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.

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.

Reliability of Early and Late Testing for Suspected Perioperative Hypersensitivity.

BACKGROUND: The optimal timing of diagnostic testing for perioperative hypersensitivity (POH) remains unknown. It has been recommended that investigation is best carried out at least 4 to 6 weeks after the event. On the other hand, guidelines discourage the use of in vitro tests later than 3 years after the index reaction. OBJECTIVE: This retrospective study aimed to assess the reliability of early and late skin tests (STs). It also attempted to verify whether discouraging late ex vivo and in vitro tests is substantiated. METHODS: For the first aim, patients were stratified over three epochs: an early timing group, with investigations performed within 6 weeks; a recommended timing group, with tests performed between 6 weeks and 6 months; and a late timing group, tested later than 6 months after the event. For the second study purpose, we studied the reliability of specific IgE quantification and basophil activation test rocuronium within 6 weeks and after 3 years in patients who experienced an ST-proven POH to rocuronium. RESULTS: A total of 677 patients were included. Based on a positive ST result, a causative agent was found in 74.2% of the early timing group, 62.6% of the recommended timing group, and 50% of the late timing group. A positive specific IgE for rocuronium or morphine was found in 80% of patients tested within 6 weeks, 63% of patients tested between 6 weeks and 3 years, and 50% of patients tested more than 3 years after the event. A positive basophil activation test was found in 83.3%, 51%, and 20%, respectively, of patients. CONCLUSIONS: Our data confirm that evaluation of drug allergy for suspected POH can be performed before 6 weeks after the event, and there is no maximal upper time limit disclosing ex vivo and in vitro testing.

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.

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.

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.

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.

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.

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

BACKGROUND: 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. AIM: 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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.

Serum specific IgE antibodies in immediate drug hypersensitivity.

Immediate drug hypersensitivity reactions (IDHRs) constitute a significant health problem that may be compounded by consequences of diagnostic error. In daily practice, IDHR diagnostic work up starts with drug-specific skin testing and/or quantification of specific IgE (sigE) antibodies in serum. Here we critically review the performance, i.e. potential and limitations of sIgE to ß-lactam antibiotics (ß-LABs), curarizing neuromuscular blocking agents (NMBAs), opiates and (semi)synthetic opioids, fluoroquinolones (FQs), poppy seed (papaver somniferum), chlorhexidine and finally Hevea latex. Quintessence of these studies is clear. Quantification of these drug-specific sIgE should not be used in isolation to document or exclude diagnosis of an IDHR. False negative results entail the risk for subsequent potentially life-threatening anaphylaxis upon re-exposure. False positive results lead to erroneous avoidance and unnecessary substitutions and fails to identify the true etiology.

Cannabis allergy: what the clinician needs to know in 2019.

INTRODUCTION: Although the use of cannabis dates back millennia, the first description of cannabis allergy is relatively recent (1971). Recent large-scale data show that cannabis allergy can manifest severe and generalized symptoms with extensive cross-reactions. Thus, it is essential to become familiarized with its clinical presentation, diagnostic aids, and adequate therapeutic guidance. Areas covered: Here we provide a hands-on overview on cannabis allergy focusing on symptomatology and the reliability of diagnostic options. Recent advances in proteomics are discussed in detail, elucidating the link with nsLTP-related allergies. The proteomics advancements have paved the way for more reliable diagnostics, especially component-based tools. Finally, the current experience in treatment options is highlighted. Expert opinion: Cannabis allergy is an allergy entity which can significantly impact the quality of life. For optimal diagnosis, we advise to start with a validated and standardized crude-extract based test such as sIgE hemp complemented by component-based diagnostics such as sIgE Can s 3 quantifications where available. Future research should lift the veil on the true prevalence of cannabis allergy and the importance of other cannabis allergens to further guide our practice.