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.

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.

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.

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.

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.

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 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.