Biomarkers of anaphylaxis

Introduction

According to the international and domestic conciliatory documents that exist today, anaphylaxis (AP) is a severe, life-threatening, generalized or systemic hypersensitivity reaction that develops rapidly and can lead to death [1-3]. The diagnosis of AP continues to be primarily clinical and is verified if the symptoms of a systemic reaction correspond to one or more clinical criteria of AP [1-3]. Every year, a significant number of research works are initiated aimed at a deeper study of the mechanisms of AP development and the search for its biomarkers, which could with a high degree of probability not only confirm the diagnosis of anaphylactic reaction but also be effectively used as predictors of the risk of its development, severe course, and fatal outcome [4-7]. The term “biomarker” has various definitions; most often, it is understood as quantitative characteristics of physical data and/or laboratory parameters for which the boundaries of “norm” and “pathology” are defined [8]. The optimal biomarker should be highly specific, sensitive, predictive, fast and easy to use, affordable, and minimally non-invasive. Currently, specific immunoglobulins of class E (SiGe) can be identified as AP biomarkers, which play a key role in the development of immune IgE-mediated AP, as well as a number of potential nonspecific biomarkers – AP mediators synthesized and released by various populations of effector cells involved in the pathogenesis of immune and non-immune systemic reactions.

Specific immunoglobulins of class E

The mechanism of development of IgE-mediated AP is based on a typical allergic reaction of the immediate type caused by the interaction of SiGe antibodies with relevant allergens. The determination of SiGe in the blood serum of patients with suspected AP is an important and integral stage of allergy examination in order to identify possible clinically significant sensitization and identify the trigger of a systemic reaction. The determination of SiGe is carried out to selective sources (for example, cow's milk, chicken egg, etc.) and/or selected allergen molecules (preferably recombinant) using the ImmunoCAP test system, as well as in some cases to an extensive panel of allergenic molecules (Immuno Solid-phase Allergy Chip (ISAC). It is these diagnostic systems that are used in the vast majority of both clinical and experimental studies on AP, which is determined by their exceptionally high sensitivity and specificity, as well as reproducibility. As a rule, SiGe assessment is carried out no earlier than 4–6 weeks after the postponed episode of a systemic reaction. The listed tests are the most informative in the framework of the diagnosis of food AP; they are also actively used in drug, insect, and other types of AP [4][9][10]. According to the authors’ data, in almost all the patients with food AP, SiGe levels were detected above the threshold values (≥0.35 kU/L) for the presumed food trigger; the concentration of this biomarker varied significantly, but did not correlate with the severity of reactions [11][12]. At the same time, a high degree of sensitization (>100 kU/L) to fish/seafood was associated with inhalation hypersensitivity to this allergen and determined the features of elimination measures (excluding being in a room where fish/seafood is fried, boiled, butchered) to prevent repeated AP episodes in this group of patients [12][13]. In the case of idiopathic AP, the determination of SiGe using the ISAC platform is an effective tool for identifying such hidden triggers as alpha-Gal, omega-5-gliadin of wheat, protein-a non-specific lipid transporter, oleosin (sesame seeds, almonds, peanuts, hazelnuts), and sensitization to allergens of nematodes of the Anisakidae family [14][15]. According to Heaps et al., 20% of patients with idiopathic AP were able to identify causally significant triggers using the ISAC allergy chip [16]. In addition, the important role of determining the level of SiGe to “dangerous” major molecules of a number of food allergens is its effective use in the formation of high-risk groups of patients for the development of systemic (including cross-) reactions, control of the dynamics of sensitization increase or, conversely, the development of tolerance. In particular, a predictive marker of the development of severe, including systemic, reactions to milk is high or increasing sensitization to casein (Bos d 8) [17], chicken egg – sensitization to ovomucoid (Gal d1) [18], peanuts – sensitivity to its recombinant allergens (Aga h1, Aha h2, Aha h3, Aha h6, Aha h9) [19], etc. In turn, with a significant decrease in the dynamics of the level of SiGe to a causally significant food allergen and its specific components, taking into account the absence of AP episodes over the past few years, as well as a complex of other clinical symptoms, in some cases, it is possible to consider the introduction of the product by conducting a provocative test in a hospital.

Thus, SiGe are undoubtedly the key biomarkers of IgE-mediated AP, which are currently actively used in clinical practice not only for differential diagnosis and identification of the trigger of systemic reactions but also for determining the overall risk of AP, including inhalation hypersensitivity to a number of different allergens, as well as the prognosis of tolerance and the possibility of introducing a causally significant food product. At the same time, there are currently many open questions about the relationship between the presence of IgE-mediated sensitization and AP. So, it is unclear why AP occurs only in a part of people with detected sensitization to a certain allergen, which determines the absence of a close correlation between the concentration of SiGe and the severity of the systemic reaction, etc. Of course, these and other clinically significant facts indicate the role of not only SiGe but also other factors in the development of AP.

Non-specific biomarkers of anaphylaxis

Nonspecific AP markers are released by the effector cells involved (mast cells, basophils, macrophages, neutrophils, etc.) directly during the acute period of systemic reactions (Table 1). These mediators have different characteristics with respect to their clinical significance (improved diagnosis, stratification of the severity and risk of AP, response to therapy), the half-life of the features of collecting biomaterial for analysis and subsequent interpretation of the results, which significantly affects the possibility of their application in real clinical practice.

Tryptase is a neutral serine protease contained in the secretory granules of mast cells. Tryptase is produced mainly by mast cells, so the level of this enzyme in plasma is a key biomarker reflecting their activation, including in the acute period of AP, basophils can also release tryptase, but in much smaller quantities. There are two main isoforms of tryptase (α and β). The first (α-tryptase) is released constantly, its concentration in the body is quite stable, and it increases with an increase in the number of mast cells, in particular with mastocytosis, and in the case of activation (degranulation) of mast cells with AP, β-tryptase is released [6]. The content of tryptase in mast cells of various organs varies, which can determine the dependence of the severity of an acute reaction on the pathway of the allergen into the body. Today, diagnostic commercial tests are available to determine total tryptase (α/β-tryptase), which allows evaluating this biomarker in clinical practice. The method of measuring serum tryptase is stable, reliable, and easy to process, but there are limitations for the timing of taking biomaterial. The basal tryptase level in healthy people in the blood serum ranges from 1 to 11 ng/ml (ImmunoCAP).

From the first minutes of the development of AP symptoms, the concentration of tryptase in the blood serum increases sharply, then its increase reaches peak values within 60–90 minutes, followed by its steady decline, followed by disappearance within a few hours [5]. If AP is suspected, in accordance with the recommendations of the conciliation documents, blood samples for the determination of tryptase should be collected twice: the first – in the time interval from 15 minutes to 3 hours after the onset of the first AP symptoms, the second – 24 hours or more after the disappearance of AP symptoms to determine the basal level of the marker [1][2]. Measurement of the basal level of tryptase outside of an acute AP episode is especially relevant for patients in whom, during the acute period of systemic reaction, the concentration of tryptase is fixed in the redistribution of reference values and if it turns out to be higher than the values obtained by the formula [1.2 x baseline level + 2 ng/ml], it can be considered diagnostically significant for AP [1][2].

Based on the results of the research, the clinical significance of determining the level of tryptase as a biomarker of AP is considered in the context of diagnosis, risk of development, and stratification of the severity of AP and depends on a number of factors (trigger, age of patients, symptoms). An increase in the concentration of tryptase is most often observed in the case of insect and medicinal types of AP, while in the case of food AP, the biomarker in question is less potential and is often determined within the normal range, even if blood sampling is carried out at the optimal time. Thus, with the development of perioperative AP in children, a high concentration of blood tryptase is observed in more than half of patients [20][21]. In the case of drug AP, a more significant and prolonged increase in the level of this marker may be noted [22]. According to Ruëff et al., in patients with insect allergy, a high concentration of basal tryptase increases the risk of developing AP during allergen-specific immunotherapy [23]. Serum tryptase levels have been shown to positively correlate with histamine concentration, AP severity, and the development of severe hypotension and skin symptoms (erythema and urticaria) [22][24]. In some patients, the absence of an increase in the level of tryptase in the acute period of AP, but at the same time an increase in histamine may indicate the predominant involvement of basophils in the development of a systemic reaction [25]. When interpreting the results of the tryptase level, it is important to take into account that its normal or slightly elevated concentration may be observed in 36–40% of patients in the acute AP period, which does not exclude this diagnosis [24][26]. In young children, the basal level of tryptase is initially elevated, and only by 9–12 months, it reaches the level of normal values [27]. In adult patients, the concentration of tryptase tends to increase with age [28], and, according to some data, its level in men is higher than in women [29]. In addition, tryptase may increase in patients with an acute allergic reaction occurring in the form of isolated skin symptoms, with systemic mastocytosis, oncological diseases, and a number of other conditions not related to AP [22].

In general, tryptase today is a recognized pathogenetically significant, accessible, and recommended for measurement in routine practice AP biomarker, which has a certain evidence base. However, the existing limitations of the sensitivity of the marker and the period of blood sampling significantly limit and complicate its use.

Histamine is the next important mediator of IgE-mediated allergic reactions, the source of which are granules of mast cells and basophils. The effect of histamine determines the development of most AP symptoms (edema, hyperemia, urticaria, itching, etc.) and correlates with its severity [24], but the informative value of determining the concentration of histamine in plasma and/or its metabolites (N-methylhistamine and N-methylimidazole acetate) in urine as biomarkers of AP remains questionable to date. With the development of AP, the level of histamine in the blood plasma reaches its peak 5 minutes after exposure to the allergen and returns to basal values after 15–30 minutes [30]. In this regard, the use of histamine as a nonspecific marker of AP is possible only when collecting blood samples within the first 15 minutes from the start of the reaction, which is extremely difficult in real practice. A rapid decrease in the concentration of histamine in plasma is associated with its sequential methylation by N-methyltransferase to form N-methylhistamine and its further oxidative deamination under the action of diamine oxidase into N-methylimidazole acetate. The resulting metabolites are stable and can be detected in the urine for 24 hours or more from the onset of AP symptoms and, according to some data, correlate with its severity [26]. Currently, there are commercial kits for this study, but their use in clinical practice is unpopular. In addition, the normal level of these markers does not exclude the diagnosis of AP; false positive test results may be associated with the preliminary consumption of histamine-liberating foods, with systemic mastocytosis [31].

Chymase belongs to a group of relatively new and still poorly studied biomarkers of AP. Chymase is a serine protease that is found in the secretory granules of mast cells. This marker is quite stable in blood serum; it was detected in eight cases of fatal AP at autopsy, its concentration positively correlated with the level of serum tryptase [32]. In a similar work, Osawa et al. identified chymase in lung mast cells during the autopsy of 3 cases of fatal AP, along with the absence of this marker in control autopsy cells without AP [33]. The level of chymase in the blood serum of patients with food, drug, and insect AP was determined higher relative to its concentration in blood samples of the control group within 8 hours from the moment of the development of the first symptoms of AP, and it continued to remain high for at least 24 hours [34].

Along with the listed markers, carboxypeptidase A3 is another potential mediator of AP released by activated mast cells. Brown et al. in their work analyzed the levels of carboxypeptidase A3 in the blood and saliva of 33 patients with suspected drug allergy undergoing provocative tests [35]. Baseline basal levels of carboxypeptidase A3 were higher in the blood serum and saliva of patients with positive provocative tests and in patients who had a history of severe allergic reactions with cardiovascular and/or respiratory symptoms. When analyzing this marker directly at the time of provocation, its level increased only in the saliva of patients with a positive sample. In turn, according to Zhou et al., high levels of carboxypeptidase A3 were also detected in plasma/serum samples of patients collected within eight hours after the onset of a systemic allergic reaction, which in 70% of cases were characterized by normal tryptase concentration [36]. In general, the stability of carboxypeptidase A3, the wide time range for its determination, the prospects of its use in patients with normal tryptase levels, and the noninvasiveness of the method for determining carboxypeptidase A3 in saliva justify further studies regarding the significance of this marker in the framework of AP.

In addition to mast cell mediators, there are promising data on the main factor of basophil chemotaxis – CCL2, responsible for ensuring the influx of basophils to the focus of inflammation after exposure to an allergen. The potential use of CCL-2 as an AP biomarker was studied in the work by Korosec et al., where it was evaluated in blood samples taken directly during the AP episode, 7 and 30 days after the systemic reaction [37]. The study showed that the absolute number of circulating basophils was significantly lower in the acute AP period relative to the non-reaction period. The concentration of CCL-2, on the contrary, was significantly higher in the acute period of AP in comparison with samples taken outside the reaction, while the level of CCL-2 decreased to baseline values within 2 hours after the onset of AP symptoms. The sensitivity and specificity of the test were estimated as 94% and 96%, respectively. The results of a similar study demonstrated not only an increase in CCL-2 in blood serum during the acute period of systemic reactions but also a pronounced positive correlation of this marker with their severity [38].

According to a number of studies, one of the key functions in the pathogenesis of AP is played by platelet activation factor (PAF) and the enzyme acetylhydrolase of platelet activation factor (PAF-AG) that cleaves it. PAF is a pro-inflammatory phospholipid that is synthesized and secreted by mast cells, monocytes, and tissue macrophages. The effects of PAF in AP largely determine the symptoms of the cardiovascular system (hypovolemia, increased vascular permeability, myocardial dysfunction, etc.). According to a series of research papers, at the time of an anaphylactic attack, high levels of PAF and low levels of PAF-AG are detected in the blood serum of patients, which correlate with the severity of AP [39-41] to a greater extent than tryptase and histamine. While analyzing the basal level of PAF-AG in patients with insectic AP (outside of acute reaction), similar results were demonstrated. One of the important disadvantages of PAF is an extremely short period of life (3–15 minutes) from the moment of the AP symptoms development, which makes it almost impossible to use this marker in clinical practice.

To date, there are reports demonstrating the protective role of angiotensin-converting enzyme (ACE) in relation to the development of life-threatening edema in AP [42][43]. Summers et al. note that in patients with a history of severe pharyngeal and laryngeal edema with the development of acute allergic reactions to food allergens, basal ACE levels in blood serum significantly reduced compared to patients without a history of similar symptoms [44]. A decrease in ACE concentration in blood serum increased the relative risk of severe, life-threatening edema of the pharynx and larynx tenfold in these patients.

Among the less studied markers of the acute period of AP, dipeptidyl peptidase 1 (DPP1) can be distinguished, since the concentration of it in the blood serum in AP positively correlates with the level of chymase and presumably activates it [34][45]. As an alternative to histamine as a serum marker of AP, the role of the functionally similar protein of basogranulin basophil granules is being actively studied [46]. In the pathogenesis of anaphylactic reaction, various processes of heparin release are involved, which leads to the activation of the complement system and factor XII, determines kininogen proteolysis and bradykinin release, while the degree of activation of the complement system and the level of bradykinin correlate with the severity of AP [26][47]. In addition, there is evidence indicating the potential significance of determining the concentration of leukotriene E4 (LTE4) and 11-beta-prostaglandin F2-alpha in urine [48]. The level of a number of other inflammatory mediators in blood plasma, such as IL-2, IL-4, IL-5, IL-6, IL-10, IL-13, interferon-gamma (IFN-γ), tumor necrosis factor alpha (TNF-α), may be increased in patients with AP and correlate with its severity, but the sensitivity and specificity of these markers have not yet been established [24]

The significant disadvantages of most of the above-mentioned nonspecific biomarkers of an acute AP episode are their conditional stability and a limited time period for collecting biomaterial samples, which introduces certain difficulties for their application and interpretation. In this regard, potential genetic AP biomarkers are being actively studied and identified, which have good stability, have no time limits, can be measured outside the acute period of AP, and, therefore, are potentially relevant for the prevention of systemic reactions and the formation of groups of patients with a high risk of severe AP. In particular, mutations of the C-KIT gene regulating mast cell function were associated not only with mastocytosis but also with cases of idiopathic AP [49]. Górska et al. revealed an association of increased expression of the factor 4 gene associated with the tumor necrosis factor receptor (TRAF-4) with the development of nutritional AP in patients with mastocytosis [50]. Some studies have demonstrated the connection of AP with changes in the functioning of genes encoding interleukins, which are crucial in regulating the balance of Th1/Th2 cellular response. Thus, single nucleotide polymorphisms of genes encoding IL4 and IL-10 were detected in patients with AP to penicillin [51][52]. According to the studies conducted by the authors of this paper, in children with nutritional AP (outside the acute episode), a significant decrease in the expression of the platelet activation factor acetylhydrolase gene (PLA2G7) was determined in comparison with the control group without AP [53]. In addition, a negative correlation was found between the severity of AP and the expression of the gene under study, and a relationship was established in the form of a significant decrease in its expression in patients with a history of clinical symptoms from the cardiovascular system (drop in blood pressure, pronounced tachycardia/bradycardia) with the development of AP. In the course of this work, interesting data were obtained regarding the expression of the ACE gene [53]. The frequency of determining the expression of this gene in patients with a history of mild and localized edema with or without AP was significantly higher in comparison with patients with AP accompanied by life-threatening edema of the tongue and soft tissues of the oropharynx.

Conclusion

The results of numerous studies obviously confirm the fact that the spectrum of immune cells and mediators involved in the pathogenesis of anaphylactic reactions is extremely complex and diverse. The search and introduction into clinical practice of potential AP biomarkers is undoubtedly an important tool to facilitate the verification of diagnosis, prevention and risk assessment of repeated episodes of AP, stratification of the severity of its course, the risk of life-threatening/lethal episodes of systemic reactions and may be of key importance in the development of new therapeutic strategies.