemented by the authors' own expertise., Results: At present BAT has mainly been employed as a diagnostic aid. However, evidence is emerging that the technique might also deepen our insights in immune (allergic) and nonimmune (nonallergic) mechanistic processes of IDHR. It is anticipated that BAT might also benefit the identification of antibody recognition sites and benefit our understandings of desensitization strategies., Conclusion: Although the nondiagnostic application of BAT in IDHR is still in its infancy, with increasing employment, we can expect the technique to become a valuable asset to study many domains of IDHR that remain poorly understood.

The basis of flow cytometric allergy diagnosis is the quantification of changes in the expression of basophilic surface membrane markers (Ebo et al., Clin Exp Allergy 34: 332-339, 2004). Upon encountering specific allergens recognized by surface receptor FcεRI-bound IgE, basophils not only secrete and generate quantifiable bioactive mediators but also upregulate the expression of different markers (e.g., CD63, CD203c) which can be detected by multicolor flow cytometry using specific monoclonal antibodies (Ebo et al., Cytometry B Clin Cytom 74: 201-210, 2008). Here, we describe two flow cytometry-based protocols which allow the detection of surface marker activation (Method 1) and changes in intragranular histamine (Method 2), both reflecting different facets of basophil activation.

Histamine and its release can be studied by multicolor flow cytometry on a single cell level by an enzyme affinity method (HistaFlow®). However, for the time-being, the clinical and scientific application of the HistaFlow® technique remains limited. This study aims at verifying the reliability of the HistaFlow® as an instrument to quantify IgE-mediated basophil responses to drugs, i.e., rocuronium, which are believed to be less potent basophil activators than large proteinaceous allergens. Ten patients and three exposed control individuals were included in this study. Each subject underwent in vitro basophil activation tests (HistaFlow®) with 0.16 and 1.6 mmol/L rocuronium. Patients showed an activation of basophils ranging from 11 to 86% of CD63 positive basophils and a median histamine release per cell from 68 to 100% after stimulation with an optimal concentration of 1.6 mmol/L rocuronium. For the control individuals no activation was demonstrable. This study confirms that the HistaFlow® technique is a reliable tool to study histamine release by individual cells in response to drugs. Although the HistaFlow® technique will probably not add to the diagnostic management of rocuronium allergy, our findings suggest that the technique could constitute an important asset for future studies on the pathomechanism(s) of immediate drug hypersensitivity reactions. © 2015 International Clinical Cytometry Society., (© 2015 International Clinical Cytometry Society.)

The basis of flow cytometric allergy diagnosis is quantification of changes in expression of basophilic surface membrane markers (Ebo et al., Clin Exp Allergy 34: 332-339, 2004). Upon encountering specific allergens recognized by surface receptor FcεRI-bound IgE, basophils not only secrete and generate quantifiable bioactive mediators but also up-regulate the expression of different markers (e.g., CD63, CD203c) which can be detected by multicolor flow cytometry using specific monoclonal antibodies (Ebo et al., Cytometry B Clin Cytom 74: 201-210, 2008). Here, we describe two flow cytometry-based protocols which allow detection of surface marker activation (Method 1) and changes in intragranular histamine (Method 2), both reflecting different facets of basophil activation.

Basophils are key effector cells in allergic inflammatory reactions. However, the mechanisms of FcεRI-induced degranulation are complex and remain to be disentangled. Signal transducer and activator of transcription (STAT) molecules modulate various cell functions. STAT5 appears to be essential for IgE-mediated mast cell function, but its role in human basophils after cross-linking FcεRI is unknown. In this study, STAT5 phosphorylation was investigated by flow cytometry, and combined with analyses of the degranulation marker CD63 at single cell level. Kinetics of STAT5 phosphorylation were studied in basophils of birch pollen allergic patients and showed a fast phosphorylation induced by interleukin (IL)-3, but not with antigen alone. Stimulating basophils with a mixture of allergen and IL-3 resulted in a two to three fold higher phosphorylation of STAT5 than induced by IL-3 alone. In the presence of IL-3, antigen elicited a dose-dependent STAT5 response. In conclusion, this study demonstrates that STAT5 in human basophils is activated through both the IL-3 and the FcεRI signaling pathway., (Copyright © 2011 International Clinical Cytometry Society.)

Background: Stimulated human basophils exhibit different degranulation patterns with release of mediators and appearance of activation markers such as CD63 and CD203c. Traditionally, released mediators are quantified in the supernatant of activated cells, whereas the expression of activation markers by individual cells is analyzed by flow cytometry. Alternatively, intracellular histamine and its release by basophils and mast cells have been repeatedly studied applying an enzyme-affinity-gold method based on the affinity of the histaminase diamine oxidase for its substrate histamine., Objective: To develop a flow cytometric technique enabling to study histamine release by individual basophils in combination with the expression of activation markers. To elucidate the principles of basophil degranulation on a single cell level., Methods: Intracellular histamine and its release is analyzed flow cytometrically by an enzyme-affinity method using diamine oxidase conjugated to laser-excitable fluorochromes. Phenotyping of cells implied flow cytometric quantification of CD63 and CD203c. Stimuli such as allergen, anti-IgE, N-formyl-met-leu-phe (fMLP), phorbol 12-myristate 13-acetate (PMA), ionomycin and interleukin (IL-)3 are applied to obtain different degranulation profiles., Results: Stimulation with anti-IgE, allergen, fMLP and PMA±ionomycin induces a rapid release of histamine that can be analyzed flow cytometrically. Analyses on a single cell level reveal that histamine release is not restricted to cells showing significant up-regulation of CD63. Alternatively, up-regulation of CD203c does not per se indicate histamine release. In some patients, priming of cells with IL-3 not only facilitates basophil responsiveness but also implies an increased ability of DAO to label the cells., Conclusion: This study provides the proof-of-concept that histamine and its release can be studied by multicolor flow cytometry on a single cell level (HistaFlow). Coupling the data to simultaneous phenotyping of activated basophils confirms that histamine release principally results from anaphylactic degranulation and in a lesser extent from piecemeal degranulation., (Copyright © 2011 Elsevier B.V. All rights reserved.)

The diagnosis of allergic reactions in clinical practice rests upon both clinical history and the demonstration of specific immunoglobulin E (sIgE), either in the serum or via skin tests. However, for various reasons, identification of the offending allergen(s) is not always possible. Moreover, not all allergies are IgE-mediated. In an attempt to find reliable methods to investigate hypersensitivity reactions, histamine and sulfidoleukotriene release tests have long been introduced. However, relatively few comprehensive quality reports have been published so far. Upon challenge with a specific allergen, basophils not only secrete quantifiable bioactive mediators but also upregulate the expression of different markers which can be detected efficiently by flow cytometry using specific monoclonal antibodies. This review addresses the principals, particular technical aspects and pitfalls as well as the clinical and research applications of flow-assisted analysis of in vitro activated basophils., ((c) 2008 Clinical Cytometry Society)

Background: The signal transduction pathways and control mechanisms involved in IgE-mediated basophil activation remain incompletely understood., Objectives: To investigate whether basophilic intracellular signal transduction and immunophenotype can be analysed simultaneously by flow cytometry., Methods: Basophils in whole blood were stimulated with anti-IgE and latex antigen at various concentrations and during different time courses. Phosphorylation of p38 mitogen-activated protein kinase (MAPK) as a representative of the intracellular signal transduction pathway and surface expression of CD63 was assessed simultaneously flow cytometrically. The effect of pre-incubation with IL-3 was assessed., Results: Stimulation of the basophils with anti-IgE and allergen induces a rapid phosphorylation of p38 MAPK that peaks between 1 and 5 min and returns to baseline levels after 60 min. In contrast, CD63 up-regulation demonstrates a maximal but more continuous expression that peaks approximately 5 min later than phosphorylation of p38 MAPK. Specific inhibition of p38 MAPK reduced or almost completely abrogated up-regulation of CD63. Pre-incubation of the basophils with IL-3 produces a rapid p38 MAPK phosphorylation over basal levels, but this was weaker and shorter than for anti-IgE stimulation. Pre-incubation of the basophils with IL-3 did not potentiate anti-IgE-induced phosphorylation of p38 MAPK and did affect spontaneous or IgE-mediated CD63 up-regulation., Conclusions: This study provides the proof that the flow cytometer allows an integrated analysis of basophilic intracellular signalling and immunophenotyping. Owing to its technical simplicity, the low number of cells required and rapid analysis, the technique seems promising for use in the clinic as a diagnostic tool or to monitor therapy., Capsule Summary: This study is the first to provide evidence for a combined analysis of basophilic intracellular signalling and immunophenotyping by flow cytometry. Owing to its technical simplicity, the low number of cells required and rapid analysis, the technique seems promising for use in the clinic as a diagnostic tool or to monitor therapy.

Background: Correct identification of the culprit venom is a prerequisite for specific venom immunotherapy (VIT). Despite the efficacy of VIT, issues as how to monitor treatment and when to discontinue maintenance therapy remain to be established., Methods: To evaluate diagnostic performances of the basophil activation test (BAT) in wasp venom allergy, 80 patients with a definite history of wasp venom anaphylaxis (systemic reactors) and 14 wasp-stung asymptomatic controls (stung controls) were enrolled. Venom-induced basophil activation was analyzed flow cytometrically by double-labeling with anti-IgE and anti-CD63. Results were compared to wasp IgE levels and results of a venom skin test (VST). To establish whether the BAT constitutes a candidate marker to monitor VIT, the BAT was repeated in 22 patients on the 5th day of a build-up course and after 6 months of maintenance VIT. Whether the BAT could contribute in the decision of discontinuing VIT was assessed in a cross-sectional analysis in 30 patients receiving treatment for 3 years., Results: Comparison between systemic reactors and stung controls revealed a sensitivity of 86.4% and specificity of 100% for venom IgE, and sensitivity of 81.8% for VST, respectively. In contrast to stung controls, patients demonstrated dose-dependent venom-induced basophil activation. The BAT attained a sensitivity of 83.8% and specificity of 100%. At the end of the build-up course, no effect of VIT on the BAT was demonstrable. When the BAT was repeated after 6 months of treatment, submaximal stimulation of the cells demonstrated a significant decreased CD63 expression (P < 0.04). Patients having VIT for 3 years also demonstrated significantly lower venom-induced CD63 expression (P < 0.001). After 3 years, 60% of the patients had a negative BAT for submaximal stimulation of the cells whereas only 17.9% of the patients had negativation of wasp IgE., Conclusions: The BAT is a reliable instrument for the diagnosis of wasp venom anaphylaxis and might constitute an instrument to monitor wasp VIT., (Copyright 2006 International Society for Analytical Cytology)

Physicians predominantly rely upon quantification of serum-specific immunoglobulin E (IgE) and/or skin test to confirm clinically suspected IgE-mediated allergy. However, for various reasons, identification of the offending allergen(s) and potentially cross-reactive structures is not always straightforward. Flow-assisted allergy diagnosis relies upon quantification of alterations in the expression of particular basophilic activation markers. Actually, upon challenge with a specific allergen, basophils not only secrete quantifiable bioactive mediators but also upregulate the expression of different markers which can be detected efficiently by flow cytometry using specific monoclonal antibodies. Currently, the technique has been applied in the investigation of IgE-mediated allergy caused by classical inhalant allergens, food, Hevea latex, hymenoptera venoms and drugs. It is also appreciated; the technique proves valuable in the diagnosis of non-IgE-mediated (anaphylactoid) reactions such drug hypersensitivity and the detection of autoantibodies in certain forms of chronic urticaria. This review will not address immunologic features, characteristics and general pitfalls of flow-assisted analysis of in vitro-activated basophils as summarized elsewhere. After a recapitulation of the principles and some specific technical issues of flow-assisted analysis of in vitro-activated basophils, we principally focus on the current clinical and research applications of the basophil activation tests. Personal experience of both research groups is provided, where appropriate. Finally, a viewpoint on how the field might evolve in the following years is provided.

Background: Diagnosis of anaphylaxis from neuromuscular blocking agents (NMBA) is not always straightforward., Objectives: To assess flow cytometric analysis of activated basophils (BAT) as a diagnostic instrument in anaphylaxis from rocuronium. To investigate whether the technique might help to identify cross-reactive and safe alternative compounds., Methods: For validation of the BAT, 14 patients with perioperative anaphylaxis demonstrating a positive skin test (ST) for rocuronium and eight individuals that tolerated rocuronium and a negative ST for this drug were enrolled. To confirm specificity of the BAT, five patients that tolerated atracurium or cisatracurium with a negative ST for rocuronium were tested. Basophil activation with rocuronium, vecuronium, atracurium, cisatracurium and suxamethonium was analysed flow cytometrically by labelling with anti-CD123/anti-HLADR/anti-CD63., Results: Sensitivity of BAT for rocuronium was 91.7% and specificity 100%. However, in two patients the BAT was lost as a diagnostic tool, as their cells were nonresponsive to positive control stimulation and allergen. Seven from the 12 responsive patients also demonstrated a clear basophilic activation for vecuronium. Moreover, according to ST and/or BAT cross-reactivity between rocuronium and vecuronium was suspected in 10/14 patients. Except one patient, all patients had negative BAT and ST investigations for atracurium and cisatracurium. Currently, five patients tolerated administration of cisatracurium. All control individuals demonstrated negative ST and BAT for all tested NMBA., Conclusions: The BAT constitutes a reliable instrument to diagnose anaphylaxis from rocuronium. The technique also allows quick and simultaneous testing of different potential cross-reactive NMBA and to tailor a safe alternative.

Background: Specific immunoglobulin E (IgE) and commercially available skin prick tests have been demonstrated to be unreliable methods to diagnose pollen-associated food allergy. To evaluate the predictive value of the basophil activation test (BAT) in pollen-associated food allergy, the apple-mediated oral allergy syndrome (OAS) in patients with birch pollinosis was chosen as a representative model., Methods: Patients with birch pollen allergy and a history of apple-mediated OAS (OAS(+), n = 29), patients with birch allergic without OAS (OAS(-), n = 22), and healthy controls (HC, n = 10) without birch pollen allergy and OAS were included. Apple IgE was quantified by the CAP FEIA method. Skin prick tests were performed with a Jonagold apple extract. Flow cytometric analysis of basophils activated with the same Jonagold extract was based on double staining with anti-IgE/anti-CD63 monoclonal antibodies., Results: Comparison between OAS(+) subjects and HC showed sensitivities and specificities of 96% and 100% for apple IgE and 88% and 100% for the apple skin prick test, respectively. For the BAT, sensitivity and specificity were 100%. In contrast, when nonresponders on the BAT were considered, sensitivity decreased to 90%. In a separate analysis between OAS(+) and OAS(-) subjects, specificities decreased to 30% for apple IgE and to 80% for the apple skin test, respectively. The BAT reached a sensitivity of 88% and a specificity of 75%., Conclusion: Flow cytometry-assisted quantification of in vitro basophil activation seems to be a reliable instrument in the diagnosis of this model of pollen-associated food allergy. In addition, this study reemphasizes that the specificity of diagnostic allergy tests decreases considerably when, apart from HC, control individuals with cross-reactive antibodies are included.

Background: We investigated the relation between intracellular cytokine production and extracellular cytokine levels by using two flow cytometric techniques., Methods: A two-color flow cytometric technique was used to measure interleukin (IL)-1beta, IL-6, tumor necrosis factor alpha (TNF-alpha), IL-10, and IL-12 production blocked intracellularly with brefeldin A in lipopolysaccharide (LPS)-stimulated CD14(+) monocytes and IL-2, IL-4, and IFN-gamma production in phorbol-12-mirystate-13-acetate (PMA)-stimulated CD3(+) T lymphocytes in samples from patients with rheumatoid arthritis. A flow cytometric microsphere-based immunoassay was performed to detect cytokine secretion in plasma of PMA- and LPS-stimulated whole blood samples., Results: There was a strong linear correlation between extracellular quantitative (pg/ml) and intracellular semiquantitative detection of LPS-stimulated IL-1beta, IL-6, IL-10, and IL-12 production (r > 0.9). For lymphocytes, extracellularly detected IL-2 and IFN-gamma correlated well with percentages of cytokine-producing cells (r > 0.8). The percentages of IL-4-positive T cells were moderately correlated with the secreted amounts of IL-4 as detected with the microsphere-based immunoassay (r = 0.7)., Conclusion: Overall, there was a good correlation between semiquantitative intracellular detection of cytokines and the secreted amounts of cytokines detected with the microsphere based immunoassay., (Copyright 2003 Wiley-Liss, Inc.)

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.

Flow cytometry has become a powerful technique to measure intracellular cytokine production in lymphocytes and monocytes. Appropriate inhibition of the secretion of the produced cytokines is required for studying intracellular cytokine expression. The aim of this study was to compare the capacity of cytokine secretion inhibitors, monensin and brefeldin A, in order to trap cytokine production (interleukin-1 beta [IL-1beta], IL-6, tumor necrosis factor-alpha [TNF-alpha]) within peripheral blood monocytes. A two-color flow cytometric technique was used to measure intracellular spontaneous and lipopolysaccharide (LPS)-stimulated IL-1beta, IL-6, and TNF-alpha production in monocytes (CD14+) of whole blood cultures. The viability of monensin-treated monocytes was slightly lower than that of brefeldin A-inhibited monocytes, as measured with propidium iodide (PI). The percentage of IL-6 and TNF-alpha-producing monocytes after 8 h of culture without stimulation revealed significant lower values for monensin-treated than for brefeldin A-treated monocytes. The percentages for stimulated cells did not differ. The spontaneous intracellular production in molecules of equivalent soluble fluorochrome units (MESF) of IL-1beta, IL-6, and TNF-alpha after 8 h of culture was higher in brefeldin A than in monensin-inhibited monocytes. The LPS-stimulated intracellular production of IL-1beta, IL-6, and TNF-alpha was increased in brefeldin A-inhibited monocytes. In conclusion, for flow cytometric determination of intracellular monocytic cytokines (IL-1beta, IL-6, and TNF-alpha), brefeldin A is a more potent, effective, and less toxic inhibitor of cytokine secretion than monensin.