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.

Bariatric Surgery: A Novel Risk Factor for Food Allergy?

In this case report, we describe a birch pollen-allergic patient in whom a Fobi pouch gastric bypass was associated with the transition from a mild, localized, birch pollen-related oral allergy syndrome to more severe, generalized allergic reactions to peach.

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.

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.

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.

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.

Novel Insights on MRGPRX2-Mediated Hypersensitivity to Neuromuscular Blocking Agents And Fluoroquinolones.

Neuromuscular blocking agents (NMBAs) like atracurium and rocuronium as well as fluoroquinolones (FQs) cause mast cell-mediated anaphylaxis by activating Mas-related G protein-coupled receptor X2 (MRGPRX2), but many questions remain unanswered. Here, we address three of them, namely whether primary human mast cells show similar activation by these drugs as murine mast cells and mast cell lines, how sugammadex protects from atracurium-induced MRGPRX2-mediated mast cell activation, and why some but not all patients treated with rocuronium develop anaphylaxis. We used peripheral blood-derived cultured mast cells from healthy donors and patients, assessed mast cell activation and degranulation by quantifying intracellular calcium and CD63 expression, respectively, and made use of MRGPRX2-silencing, via electroporation with Dicer-substrate small interfering RNAs, and single cell flow cytometric analyses. Atracurium, ciprofloxacin, and levofloxacin activated and degranulated primary human mast cells, but only MRGPRX2-positive and not MRGPRX2-negative or -silenced mast cells. Sugammadex attenuated the atracurium-induced and MRGPRX2-mediated activation and degranulation of human mast cells by reducing free atracurium levels. The mast cells of patients with IgE-independent anaphylaxis to rocuronium were similar, in their MRGPRX2 expression and function, to those of patients with IgE-mediated anaphylaxis. These findings further improve our understanding of the role and relevance of MRGPRX2-driven mast cell activation in anaphylactic reactions to NMBAs and FQs and may help to improve their prediction, prevention, and treatment.

Mast Cell Activation During Suspected Perioperative Hypersensitivity: A Need for Paired Samples Analysis.

BACKGROUND: Perioperative hypersensitivity (POH) reactions constitute a significant clinical and diagnostic challenge. A transient increase in serum tryptase during POH reflects mast cell activation (MCA) and helps to recognize an underlying hypersensitivity mechanism. OBJECTIVE: To determine the diagnostic performance of different tryptase decision thresholds based on single and paired measurements to document MCA in suspected POH. METHODS: Acute serum tryptase (aST) and baseline serum tryptase (bST) samples were obtained from patients referred to our outpatients clinic because of clinical POH. Tryptase samples from controls were obtained before induction (Tt0) and 1.5 hours after induction (Tt1) in uneventful anesthesia. Different cutoff points for tryptase increase over bST and the percentage increase in tryptase (%T) were calculated and compared with existing thresholds: aST > [1.2 × (bST) + 2] (consensus formula), aST higher than 11.4 ng/mL, and aST higher than 14 ng/mL. RESULTS: Patients with POH had higher bST and aST levels compared with controls (respectively 5.15 vs 2.28 ng/mL for bST and 20.30 vs 1.92 ng/mL for aST). The consensus formula and a tryptase increase over bST of greater than or equal to 3.2 ng/mL held the highest accuracies to document MCA in POH (respectively 81% and 82%). A bST of higher than 8 ng/mL was present in 4% of controls, 5% of patients with grade 1 POH, 24% of patients with grade 2 POH, 15% of patients with grade 3 POH, and 17% of patients with grade 4 POH. CONCLUSIONS: Our data endorse the consensus formula for detection of MCA in POH. Furthermore, it shows that a bST of higher than 8 ng/mL was associated with occurrence of anaphylaxis.

Culturing cells with mast cell phenotype and function: Comparison of peripheral blood and bone marrow as a source.

BACKGROUND: Studies on the mechanisms that govern mast cell (MC) functions are hindered by the difficulties in isolating sufficient numbers of these tissue-resident cells. Therefore, many research groups use cultured human MCs obtained out of progenitor cells. However, these culture methods significantly differ regarding primary source material, culture durations and conditions. Consequently, the finally obtained cells are likely to exhibit morphological, phenotypical and/or functional heterogeneity. OBJECTIVE: To compare the phenotype and functionality of cells cultured from peripheral blood and bone marrow progenitor cells from patients with suspected clonal MC disease. These cells are designated as PBCMCs and BMCMCs, respectively. METHODS: Twenty paired PBCMCs and BMCMCs cultures starting from CD34+ progenitor cells were compared. Cells were cultured for 4 weeks. Phenotyping included Giemsa and CD117 staining and flow cytometric staining for CD117, CD203c, FcεRI, MRGPRX2, CD300a, CD32, CD63 and CD25. Functional assessment included measurement of the up-regulation of CD63 after cross-linking of the high affinity receptor for IgE (FcεRI) with anti-FcεRI and ligation of MRGPRX2 with substance P. RESULTS: PBCMCs and BMCMCs are phenotypically comparable. Functionally, after activation with anti-FcεRI and substance P, PBCMCs and BMCMCs show similar up-regulation of the lysosomal degranulation marker CD63. However, the yield of PBCMCs is higher than BMCMs and peripheral blood cultures are purer than bone marrow cultures. CONCLUSION: PBCMCs are an attractive alternative to the more difficult to obtain BMCMCs for the exploration of the complex mechanisms that govern IgE- and MRGPRX2-dependent MC activation and degranulation. Unlike BMCMCs, PBCMCs are easily accessible and enable repetitive analyses.

Non-specific lipid-transfer proteins: Allergen structure and function, cross-reactivity, sensitization, and epidemiology.

BACKGROUND: Discovered and described 40 years ago, non-specific lipid transfer proteins (nsLTP) are present in many plant species and play an important role protecting plants from stressors such as heat or drought. In the last 20 years, sensitization to nsLTP and consequent reactions to plant foods has become an increasing concern. AIM: The aim of this paper is to review the evidence for the structure and function of nsLTP allergens, and cross-reactivity, sensitization, and epidemiology of nsLTP allergy. MATERIALS AND METHODS: A Task Force, supported by the European Academy of Allergy & Clinical Immunology (EAACI), reviewed current evidence and provide a signpost for future research. The search terms for this paper were "Non-specific Lipid Transfer Proteins", "LTP syndrome", "Pru p 3", "plant food allergy", "pollen-food syndrome". RESULTS: Most nsLTP allergens have a highly conserved structure stabilised by 4-disulphide bridges. Studies on the peach nsLTP, Pru p 3, demonstrate that nsLTPs are very cross-reactive, with the four major IgE epitopes of Pru p 3 being shared by nsLTP from other botanically related fruits. These nsLTP allergens are to varying degrees resistant to heat and digestion, and sensitization may occur through the oral, inhaled or cutaneous routes. In some populations, Pru p 3 is the primary and sole sensitizing allergen, but many are poly-sensitised both to botanically un-related nsLTP in foods, and non-food sources of nsLTP such as Cannabis sativa, Platanus acerifolia, (plane tree), Ambrosia artemisiifolia (ragweed) and Artemisia vulgaris (mugwort). Initially, nsLTP sensitization appeared to be limited to Mediterranean countries, however more recent studies suggest clinically relevant sensitization occurs in North Atlantic regions and also countries in Northern Europe, with nsLTP sensitisation profiles being broadly similar. DISCUSSION: These robust allergens have the potential to sensitize and provoke symptoms to a large number of plant foods, including those which are raw, cooked or processed. It is unknown why some sensitized individuals develop clinical symptoms to foods whereas others do not, or indeed what other allergens besides Pru p 3 may be primary sensitising allergens. It is clear that these allergens are also relevant in non-Mediterranean populations and there needs to be more recognition of this. CONCLUSION: Non-specific LTP allergens, present in a wide variety of plant foods and pollens, are structurally robust and so may be present in both raw and cooked foods. More studies are needed to understand routes of sensitization and the world-wide prevalence of clinical symptoms associated with sensitization to these complex allergens.