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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., (Copyright © 2021 Elsevier B.V. All rights reserved.)

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.

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. [ABSTRACT FROM AUTHOR]

The article in Clinical & Experimental Allergy discusses the importance of protonation status in IgE-mediated mast cell responses to rocuronium. The study found that acidification to pH 6.9 during stimulation may enhance degranulation in low responders but dampen responsiveness in high responders. The research highlights the need for individualized protocols in IgE-mediated drug allergy testing, emphasizing the impact of acidification on mast cell responsiveness to rocuronium under suboptimal protonation conditions. [Extracted from the article]

Dual intracellular staining of CD154 (CD40L) and cytokines at single-cell level: A novel aid to document immediate hypersensitivity to amoxicillin Actually, 11/15 (73%) patients demonstrated a positive CD154/IL-4 LAT (e.g. a net expression of CD154 and/or IL-4 higher than the respectively maximal values observed in tolerant controls). Keywords: amoxicillin; CD154; cytokines; flow cytometry; IFN- ; IL-4; immediate drug hypersensitivity EN amoxicillin CD154 cytokines flow cytometry IFN- IL-4 immediate drug hypersensitivity 679 682 4 06/09/23 20230601 NES 230601 Key messages Multichromatic flow cytometry enables simultaneous analysis of intracellular CD154, IL-4 and IFN- in drug-reactive T lymphocytes. In conclusion, flow-based combined analysis of intracellular expression of CD154, IL-4 and IFN- by AXE-reactive T lymphocytes is an attractive approach for safe diagnosis of immediate amoxicillin hypersensitivity reactions. [Extracted from the article]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. © 2015 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Rocuronium is a major cause of perioperative hypersensitivity (POH). Skin tests (STs) and quantification of specific immunoglobulin E antibodies (sIgEs) can yield incongruent results. In such difficult cases, the basophil activation test (BAT) can be helpful. Here, we evaluated the passive mast cell activation test (pMAT) as a substitute of BAT as part of the diagnostic tests for rocuronium allergy. Sera from patients with a suspected POH reaction potentially related to rocuronium were included. All patients had a complete diagnostic investigation, including STs, quantification of sIgEs to morphine and rocuronium, and BAT. For execution of pMAT, human mast cells were generated from healthy donor peripheral blood CD34+ progenitor cells and sensitised overnight with patient sera. In total, 90 sera were studied: 41 from ST+sIgE+ patients, 13 from ST–sIgE– patients, 20 from ST+sIgE– patients, and 16 from ST–sIgE+ patients. According to BAT results, patients were further allocated into subgroups. Of the 38 BAT+ patients, 25 (66%) showed a positive pMAT as well. Of the 44 BAT– patients, 43 (98%) also showed a negative pMAT. Mast cells that were not passively sensitised did not respond to rocuronium. We show that the pMAT, in many cases, can substitute for BAT in the diagnosis of rocuronium hypersensitivity and advance diagnosis in difficult cases with uncertain ST or sIgE results when BAT is not locally available. [ABSTRACT FROM AUTHOR]

• The basophil activation test (BAT) has evolved to a safe and reliable diagnostic. • The BAT has some limitations hindering wider application. • The mast cell activation test (MAT) appears a promising adjunct diagnostic. • The MAT does not necessitate viable patients' cells and allows lager batch analyses. • Unlike BAT, MAT can directly reveal potential MRGPRX2 agonistic activity. The basophil activation test (BAT) has emerged as a reliable complementary diagnostic to document IgE-dependent allergies and to study cross-reactivity between structural homologues. However, the BAT has some weaknesses that hinder a wider application. The BAT requires fresh blood samples and is lost as a diagnostic in patients showing a non-responder status of their cells. The BAT is difficult to standardize mainly because of the difficulty to perform batch analyses. In contrast, mast cell activation tests (MATs), using passively sensitized mast cells (MCs) with patients' sera (henceforth indicated as passive MAT; pMAT), use serum samples that can be frozen, stored, and shipped to a reference center experienced in MC lines and/or cultures and capable of offering batch testing. With the recent recognition of the Mas-related G protein-coupled receptor X2 (MRGPRX2) occupation as a putative mechanism of immediate drug hypersensitivity reactions, the MAT has another advantage compared to the BAT. MCs, in contrast to resting basophils, express the MRGPRX2 and can therefore be used to study this IgE-independent mechanism. This review provides a status update of pMAT in the diagnosis of allergic IgE-mediated hypersensitivity and speculates how direct activation of MCs via the MRGPRX2 receptor could advance paradigms for this non-allergic hypersensitivity. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. 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. 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. 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. 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. [ABSTRACT FROM AUTHOR]

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. To compare different protocols for the generation of human MCs from peripheral blood progenitors. 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. 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. 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. • Primary human mast cells can be cultured in a four-week culture system. • IL-6 increases the mast cell yield and improves functionality. • The mast cells enable exploration of sIgE/FceRI- and MRGPRX2-dependent activation. [ABSTRACT FROM AUTHOR]