Christel Mertens

Flow-assisted allergy diagnosis: current applications and future perspectives

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.

Flow-assisted diagnostic management of anaphylaxis from rocuronium bromide

Background:  Diagnosis of anaphylaxis from neuromuscular blocking agents (NMBA) is not always straightforward.

Analyzing histamine release by flow cytometry (HistaFlow): A novel instrument to study the degranulation patterns of basophils

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.

In Vitro Diagnosis of Immediate Drug Hypersensitivity Anno 2017: Potentials and Limitations

BackgroundFor most physicians, quantification of drug-specific immunoglobulin E (drug-sIgE) antibodies constitutes the primary in vitro measure to document immediate drug hypersensitivity reactions (IDHR). Unfortunately, this is often insufficient to correctly identify patients with IgE-mediated IDHR and impossible for non-IgE-mediated IDHR that result from alternative routes of basophil and mast cell activation. In these difficult cases, diagnosis might benefit from cellular tests such as basophil activation tests (BAT).AimThe aim was to review the potential and limitations of quantification of sIgE and BAT in diagnosing IDHR. The utility of quantification of serum tryptase is discussed.MethodsA literature search was conducted using the key words allergy, basophil activation, CD63, CD203c, diagnosis, drugs, hypersensitivity, flow cytometry, specific IgE antibodies; this was complemented by the authors’ own experience.ResultsThe drugs that have been most studied with both techniques are β-lactam antibiotics and curarizing neuromuscular blocking agents (NMBA). For sIgE morphine, data are available on the value of this test as a biomarker for sensitization to substituted ammonium structures that constitute the major epitope of NMBA, especially rocuronium and suxamethonium. For the BAT, there are also data on non-steroidal anti-inflammatory drugs (NSAIDs) and iodinated radiocontrast media. For β-lactam antibiotics, sensitivity and specificity of sIgE varies between 0 and 85% and 52 and 100%, respectively. For NMBA, sensitivity and specificity varies between 38.5 and 92% and 85.7 and 100%, respectively. Specific IgE to morphine should not be used in isolation to diagnose IDHR to NMBA nor opiates. For the BAT, sensitivity generally varies between 50 and 60%, whereas specificity attains 80%, except for quinolones and NSAIDs.ConclusionsAlthough drug-sIgE assays and BAT can provide useful information in the diagnosis of IDHR, their predictive value is not absolute. Large-scale collaborative studies are mandatory to harmonize and optimize test protocols and to establish drug-specific decision thresholds.

Flow Cytometric Allergy Diagnosis: Basophil Activation Techniques

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.

Cannabis sativa allergy: looking through the fog.

IgE‐mediated Cannabis (C. sativa, marihuana) allergy seems to be on the rise. Both active and passive exposure to cannabis allergens may trigger a C. sativa sensitization and/or allergy. The clinical presentation of a C. sativa allergy varies from mild to life‐threatening reactions and often seems to depend on the route of exposure. In addition, sensitization to cannabis allergens can result in various cross‐allergies, mostly for plant foods. This clinical entity, designated as the ‘cannabis‐fruit/vegetable syndrome’, might also imply cross‐reactivity with tobacco, natural latex and plant‐food‐derived alcoholic beverages. Hitherto, these cross‐allergies are predominantly reported in Europe and appear mainly to rely upon cross‐reactivity between nonspecific lipid transfer proteins or thaumatin‐like proteins present in C. sativa and their homologues, ubiquitously distributed throughout plant kingdom. At present, diagnosis of cannabis‐related allergies predominantly rests upon a thorough history completed with skin testing using native extracts from crushed buds and leaves. However, quantification of specific IgE antibodies and basophil activation tests can also be helpful to establish correct diagnosis. In the absence of a cure, treatment comprises absolute avoidance measures. Whether avoidance of further use will halt the extension of related cross‐allergies remains uncertain.

Diagnosing cefazolin hypersensitivity: Lessons from dual-labeling flow cytometry

In the absence of a cefazolin-specific IgE assay and provocation tests with this intravenous cephalosporin being hazardous, diagnosis of immediate cefazolin hypersensitivity predominantly relies on skin tests. However, validation of cefazolin skin tests has focused on assessing the irritating potential in healthy (exposed) control individuals and data on sensitivity of cefazolin skin testing remain scarce. Therefore, the availability of another diagnostic test could be of interest to further evaluate the reliability of cefazolin skin tests and to add to the diagnosis. Upon encounter of allergen crosslinking surface-bound IgE antibodies, basophils upregulate activation and degranulation markers and release bioactive mediators such as histamine. Both the immunophenotypic alterations and the release of histamine are quantifiable on a single-cell level by multicolor flow cytometry in the basophil activation test (BAT/HistaFlow). BAT/Histaflow is being increasingly introduced in the diagnostic approach of immediate drug hypersensitivity reactions (IDHRs), and have mainly been applied to document IDHRs to b-lactam antibiotics, particularly aminopenicillins and to a lesser extent quinolones. For the time being, BAT has not been thoroughly validated in immediate cephalosporin hypersensitivity. In this study, we sought to evaluate the BAT in cefazolin hypersensitivity because allergy to this first-generation cephalosporin can constitute an important cause of perioperative anaphylaxis with serious consequences of diagnostic error. Patients and controls were selected as described in Uyttebroek et al. Eighteen patients suffering from perioperative anaphylaxis after intravenous injection of cefazolin demonstrating a positive skin test result for the drug and all other possible causes excluded were selected. Seventeen individuals exposed to cefazolin during anesthesia with a negative skin test result and another identifiable cause served as exposed control individuals. Investigations were performed between 0.3 and 38 months (median, 3 months) after the reaction. Patients provided informed consent in accordance with the Declaration of Helsinki. Skin testing with cefazolin (Cefazoline 1 g, Sandoz, Vilvoorde, Belgium) was performed according to the European Network and European Academy (ENDA) recommendations, but a maximum concentration of 20 mg/mL was applied. The BAT was performed as described elsewhere. Briefly, within 3 hours of sampling in endotoxin-free heparinized tubes, aliquots of 100 mL whole blood were incubated (20 minutes, 37 C) with dilution buffer (negative control), positive control (anti-IgE 10 mg/mL, BD Biosciences, Erembodegem, Belgium), or serial dilutions of cefazolin ranging from 11 to 1100 mmol/L. Cells were stained with 20 mL of monoclonal anti-human IgE (clone GE-1, Sigma Aldrich GmBH, Steinheim, Germany) labeled with Alexa Fluor 488 (Molecular Probes, Invitrogen, Paisley, UK) and 10 mL of monoclonal anti-human CD63-PE (clone H5C6, BD Biosciences) and 10 mL CD203c-APC (clone NP4D6, BD Biosciences). Flow cytometric characterization of basophils relied on a combination of side scatter, anti-IgE, and CD203c positivity. Results were expressed as %CD63 and %CD203c upregulated basophils. In all patients, diagnosis of cefazolin hypersensitivity was established with intradermal skin testing (IDT). Two patients were nonresponsive to positive control stimulation in the BAT and were withdrawn from further analysis. In the 16 responders, spontaneous and anti-IgEeinduced appearance of CD63 and upregulation of CD203c was comparable to results in exposed control individuals (data not shown). As displayed in Figure 1, A and B, in exposed control individuals, cefazolin-induced activation showed no upregulation for CD203c and CD63 on basophils. In contrast, patients demonstrated a dose-dependent upregulation of both surface markers. For both read-outs, 2-graph receiver-operating characteristic analysis revealed stimulation with 1100 mmol/L to be most discriminative between patients and exposed control individuals. For this stimulation concentration, 2-graph receiveroperating characteristic analysis generated a diagnostic threshold value of 5% for CD63 and CD203c net upregulation (Figure 1, B and D). However, for this threshold, the CD63-BAT was positive in only 6 of 16 patients (sensitivity, 38%; 95% CI, 15%-65%) and 1 of 17 control individuals (specificity, 94%; 95% CI, 71%-100%), whereas the CD203c read-out was positive in 12 of 16 patients (sensitivity, 75%; 95% CI, 47%-98%) and 1 of 17 control individuals (specificity, 94%; 95% CI, 71%-100%). Figure 1, E and F, displays the individual percentages of CD63 and CD203c upregulation and number of positive BAT in responsive patients and exposed control individuals. The potentials and limitations of BAT in the diagnosis of IDHR to b-lactam antibiotics has mainly been investigated in penicillins, particularly amoxicillin. To our knowledge, this is the first study that assesses the applicability of BAT with dual labeling of CD63 and CD203c in immediate cefazolin hypersensitivity. Our data confirm that the diagnostic outcome of the BAT significantly varies according to the applied read-out with upregulation of the lineage-specific ectoenzyme nucleotide pyrophosphate phosphodiesterase-3 (CD203c) to be more sensitive than the appearance of lysosome-associated membrane glycoprotein-3 (CD63). Actually, the IL-3efree CD63-BAT

Immediate moxifloxacin hypersensitivity: Is there more than currently meets the eye?

Immediate drug hypersensitivity reactions (IDHR) to moxifloxacin constitute a pathomechanistic conundrum and a diagnostic challenge. Our objective was to study whether simultaneous phenotyping and quantification of histamine release might add to our knowledge about the basophil activation properties of moxifloxacin and constitute a reliable diagnostic aid. Fifteen patients with an IDHR to moxifloxacin and nine moxifloxacin challenged controls were selected. All had a basophil activation test (BAT) with moxifloxacin. Flow cytometric analysis of basophil responses implied labeling for CD63, CD203c, and intracellular histamine. Unlike tolerant challenged controls, basophilic upregulation of CD203c in response to moxifloxacin was observed in seven of 15 patients. Only two of these seven patients demonstrated appearance of CD63 and release of histamine. In the remainder eight patients, no basophil responses were demonstrable. In conclusion, immediate hypersensitivity to moxifloxacin might involve mechanisms difficult to capture by traditional CD63‐/CD203c‐based BAT. Deciphering the complexity of quinolone IDHR seems mandatory.

Cross-reactive aeroallergens: which need to cross our mind in food allergy diagnosis?

Secondary food allergies due to cross-reactivity between inhalant and food allergens are a significant and increasing global health issue. Cross-reactive food allergies predominantly involve plant-derived foods resulting from a prior sensitization to cross-reactive components present in pollen (grass, tree, weeds) and natural rubber latex. Also, primary sensitization to allergens present in fungi, insects, and both nonmammalian and mammalian meat might induce cross-reactive food allergic syndromes. Correct diagnosis of these associated food allergies is not always straightforward and can pose a difficult challenge. As a matter of fact, cross-reactive allergens might hamper food allergy diagnosis, as they can cause clinically irrelevant positive tests to cross-reacting foods that are safely consumed. This review summarizes the most relevant cross-reactivity syndromes between inhalant and food allergens. Particular focus is paid to the potential and limitations of confirmatory testing such as skin testing, specific IgE assays, molecular diagnosis, and basophil activation test.

Rocuronium hypersensitivity: Does off-target occupation of the MRGPRX2 receptor play a role?

BACKGROUND The neuromuscular blocking agent (NMBA) rocuronium is a relevant cause of perioperative hypersensitivity (POH) with a significant risk of diagnostic error. Recently, it has been suggested to reclassify hypersensitivity to NMBA as type A reactions resulting from off-target occupation of the nonimmune MRGPRX2 receptor. OBJECTIVE To investigate whether basophil activation experiments can benefit diagnosis and add to the insights in the pathomechanisms of rocuronium hypersensitivity. METHODS A total of 140 patients with a suspected POH to rocuronium in whom peak tryptase was available had complete diagnostic workup for all potential culprits including triple confirmatory testing with skin tests, basophil activation test (BAT), and quantification of specific IgE (sIgE) antibodies to rocuronium and morphine. To further analyze the clinical relevance of sIgE antibodies, quantitative basophil inhibition experiments were performed by coincubation of the cells with rocuronium and morphine, an opiate known to harbor a substituted ammonium structure. RESULTS Diagnosis of rocuronium hypersensitivity was established in 72 of 140 patients (51.4%), of whom 65 (90.3%) demonstrated mast cell activation. Of the 72 patients, 64 displayed a positive skin test, 8 (11.1%) had their diagnosis documented only by BAT. Coincubation of morphine and rocuronium induced a dose-dependent inhibition of BAT with rocuronium that was restricted to 4 of 6 patients with IgE reactivity to rocuronium and/or morphine. CONCLUSIONS BAT can benefit diagnosis of rocuronium hypersensitivity. As basophils barely express MRGPRX2 and BAT rocuronium can be inhibited by morphine, we believe that hypersensitivity to rocuronium still mainly results from IgE/high-affinity receptor for sIgE (FcεRI)-dependent effector cell activation. However, it cannot be excluded that in a few patients rocuronium hypersensitivity results from off-target occupation of the MRGPRX2 receptor.