AllergoOncology: Emerging Translational and Clinical Significance of Basophils and Mast Cells in Cancer.

In the past two decades, we witnessed the evolution of the basophil activation test (BAT) from mainly research applications to a potential complementary diagnostic tool to document IgE-dependent allergies. However, BAT presents some technical weaknesses. Around 10%-15% of tested patients are non-responders, BAT can be negative immediately post-reaction and the use of fresh basophils, ideally analysed within 4 h of collection, restricts the number of tests that can be performed per sample. The need for fresh basophils is especially limiting when conducting batch analyses and interlaboratory comparisons to harmonize BAT methodology. These limitations significantly hinder the wider application of BAT and urge the development of alternative testing, such as the mast cell activation test (MAT). The essential difference between BAT and MAT is the heterogeneity of the starting material used to perform the assays. Mast cells are tissue-resident, so cannot be easily accessed. Current alternative sources for functional studies are generating primary human mast cells, differentiated from donor progenitor cells, or using immortalized mast cell lines. Hence, the methodological approaches for MAT are not only vastly different from BAT, but also different among MAT protocols. This review summarizes the advantages and disadvantages of BAT and MAT assays, dedicating special attention to elucidating the key differences between the cellular sources used and provides an overview of studies hitherto performed comparing BAT and MAT in the diagnosis of IgE-mediated food and drug allergies.

Background: Mast cells, are the primary responder cells of IgE-mediated allergy and while extensive research has been done to elucidate their precise structural and functional characteristics, mast cells are a type of tissue resident cell which makes their isolation and use for various research and diagnostic procedures in the field of allergy and immunology very complicated. Recently, attempts have been made to devise a method measuring mast cell degranulation through allergen stimulation for allergy diagnosis – Mast cell activation test (MAT). The aim of this article is to summarize existing knowledge on mast cells, the MAT, and the related Basophil activation test (BAT) on which the MAT is based on. Methods: We analyzed data from 51 relevant articles. Results: We made an extensive literature review of available data on the cytology, biochemistry and role of mast cells in the pathogenesis of allergic diseases-anaphylaxis, allergic rhinitis, and bronchial asthma, as well as available data on the MAT andhow it compares to the BAT. Conclusion: While much key information is available on mast cells, further research is required to expand on their function and role in allergy. Further research is needed to adequately compare MAT and BAT and create standardized protocols for the application of MAT in clinical practice and allergy research.

An accurate diagnosis of egg allergy is extremely important. Understanding egg-specific antibody function can inform future therapeutic strategies. We aimed to assess the diagnostic performance of the mast cell activation test (MAT) to egg and how IgE characteristics and IgG4 modulate mast cell response to egg allergens. BAT2 egg study participants with complete clinical outcomes and samples available for MAT were grouped based on oral food challenges (OFC) to baked egg (BE) and loosely cooked egg (LCE). Raw egg allergy was not assessed. IgE levels, specific activity, diversity, avidity for egg allergens and ratios of egg-specific IgG4/IgE were compared between baked egg allergic (BEA) and baked egg tolerant (BET) children and related to MAT and basophil activation test (BAT) to egg. BAT2 egg study participants studied (n = 133) included 58 BEA and 75 BET (16 LCE allergic and 59 LCE tolerant). MAT to egg showed an area under the ROC curve (95% CI) of 0.749 (0.663; 0.835) for BEA and 0.704 (0.616; 0.793) for LCE allergy. Sensitivity/specificity of MAT's optimal cut-off was 52%/89% for BE allergy and 70%/66% for LCE allergy. Diagnostic accuracy of MAT was 68% for both forms of egg allergy, which was lower than that of other tests. MAT was dependent on the presence of allergen-sIgE and had superior diagnostic accuracy to sIgE only when levels sIgE ≥ 1 KU/L. BAT and MAT results were moderately correlated (Rs = 0.431, p < 0.001). IgE levels, specific activity and avidity for egg allergens were directly correlated, and egg-specific IgG4/IgE ratios were inversely correlated with MAT to egg. MAT distinguishes BEA from BET children and is a suitable tool to test IgE function and the interference of IgG4 in mast cell response to egg allergens.

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Basophil activation test (BAT) or the mast cell activation test (MAT) are two invitro tests that are currently being studied in food allergy as diagnostic tools as an alternative to oral food challenges (OFCs). We conducted a meta-analysis on BAT and MAT, assessing their specificity and sensitivity in diagnosing peanut allergy. Six databases were searched for studies on patients suspected of having peanut allergy. Studies using BAT or MAT to peanut extract and/or component as diagnostic tools with results given in percentage of CD63 activation were included in this meta-analysis. Study quality was evaluated with the QUADAS-2 tool. On the 11 studies identified, eight focused exclusively on children, while three included a mixed population of adults and children. Only one study provided data on MAT, precluding us from conducting a statistical analysis. The diagnostic accuracy of BAT was higher when stimulated with peanut extract rather than Ara h 2 with a pooled specificity of 96% (95% CI: 0.89-0.98) and sensitivity of 0.86 (95% CI: 0.74-0.93). The sensitivity and specificity of BATs in discriminating between allergic and sensitized patients were studied as well, with pooled analysis revealing a sensitivity of 0.86 (95% CI: 0.74; 0.93) and a specificity of 0.97 (95% CI: 0.94, 0.98). BATs, when stimulated with peanut extracts, exhibit a satisfactory sensitivity and specificity for the diagnosis of peanut allergy and can help to discriminate between allergic individuals and those only sensitized to peanuts. More investigations on the potential for MATs diagnostic methods are warranted.

BackgroundFood allergy is an increasing public health issue and the most common cause of life-threatening anaphylactic reactions. Conventional allergy tests assess for the presence of allergen-specific IgE, significantly overestimating the rate of true clinical allergy and resulting in overdiagnosis and adverse effect on health-related quality of life.ObjectiveTo undertake initial validation and assessment of a novel diagnostic tool, we used the mast cell activation test (MAT).MethodsPrimary human blood-derived mast cells (MCs) were generated from peripheral blood precursors, sensitized with patients' sera, and then incubated with allergen. MC degranulation was assessed by means of flow cytometry and mediator release. We compared the diagnostic performance of MATs with that of existing diagnostic tools to assess in a cohort of peanut-sensitized subjects undergoing double-blind, placebo-controlled challenge.ResultsHuman blood-derived MCs sensitized with sera from patients with peanut, grass pollen, and Hymenoptera (wasp venom) allergy demonstrated allergen-specific and dose-dependent degranulation, as determined based on both expression of surface activation markers (CD63 and CD107a) and functional assays (prostaglandin D2 and β-hexosaminidase release). In this cohort of peanut-sensitized subjects, the MAT was found to have superior discrimination performance compared with other testing modalities, including component-resolved diagnostics and basophil activation tests. Using functional principle component analysis, we identified 5 clusters or patterns of reactivity in the resulting dose-response curves, which at preliminary analysis corresponded to the reaction phenotypes seen at challenge.ConclusionThe MAT is a robust tool that can confer superior diagnostic performance compared with existing allergy diagnostics and might be useful to explore differences in effector cell function between basophils and MCs during allergic reactions.

Immediate drug allergic reactions (IDAR) to betalactams are frequent, yet mislabelling remains common and negatively impacts clinical decisions. Conventional diagnostics such as STs and drug provocation are effective but limited by time, risk, and contraindications in severe cases. Invitro alternatives-sIgEquantification and basophil activation tests (BAT)-offer safer options, although performance may be affected by biological variability and suboptimal sensitivity with an important drawback for the latter in patients with non-releaser basophils. This study aimed to evaluate a mast cell activation test (MAT) based on human CD34+-derived mast cells (dMCs) for IDAR diagnosis to amoxicillin (AX) using both free AX and dendrimeric amoxicilloyl conjugates (G4/G5-AXO). CD34+ cells were cultured for 10-12 weeks to generate dMCs. After passive sensitization with sera from AX-allergic patients (N = 28) or tolerant controls (N = 11), dMCs were stimulated with free AX, G4-AXO, or G5-AXO. Activation was quantified by CD63 expression via flow cytometry. Diagnostic performance was compared with BAT and sIgE determination by ImmunoCAP. MAT with free AX achieved 53.57% sensitivity, G4/G5-AXO reached 46.43%, and all methods maintained 100% specificity. Combining results with AX or dendrimeric stimuli increased sensitivity to 75% while preserving specificity. Importantly, MAT identified positive cases among BAT non-releasers and patients with undetectable sIgE. Under optimized conditions, MAT using dMCs and different AX determinants reached 75% sensitivity and 100% specificity. The complementary use of free AX and dendrimeric conjugates expands detection across heterogeneous IgE reactivity profiles from AX-allergic patients, reinforcing the diagnostic value of advanced cellular models and engineered allergens.

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 4h 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.

The usefulness of the mast cell activation test (MAT) in diagnosing patients with uninterpretable basophil activation test (BAT) results caused by nonresponding basophils has not been addressed. Our study evaluated whether the results of the MAT were associated with the severity of the allergic reaction. We recruited 39 patients with Hymenoptera venom allergy (HVA), 22 nonsensitized controls, and 37 BAT nonresponding HVA patients. Specific IgE levels for honeybee venom (HBV) and yellow jacket venom (YJV) and total IgE were quantified using the IMMULITE® system. We performed a BAT and a MAT, which was based on the response of LAD2 cells to HBV and YJV. We first optimized the susceptibility of LAD2 cells to IgE-mediated degranulation in HVA and showed that prestimulation with IL-33 and IL-6 significantly increased the responsiveness of LAD2 cells to allergen stimulation (P<.01). The LAD2 MAT results correlated with the BAT results, and patients with severe sting reactions (Mueller grades III and IV) had a median 2-fold higher LAD2 MAT score than patients with nonsevere sting reactions (large local reaction or Mueller grades I and II) (P<.05). Furthermore, LAD2 MAT provided conclusive results in 20 of the 37 HVA patients (54.1%) with nonresponding basophils in the BAT. The LAD2 MAT is a new diagnostic tool for HVA patients with nonresponding basophils. LAD2 MAT can identify patients at risk of severe sting reactions and can thus help guide recommendations for venom immunotherapy and improve the management of patients with HVA.

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

ABSTRACTTo date, the exact impact of mast cells in tumor microenvironment is still controversial because of inconsistency in observations regarding the relationship between mast cell infiltrates and cancer development and prognosis. The discrepancies in previous studies have motivated us to examine the roles of mast cells in cancer pathology from different perspectives. Here, we investigated the impact of mast cells on transcriptomic profiles in the tissue microenvironment. Mice carrying the W-sh mutation in c-kit (KitW-sh) are deficient in mast cell production and were used to assess the influence of mast cells on gene expression. By examining the transcriptomic profile among wild-type mice, KitW-sh mice, and KitW-sh mice with mast cell engraftment, we identified a list of “mast cell–dependent genes,” which are enriched for cancer-related pathways. Utilizing whole-genome gene expression data from both mouse models and human cancer patients, we demonstrated that the expression profile of the mast cell–dependent genes differs between tumor and normal tissues from lung, breast, and colon, respectively. Mast cell infiltration is potentially increased in tumors compared with normal tissues, suggesting that mast cells might participate in tumor development. Accordingly, a prognostic molecular signature was developed based on the mast cell–dependent genes, which predicted recurrence-free survival for human patients with lung, breast, and colon cancers, respectively. Our study provides a novel transcriptomic insight into the impact of mast cells in the tumor microenvironment, though further experimental investigation is needed to validate the exact role of individual mast cell–dependent genes in different cancers.

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The major challenge in allergy diagnosis is the development of accessible and reliable diagnostics that can predict the clinical outcome upon the exposure to culprit allergen(s) or cross-reactive molecules and the identification of safer alternatives compared to current state-of-the-art methods. There is accumulating evidence that flow-based analyses for the quantification of activated basophils and mast cells upon in vitro challenge (Basophil and Mast Cell Activation Test, or BAT and MAT) could meet the diagnostic requirements for IgE-dependent allergies, drug hypersensitivities and subsets of autoimmune urticaria. Furthermore, BAT and MAT found application in research and other non-diagnostic fields. However, the appropriate use of BAT and MAT requires understanding of the diversity of the source materials used and degranulation metrics to ensure correct test performance and interpretation of results. In this review, we provide the main applications and limitations of BAT and MAT hitherto performed.

Comparison of the passive mast cell activation test with the basophil activation test for diagnosis of perioperative rocuronium hypersensitivity

Immediate drug hypersensitivity reactions (IDHRs) pose significant diagnostic challenges, often requiring potentially hazardous drug challenge testing (DCT). Flow cytometry-based cellular tests including the basophil activation test (BAT), the mast cell activation test (MAT) and the T cell activation test (TAT) offer promising alternatives to reduce DCT reliance. While these tests are still in development, they demonstrate potential to compete with skin tests by providing superior diagnostic performance and improved patient safety by reducing the need for DCT. Furthermore, it is encouraging that these flow cytometry-based tests are also suitable for challenging populations, such as children. Despite requiring specialised infrastructure, these tests have the potential to be cost-effective when performed in reference centres and may offer unique mechanistic insights into immediate drug hypersensitivity reactions. However, further research is needed to validate their reliability, address pharmaceutical-specific testing considerations, and potentially integrate them into clinical guidelines.