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Human mast cell tryptase in biology and medicine夽 Joana Vitte ∗ Laboratoire d’Immunologie Hôpital de la Conception, 147 Bd Baille, 13005 Marseille, France; Aix-Marseille University, INSERM UMR 1067/CNRS UMR 7333, Marseille, France

a r t i c l e

i n f o

Article history: Received 15 December 2013 Received in revised form 1 April 2014 Accepted 2 April 2014 Available online xxx Keywords: Tryptase Mast cell Mast cell degranulation Anaphylaxis Mastocytosis

a b s t r a c t The most abundant prestored enzyme of human mast cell secretory granules is the serine-protease tryptase. In humans, there are four tryptase isoforms, but only two of them, namely the alpha and beta tryptases, are known as medically important. Low levels of continuous tryptase production as an immature monomer makes up the major part of the baseline serum tryptase levels, while transient release of mature tetrameric tryptase upon mast cell degranulation accounts for the anaphylactic rise of serum tryptase levels. Serum tryptase determination contributes to the diagnosis or monitoring of mast cell disorders including mast cell activation – induced anaphylaxis, mastocytosis and a number of myeloproliferative conditions with mast cell lineage involvement. Baseline serum tryptase levels are predictive of the severity risk in some allergic conditions. © 2014 Elsevier Ltd. All rights reserved.

1. Introduction Mast cells (MC) are immune cells of haemopoietic origin. They belong to the innate arm of the immune system and display a number of morphological and functional similarities with basophils, including a common tryptase-releasing precursor cell in 14-million year old Urochordates (Voehringer, 2013). In modern mammals, most MC line up interfaces between the organism and the environment, such as skin, bronchi, and gut, which are an ideal location for MC performing their pathogen-sensing sentinel functions. MC contribute to immune regulation, spanning from the initiation of pathogen clearance to the resolution of inflammation (StJohn and Abraham, 2013). Despite these very recent findings, MC most prominent involvement in current medical practice is still represented by the effector phase of immediate allergic responses: massive degranulation with release of prestored mediators, in response to a potent stimulus whose paradigm is the aggregation of immunoglobulin (Ig) E-bearing Fc␧RI by a multivalent allergen, followed by a second-wave of delayed mediator secretion (Kumar and Sharma, 2010). During IgE-induced degranulation, mast cell secretory granules (SG) fuse among them and with the plasma membrane, thus opening into the extracellular space (Lorentz et al., 2012). SG

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release dozens of preformed mediators, including mature tryptase (Theoharides et al., 2007; Lundequist and Pejler, 2011). SG storage of tryptase and other MC proteases establishes a physical separation between (i) IgE-induced MC degranulation, which engages the SG compartment of very large granules (up to 0.5–1 ␮m in diameter), and (ii) cytokine production and exocytosis, a process that responds to distinct stimuli and involves small secretion vesicles, usually 80 nm or less (Blank, 2011). Tryptases are a family of trypsin-like serine-proteases present in large amounts in the secretory granules of human and animal MC (McNeil et al., 2007; Trivedi and Caughey, 2010; Schwartz, 2006). Similar to chymases, another group of MC serine-protease, tryptases are highly specific of MC. Detection of tryptase or chymase provides information about MC distribution, numbers, activation status or releasability, depending upon the clinical or experimental context (Valent, 2013). Currently, in human medicine there is only one immunoassay for in vitro diagnosis allowing tryptase detection and measurement in body fluids (Schwartz et al., 1994), while no equivalent exists for chymase. Given the tissue resident situation of mast cells, this paucity of specific humoral markers lends value to tryptase measurement and explains current focus on its pathophysiology. 2. Tryptase genetics, structure, and functions Human tryptase genes are clustered on chromosome 16p13.3 and comprise five loci. TPSG1 encodes gamma-tryptase, TPSB2 encodes betaII and betaIII-tryptase, TPSAB1 codes for alphatryptase and betaI-tryptase, TPSD1 accounts for delta-tryptase, and TPSE1 encodes epsilon-tryptase (Fukuoka and Schwartz, 2007;

http://dx.doi.org/10.1016/j.molimm.2014.04.001 0161-5890/© 2014 Elsevier Ltd. All rights reserved.

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Trivedi et al., 2008). In the great ape lineage including humans, TPSD1 is inactivated, and the human ␧-tryptase is biochemically and immunologically distinct from ␣ and ␤ tryptases. TPSAB1, TPSB1, and TPSG1 genes are transcribed and translated as monomeric zymogens. These zymogens need proteolytic activation in order to become functional. ␥-Tryptase presents as a SG membrane-anchored protease, reaching the plasma membrane upon MC activation and degranulation. ␣- and ␤-tryptase are soluble proteases, and proteolytic activation leads them to assemble into a mature tetrameric form. This process takes place normally with ␤-tryptases, whose mature, active tetramers are packed in SG together with a scaffold of proteoglycans, mainly heparin. A small portion of the ␤-tryptase monomer production escapes proteolytic activation and SG storage. Immature, monomeric ␤tryptase then enters the constitutive secretion pathway and is released by unstimulated MC, thus contributing to each individual’s baseline systemic tryptase level (Caughey, 2006). In some cases, ␤tryptase monomers exposed to acidic pH may exhibit enzymatic activity, but as a rule ␤-tryptase activity is limited to tetrameric forms, whose catalytic site is protected from the action of natural inhibitors, embedded at the junction of the four subunits (Fukuoka and Schwartz, 2006, 2007). In contrast, production and maturation of ␣-tryptase is constantly hampered, as the ␣-tryptase gene is subject to various mutations, resulting in deficient transcription, zymogen activation, catalytic site conformation, and even deletion of the gene as a whole. Indeed, ␣-tryptase deletion was reported in 30% of a study population of 274 individuals (Soto et al., 2002), and up to 57% in a UK-based population (Abdelmotelb et al., 2013). ␣- and ␤-tryptase gene polymorphisms are numerous, and the number of functional tryptase alleles an individual may carry varies from two to four (Trivedi et al., 2009). Although it seems that complete tryptase deficiency is not seen in human populations, the number and type of functional alleles carried by an individual may alter the baseline systemic tryptase levels. Frequencies of haplotypes of the two loci on chromosome 16 were 50% for ␤␤/␤␣, 29–25% ␤␤/␤␤ and 21–25% of ␤␣/␤␣ (Soto et al., 2002; Schwartz, 2006). Baseline tryptase levels are often used as a surrogate for an individual’s mast cell burden, and therefore genetic variations may interfere with assumptions made on this basis (Soto et al., 2002; Trivedi et al., 2008, 2009). In particular, the diagnosis of mastocytosis may be more difficult in patients with less functional alleles, especially for alpha tryptase. Genetics influences indeed the baseline serum levels of tryptase (Min et al., 2004; Sverrild et al., 2013; Lyons et al., 2014). Mature ␤-tryptase is stabilized by heparin packing in SG (Sakai et al., 1996). Abnormal heparin formation alters mature tryptase contents of mast cells. In experimental murine models of heparanase overexpression, shorter heparin molecules were associated with a diminished content of MC proteases and a diminished release of proteases upon MC degranulation. Conversely, heparanase deficiency was associated with bigger heparin chains and an increased storage of MC proteases (Wang et al., 2011). Upon MC activation, release of SG contents into the extracellular space is a matter of minutes. Histamine, the historical hallmark of SG degranulation from mast cells, may be detected in peripheral blood in the first five minutes of MC degranulation-induced symptoms, while tryptase detection is delayed by 15 or 20 min due to its cumbersome heparin scaffold. This distinction must not be overlooked in human medicine, because it explains why histamine and tryptase are not optimally measured in the same blood sample. Indeed, while histamine may have raised to its acme by 5–10 min after the onset of anaphylaxis symptoms, tryptase measurement at such early time points often results in values of less than 12 ␮g/L, which are erroneously considered as “normal” or even “negative”.

Fig. 1. Production and intracellular trafficking of human ␣-, ␤- and ␥-tryptases in mast cells. Baseline serum tryptase levels are the result of continuous release of immature ␣- and ␤-tryptase monomers; mature tetrameric ␤-tryptase is stored in specialized compartments called secretory granules, where it is stabilized by means of proteoglycan, mostly heparin, interaction. Mature ␤-tryptase is not released continously, but as a result of mast cell activation. Thus, serum tryptase levels measured after mast cell degranulation comprise both immature and mature forms of ␣- and ␤-tryptase. ␥-tryptase is a membrane-bound monomer.

Production, intracellular trafficking and relative contribution of mast cell tryptases to serum levels are summarized in Fig. 1. From a functional viewpoint, mature tryptase exerts sequential actions. Following MC degranulation, tryptase acts as a vasoactive, proinflammatory, chemotactic and priming agent, but at the end of the process, tryptase stimulates tissue repair (Miller and Pemberton, 2002; Heutinck et al., 2010; MacLachlan et al., 2008; McNeil et al., 2007; Van der Linden et al., 1992). Under the effect of degranulated tryptase, generation of bradykinins from kininogens promote vascular permeability, while extracellular matrix is degraded, thus facilitating cellular migration. Tryptase induces leukocyte recruitment and activation, with special chemotactic effects on neutrophils and eosinophils, both involved in allergic-related late phase inflammation. Tryptase contributes to the crosstalk between mast cells and the mononuclear phagocytic system, leading to monocyte and macrophage activation. Finally, tryptase stimulates fibroblast proliferation and collagen synthesis, which contribute to tissue repair and restitutio ad integrum (Frungieri et al., 2002; Chen et al., 2014). More recent data indicate a role for tryptase in pain triggering, such as post-operative pain due to nociceptive protease-activated receptors activation (Oliveira et al., 2013). Functional tryptase is mostly tetrameric, a condition that is generally fulfilled by retaining the proteoglycan association (Miller and Pemberton, 2002). Research has been traditionally focusing almost exclusively on tryptase as an extracellular mediator, but very recent data indicate a role for tryptase in the homeostasy of mast cell nuclear histones and in the desorganisation of histone scaffolds during cell death (Melo et al., 2014). Human tryptase is considered as virtually specific of mast cells, which may contain high amounts, up to 35 pg per cell (Schwartz et al., 1987). Basophils also contain and release tryptase (Castells et al., 1987), but they do not represent important contributors to tryptase levels, according to reports which showed their tryptase contents was one hundred times less than that of mast cells (Foster et al., 2002; Jogie-Brahim et al., 2004). Tryptase load is highly variable both in mast cells depending on their microenvironment, and in basophils, with a range from 1 to 100 in different human donors (Foster et al., 2002). The latter study demonstrated the ability of human basophils to release tryptase following IgE-mediated

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activation, but the pathophysiological implications of this issue have not been clarified yet. According to tryptase synthesis and exocytosis as described above, it is important to keep in mind that the circulating tryptase level measured at any time point in a given individual is the combined result of the total number of mast cells (“mast cell burden”), of their genetically determined level of alpha and beta tryptase production and of their activation status resulting in mature tryptase release. In the next chapter I will explain why the only available current tryptase immunoassay fails to address precisely any of these three points, yet is gaining importance in laboratory work-up of an increasing number of clinical conditions.

3. Tryptase as a mast cell laboratory marker in human disease 3.1. Tryptase determination and reference values There is only one commercial method currently available for the determination of human tryptase in body fluids (ImmunoCAP Tryptase, ThermoFisher Scientific Diagnostics, Phadia, Sweden). Determination is certified for serum and plasma, although other fluids may be assayed (such as tears, cell culture supernatants, etc.). This method measures the total concentration of ␣- and ␤-tryptases in both mature (tetrameric) and immature (monomeric) forms (Schwartz et al., 1994). Since there is no distinction between alpha and beta, neither between mature and immature forms, current tryptase determination depends on both the size and the activation status of an individual’s mast cell population but is not directly informative on the contribution of any of these factors. With the current method, the median value of serum baseline tryptase in adults is assumed to be 3.8 ␮g/L, with 95% of healthy individuals displaying tryptase values of 11.4 ␮g/L or less, according to ThermoFisher data obtained in 126 apparently healthy donors (61 males and 65 females, aged 12–61 years) (http://www.phadia.com/en-GB/5/Products/ImmunoCAP-Assays/ ImmunoCAP-Tryptase/). Tryptase values are stable in a given donor over time, in the absence of mast cell activation or mast cell disease. In children, reference values for tryptase were established only recently. Children aged 6 months–18 years display a median tryptase value of 3.5 ␮g/L, as assessed by a study conducted on 197 US children (Komarow et al., 2009), and by two others conducted on a total of 213 French children (Ibrahim et al., 2009; Belhocine et al., 2011). Serum tryptase levels seem to be very similar in adults and in children older than 6 months, but this is not the case in young infants and newborns. According to a study from our team, infants display significantly higher levels of serum tryptase during their first three months of life, with a median level of 6.1 ␮g/L, then tryptase levels gradually decrease (Belhocine et al., 2011). In elderly patients, tryptase levels were reported to be slightly but significantly increased, with a median of 4 ␮g/L in donors aged 18–30 years and 6.6 ␮g/L in those older than 80 years (GonzalezQuintela et al., 2010; Blum et al., 2011). The reason for this increase is currently unknown. The first hypothesis was a diminished renal excretion of tryptase, which seemed a good hypothesis given that tryptase levels increase during renal failure (Sirvent et al., 2010), a common condition among elderly patients, but no tryptase urinary excretion was found in a subsequent study (Simon et al., 2010). While the causes of such an increase during aging are still to be found, tryptase elevation with age should be taken into account when tryptase levels are used as a risk marker for anaphylaxis or other severe allergic symptoms (see Section 3.3 below). Reports on the effect of gender are conflicting; a higher body mass index is associated with a slight increase in levels of serum baseline tryptase (Min et al., 2004; Gonzalez-Quintela et al., 2010).

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Technically, tryptase measurement is highly specific, although falsely increased tryptase levels in sera containing heterophilic antibodies such as rheumatoid factor have been reported (Sargur et al., 2011). Such interferences are rare events and have a minor impact on measured tryptase levels (Schliemann et al., 2012). 3.2. Clinical implications of mast cell tryptase determination For some 20 years, tryptase has been looked at mainly as a dual marker of either acute allergic reactions or baseline mast cell status, besides a more restricted use in the field of haematologic malignancies. According to the recently released global unifying classification of mast cell disorders (Valent et al., 2012), diagnostic frameworks have been reshaped into four major categories: mast cell activation syndrome, mastocytosis, mast cell hyperplasia, and myelomastocytic conditions (Valent et al., 2012). The mast cell activation syndrome (MCAS) is defined as a recurrent association of (i) typical symptoms, (ii) a transient increase in serum tryptase levels (or another established mast cell mediator) and (iii) clinical responsiveness to mediator-targeting or mast cell-stabilizing drugs (Valent et al., 2012; Valent, 2013). MCAS are further divided into primary MCAS (monoclonal KIT-mutated mast cells), secondary MCAS (IgE-dependent allergy or another inflammatory disease), and idiopathic MCAS (no underlying disease and no clonal mast cell abnormality). Clonal mast cells proliferation and accumulation in various tissues and organs (skin, bone marrow, etc.) is typical of mastocytosis. Cutaneous and systemic mastocytosis are best known forms, but there is great heterogeneity among clinical presentations, underlying mast cell mutations, and laboratory findings including serum tryptase (Bonadonna et al., 2009; Valent et al., 2012; Alvarez-Twose et al., 2014). In the light of this classification, the pathophysiological link between serum baseline tryptase and the risk of severe mast cell activation symptoms is emphasized (e.g., association of two distinct mast cell disorders, such as mastocytosis and a secondary MCAS). Serum tryptase is confirmed as the indispensable laboratory marker of mast cell disorders; moreover, its chronology and interpretation are rigorously defined in order to provide full and appropriate information (Valent et al., 2011). These advances in mast cell and serum tryptase understanding are presented below. A simple diagram of serum tryptase determination in medical practice is presented in Fig. 2. 3.3. Serum baseline tryptase and risk of severe allergic symptoms As predicted by Schwartz in early papers (Schwartz et al., 1994), serum baseline tryptase determination is indicative of an individual’s risk of severe allergic manifestations. The pathophysiologic culprit seemed to be beta tryptase in early years (Soto et al., 2002); recently, immature tryptase has been demonstrated in a novel, nonclonal, genetically inherited association of elevated serum tryptase and atopy (Lyons et al., 2014). Nevertheless, in an overwhelming majority of patients, clonal mast cell disorders are the underlying condition of an elevated serum baseline tryptase (Metcalfe and Schwartz, 2009; Bonadonna et al., 2009; Alvarez-Twose et al., 2010, 2011, 2014; Valent et al., 2012). The link between mastocytosis and severe Hymenopterainduced mast cell activation symptoms was first reported in 1983 (Muller et al., 1983), and the first cohort study was published in 2001 (Ludolph-Hauser et al., 2001). In this study, 9/12 (75%) patients with raised serum baseline tryptase and 28/102 (28%) patients with normal serum baseline tryptase, the cut-off level being set at 13.5 ␮g/L, experienced severe anaphylactic reactions after Hymenoptera stings. Careful skin re-examination of patients with severe anaphylaxis resulted in a diagnosis of cutaneous

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Fig. 2. Human serum tryptase determination: when, how, why. Serum tryptase determination in mast cell disorders is a key finding, provided it is measured at the right time and interpreted according to clinical data. Transient elevation of serum tryptase is pathognomonic of mast cell activation, while serum baseline tryptase is useful in mastocytosis work-up, in assessing the risk of severe allergic reactions, in establishing the diagnosis and prognosis of haematologic disorders with mast cell lineage involvement.

mastocytosis in 11/12 patients with raised serum baseline tryptase and in 2/28 with normal serum baseline tryptase. The main findings of this study, namely the importance of mastocytosis as a risk factor for severe Hymenoptera sting IgE-dependent mast cell activation symptoms, including cases when serum baseline tryptase is not elevated, were confirmed by subsequent publications in both adults and children (Haeberli et al., 2003; Bonadonna et al., 2009; AlvarezTwose et al., 2010, 2011, 2014). One of the most recent papers in the field (Alvarez-Twose et al., 2014) described the privileged links between Hymenoptera and other insect-induced anaphylaxis and indolent systemic mastocytosis without cutaneous involvement. This association is often difficult to recognize, because such patients usually present with a proteiform array of mast cell activation symptoms, frequent venom IgE sensitization, and no cutaneous sign of mastocytosis (Alvarez-Twose et al., 2014). Currently, the understanding of serum baseline tryptase as a factor predictive of severe allergic reactions is evolving to consider as a continuous variable, rather than a discrete one. In other words, serum baseline tryptase distributed along the whole range of measurable values will define low, medium, and high risk patients. The idea of a cut-off between “normal” and “raised” serum baseline tryptase may well become obsolete. For example, considering an odds ratio of 1 for a serum baseline tryptase level of 5.5 ␮g/L, Ruëff and coworkers found an odds ratio of 2 associated with a serum baseline tryptase level of 12 ␮g/L (Ruëff et al., 2009). A higher risk is reported for much lower tryptase values during the induction phase of specific immunotherapy for Vespula: odds ratios reach 2 for a tryptase level of 7.5 ␮g/L, 5 for 10 ␮g/L, and 10 for 20 ␮g/L (Ruëff et al., 2010). These findings also hold true for children, with baseline tryptase levels of 4.8 ␮g/mL and higher predictive of a 50% probability of severe sting-induced systemic reaction, and levels of 8 ␮g/L and higher associated with a 75% probability (Yavuz et al., 2013). This values are reminiscent of those established in a pediatric population of mastocytosis patients, with a serum baseline tryptase cut-off of 6.6 ␮g/L predictive of grade 2 or higher mast cell activation symptoms, while a cut-off at 16 ␮g/L was predictive of grade 4 symptoms (Alvarez-Twose et al., 2012). In elderly, whose baseline tryptase levels are increased as compared with younger patients and continue to increase with age, the severity of sting reactions follows a parallel pattern (Guenova et al., 2010), suggesting the need for therapeutic adjustments as a function of age-related tryptase changes.

The link between serum tryptase levels and severity risk in Hymenoptera allergy was puzzled by a recent publication showing that the prevalence of Hymenoptera sting-induced anaphylaxis in mastocytosis patients is bell-shaped, with a maximum of about 50% for a serum tryptase level of 28 ␮g/L (Van Anrooij et al., 2013). This finding suggests a complex regulation of mast cell activation during mastocytosis. Interestingly, the bell-shaped curve is similar to that of IgE-induced mast cell activation, where changes in signal transduction result in diminished mast cell mediator release following supraoptimal allergen stimulation (Huber, 2013). Serum baseline tryptase level as a predictor of clinical severity is not restricted to insect sting. Food allergy in children has been related to higher levels of baseline tryptase (Sahiner et al., 2014). Notably, in food-allergic pediatric patients, serum baseline tryptase levels of 5.7 ␮g/L or higher predict a 50% probability of food-induced anaphylaxis, with an allergen-dependent pattern involving peanut or tree nut allergy more often than milk or egg allergy (Sahiner et al., 2014). These findings, which are expected to bring aid to monitoring and therapeutic decision in food allergic patients, are contrasting with common findings in food-induced systemic allergic reactions. Indeed, serum tryptase level elevation from baseline is generally not prominent and may be lacking in such cases. This may be a paradigm of primarily organ-based allergic reactions (gastrointestinal, bronchial, conjunctival, etc.) as opposed to primarily systemic ones (insect sting, injected drugs). Support for this view comes from a number of studies reporting local tryptase elevation in the affected tissue. For instance, tissue tryptase contents is increased in gut mucosa of food allergic patients (Hagel et al., 2013), in effusion of allergic otitis media patients (Jang and Jung, 2003), in nasal secretion of allergic rhinitis patients (Rondon et al., 2012) and in tears (Leonardi, 2013). Similar to other clinical settings, tryptase measurements correlate with mast cell activation status and are found to diminish during allergy remission (Hagel et al., 2013). 3.4. Serum tryptase in anaphylaxis Anaphylaxis is a medical emergency defined as a serious, rapid in onset, life-threatening, generalized or systemic hypersensitivity reaction, whose diagnosis is based on clinical criteria (Simons et al., 2011, 2012, 2013; Simon et al., 2012). In the light of the global classification (Valent et al., 2012) of mast cell disorders, anaphylaxis, which in most human cases is assumed to be IgE mediated, reflects severe mast cell activation. Laboratory tests are not universally available, not performed on an emergency basis, and not specific for anaphylaxis (Simons et al., 2011). Serum tryptase determination as a support of the clinical diagnosis of anaphylaxis is highly valuable if correctly performed (Valent et al., 2011, 2012). Conversely, tryptase determination may be completely useless if blood collection timing is inadequate. Therefore, the two keys for informative serum tryptase determination as an anaphylaxis marker are (i) comparison of each patient’s peak serum tryptase level with his (or her) own baseline tryptase levels and (ii) knowledge of serum tryptase kinetics. The first key is the comparison of each patient’s peak serum tryptase level with his or her own baseline tryptase levels (Schwartz et al., 1989, 1994; Stone et al., 2009; Borer-Reinhold et al., 2011; Valent et al., 2012; Vitte and Bongrand, 2013) and not with the 11.4 ␮g/L upper limit of current reference values (Vadas et al., 2013; Sala-Cunill et al., 2013). This is a crucial point, because a “normal” tryptase level in an anaphylactic patient may conceal an important rise when compared with the baseline value of that precise patient. Therefore, when assessing serum tryptase for anaphylaxis diagnosis, sequential tryptase determination is mandatory, with at least two blood samples: peak and baseline.

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Table 1 Examples of tryptase measures in anaphylactic patients. Peak and baseline tryptase levels are indicated, together with the etiology as established after allergologic work-up. Age

Gender

Initial healthcare setting

Peak serum tryptase (30 min to 2 h after the onset of symptoms) (␮g/L)

Baseline serum tryptase (24 h after the onset of symptoms) (␮g/L)

Etiologic agent as established after allergologic assessment

9 4 58 31 22 55

F F M F M M

Emergency Emergency Anesthesia Obstetrics Oncology Oncology

5.36 14.4 92 2.1 7.2 41

3.84 4.2 6 1 3.5 7

Soy Kiwifruit Suxamethonium Latex Omeprazole Irinotecan

An elevation of serum tryptase of no more than 135% or 2 ␮g/L may predict true anaphylaxis (Stone et al., 2009; Borer-Reinhold et al., 2011). A more stringent criterion of a two-fold increase is also in use (Schwartz et al., 1994; Berroa et al., 2014), giving the advantage of increased specificity. The other side of the coin is decreased sensitivity, which in this case may leave as many as 60% of the general population underdiagnosed or misdiagnosed in case of true mast cell activation reactions (Borer-Reinhold et al., 2011). Finally, the consensus meeting on mast cell disorders has proposed a value of 120% baseline plus 2 ␮g/L (Valent et al., 2012). For example, anaphylaxis in an individual with a peak tryptase value of 8 ␮g/L and a baseline value of 5 ␮g/L is accompanied by confirmed mast cell activation (Valent, 2013). The amplitude of tryptase elevation varies as a function of anaphylaxis etiology and severity. Tryptase elevation is usually more pronounced in drug, anesthetic and insect sting-induced anaphylaxis than in food-induced anaphylaxis. Severe anaphylaxis is more likely to be associated with higher tryptase levels (Van der Linden et al., 1992; Sala-Cunill et al., 2013; Vadas et al., 2013). Serum baseline tryptase is extremely stable over time in each individual, allowing for comparison between peak and baseline. The second paramount point is serum tryptase kinetics, which needs to be considered for blood collection timing. MC degranulation results in the release of mature tryptase tetramers in a proteoglycan frame. Diffusion of these complexes from tissueresident MC to peripheral blood is slower than that of small molecules such as histamine, a single aminoacid derivative. Therefore, elevation of serum tryptase concentrations may not be detectable during the first 15 or 30 min after the onset of an allergic systemic reaction (Schwartz et al., 1989, 1994). Once in the blood flow, the half-life of tryptase is about two hours (Schwartz et al., 1989). Thus, the optimal delay for peak tryptase collection is best estimated between 30 min and 2 h after the onset of anaphylaxis. This time interval is long enough to allow degranulated tryptase to reach peripheral blood, and short enough to avoid missing an elevated serum tryptase due to serum clearance. Peak serum tryptase determination in this time frame reduces the risk of false negative results yielded by too early or too late peak serum tryptase measurements. Because anaphylaxis is usually unexpected, most patients do not have a pre-anaphylaxis tryptase determination. It is therefore important to draw a second blood sample for comparison. Considering a serum tryptase half-life of 2 h and a peak serum tryptase level that may reach 100 ␮g/L or more, a significant decrease in tryptase levels is usually seen 24 h after the anaphylactic episode. Drawing a second blood sample at 24 h is easy to remember, may coincide with the end of clinical post-anaphylaxis monitoring in a healthcare setting, and may allow simultaneous measurement of paired peak and delayed tryptase levels in the laboratory, which minimizes technical variations. Nevertheless, serum tryptase may not reach the “true” baseline values until 48–72 h after the onset of symptoms, due to very high peak values or prolonged mast cell activation. Therefore, accurate baseline tryptase assessment is ideally performed more than 48–72 h after the end of mast cell activation induced symptoms. In practice, determination of serum tryptase

baseline value can also be done during the allergy/immunology follow-up which is strongly recommended for patients who have experienced an anaphylactic episode (Simons et al., 2011). In some cases, serum tryptase levels remain elevated. This may be a circumstance of discovery of mastocytosis or another mast cell disorder. In short, serum tryptase determination in anaphylaxis must be done on a sequential basis. The peak tryptase value is measured optimally between 30 min and 2 h after the onset of anaphylaxis symptoms, and must be compared to a delayed measure, for instance at 24 h in the absence of symptoms. The accurate baseline value is best measured at least 48–72 h after the anaphylaxis episode. There must be no such conclusion as “negative tryptase” if the peak value is 11.4 ␮g/L or less in the absence of a baseline determination, since each individual’s peak tryptase value has to be compared to his or her own baseline value. The relative elevation of serum tryptase level that is predictive of anaphylaxis may be as low as 135%, or 120% + 2 ␮g/L. Consensual work in the field is needed, but in the meantime each team should use sequential tryptase measurement protocols based on local experience and feasibility. Table 1 shows examples of tryptase values in anaphylactic patients from our experience.

3.5. Serum tryptase in haematologic disorders Systemic mastocytosis is a hematological disorder characterized by the clonal proliferation of abnormal mast cells (Carter et al., 2014). Basally secreted tryptase from these cells may induce an elevation of serum baseline tryptase levels in systemic mastocytosis patients, which is a minor diagnostic criteria for mastocytosis. It is widely accepted that increased serum baseline tryptase levels over 20 ␮g/L are suggestive of mastocytosis (Horny et al., 2008; Valent et al., 2012); nevertheless, as previously explained, serum baseline tryptase may be viewed as a continuous variable, with recent data suggesting that a lower threshold of 11.4 ␮g/L may be more efficient (Metcalfe and Schwartz, 2009). Finally, 5–7% of indolent systemic mastocytosis patients display serum baseline tryptase levels under 11.4 ␮g/L (Alvarez-Twose et al., 2014). During mastocytosis, serum tryptase levels reflect the burden of mature mast cells, while the other common marker of the disease, the mutation D816V+ carried by the KIT gene (CD117), is less specific of mature mast cells (Kristensen et al., 2013). Monitoring of serum baseline tryptase evolution has prognostic value, either good when tryptase levels are stable over time or poor in case of increasing tryptase levels (Matito et al., 2013). Elevated tryptase levels are associated with bone sclerosis in mastocytosis patients (Kushnir-Sukhov et al., 2006). Increase of serum tryptase levels in systemic mastocytosis may reflect transition to more aggressive forms, including mast cell leukemia. Other myeloid leukemias may bear the D816V+ mutation and present with increased serum tryptase levels, whose monitoring contributes to the diagnosis and follow-up of such malignancies (Valent et al., 2012; Sperr et al., 2009). Finally, due to the involvement of mast cells, elevated sBT and imatinib responsiveness have been demonstrated in a subset

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of idiopathic hypereosinophilic syndromes with myeloproliferative features (Klion et al., 2003). 3.6. Post mortem serum tryptase determination Elevated levels of tryptase in postmortem sera were reported as early as 1991 (Yunginger et al., 1991), raising the possibility of postmortem diagnosis of anaphylaxis and suggesting that a number of unexplained deaths, including some sudden infant deaths, may be due to mast cell activation (Pumphrey and Roberts, 2000; Buckley et al., 2001; Edston et al., 2007). This still holds true today, although it has been demonstrated that postmortem tryptase levels may be raised in cases of pre-mortem heavy trauma, myocardial infarction, death due to asphyxia, and pulmonary damage. The postmortem tryptase level considered to be predictive of an anaphylactic cause of death is constantly rising, from 44.5 ␮g/L (Edston et al., 2007) to 110 ␮g/L (McLean-Tooke et al., 2014). 4. Concluding remarks and future perspectives Mast cell tryptases belong to the family of mast cell serine proteases. In healthy individuals, serum baseline tryptase levels are very stable over time. Serum baseline tryptase levels provide information about potential underlying mast cell disorders which increase the risk of severe allergic symptoms. Even tryptase levels within the conventional “normal range” have an impact on allergic risk; moreover, such tryptase levels may also correspond to peak anaphylaxis values. Therefore, it is of paramount importance to proceed to sequential peak and baseline serum tryptase measurements in acute situations and especially during anaphylaxis assessment. Tryptases display a wide array of genetic, functional, and pathophysiological variations which are being unraveled at a rapid pace. In contrast, the only current means of exploring human tryptases is the very limited, although robust, ELISA assay of 1994 (Schwartz et al., 1994). In the future, progress accomplished on the front of clinical mast cell disorders needs to be accompanied by novel laboratory methods, allowing for more accurate clinical assessment of the multifaceted complexity of human tryptase biology. Acknowledgement The author is very grateful for the Reviewers’ comments and suggestions, which helped her correct and improve the manuscript. References Abdelmotelb, A.M., Rose-Zerilli, M.J., Barton, S.J., et al., 2013. Alpha-tryptase gene variation is associated with levels of circulating IgE and lung function in asthma. Clin. Exp. Allergy, http://dx.doi.org/10.1111/cea.12259 (online). Alvarez-Twose, I., Gonzalez de Olano, D., Sanchez-Munoz, L., et al., 2010. Clinical, biological and molecular characteristics of systemic mast cell disorders presenting with severe mediator-related symptoms. J. Allergy Clin. Immunol. 125, 1269–1278. Alvarez-Twose, I., Gonzalez-de-Olano, D., Sanchez-Munoz, L., et al., 2011. Validation of the REMA score for predicting mast cell clonality and systemic mastocytosis in patients with systemic mast cell activation symptoms. Int. Arch. Allergy Immunol. 157, 275–280. Alvarez-Twose, I., Vano-Galvan, S., Sanchez-Munoz, L., et al., 2012. Increased serum baseline tryptase levels and extensive skin involvement are predictors for the severity of mast cell activation episodes in children with mastocytosis. Allergy 67, 813–821. Alvarez-Twose, I., Zanotti, R., Gonzalez-de-Olano, D., et al., 2014. Nonaggressive systemic mastocytosis (SM) without skin lesions associated with insect-induced anaphylaxis shows unique features versus other indolent SM. J. Allergy Clin. Immunol. 133, 520–528. Belhocine, W., Ibrahim, Z., Grandné, V., et al., 2011. Total serum tryptase levels are higher in young infants. Pediatr. Allergy Immunol. 22, 600–607. Berroa, F., Lafuente, A., Javaloyes, G., et al., 2014. The usefulness of plasma histamine and different tryptase cut-off points in the diagnosis of peranesthetic hypersensitivity reactions. Clin. Exp. Allergy 44, 270–277. Blank, U., 2011. The mechanisms of exocytosis in mast cells. Adv. Exp. Med. Biol. 716, 107–122.

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