Non-IgE-mediated Immediate Drug-Induced Hypersensitivity Reactions

Abstract

Immediate drug-induced hypersensitivity reactions (IDHSRs) have conventionally been attributed to an IgE-mediated mechanism. Nevertheless, it has now been acknowledged that IDHSRs can also occur independently of IgE involvement. Non-IgE-mediated IDHSRs encompass the activation of effector cells, both mast cell (MC) dependent and independent and the initiation of inflammatory pathways through immunogenic and non-immunogenic mechanisms. IDHSRs involve inflammatory mediators beyond histamine, including the platelet-activating factor (PAF), which activates multiple cell types, including smooth muscle, endothelium, and MC, and evidence supports its importance in IgE-mediated reactions in humans.

Clinically, distinguishing IgE from non-IgE mechanisms is crucial for future treatment strategies, including drug(s) restriction, re-administration approaches, and pretreatment considerations. However, this presents significant challenges, as certain drugs can trigger both mechanisms, and their presentations can appear similarly, ranging from mild to life-threatening symptoms. Thus, history alone is often inadequate for differentiation, and skin tests lack a standardized approach. Moreover, drug-specific IgE immunoassays have favorable specificity but low sensitivity, and the usefulness of the basophil activation test remains debatable. Lastly, no biomarker reliably differentiates between both mechanisms. While non-IgE-mediated mechanisms likely predominate in IDHSRs, reclassifying most drug-related IDHSRs as non-IgE-mediated, with suggested prevention through dose administration adjustments, is premature and risky. Therefore, continued research and validated diagnostic tests are crucial to improving our capacity to distinguish between these mechanisms, ultimately enhancing patient care.

INTRODUCTION

Annually, there are 221,600 cases of immediate drug-induced hypersensitivity reactions (IDHSRs) in the United States (U.S.), with 6% of them escalating to anaphylaxis.^1–3 Anaphylaxis is typically understood as an allergic response facilitated by specific IgE (sIgE), activating mast cells (MCs) and basophils through the cross-linking of the high-affinity IgE receptor (FcεRI).⁴ However, non-IgE-mediated mechanisms can also lead to anaphylaxis.⁵ In most IDHSRs, while available tests may have limited sensitivity in ruling out IgE-mediated mechanisms, non-IgE mechanisms are likely more common. Identifying the underlying mechanism of IDHSR is crucial, as it significantly influences management approaches, including drug avoidance or reintroduction decisions.⁶

IgE-mediated anaphylaxis demands strict avoidance of the triggering drug and immunological cross-reactive drugs.^{6, 7} Alternatively, drug desensitization is an option, but it comes with a risk of severe and potentially fatal reactions, necessitating intensively monitored settings.⁸ Consequently, these procedures are labor-intensive and costly.^{9, 10} In contrast, most non-IgE-mediated IDHSRs vary widely and can be managed with both drug dose adjustments and pre-medications like antihistamines. Still, the precise role of premedication in certain non-IgE-mediated IDHSRs remains unclear.^{6, 7, 11}

Differentiating IgE- and non-IgE-mediated IDHSRs presents a significant challenge, as validated diagnostic tests are unavailable.^12–14 As a result, IDHSRs are frequently misperceived as IgE-mediated, prompting unnecessary lifelong drug avoidance.^{15, 16} This misclassification can result in a reliance on alternative drugs that may be less safe and effective.^{11, 17, 18} Continued research and validated diagnostic tests are crucial to improving our capacity to distinguish between these mechanisms, ultimately enhancing patient care. In this review, we review recent advancements in non-IgE-mediated IDHSR, exploring the underlying mechanisms leading to IDHSRs without sIgE allergen activation, those that function independently of MCs and basophils, and describing associated risk factors.

PATHOPHYSIOLOGY

IDHSRs entail the activation of effector cells like MCs or basophils, along with the initiation of inflammatory pathways via immunogenic and non-immunogenic mechanisms (Figure 1).^{10, 19} The mechanisms underlying immunogenic-mediated IDHSR involve the adaptive immune system and comprise both IgE- and non-IgE-mediated mechanisms. In contrast, non-Immunogenic-mediated IDHSR mechanisms do not involve the adaptive immune system.

Figure 1. Mechanisms of IDHSRs:

Immunologic and non-immunologic pathways, MC-dependent and independent mechanisms, and associated mediators.

*Drugs reported to trigger both IgE and non-IgE-mediated IDHSR mechanisms.

**Tryptase biological roles are unclear. Elevated tryptase indicates MC and basophil involvement; however, it does not reliably distinguish between IgE- and non-IgE-mediated IDHSRs.

Abbreviations: FcεRI, high-affinity IgE receptor; FcγR, Fc gamma receptor; IDHSR, immediate drug-induced hypersensitivity reactions; MC, mast cell; MRGPRX2, mas-related G-protein coupled receptor X2; NMBA, neuromuscular blocking agent; PAF, platelet-activating factor; PMNs, polymorphonuclear neutrophils.

Immunogenic Non-IgE-mediated IDHSR Mechanisms

Immune complex-mediated IDHSRs

Complement activation leads to the release of anaphylatoxins that can activate MCs and basophils and directly affect smooth muscle and endothelium, causing contraction and increased vascular permeability.²⁰ This activation can be triggered by immune complexes (ICs), often induced by drug-specific IgG (sIgG), characterized classically as Gell-Coombs type III reactions.^{19, 21} These ICs can activate complement and Fc gamma receptors (FcγR) on MCs, resulting in a synergistic interplay between these mechanisms.²² IC-mediated IDHSRs have been documented with drugs like dextran and von Willebrand factor.^{20, 23}

IgG-mediated IDHSRs

Insights from animal models highlight the significance of IgG-mediated mechanisms.^24–28Across the varying interactions of IgG and FcγR expressed on different cells, various potential roles of IgG have been identified.^{22, 29} FcγR expression has been demonstrated on the surface of key cellular mediators of anaphylaxis. FcγRI expression is inducible on human MCs and neutrophils, and binding with IgG1 and IgG ICs can result in their activation.^{20, 29, 30} Also, activation of FcγRIII on murine macrophages and neutrophils results in platelet-activating factor (PAF) release, which plays a significant role in anaphylaxis by inducing mediators like prostaglandin E2 and nitric oxide, derived from MCs.²²

In murine anaphylaxis models, the absence of FcγRs resulted in the elimination of anaphylactic responses. However, when human FcγRIIA was reintroduced into a mouse model with neutrophils as effector cells, anaphylaxis was restored.^{31, 32} Various models have highlighted the role of different cell types (e.g., macrophages, basophils, and neutrophils) in driving IgG-mediated anaphylaxis.^{22, 33, 34} Platelets also play a significant role, as their activation through ICs can contribute to anaphylaxis and may be mitigated by inducing thrombocytopenia.^{35, 36} Notably, in mice, IgG-mediated anaphylaxis needs higher antigen and sIgG levels than IgE-mediated reactions.^{22, 37} At low antigen doses, sIgG acts as an inhibitor of IgE-mediated anaphylaxis by competitively interacting with sIgE and binding to inhibitory FcγRIIB. On the contrary, ICs can induce anaphylaxis at higher doses through FcγRs. Additionally, in a study examining IgG-mediated anaphylaxis to antiprogrammed cell death immune checkpoint inhibitor in tumor-bearing mice, fatal anaphylaxis was linked to elevated macrophages and neutrophils, and anaphylaxis was prevented by depleting these cells.³⁸ Lastly, the uneven distribution of ICs, with enhanced accumulation in the lungs following a challenge, raises questions about its impact on the development and severity of anaphylaxis.³⁹

Human data on IgG-mediated anaphylaxis is limited, indirect, and significantly lags behind mouse model findings. Challenges include the absence of validated markers and the presence of drug-sIgG, not necessarily proving causality. In a multicenter study comparing patients with suspected anaphylaxis to neuromuscular blocking agents (NMBAs) to NMBA-tolerant controls, researchers assessed markers for IgG-mediated anaphylaxis.⁴⁰ In this cohort, 72% of anaphylaxis patients lacked anti-NMBA-sIgE. All patients and controls had detectable anti-NMBA-sIgG, but anaphylaxis severity correlated significantly with sIgG levels. Greater anaphylaxis severity was also linked to increased FcγR activation, PAF, and neutrophil activation markers. In one patient, anti-rocuronium-sIgG ICs were observed ex vivo and resulted in neutrophil activation of healthy control neutrophils through activation of FcγR.⁴⁰ While anti-NMBA-sIgG may be necessary for IgG-mediated anaphylaxis, it is insufficient, as all control subjects had measurable anti-NMBA-sIgG. This study aligns with murine model findings but awaits replication with other drugs. There is emerging evidence that asparaginase can induce basophil and macrophage activation through anti-asparaginase-sIgG FcγR interactions.⁴¹ Nevertheless, although there is an association between anti-asparaginase-sIgG and hypersensitivity reactions in humans, it does not appear to be either strictly necessary or sufficient for causing such reactions.⁴² Further research is required to understand the contributions of other factors to anaphylaxis in these patients.

Non-Immunogenic IDHSR Mechanisms

These mechanisms involve activating effector cells without IgE, IgG, or ICs. Drugs have long been known to induce non-IgE-IDHSR MC activation. Still, the mechanism remained largely unknown until the recent discovery of the Mas-related G-protein coupled receptor X2 (MRGPRX2), providing some understanding of the non-IgE-mediated activation of MCs.^{4, 43, 44}

MRGPRX2-mediated IDHSRs

MRGPRX2, primarily found in skin MCs, is a distinctive class A G protein-coupled receptor known for its unique structural features, genetic diversity, and responsiveness to a wide range of positively charged endogenous and exogenous molecules.^45–50 From an evolutionary standpoint, MRGPRX2 activates mast cells during host defense, immune modulation, and wound healing through interactions with antimicrobial peptides (e.g., defensins, catestatin, LL-37) and neuropeptides (e.g., substance P).⁴³ In mice, MRGPRB2 and MRGPRA1 serve as functional counterparts.^{46, 51} The recent discovery of MRGPRX2’s 3D structure has provided critical insights into its ligand binding and signaling mechanisms, revealing why it reacts with multiple triggers.^{52, 53} This is attributed to two sub-pockets within the receptor that interact differently with ligands. The negatively charged surface of “sub-pocket 1” can recognize several positively charged small molecule drugs, while large peptide drugs can bind to both sub-pockets. The charge interaction with amino acids Asp184 and Glu164 at sub-pocket one has been identified as crucial for MRGPRX2 activation.^{52, 53}

MRGPRX2 shows several genetic polymorphisms, which may explain why some individuals are more susceptible to IDHSRs (Table 1).⁵⁴ Mutations, such as Gly165Glu, Asp184His, Trp243Arg, and His259Tyr, abolished NMBA, icatibant, and opioids MRGPRX2-mediated activation.^52–56 This holds potential significance for individuals undergoing surgery, where conducting a drug challenge is often unfeasible.

Table 1:

MRGPRX2 genetic variations, including amino acid changes and SNPs, lead to specific drug(s) loss of function and altered EC50, resulting in MRGPRX2 activation.

Drug name	Amino acid mutations (SNPs)	EC50 (μg/mL)56			Peak Plasma Concentration (μg/mL)56
		MM	TLC	HMC
Atracurium	Asp184His (rs372988289)51	4542	2942	36366	<10
	Trp243Arg (rs150365137)51
Tubocurarine	Asp184His (rs372988289)51
	Trp243Arg (rs150365137)51
Rocuronium	Asp184His (rs372988289)	2242	26142		6–15
	Trp243Arg (rs150365137)51
Icatibant	Gly165Glu (rs141744602)		15.8 ± 2,7		0.979 ± 0.262
	Asp184His (rs372988289)53
	Trp243Arg (rs150365137)53
	His259Tyr (rs140862085)53
Dextromethorphan	Glu164Gln72*		0.46¶		PO: 0.002006
	Asp184Asn72*
Morphine	Glu164Gln72*		272	88126	PO: 0.004
	Asp184Asn72*				IV: <0.570
Dynorphin A	Asp184Asn72*		278¶
Vancomycin	ND		60127		70
Levofloxacin	ND	80842	2342	56266	Single dose: 6 μg/mL
					Multiple doses: 20 μg/mL
Moxifloxacin	ND	1442	9.942	102126	6 μg/mL
Ciprofloxacin	Asp184His (rs372988289)	12742	6.8	106	Single dose: 5 μg/mL
	Trp243Arg (rs150365137) 51				Multiple doses: 10 μg/mL

^*SNPs have not been determined for these mutations

^¶Calcium release

^§Levels up to 70 μg/mL described¹²⁸

Abbreviations: EC50, half-maximal effective concentration; HMC: human mast cells; IV, intravenous; MM, mouse model; ND, not determined; PO, per os; SNPs, single nucleotide polymorphisms; TCL, transfected cell lines.

Variable MRGPRX2 response

MRGPRX2-mediated signaling requires higher and sustained drug plasma and tissue concentrations. This underlines the potential for modulation through rate and dosage adjustments.^{5, 43, 57} In vitro, EC50 values for many drugs surpass in vivo peak plasma concentrations, highlighting the complexity of this interaction (Table 1).^{43, 58} Plasma concentrations may not accurately reflect tissue concentrations, and variability exists in drug-triggered MRGPRX2 responses among different populations.

Various factors can affect receptor occupancy, including quantitative and qualitative aspects. Quantitative factors encompass receptor expression levels and drug dosage. In specific conditions, such as chronic urticaria MCs display an increased MRGPRX2 expression and enhanced MRGPRX2 sensitivity to drug ligands.^{59, 60} In mastocytosis, expression of MRGPRX2 is raised in the skin but not in the bone marrow.^61–63 Research is ongoing to determine whether these conditions heighten susceptibility to MRGPRX2-mediated IDHSRs. Conversely, qualitative factors play an important role, as polymorphisms significantly impact MRGPRX2-mediated reactions, with their presence at specific receptor sites determining responsiveness to MRGPRX2 agonists.^{46, 54} This intricate interplay may explain why individuals react to some MRGPRX2 agonists but not others, revealing that “cross-reactivity” patterns extend beyond structural homology.⁴⁶ Lastly, infections can attenuate MCs’ response to certain drugs, such as vancomycin.⁶⁴

Additionally, distinct secretion patterns of granules are observed in response to IgE- and MRGPRX2-mediated MC activation. IgE-mediated MC activation exhibits a slower compound exocytosis, marked by the delayed release of large and stable granules. In contrast, MRGPRX2 is associated with reduced inflammatory mediator production and triggers a unique “kiss-and-run” phenomenon with the rapid release of small and less stable granules.⁵⁷

Drugs as MRPRX2-ligands

Neuromuscular Blocking Agents

Recent research has examined drugs like rocuronium and atracurium, often linked to MRGPRX2-mediated IDHSRs. However, the connection between rocuronium-induced IDHSRs and MRGPRX2 remains debated.^65–67 Rocuronium differs from most MRGPRX2 drug ligands by lacking the common tetrahydroisoquinoline motif, making it a weak ligand. In vitro studies have revealed that rocuronium-induced MRGPRX2 activation occurs at concentrations well above the in vivo EC50.^{43, 68} A study by Suzuki et al. proposed that amino acid mutations (Leu237Pro, Leu226Pro, Met196De) in MRGPRX2 could connect these IDHSRs to rocuronium. However, the chosen confirmatory skin test concentration exceeded the recommended non-irritative maximum, raising concerns about false-positive results.^69–71 The interplay between physiological concentrations, genetic variations, and post-transcriptional modifications of MRGPRX2 emphasizes the complexities of rocuronium-induced IDHSRs and MRGPRX2 associations.^{66, 71}

Fluoroquinolones

Fluoroquinolones like ciprofloxacin and levofloxacin are commonly linked to IDHSRs.^{5, 72} The MRGPRX2-mediated mechanism has emerged as an underlying explanation.^{73, 74} Several studies have reported the MRGPRX2-mediated MC degranulation activity of ciprofloxacin and levofloxacin.^{68, 75} Liu, Hu et al. reported the in vitro and in vivo evidence of MRGPRX2-mediated MC activation of nine fluoroquinolones.⁷⁵ In addition, key amino acid residues of MRGPRX2 responsible for binding with fluoroquinolones can help predict the risks of IDHSRs.⁷⁶ This difference in the MRGPRX2 gene and amino acids could explain why some individuals are more susceptible to fluoroquinolone and NMBAs than others.

Vancomycin

Vancomycin is known for its specific IDHSR.^{77, 78} These IDHSRs are believed to be mediated by MRGPRX2, supported by the lack of sIgE and negative skin tests at non-irritative doses, along with in vitro MC activation in MRGPRX2-positive MCs.⁷⁹ In a recent retrospective observational cohort study, 19.7% of the population (3,165 vancomycin-exposed patients) were potentially MRGPRX2 reactors, and comorbid allergies likely exacerbated MRGPRX2-mediated IDHSRs.⁸⁰ However, further research is needed to understand the association between MRGPRX2 and vancomycin-related IDHSRs comprehensively.

Opioids

Opioids are often associated with IDHSRs.⁸¹ Research has demonstrated that small-molecule opioids like morphine activate MCs through MRGPRX2, while larger opioid molecules, such as fentanyl, do not.^{74, 81, 82} Navinés-Ferrer et al. demonstrated that morphine activates MRGPRX2 in LAD2 cells, with activation levels significantly reduced following siRNA knockdown of MRGPRX2.⁷⁹ Notably, cutaneous MCs lack opioid receptors, suggesting that opioid-induced MC activation possibly relies on MRGPRX2. Nonetheless, opioid-associated itching may include an MC-independent mechanism, as neurons responsible for itch sensation express opioid receptors, and their activation alone can elicit itching.⁷⁴

Other MRGPRX2 drug ligands

Icatibant activates MCs through MRGPRX2, supported by evidence connecting MRGPRX2 and MRGPRB2 to icatibant-induced IDHSRs.⁴⁴ This activation leads to injection site reactions in most patients.^83–85 Recent studies have found that meglumine gadoterate (Gd-DOTA), a gadolinium-based contrast agent, activates LAD2 mast cells through MRGPRX2.⁸⁶ Furthermore, iohexol, an iodine-based contrast media, induced allergic reactions in mice via MCs and activated LAD2 cells via MRGPRX2.⁸⁷ Research has indicated that antidepressant drugs (e.g., clomipramine, paroxetine, desipramine) can trigger IDHSRs by activating MRGPRX2.⁸⁸ Notably, systemic anaphylactic reactions to these antidepressants are rare or unreported.

Novel MRGPRX2 antagonists

MRGPRX2 antagonists have emerged as potential candidates for attenuating MC activation. Efforts have been made to develop novel MRGPRX2 antagonists encompassing diverse structures, including peptides, DNA aptamers, and small molecules.^{46, 89–93} The recent breakthrough in obtaining the MRGPRX2’s 3D structure has expedited the development of MRGPRX2 antagonists.^{52, 53} Kumar et al.’s work has demonstrated the in vitro antagonistic activity of small molecule MRGPRX2 antagonists on ciprofloxacin-induced MC activation.⁹² Ogasawara et al. have shown in vitro MRGPRX2 antagonists with inhibitory activity on human cord blood-derived MCs, effectively reducing MC activation.^{92, 93} A specific and efficient MRGPRX2 antagonist can help validate the involvement of suspected drugs in MC activation through MRGPRX2. Moreover, MRGPRX2 antagonists hold the potential to advance treatment. Nonetheless, most antagonist research has explored their effectiveness in mice or in vitro human MCs, providing limited insights into their clinical application.

Differences between IgE- and MRGPRX2

Currently, no definitive clinical evidence establishes the significance of the MRGPRX2 mechanism in IDHSRs. Several challenges contribute to this uncertainty, including the oversimplified dichotomy of IgE vs. MRGPRX2 and the ability of certain drugs to activate MC through both mechanisms, resulting in a potential amplification of anaphylaxis.⁹⁴ Notably, the clinical presentation of MRGPRX2-mediated IDHSRs is debatable, with questions about whether they are primarily confined to cutaneous manifestations.^{43, 95, 96} However, recent studies indicate that rocuronium- and moxifloxacin-induced MRGPRX2-mediated IDHSRs present predominantly as anaphylaxis.^{97, 98} Conversely, isolated urticaria is a common manifestation of IgE-mediated IDHSRs to chlorhexidine, a biguanide lacking MRGPRX2-activating capacity.^99–101 Both mechanisms are associated with a broad clinical presentation spectrum (Table 2). Notably, cutaneous symptoms are a common feature in MRGPRX2, potentially serving as a distinguishing parameter based on in vitro kinetics, as MRGPRX2-mediated IDHSRs often resolve more rapidly than IgE-mediated ones.⁵⁷ Moreover, unlike IgE, MRGPRX2-mediated IDHSRs do not require prior exposure to the culprit. Therefore, drug naivety should raise the suspicion of an MRGPRX2-mediated mechanism. Likewise, IgE-mediated IDHSRs in drug-naïve patients can be attributed to IgE cross-sensitization, as observed in some NMBAs-induced IDHSRs.^{68, 102, 103} Despite these potential clinical differences, none of these parameters have been validated to reliably distinguish between these mechanisms. Therefore, a personalized and comprehensive evaluation remains crucial for patient safety.

Table 2.

Potential distinctions between IgE and suspected MRGPRX2-mediated IDHRS.

Characteristics	IgE-mediated IDHSR	MRGPRX2-mediated IDHSR
Clinical presentation	Entire spectrum IDHR	Entire spectrum IDHR
	Inconsistent skin symptoms with presumably slower resolution	Presumably, consistent skin symptoms with rapid resolution
Prior exposure	Necessary	Not required
	IgE cross-sensitization
Dose	Low-dose	Higher and sustained drug plasma/tissue concentrations
Affinity	High	Low
Comorbidities	No influence on the outcome	Likely impact on the outcome
Genetics	Limited role	Polymorphisms affect affinity for specific ligands
Biomarkers	Tryptase (non-specific)	Tryptase (non-specific)
Cross-reactivity	Structural homology	Related on ability to bind the receptor
Diagnosis	ST (SPT, IDT) positive at non- irritative low dose/concentration	ST (IDT) positive at a higher non- irritative dose/concentration
	ST positivity is inhibited by BTKi	ST positivity is not inhibited by BTKi
	Traditional sIgE - BAT	Conditioned basophil functional assay*
	Challenge positive at low doses	Challenge positive at high doses
Slow administration	No	Possible
Pre-treatment	No	Possible
Desensitization	Possible	Questionable

^*Insufficient evidence supports conditioned BAT for assessing MRGPRX2-mediated effector cell activation.

Abbreviations: BAT, basophil activation test; BTK, Bruton's tyrosine kinase; IDHSR, immediate drug hypersensitivity reaction; IDT, intradermal test; MRGPRX2, Mas-related G-protein coupled receptor X2; sIgE, specific IgE; SPT, skin prick test; ST, skin test.

Updated from Sabato et al, JACIP 2022

Non-immunogenic complement-mediated IDHSR

Complement activation may also result from non-adaptive activation through the recognition by pattern recognition receptors or pathogen-associated molecular patterns of liposome epitopes, classified as “non-immunogenic.” However, this classification is not mutually exclusive, as both mechanisms of complement activation have been attributed to liposomal drugs.¹⁰⁴ Furthermore, the term complement activation-related pseudo-allergy (CARPA) has been used for reactions driven by either mechanism of complement activation (Figure 2).²³ The exact contributions of the inciting mechanisms of complement activation for different drugs remain unclear.

Figure 2. CARPA Cascade:

Pathway of complement-mediated IDHSRs. Numerous drugs can activate complement leading to C3a, C5a anaphylatoxin release, comprising signal or “hit” 1. In addition, pegylated liposomes can directly bind to pattern recognition receptors on macrophages, MCs, and basophils, comprising signal or “hit” 2. These two signals contribute to MC, basophil, and macrophage activation and mediator release resulting in end-physiologic changes. Concomitant activation of endothelial and smooth muscle directly by C3a and C5a compound these physiologic changes.

Abbreviations: CARPA, complement activation-related pseudoallergy; IDHSR, immediate drug-induced hypersensitivity reactions; MC, mast cell; PAF, platelet activating factor.

Other non-Immunogenic-mediated IDHSR

There are other mechanisms through which drugs can trigger immediate reactions, including nonsteroidal anti-inflammatories through cyclooxygenase-1 inhibition and drugs with inherent immunomodulatory properties, such as monoclonal antibodies, leading to cytokine release reactions.^105–107 Furthermore, alternative non-immunogenic mechanisms are capable of triggering anaphylaxis, even without MCs and basophils involvement, including coagulation, kallikrein, and platelet-mediated IDHSRs. MC activation can additionally attract and activate these pathways, resulting in an additive amplification of anaphylaxis.¹⁰⁸

MEDIATORS

In the broader context of IDHSRs, MCs, basophils, and other immune cells like T lymphocytes and macrophages can be activated through non-IgE-mediated mechanisms. These mechanisms involve the release of cytokines along with vasoactive mediators.¹⁰⁹ In addition to histamine, several mediators have been identified with anaphylaxis, including preformed mediators like proteases, tumor necrosis factor, and PAF, and newly formed mediators like leukotrienes and prostaglandins (Figure 1).^{6, 10, 19} PAF is a potent phospholipid-derived mediator that can activate various cells expressing the PAF receptor (PAF-R), such as endothelium, smooth muscle, and MCs.^{110, 111} PAF can induce vascular smooth muscles relaxation, increase vascular permeability, reduce cardiac output, and lead to circulatory collapse.¹¹² Studies have shown that PAF is important in IgE-mediated reactions in humans.¹¹³ The activity of PAF is regulated by PAF acetylhydrolase (PAF-AH), and patients with high levels of PAF and decreased PAF-AH activity are at a greater risk of severe anaphylaxis than those with normal PAF-AH activity.¹¹⁴

While the exact effects of certain cytokines and chemokines in anaphylaxis are not fully understood, some of these molecules have been associated with the severity of anaphylactic episodes. Further research is needed to comprehend better the intricate interplay of these mediators and cytokines in the pathophysiology of anaphylaxis.

DIAGNOSIS

Biomarkers

Currently, no definitive acute biomarker can differentiate between IgE- and non-IgE-mediated MC activation reliably. Clinically, analyzing paired acute and baseline serum tryptase levels is crucial for accurate MC activation assessment.¹¹⁵ Some studies have suggested that changes in acute serum tryptase levels might distinguish between IgE- and MRGPRX2-mediated reactions; however, this distinction even includes patients with negative skin tests, which contradicts an MRGPRX2 mechanism.^{79, 116–119} Recent in vitro studies have raised doubts about the discriminatory capacity of tryptase levels in this context.¹²⁰ Furthermore, in a recent analysis of rocuronium IDHSRs, no significant difference in acute tryptase titters was observed between IgE- and suspected MRGPRPX2-mediated IDHSRs.⁹⁸

Diagnostic Tools

Drug Challenge

Clinically, drug challenges have a primary role in excluding an IDHSR. They are indicated for patients who are considered unlikely to be allergic to the drug in question based on history or negative skin or in vitro tests.¹²¹ As a diagnostic method, drug challenges are theoretically the gold-standard method for confirming an IDHSR, particularly an IgE-mediated IDHSR. However, they still do not provide reliable insight into the underlying IDHSR mechanisms. The fundamental concept behind these procedures is determining the specific dosage or threshold required to trigger allergic symptoms. While IgE-mediated reactions can display dose-dependency, influenced by factors like sIgE titers, surface density on effector cells, and allergen affinity, a significant difference in the required dosage to elicit symptoms can differentiate between IgE- and MRGPRX2-mediated IDHSR. MRGPRX2 typically requires significantly higher doses to reach an EC50 threshold needed for triggering a response. Nonetheless, further research in this area is necessary, and the limitations become apparent when dealing with cases where patients cannot be re-challenged due to their initial high pre-test probability.¹²²

Skin test

The skin test for identifying sIgE faces several challenges, primarily due to potential false positives from non-specific histamine release.¹²³ Moreover, the dogma that a positive skin test indicates an IgE-mediated reaction has been repeatedly challenged, especially for MRGPRX2 agonists, such as opiates, vancomycin, and fluoroquinolones.¹²⁴ A significant obstacle is the lack of a standardized approach to skin testing, which includes the absence of a well-defined range of concentrations for the different drugs.^{121, 125, 126} Existing published methods often rely on case reports with varying methodologies.¹²⁶ This has resulted in uncertainties surrounding test results’ positive and negative predictive values. Additionally, the sensitivity, specificity, and diluent effects in skin tests for drugs with high irritant potential are poorly understood.^{124, 125, 127}

It is worth examining whether skin test concentrations and methodologies can aid in identifying specific mechanisms. In patients with IgE-mediated IDHSRs, skin tests yield positive results at lower concentrations required to trigger nonspecific skin test responses through MRGPRX2 activation.⁹⁸ Alvarez-Arango et al. reported a 30-fold variability in vancomycin skin test response EC50 among individuals without prior vancomycin exposure.¹²⁴ This heterogeneity may reflect variable MRGPRX2-mediated reactions to vancomycin. However, further research is needed to prove this hypothesis. Krantz et al. have proposed updated criteria for fluoroquinolone allergy testing, considering the complexity of immune activation and selective reactions to fluoroquinolones. The aim is to establish sufficiently sensitive criteria to rule out an IgE-mediated IDHSR while avoiding a provoked dose-related reaction consistent with non-IgE-mediated MC activation.¹²⁸ Lastly, inhibitors of Bruton’s tyrosine kinase (BTK), an essential enzyme for FcεRI signaling, hold the potential to distinguish the underlying IDHSR mechanism in a positive skin test. However, there is a lack of clinical feasibility and evidence supporting BTK inhibitors use for this purpose.

Drug-specific IgE assays

Recommendations for drug-sIgE assays vary depending on the geographic area. European countries recommend sIgE immunoassays for evaluating IDHSRs to specific drugs. In the U.S., immunoassays for penicillin sIgE are suboptimal due to their low sensitivity. Drug-sIgE assays generally demonstrate favorable specificity, but false-positive results can occur depending on the drug. Such false positives can be influenced by high titers of total IgE, resulting in nonspecific binding of IgE to certain drug components and limiting the diagnostic value of these tests.

Functional basophil and mast cell test assays

The basophil activation test (BAT) is recommended in some European countries, mainly when skin tests yield inconclusive results. In the U.S., no commercially available BAT assay has demonstrated validity. BAT can be valuable in identifying IgE-mediated IDHSRs; however, there is controversy surrounding the expression of MRGPRX2 on resting basophils.^{45, 47, 129} Conversely, this receptor has been reported to be upregulated after stimulation with anti-IgE, N-Formylmethionyl-leucyl-phenylalanine (fMLP), or purification, potentially making basophils responsive to MRGPRX2 activation. These “conditioned” basophils may hold promise for evaluating IDHSRs stemming from MRGPRX2.⁴⁵ Nonetheless, conditioned basophils in clinical practice lack robust supporting evidence and clinical feasibility. Moreover, the choice of basophil activation marker (CD63 or CD203c) is also contentious, given that different drugs may induce the expression of distinct activation markers. Lastly, the MC activation test (MAT) is a promising diagnostic tool as it allows simultaneous analysis of MRGPRX2+ and MRGPRX2- subpopulations and, importantly, silencing of the receptor.¹⁰¹ However, currently, it primarily serves as a research tool.

CONCLUSION

Our understanding of IDHSRs, including antibodies, effector cells, and mediators, has continuously evolved. It has become increasingly apparent that these reactions can manifest through various mechanisms, whether additive or independent of IgE mediation, and may involve MCs in a dependent or independent manner.¹³⁰ Discerning between IgE- and non-IgE-mediated IDHSRs will be crucial in the context of precision medicine, as it will directly influence patient management and outcomes. The lack of validated diagnostic tests and similar clinical presentations pose significant challenges in distinguishing between these mechanisms. Without a definitive test to confirm non-IgE-mediated IDHSRs, a combination of history and available tests may offer some insights into the potential underlying mechanism.⁵⁸ However, as our understanding of the intricate pathophysiology of IDHSRs and their interconnected mechanisms continues to advance, there is a growing need for more effective diagnostic options to facilitate prompt and accurate diagnosis and, consequently, more efficacious treatment approaches.

Abbreviations:

AH

Acetylhydrolase

BAT

Basophil activation test

BTK

Bruton’s tyrosine kinase

CARPA

Complement activation-related pseudoallergy

EC50

Half-maximal effective concentration

IDHSR

Immediate drug-induced hypersensitivity reactions

FcεRI

High-affinity IgE receptor

FcγR

Fc gamma receptor

IC

Immune complexes

IgE

Immunoglobulin E

sIgE

Specific IgE

MC

Mast cell

MRGPRX2

Mas-related G-protein coupled receptor X2

NMBA

Neuromuscular blocking agent

PAF

Platelet-activating factor

PEG

Polyethylene glycol

SNP

Single nucleotide polymorphism

U.S.

United States