Mast cell activation test: A new asset in the investigation of the chlorhexidine cross-sensitization profile.

BACKGROUND: Insights into the IgE cross-sensitization and possible cross-reactivity patterns of sera reactive to chlorhexidine (CHX) are still incomplete and are likely to benefit from a functional exploration using a passive mast cell activation test (pMAT). Therefore, we want to study whether the pMAT with CHX-specific IgE (sIgE) enables to depict effector cell degranulation in response to alexidine (ALX), octenidine (OCT) and/or polyhexamethylene biguanide (PHMB) indicative of cross-reactivity between these compounds and CHX. METHODS: Serum of 10 CHX-allergic patients, nine individuals with an isolated sIgE CHX and five healthy controls were included. Human cultured mast cells (MCs) were, before and after sensitization, challenged with CHX, ALX, OCT or PHMB. Degranulation was measured via quantification of upregulation of CD63. RESULTS: Mast cell responsiveness to ALX and OCT was demonstrable with 4/10 and 3/10 of the sera of CHX-allergic patients respectively. Percentage of degranulation varied between 12 and 34% for ALX-reactive MCs and between 4 and 22% for OCT-reactive MCs. No reactivity to ALX or OCT was demonstrable when using sera obtained from individuals with an isolated sIgE CHX or from healthy controls. Unlike CHX, ALX and OCT, PHMB turned out to be a direct MC activator via occupation of MRGPRX2. PHMB-reactive sIgEs were demonstrable in some patients with an isolated sIgE CHX but were unable to trigger PHMB-induced degranulation in MRGPRX2 knockdown MCs. CONCLUSION: Mast cells constitute an attractive tool to explore cross-reactivity between structurally similar compounds. Along with the identification of safe alternatives for the individual patient, the pMAT can advance our insights into sIgE cross-reactivity patterns including assessment of molecules not yet approved for human use.

Principles, potential, and limitations of ex vivo basophil activation by flow cytometry in allergology: A narrative review.

The major challenge of allergy diagnosis lies in the development of accessible and reliable diagnostics allowing correct prediction of the clinical outcome following exposure to the offending allergen(s) and cross-reactive structures. Since the late nineties, evidence has accumulated that flow-assisted analysis and quantification of ex vivo-activated basophils (according to the basophil activation test [BAT]) might meet this requirement for different IgE-dependent allergies and particular forms of autoimmune urticaria. Other so-called nondiagnostic applications of the BAT involve therapeutic monitoring, follow-up of natural histories, and identification of allergenic recognition sites. However, it has also become clear that appropriate use of the BAT necessitates knowledge about degranulation metrics and guidance to guarantee correct execution and interpretation of the results. Here, we have reviewed the most relevant applications and limitations of the BAT. Some personal statements and views about its perspectives are made.

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 4 h 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.

Performance of basophil activation test and specific IgG4 as diagnostic tools in nonspecific lipid transfer protein allergy: Antwerp-Barcelona comparison.

BACKGROUND: Recent studies show that nsLTP sensitization is not limited to the Mediterranean basin and can present diverse clinical phenotypes. It remains challenging to predict clinical outcome when specific IgE antibodies (sIgE) to nsLTPs are present. This study compares both clinical and in vitro allergy characteristics but also diagnostic performance of a basophil activation test (BAT) and sIgG4 in nsLTP-sensitized patients from Antwerp (ANT, Belgium) and Barcelona (BCN, Spain). METHODS: Adult subjects with positive sIgE rPru p 3 and/or rMal d 3 ≥ 0.10 kUA /L (n = 182) and healthy controls (n = 37) were included. NsLTP-sensitized individuals were stratified according to clinical symptoms with peach/apple, respectively. BAT rPru p 3 and rMal d 3 were performed and sIgG4 antibodies to both components quantified. RESULTS: In BCN, only ratios of sIgG4/sIgE rMal d 3 and BAT rMal d 3 (0.001 µg/mL) can identify clinically relevant Mal d 3 sensitization (sensitivity of 60%-63% and a specificity of 75%-67%, respectively). In ANT, only the sIgE/total IgE rPru p 3 ratio shows added value (sensitivity 60% and specificity 83%). Finally, it appears that symptomatic patients in BCN are more sensitive to lower allergen concentrations compared to ANT. In addition, it was shown that ANT patients were more often sensitized to pollen and that specific pollen sources differed between regions. CONCLUSIONS: NsLTP-related allergy profiles and diagnostic performance differ significantly between regions and are component-specific, which makes extrapolation of data difficult to do. In addition, it seems that basophil sensitivity might show geographical differences. Additional research is needed to confirm these findings.

Mast cell activation test in chlorhexidine allergy: a proof of concept.

BACKGROUND: Immediate drug hypersensitivity reactions are an increasing public health issue and a frequent cause of life-threatening anaphylaxis. Conventional confirmatory testing include skin tests and, for a few drugs, quantification of drug-specific immunoglobulin E (IgE) antibodies. However, none of these tests are absolutely predictive for the clinical outcome, and can yield false-negative and false-positive results. We performed a proof-of-concept study to assess whether a mast cell activation test could improve diagnosis of IgE-mediated chlorhexidine hypersensitivity, a common cause of perioperative anaphylaxis. METHODS: Human mast cells were generated from CD34+ progenitor cells and sensitised with patients' sera to become IgE+ human mast cells (dMCIgE+), and then incubated with chlorhexidine to assess degranulation. We compared the diagnostic performance of this mast cell activation test with serum from patients with and without positive skin test and basophil activation test to chlorhexidine. RESULTS: In dMC sensitised with sera from patients with a positive skin test and basophil activation test to chlorhexidine showed drug-specific and concentration-dependent degranulation upon stimulation with chlorhexidine, determined by surface upregulation of the degranulation marker CD63. In contrast, dMC sensitised with sera from patients with a negative skin test and basophil activation test to chlorhexidine were unresponsive in the mast cell activation test. CONCLUSIONS: Our study suggests that the mast cell activation test can be used to diagnose IgE/FcεRI-dependent immediate drug hypersensitivity reactions. It also shows potential to assess the clinical relevance of drug-specific IgE antibodies in their ability to elicit mast cell degranulation, and therefore discriminate between allergy and sensitisation. Extended studies are required to verify whether this technique can be used in other causes of perioperative anaphylaxis.

In vitro Diagnosis of Immediate Drug Hypersensitivity: Should We Go with the Flow.

BACKGROUND: Diagnosis of immediate drug hypersensitivity reactions (IDHRs) is based upon history taking, skin prick or intradermal tests and quantification of specific immunoglobulin E (IgE) antibodies. Unfortunately, this is often insufficient to correctly identify patients with IgE-mediated IDHRs and is impossible in the case of non-IgE-mediated IDHRs. Drug provocation tests (DPT) are considered the 'gold standard' diagnostic but are not always possible, for ethical and practical reasons. Therefore, the validation of new cellular tests such as basophil activation testing (BAT) was necessary. This review focuses on the applications of BAT in IDHRs. METHODS: A literature search was conducted, using the words basophil, flow cytometry, immediate drug allergy and drugs; this was complemented by the authors' own expertise. RESULTS: BAT/HistaFlow® is a useful diagnostic tool in IDHRs, mainly used to diagnose allergy to neuromuscular blocking agents (NMBAs), antibiotics, nonsteroidal anti-inflammatory drugs (NSAIDs) and iodinated radiocontrast media. Its sensitivity varies between 50 and 60%, and specificity attains 80%, except for with quinolones and NSAIDs. CONCLUSIONS: The diagnostic utility of BAT (and to lesser extent HistaFlow) has been demonstrated and is mostly applied in IDHRs. However, larger-scale collaborative studies are necessary to optimize test protocols and validate the entry of BAT as a diagnostic instrument in drug allergy.

Determinants of baseline lung function and bronchodilator response in 4-year-old children.

The prolonged period required for maturation of the respiratory system makes it vulnerable to environmental exposure. This study aimed to investigate the association between early-life factors and lung function in preschool children. Children aged 4 years, who were included in a prospective birth cohort, underwent lung function testing at baseline (n=535) and after bronchodilation (n=498) by forced oscillations. Information on symptoms and exposures was collected through half-yearly questionnaires. Allergen-specific serum IgE was quantified at 1 and 4 years. Multiple linear regression analysis showed that the baseline respiratory resistance and reactance area were larger in the children with previous wheeze, those with early-onset sensitisation to inhalant allergens and those who were smaller. Furthermore, children with previous lower respiratory tract infections exhibited higher baseline resistance values. The baseline resistance was the only independent determinant of the bronchodilator-induced change in resistance, whereas current height and baseline reactance area were independently associated with the change in reactance area. In conclusion, previous lower respiratory tract infections, the timing of previous wheeze, inhalant sensitisation and current height independently influence the baseline lung function of 4-year-old children, whereas baseline lung function is the principal determinant of the bronchodilator response.

Cor a 14: missing link in the molecular diagnosis of hazelnut allergy?

BACKGROUND: Hazelnut allergy shows distinct clinical patterns that can be predicted through component-resolved diagnosis. However, identification of sensitization profiles remains incomplete. METHODS: Sera of 75 patients allergic to hazelnuts, 14 infants with atopic dermatitis (AD) sensitized to hazelnuts, 15 hazelnut-tolerant individuals with specific IgE (sIgE) to hazelnuts and 15 healthy control individuals were tested for sIgE reactivity to rCor a 1.04, rCor a 8, nCor a 9, nCor a 11, rCor a 14, rBet v 1, rBet v 2 and cross-reactive carbohydrate determinants (CCDs). RESULTS: Sensitization to Cor a 14 was observed in 18 out of 20 preschool children, 8 out of 10 school-aged children and 2 out of 7 adults with generalized reactions and in 3 out of 14 infants with AD. Only 2 out of 38 patients with an oral allergy syndrome (OAS) were sensitized to Cor a 14. No sensitization to Cor a 14 was observed in the group of hazelnut-tolerant and healthy control individuals. Sensitization to Cor a 1.04 was seen in 36 out of 38 OAS patients and in 14 out of 37 patients with generalized reactions. However, only 3 patients with generalized reactions were monosensitized to Cor a 1.04. Sensitization to Cor a 9 was observed in 26 out of 37 patients with generalized reactions and in 4 out of 14 infants with AD. Sensitization to Cor a 11, Cor a 8, rBet v 2 and CCDs was rare. CONCLUSIONS: Sensitization to Cor a 14 can have early onset and shows age-related variations. Together with Cor a 9, Cor a 14 enables us to correctly identify almost 90% of children with generalized reactions to hazelnut.

Expressions and inhibitory functions of CD300a receptors on purified human basophils.

The inhibitory myeloid immunoglobulin receptor CD300a (IRp60) has been shown to downregulate mast cell and eosinophil activities, thereby serving as a potential target for inhibiting allergic effector cell input in allergy. Our aims were to study the expression and functional properties of this receptor in purified human basophils, cells that crucially contribute to Th2-type immunity and allergy. Basophils homogeneously expressed CD300a as well as the inhibitory receptor CD200R on their cell surface, and these expressions increased after anti-IgE stimulation. IgE-mediated basophil degranulation was also significantly inhibited by crosslinking of either CD200R or CD300a (by 90% and 50%, respectively). Inhibitory SHIP-1 phosphorylations were also induced by CD200R and CD300a, although they were not noticeably increased by IgE-dependent activation. We conclude that both CD200R and CD300a play a role in reducing IgE-mediated basophil function and may crucially govern the known differential activities of these cells in vivo.

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

Background: 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. Objective: To explore the expression and functionality of MRGPRX2 in and on basophils. Methods: 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. Results: 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. Conclusion: 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.

Overexpression of FcεRI on Bone Marrow Mast Cells, but Not MRGPRX2, in Clonal Mast Cell Disorders With Wasp Venom Anaphylaxis.

Background: Uncertainties remain about the molecular mechanisms governing clonal mast cell disorders (CMCD) and anaphylaxis. Objective: This study aims at comparing the burden, phenotype and behavior of mast cells (MCs) and basophils in patients with CMCD with wasp venom anaphylaxis (CMCD/WVA+), CMCD patients without anaphylaxis (CMCD/ANA-), patients with an elevated baseline serum tryptase (EBST), patients with wasp venom anaphylaxis without CMCD (WVA+) and patients with a non-mast cell haematological pathology (NMHP). Methods: This study included 20 patients with CMCD/WVA+, 24 with CMCD/ANA-, 19 with WVA+, 6 with EBST and 5 with NMHP. We immunophenotyped MCs and basophils and compared baseline serum tryptase (bST) and both total and venom specific IgE in the different groups. For basophil studies, 13 healthy controls were also included. Results: Higher levels of bST were found in CMCD patients with wasp venom anaphylaxis, CMCD patients without anaphylaxis and EBST patients. Total IgE levels were highest in patients with wasp venom anaphylaxis with and without CMCD. Bone marrow MCs of patients with CMCD showed lower CD117 expression and higher expression of CD45, CD203c, CD63, CD300a and FcεRI. Within the CMCD population, patients with wasp venom anaphylaxis showed a higher expression of FcεRI as compared to patients without anaphylaxis. Expression of MRGPRX2 on MCs did not differ between the study populations. Basophils are phenotypically and functionally comparable between the different patient populations. Conclusion: Patients with CMCD show an elevated burden of aberrant activated MCs with a significant overexpression of FcεRI in patients with a wasp venom anaphylaxis.

Young infants with atopic dermatitis can display sensitization to Cor a 9, an 11S legumin-like seed-storage protein from hazelnut (Corylus avellana).

Allergy to hazelnut (Corylus avellana) can be severe and occur at young age. Atopic dermatitis (AD) can involve sensitization to various foods. The objective is to investigate the pattern of hazelnut sensitization in infants with AD. Sera of 34 infants all under 1 year of age and suffering from AD were selected according to prior specific IgE results. Twenty-nine infants were sensitized to traditional food allergens, five were not. From the 29 infants with a sensitization to at least one food allergen, 20 demonstrated IgE reactivity to hazelnut. All sera were analyzed with the allergen microarray immunoassay (ImmunoCAP ISAC). Twelve (60%) of the children with IgE reactivity to hazelnut demonstrated sensitization to Cor a 9, the 11S legumin-like seed-storage protein from hazelnut. In these infants, no sensitization to Cor a 1, the homologue of the major birch pollen allergen Bet v 1 (Betula verrucosa), or the lipid transfer protein (Cor a 8) from hazelnut was demonstrable. Half of the children sensitized to Cor a 9 demonstrated IgE reactivity to its homologue in peanut (Arachis hypogaea; Ara h 3) from which five were also sensitized to Gly m 6 from soy (Glycine max). None of the infants with AD without IgE reactivity to hazelnut demonstrated sensitization to Cor a 1, 8, or 9. In conclusion, young infants with atopic dermatitis sensitized to hazelnut can already display IgE reactivity to Cor a 9, a potentially dangerous hazelnut component. The mechanism(s) of this early sensitization and its clinical significance remain elusive.

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.

Diagnosis of Primary Mast Cell Disorders in Anaphylaxis: Value of KIT D816V in Peripheral Blood.

BACKGROUND: Anaphylaxis is frequent in patients suffering from primary mast cell disorders (PMCDs). In patients without mastocytosis in the skin (MIS) and a baseline serum tryptase (bST) less than 30 ng/mL, the diagnosis of PMCD is challenging. In these patients, detection of the KIT D816V mutation in peripheral blood (PB) has been suggested as screening tool for a PMCD. OBJECTIVE: In this study, we investigated whether KIT D816V in PB can contribute to the decision to perform a bone marrow (BM) biopsy in patients with anaphylaxis without MIS and a bST less than 30 ng/mL. METHODS: We selected 74 patients with severe anaphylaxis without MIS and a bST less than 30 ng/mL. All underwent a BM biopsy. KIT D816V mutation was quantified in both PB and BM using digital droplet polymerase chain reaction (ddPCR). RESULTS: Diagnosis of a PMCD was established in 40 patients (54%). Median bST for patients with and without PMCD was, respectively, 9.5 ng/mL (range 4.2-27 ng/mL) and 4.9 ng/mL (range 2.2-20.3 ng/mL) (P <.001). KIT D816V in PB was detected in 16 out of 40 (40%) patients with PMCD. KIT D816V in BM was detected in 22 out of 40 (55%) patients with PMCD. CONCLUSIONS: In patients without MIS and a bST less than < 30 ng/mL who experience anaphylaxis, determination of KIT D816V mutation in PB is of limited help in deciding when to proceed to a BM biopsy. Therefore, KIT D816V in PB mutation analysis should be interpreted together with scoring tools to make a better assessment in identifying patients who should undergo BM biopsy.

Peripheral blood cultured mast cells: Phenotypic and functional outcomes of different culture protocols.

BACKGROUND: Mast cells (MCs) play a pivotal role in innate and adaptive immune responses. However, MCs are also involved in different pathologic conditions. Studies on the mechanisms that govern human MC functions are impeded by their limited and difficult recovery. Therefore, several research groups have developed protocols to culture human MCs from progenitor cells. These protocols vary with respect to culture duration and used maturation cytokines. How MCs obtained by different protocols differ in phenotype and functionality is currently unknown. OBJECTIVE: To compare different protocols for the generation of human MCs from peripheral blood progenitors. METHODS: Thirteen paired human MC cultures were investigated. MCs were cultured form CD34+ progenitors cells for 4 or 8 weeks and with or without the addition of IL-6. Phenotyping comprised staining for CD117, CD203c, FcεRI, MRGPRX2, CD300a and CD32. Functional studies included measurements of the up-regulation of CD63 and CD203c after allergen-specific cross-linking of sIgE/FcεRI complexes or ligation of MRGPRX2 with substance P and different drugs. RESULTS: Cell cultures for 4 weeks in the presence of IL-6 consistently yielded the highest numbers of MCs. MCs cultured for 8 weeks with IL-6 showed more autofluorescence significantly impeding correct analyses of FcεRI and CD32. The density of FcεRI and CD32 was comparable between the different culture conditions. MRGPRX2 expression was significantly higher in the 8 week cultures. The density of CD300a was increased in the cultures with IL-6. Cells cultured for 8 weeks were more responsive to MRGPRX2 activation. In contrast, the 4-weeks cultures with IL-6 showed significantly higher allergen-specific activation. CONCLUSION: Four weeks of culture with IL-6 are sufficient to generate sizeable numbers of human mast cells from blood progenitors, thereby enabling simultaneous exploration of allergen-specific sIgE/FcεRI cross-linking and non-specific activation via MRGPRX2.