Immediate Drug Hypersensitivity Reactions Research Articles

L’énigme IgE vs MRGPRX2 et les tests cellulaires

Since the seminal description implicating Mas-linked G protein-coupled receptor X2 (MRGPRX2) occupancy in the degranulation of mast cells (MCs) by drugs, many studies have been undertaken on this potential new endotype of immediate drug hypersensitivity reactions (IDHRs). However, current evidence for this mechanism comes mainly from animal (mutant) models or in-vitro studies. Irrefutable clinical evidence in humans is lacking. Furthermore, translating these preclinical results into clinical relevance in humans is difficult and needs to be interpreted critically. Based on our clinical priorities and our experience with functional analyses of basophils, MCs and T lymphocytes, the aim of my presentation is to propose a theoretical mechanistic algorithm that could facilitate discrimination between MC degranulation due to MRGPRX2 receptor activation and bridging of specific IgE antibodies coupled to the FceRI receptor. Special focus will be paid to the role of in vitro/ex vivo cellular tests.

Flow-based basophil activation test in immediate drug hypersensitivity. An EAACI task force position paper.

Diagnosing immediate drug hypersensitivity reactions (IDHRs) can pose a significant challenge and there is an urgent need for safe and reliable tests. Evidence has emerged that the basophil activation test (BAT), an invitro assay that mirrors the invivo response, can be a complementary test for many drugs. In this position paper, members of Task Force (TF) "Basophil activation test in the evaluation of Drug Hypersensitivity Reactions" from the European Academy of Allergy and Clinical Immunology (EAACI) present the data from a survey about the use and utility of BAT in IDHRs in Europe. The survey results indicate that there is a great interest for using BAT especially for diagnosing IDHRs. However, there are still main needs, mainly in the standardization of the protocols. Subsequently consensus-based recommendations were formulated for: (i) Technical aspects of BAT in IDHRs including type of sample, management of drugs, flow cytometry protocols, interpretation of the results; and (ii) Drug-specific aspects that should be taken into account when performing BAT in relation to betalactams, neuromuscular blocking agents, fluoroquinolones, chlorhexidine, opioids, radio contrast media, chemotherapeutics, biological agents, nonsteroidal anti-inflammatory drugs, COVID vaccine, and excipients. Moreover, aspects in the evaluation of pediatric population have also been considered. All this indicates that BAT offers the clinician and laboratory a complementary tool for a safe diagnostic for IDHRs, although its place in the diagnostic algorithm depends on the drug class and patient population (phenotype, geography, and age). The standardization of BAT is important for generalizing this method beyond the individual laboratory.

Mas-related G protein-coupled receptor MRGPRX2 in human basophils: Expression and functional studies.

Occupancy of MRGPRX2 heralds a new era in our understandings of immediate drug hypersensitivity reactions (IDHRs), but a constitutive expression of this receptor by basophils is debated. To explore the expression and functionality of MRGPRX2 in and on basophils. Basophils from patients with birch pollen allergy, IDHRs to moxifloxacin, and healthy controls were studied in different conditions, that is, in rest, after stimulation with anti-IgE, recombinant major birch pollen allergen (rBet v 1), moxifloxacin, fMLP, substance P (SP), or other potential basophil secretagogues. In a separate set of experiments, basophils were studied after purification and resuspension in different media. Resting whole blood basophils barely express MRGPRX2 on their surface and are unresponsive to SP or moxifloxacin. However, surface MRGPRX2 is quickly upregulated upon incubation with anti-IgE or fMLP. Pre-stimulation with anti-IgE can induce a synergic effect on basophil degranulation in IgE-responsive subjects after incubation with SP or moxifloxacin, provided that basophils have been obtained from patients who experienced an IDHR to moxifloxacin. Cell purification can trigger a "spontaneous" and functional upregulation of MRGPRX2 on basophils, not seen in whole blood cells, and its surface density can be influenced by distinct culture media. Basophils barely express MRGPRX2 in resting conditions. However, the receptor can be quickly upregulated after stimulation with anti-IgE, fMLP, or after purification, making cells responsive to MRGPRX2 occupation. We anticipate that such "conditioned" basophils constitute a model to explore MRGPRX2 agonism or antagonism, including IDHRs originating from the occupation of this receptor.

Synthetic antigenic determinants of clavulanic acid induce dendritic cell maturation and specific T cell proliferation in patients with immediate hypersensitivity reactions.

Immediate drug hypersensitivity reactions (IDHRs) to clavulanic acid (CLV) have increased in the last decades due to a higher consumption alongside amoxicillin (AX). Due to its chemical instability, diagnostic procedures to evaluate IDHRs to CLV are difficult, and current in vitro assays do not have an optimal sensitivity. The inclusion of the specific metabolites after CLV degradation, which are efficiently recognised by the immune system, could help to improve sensitivity of in vitro tests. Recognition by dendritic cells (DCs) of CLV and the synthetic analogues of two of its hypothesised antigenic determinants (ADs) was evaluated by flow cytometry in 27 allergic patients (AP) and healthy controls (HC). Their ability to trigger the proliferation of T cells was also analysed by flow cytometry. The inclusion of synthetic analogues of CLV ADs, significantly increased the expression of maturation markers on DCs from AP compared to HC. A different recognition pattern could be observed with each AD, and, therefore, the inclusion of both ADs achieves an improved sensitivity. The addition of synthetic ADs analogues increased the proliferative response of CD4+ Th2 compared to the addition of native CLV. The combination of results from both ADs increased the sensitivity of proliferative assays from 19% to 65% with a specificity higher than 90%. Synthetic ADs from CLV are efficiently recognised by DCs with ability to activate CD4+ Th2 cells from AP. The combination of analogues from both ADs, significantly increased the sensitivity of DC maturation and T-cell proliferation compared to native CLV.

Mast cell activation tests by flow cytometry: A new diagnostic asset?

Since the late nineties, evidence has accumulated that flow-assisted basophil activation test (BAT) might be an accessible and reliable method to explore the mechanisms governing basophil degranulation and diagnostic allowing correct prediction of the clinical outcome following exposure to the offending allergen(s) and cross-reactive structures for different IgE-dependent allergies and particular forms of autoimmune urticaria. Although the BAT offers many advantages over mediator release tests, it is left with some weaknesses that hinder a wider application. It is preferable to perform the BAT analysis within 4h of collection, and the technique does not advance diagnosis in patients with non-responsive cells. Besides, the BAT is difficult to standardize mainly because of the difficulty to perform large batch analyses that might span over several days. This article reviews the status of flow cytometric mast cell activation test (MAT) using passively sensitized mast cells (MCs) with patients' sera or plasma (henceforth indicated as passive MAT; pMAT) using both MC lines and cultured MCs in the diagnosis of IgE-dependent allergies. In addition, this paper provides guidance for generating human MCs from peripheral blood CD34+ progenitor cells (PBCMCs) and correct interpretation of flow cytometric analyses of activated and/or degranulating cells. With the recent recognition of the mas-related G protein-coupled receptor X2 (MRGPRX2) occupation as a putative mechanism of immediate drug hypersensitivity reactions (IDHRs), we also speculate how direct activation of MCs (dMAT)-that is direct activation by MRGPRX2 agonists without prior passive sensitization-could advance paradigms for this novel endotype of IDHRs.

Basophil and mast cell activation tests by flow cytometry in immediate drug hypersensitivity: Diagnosis and beyond

Immediate drug hypersensitivity reactions (IDHRs) constitute a significant health issue with serious consequences of diagnostic error. The primary diagnostics to document IDHRs usually consists of quantification of drug-specific IgE (sIgE) antibodies and skin tests. Unfortunately, the positive predictive value (PPV) and negative predictive value (NPV) of these tests are not absolutely, which leaves room for new tests. Over the last two decades, the basophil activation test (BAT), in which ex vivo activation of individual basophils is quantified by flow cytometry, has emerged as a reliable complementary diagnostic to document IDHRs, to explore allergenic recognition, to study cross-reactivity and to monitor therapy. However, the BAT is technically challenging requiring specialized personnel and equipment, fresh samples and the technique is lost as a diagnostic in patients showing a non-responder status of their cells. By consequence, the BAT has still not entered mainstream application. In contrast, mast cell activation tests (MATs) use serum samples that can be frozen, stored, and shipped to a recognized reference centre experienced in mast cell (MC) lines and/or cultures and capable of offering batch testing with necessary quality controls. This review does not only highlight the use of the BAT and MAT as diagnostics in IDHRs, but also outlines the potential of both techniques in further exploring and unveiling the mechanisms that govern drug-induced basophil and MC activation and degranulation.

Basophil Activation Experiments in Immediate Drug Hypersensitivity: More Than a Diagnostic Aid.

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. 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. 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. 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. 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.

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.

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-assisted basophil activation tests in immediate drug hypersensitivity: two decades of Antwerp experience

The last two decades have witnessed that flow-assisted analysis of in vitro-activated basophils can constitute a valuable adjunct in the in vitro diagnostic approach of immediate drug hypersensitivity reactions (IDHR). This article summarises the current experience with the basophil activation test in the diagnosis of IDHR, with particular focus on allergy to curarising neuromuscular blocking agents, antibiotics (β-lactams and fluoroquinolones), iodinated radiocontrast media and opiates.