Ine I. Decuyper

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.

Shellfish allergens: tropomyosin and beyond

IgE‐mediated shellfish allergy constitutes an important cause of food‐related adverse reactions. Shellfish are classified into mollusks and crustaceans, the latter belonging to the class of arthropoda. Among crustaceans, shrimps are the most predominant cause of allergic reactions and thus more extensively studied. Several major and minor allergens have been identified and cloned. Among them, invertebrate tropomyosin, arginine kinase, myosin light chain, sarcoplasmic calcium‐binding protein, and hemocyanin are the most relevant. This review summarizes our current knowledge about these allergens.

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.

Cannabis Allergy: What do We Know Anno 2015

For about a decade, IgE-mediated cannabis (marihuana) allergy seems to be on the rise. Both active and passive exposure to cannabis allergens may lead to a cannabis sensitization and/or allergy. The clinical manifestations of a cannabis allergy can vary from mild to life-threatening reactions, often depending on the route of exposure. In addition, sensitization to cannabis allergens can trigger various secondary cross-allergies, mostly for plant-derived food. This clinical entity, which we have designated as the “cannabis-fruit/vegetable syndrome” might also imply cross-reactivity with tobacco, latex and plant-food derived alcoholic beverages. These secondary cross-allergies are mainly described in Europe and appear to result from cross-reactivity between non-specific lipid transfer proteins or thaumatin-like proteins present in Cannabis sativa and their homologues that are ubiquitously distributed throughout plant kingdom. At present, diagnosis of cannabis-related allergies rests upon a thorough history completed with skin testing using native extracts from 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 including a stop of any further cannabis (ab)use.

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

Basophil Activation Test in IgE-Mediated Food Allergy: Should We Follow the Flow?

Opinion statementIgE-mediated food allergy constitutes an important and increasing health issue with significant impairment of quality of life and significant morbidity and mortality. It affects children, as well as adolescents, and adults. Correct diagnosis of food allergy relies upon history supplemented by quantification of specific IgE (sIgE) antibodies and/or skin tests. Unfortunately, as these tests do not demonstrate absolute predictive values, controlled oral provocation tests might be needed to confirm/exclude diagnosis. However, it is unlikely oral challenges to enter mainstream application, mainly because of obvious ethical reasons. Therefore, correct diagnosis of food allergy might benefit from novel in vitro diagnostics such as allergen component-based sIgE assays and flow cytometric quantification of in vitro activated basophils. As a matter of fact, these tests might prove to be particularly helpful in discriminating genuine allergy from merely sensitization. Furthermore, they might be useful in establishing individual risk profiles, predicting persistence of allergy, and facilitating therapeutic approach. This review focuses on the applications and limitations of the basophil activation test in IgE-mediated food allergy. Anno 2016 we believe that the utility and usefulness of basophil-activation experiments need to be reevaluated thoroughly, in view of difficulties inherent to the correct preparation and storage of allergen extracts, optimizing and standardizing stimulation conditions, and also the potential of alternative diagnostics such as component resolved diagnosis that are becoming more readily accessible.

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.

Exploring the diagnosis and profile of cannabis allergy

BACKGROUND Cannabis allergy (CA) has mainly been attributed to Can s 3, the nonspecific lipid transfer protein (nsLTP) of Cannabis sativa. Nevertheless, standardized diagnostic tests are lacking and research on CA is scarce. OBJECTIVE To explore the performance of 5 cannabis diagnostic tests and the phenotypic profile of CA. METHODS A total of 120 patients with CA were included and stratified according to the nature of their cannabis-related symptoms; 62 healthy and 189 atopic controls were included. Specific IgE (sIgE) hemp, sIgE and basophil activation test (BAT) with a recombinant Can s 3 protein from Cannabis sativa (rCan s 3), BAT with a crude cannabis extract, and a skin prick test (SPT) with an nCan s 3-rich cannabis extract were performed. Clinical information was based on patient history and a standardized questionnaire. RESULTS First, up to 72% of CA reporting likely-anaphylaxis (CA-A) are Can s 3 sensitized. Actually, the Can s 3-based diagnostic tests show the best combination of positive and negative predictive values, 80% and 60%, respectively. sIgE hemp displays 82% sensitivity but only 32% specificity. Secondly, Can s 3+CA reported significantly more cofactor-mediated reactions and displayed significantly more sensitizations to other nsLTPs than Can s 3-CA. Finally, the highest prevalence of systemic reactions to plant-derived foods was seen in CA-A, namely 72%. CONCLUSIONS The most effective and practical tests to confirm CA are the SPT with an nCan s 3-rich extract and the sIgE rCan s 3. Can s 3 sensitization entails a risk of systemic reactions to plant-derived foods and cofactor-mediated reactions. However, as Can s 3 sensitization is not absolute, other cannabis allergens probably play a role.

Letter to the Authors Concerning the Published Manuscript by Rial and Sastre: Food Allergies Caused by Allergenic Lipid Transfer Proteins: What Is Behind the Geographic Restriction?

To the Editor, We read with great interest the manuscript by M. J. Rial and J. Sastre [1] about the geographical distribution of sensitization to non-specific lipid transfer proteins (ns-LTPs). In present day, this is an interesting topic, and therefore we would like to raise some issues and questions, especially concerning the prevalence of ns-LTP sensitization outside the Mediterranean area. The authors report that sensitization to ns-LTPs is infrequent in Central Europe and other non-Mediterranean regions; however, it appears that the authors have overlooked a large Belgian survey performed in 718 patients [2]. As a matter of fact, we demonstrated that the prevalence of sIgE reactivity towards nsLTP(s) is demonstrable in about one-quarter of Belgian patients presenting with symptoms of a pollen and/or plant food allergy. In this survey, all patients were systematically screened for nsLTP sensitization using a panel of six different ns-LTPs; four food ns-LTPs respectively rPru p 3 of peach (Prunus persica), rMal d 3 of apple (Malus domestica), rCor a 8 of hazelnut (Corylus avellana), and rAra h 9 of peanut (Arachis hypogaea) and two weed pollen ns-LTPs specifically nArt v 3 of mugwort (Artemisia vulgaris) and rPar j 2 of wall pellitory (Parietaria judaica). To the best of our knowledge, this study is the largest prevalence study focusing on ns-LTP sensitization in northwestern Europe. Moreover, this study also demonstrated that in a northwestern European country, patients with ns-LTP sensitization can exhibit distinct phenotypes that are not readily predictable by the sIgE results. Although, similar to initial observations in the Mediterranean basin [3–5], some of our patients demonstrated systemic reactions, the majority of patients with sIgE reactivity towards ns-LTPs did not report any clinical reaction to the respective plant food(s). A possible explanation for the absence of overt allergy could be the high prevalence of sensitization to the major allergen of birch pollen, Bet v 1 (Betula verrucosa) [6–8]. However, for the time being, this explanation is highly speculative, but relies on observations from the Mediterranean basin on sensitization to Bet v 1 (PR10 molecule) to protect for ns-LTP-related allergies. In other words, patients co-sensitized to Bet v 1 and Bet v 1 homologues report milder clinical symptoms compared to patients without co-sensitization to PR10 molecules. Clearly, more studies are needed to fully elucidate the protective effect of PR10 molecules. The exact reason(s) for the high prevalence of ns-LTP sensitization in our country remain(s) elusive. Although we cannot exclude our findings (in part) to reflect our methodology (usage of multiple sensitive single-plexed assays), we believe that in most patients, ns-LTP sensitization is genuine and might result from various sensitization routes that extend beyond pollen and plant-derived foods. Actually, we observed that Can s 3, the ns-LTP from Cannabis sativa, is a major allergen in cannabis allergy in our regions [9]. Moreover, it appears that sensitization to Can s 3 can result from both active and passive exposure to marijuana smoke [10] and that the Can s 3 cross-reactivity syndrome extends beyond fruits and vegetables but can also involve beverages and latex [11]. In conclusion, sensitization towards ns-LTP, although historically predominantly described in the Mediterranean basin, is not uncommon in north-western Europe and can result in clinically distinct phenotypes. Further collaborative studies are required to obtain insight into sensitization routes, clinical * Margaretha A. Faber margaretha.faber@uantwerpen.be

Cannabis allergy: A diagnostic challenge

M. Saito K. Yamamoto-Hanada K. Pak T. Ayabe H. Mezawa K. Ishitsuka M. Konishi L. Yang K. Matsumoto H. Saito Y. Ohya The Japan Environment and Children’s Study (JECS) Group Medical Support Center for the Japan Environment and Children’s Study, National Center for Child Health and Development, Tokyo, Japan Department of Clinical Medicine (Biostatistics), Kitasato University School of Pharmacy, Tokyo, Japan Email: yamamoto-k@ncchd.go.jp