Astrid Uyttebroek

Predictive value of allergy tests for neuromuscular blocking agents: tackling an unmet need

Neuromuscular blocking agents (NMBAs) are a predominant cause of perioperative anaphylaxis in Europe. Diagnosis of NMBA allergy relies upon the careful review of the anaesthetic report complemented with skin tests. Additional diagnostic tests are quantification of specific IgE antibodies (sIgE) and basophil activation test (BAT). However, data on the predictive value of the skin tests, the BAT and the sIgE assays (drug‐specific and substituted ammonium structures) are limited or not available, mainly because such exploration requires dangerous NMBA provocation tests.

Basophil activation tests: time for a reconsideration

Challenges in in vitro allergy diagnostics lie in the development of accessible and reliable assays allowing identification of all offending allergens and cross-reactive structures. Flow-assisted analysis and quantification of in vitro activated basophils serves as a diagnostic instrument with increasing applications developed over the years. From the earliest days it was clear that the test could constitute a diagnostic asset in basophil-mediated hypersensitivity. However, utility of the basophil activation test should be reassessed regarding difficulties with preparation, characterization and validation of allergen extracts; availability and the potential of more accessible diagnostics. Today, the added value mainly lies in diagnosis of immediate drug hypersensitivity. Other potential indications are monitoring venom-immunotherapy and follow-up of natural history of food allergies. However, results in these nondiagnostic applications are preliminary. We review the most relevant clinical applications of the basophil activation test. Some personal comments and views about perspectives and challenges about flow-assisted allergy diagnosis are made.

Flowcytometric diagnosis of atracurium-induced anaphylaxis

Allergy to atracurium is a rare condition with serious consequences of diagnostic error. However, correct diagnosis is not always straightforward. The aim of this study is to assess the utility of the basophil activation test (BAT) in atracurium sensitization and to investigate its role in identifying cross‐reactivity between muscle relaxants.

Anaphylaxis to succinylated gelatin in a patient with a meat allergy: galactose-α(1, 3)-galactose (α-gal) as antigenic determinant.

Specific immunoglobulin E (sIgE) antibodies towards the galactose-α(1,3)-galactose (α-gal) moieties may elicit life-threatening and fatal anaphylactic reactions. Patients sensitized to α-gal moieties from mammalian meat may also react towards mammalian gelatins and gelatin-containing drugs such as bovine gelatin-based colloid plasma substitute. The case of a 56 year old woman with a meat allergy who suffered anaphylaxis to succinylated gelatin is reported.

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.

Moxifloxacin hypersensitivity: Uselessness of skin testing

Quinolones are synthetic antibiotics based on the 4-quinolone and 1,8-naphthyridine nuclei. They can cause severe hypersensitivity reactions, even after first exposure, with moxifloxacin being an important culprit. Unfortunately, a diagnosis of quinolone hypersensitivity is not straightforward, because of the absence of drug-specific sIgE immunoassays and uncertainties associated with skin testing with potentially nonspecific histamine releasers. These contradictory studies show that establishing nonirritating skin-test concentrations for ciprofloxacin is not straightforward. For example, Broz et al recommend an intradermal test (IDT) concentration of 1:300 to 1:1000 in saline for ciprofloxacin. In contrast, the Empedrad study failed to determine a nonirritating concentration for ciprofloxacin because the irritating concentration varied a 1000-fold in healthy volunteers. Accordingly, the European Network on Drug Allergy and European Academy of Allergy and Clinical ImmunologyDrugAllergy InterestGrouphasmadenodrugspecific recommendations about skin prick test (SPT) and/or IDT with quinolones. For moxifloxacin, the literature reveals “nonirritant” concentrations for SPT to vary between 1 and 20 mg/mL and for IDT between 0.004 and 0.05 mg/mL. However, inspection of the data discloses that 3/5 studies did not include controls and data gathered in patients were restricted to a maximum of 5 cases. This study assessed SPT and IDT in the diagnosis of immediate moxifloxacin hypersensitivity. A comparative study was performed between patients with histories of reactions and controls who tolerated a graded oral drug provocation test (DPT) with moxifloxacin. Patient inclusion was based on clinical history. Fourteen patients with a history of an immediate hypersensitivity reaction to moxifloxacin were enrolled. All patients suffered from urticaria/angioedema, bronchospasm/hypotension, and/or loss of consciousness within 60 minutes after intake. Other possible causes were excluded (ie, concomitant intake of other drugs). Time between reaction and investigation ranged between 0.3 and 21 months (median: 4 months) (Table I). Sixteen individuals who tolerated a graded oral DPT served as an exposed-control group. Skin testing consisted of SPTs and IDTs with a parenteral formulation ofmoxifloxacin hydrochloride (Avelox 400mg/250mL [or 1.6 mg/mL], Bayer SA-NV, Diegem, Belgium). SPT was performed using a 1:10 dilution and undilutedmoxifloxacin. Results were considered positive when wheal/flare equaled or exceeded 3/3 mm. For IDTs, serial dilutions of 1:1000, 1:100, and 1:10 were used. Results were considered positive when the wheal equaled or exceeded 5 mm. Patients with a history of betalactam hypersensitivity are more prone to develop hypersensitivity reactions to fluoroquinolones. Because the majority of subjects included as controls consulted with such a history, we preferred a graded DPT instead of a full dose provocation in search of a save alternative. All controls had a negative oral graded DPT with moxifloxacin (Avelox) up to a cumulative dose of 750 mg. Statistical analysis was performed with IBM SPSS Statistics 22 (IBM Corp, Armonk, New York). Fisher’s exact tests were used to evaluate significance differences of the skin-test results between patients and controls. SPT and IDT results are displayed in Figure 1. SPT was negative in all patients and controls. Intradermal testing yielded positivity in 10/14 patients. Two patients tested positive at the 1:1000 dilution (0.0016 mg/mL), 2 patients at the 1:100 dilution (0.016 mg/mL), and 6 patients at the 1:10 dilution (0.16 mg/ mL). Of the 14 patients, 4 displayed a negative SPT and IDT. In controls, IDT was also positive in 12 individuals. Of the 16 individuals, 2 tested positive at the 1:100 dilution. Of the 14 individuals who received the 1:10 dilution, 12 tested positive. The remaining 2 manifested a (true) negative result in both SPT and IDT. Sensitivity and specificity of the intradermal testing calculated between patients and controls in this population were 57% and 12.5%, respectively. Positive and negative predictive values were 36% and 25%, respectively. A comparison of IDT between patients and controls yieldedP-values of .192 for the 1:1000 dilution, 1.00 for the 1:100 dilution, and .209 for the 1:10 dilution. This is the first assessment of skin testing for moxifloxacin with a comparative study between patients and controls who tolerated a cumulative dose of 750 mg moxifloxacine during a graded oral DPT. We conclude that SPTs and IDTs with moxifloxacin hydrochloride are unreliable methods to establish a diagnosis of immediate moxifloxacin hypersensitivity. Our data are in line with previous findings that quinolones frequently induce skin-test responses in healthy subjects. It appears that at higher dilutions skin tests remain negative in a majority of patients, whereas higher concentrations are not discriminative between patients and exposed controls. There are studies that show that the time elapsed between the acute reaction and skin tests affects the outcome of the tests, which makes it difficult to interpret. We were unable to assess such an effect because the majority of patients received skin testing within 6 months after the acute reaction, and a longitudinal follow-up study does not seem to be warranted. A limitation of our study is the absence of controlled DPTs as previous research has shown that as few as 1/3 of patients with similar recent convincing reaction histories to quinolones reacted on being challenged with the culprit drug. However, because the time between the reactions and evaluation was relatively short

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

Flow cytometric analysis of drug‐Induced basophil histamine release

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.

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.

In Vitro Diagnosis of Immediate IgE-Mediated Drug Hypersensitivity: Warnings and (Unmet) Needs

Immediate drug hypersensitivity reactions (DHR) constitute an important health condition, with serious consequences of inadequate diagnosis. In this article, some of the most important issues related to in vitro diagnosis of IgE-mediated allergies are discussed. In vitro diagnostics will benefit from expanded and novel insights and understandings in drug chemical reactivity, protein binding, biotransformation, degradation, identification of (cross-reactive) drug antigenic determinants, and deeper understanding of sensitization routes. Collective efforts should be undertaken to activate fundamental and clinical investigations.