Tryptase in Diagnosing Adverse Suspected Anaphylactic Reaction

8/13/2019 Tryptase in Diagnosing Adverse Suspected Anaphylactic Reaction

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Tryptase in Diagnosing Adverse SuspectedAnaphylactic Reaction*

Tryptaza w diagnostyce reakcji niepodanych podejrzanych o anafilaksjpodczas znieczulenia oraz w okresie okoooperacyjnym

Division of Anesthesiology and Intensive Care, 111th

Military Hospital with Outpatient Clinic Pozna, Poland

Abstract

Determination of serum mast cell tryptase (MCT) is becoming more widely used in diagnosing allergic reactionsinvolving mast cells. It can help evaluate the allergenic effects of drugs administered during anesthesia and theperioperative period. Until now, data about the role of tryptase in the body has not been clarified yet. Patients withelevated MCT levels should undergo further testing to find out the causative agent of a potential allergic reaction.Patients with normal tryptase concentration should also undergo further diagnosis if they manifest clinical symp-toms of a severe anaphylactic reaction (Adv Clin Exp Med 2012, 21, 3, 403408).

Key words:tryptase, anaphylaxis, anesthesia, perioperative period.

Streszczenie

Oznaczenie stenia tryptazy (Mast Cell Tryptase MCT) w surowicy krwi znajduje coraz szersze zastosowaniew diagnostyce reakcji alergicznych z udziaem komrki tucznej. Moe by wykorzystywane w celu oceny alergi-zujcego dziaania lekw podawanych podczas znieczulenia oraz w okresie okoooperacyjnym. Dotychczas rolatryptazy w organizmie nie zostaa ostateczne okrelona. Pacjenci ze zwikszonym steniem MCT powinni bypoddawani dalszym badaniom w celu poszukiwania rodka sprawczego, potencjalnej reakcji alergicznej. Pacjenciz prawidowym steniem tryptazy take powinni by poddawani dalszej diagnostyce, jeeli maj kliniczne objawycikiej reakcji anafilaktycznej (Adv Clin Exp Med 2012, 21, 3, 403408).

Sowa kluczowe:tryptaza, anafilaksja, znieczulenie, okres okoooperacyjny.

Adv Clin Exp Med 2012, 21, 3, 403408ISSN 18995276

REVIEWS Copyright by Wroclaw Medical University

* This article is based on the Medline database from the years 19912010. When collecting literature, the authorused the following key words: tryptase, mast cell, anaphylaxis, mastocytosis.

A huge increase in allergic diseases observedin the last ten years significantly affects those with

allergic hypersensitivity to drugs administeredduring the perioperative period and anesthesia.According to data presented in the reference liter-ature, the incidence of anesthesia-related anaphy-laxis ranges from 1:3500 to 1:20000 anesthesias [1,2]. Despite the lack of precise epidemiological re-ports from different regions, the growing risk ofanaphylactic reactions in the perioperative periodhas been thoroughly observed. The importance ofthe emerging problem of anesthesia-related ana-phylaxis was particularly noticed in the Nordic

countries, Western Europe, Australia and NewZealand [3, 4].

In 1960, the properties of mast cell granulestermed trypsin-like activity were discovered. Theisolated enzyme was named mast cell tryptase:MCT [5]. Until now, data on the biological role oftryptase in the body has not been definitively iden-tified and clarified. At the same time, its functionin diseases involving mast cells has not yet beenfully explained. This results from the fact that thephysiological substrate which would react withtryptase has not been discovered yet. Such an abil-ity for some proteins has been demonstrated only


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under in vitro conditions. In the diagnosis of ana-phylactic reactions, tryptase has been used as anenzyme that monitors mast cell activity.

There are many chemicals involved in allergicprocesses associated with the use of neuromuscularblocking agents (NMBA) and other drugs admin-

istered in the perioperative period. Many differentdrugs are used in rapid succession: not only anes-thetics, but also antibiotics, fluids, nonsteroidal an-ti-inflammatory drugs and other compounds (e.g.disinfectants, latex, chlorhexidine, etc.). Most ofthese drugs are given intravenously and in bolus,bypassing the bodys primary immune filters andpresenting high concentrations of antigen directlyto the mast cells and basophils. So it is difficult tosay which drug caused the suspected anaphylacticreaction or if the reaction was the result of the ad-ditive side effects of several drugs injected simulta-

neously [1]. Tryptase, however, is perceived to beof the highest importance. According to Scandina-vian authors, determining the presence of this en-zyme is a key element in diagnosing anaphylacticreactions [4, 5]. The NMBA-related anaphylaxisconfirmed by the presence of tryptase in plasmais compatible with a diagnosis resulting from skinand serological tests.

Mast Cell

A mast cell (mastocyte, MC) is derived frommultipotential bone marrow cells CD34+. It ap-pears in the peripheral blood, and then is deposit-ed in tissues such as skin, lungs, intestinal mucosaand submucosa. There it matures under the influ-ence of general and local growth factors, such asthe stem cell factor (SCT), mast cell growth factor(MCGF) and c-kit ligand. The population of mastcells is not fully homogeneous. Two types of cellsare distinguished among them. The first type in-cludes MCT cells (mast cells containing tryptase)whose granules contain only tryptase (located in

pulmonary alveoli and intestinal mucosa). Thesecond type consists of MCTC cells (mast cellscontaining chymase and tryptase) whose granules,apart from tryptase, contain chymase (located inthe dermis, intestinal submucosal layer and bloodvessels) [6]. The numerical ratio of MCTC to MCTis varied. It depends on the type of tissue. The ratiois different under health and disease conditions.MCTC predominate in the skin, while MCT pre-vail in lungs and gastrointestinal mucosa. Allergicreactions involve MCT which migrate to the nasaland bronchial epithelium [6].

A mastocyte is typically identified with an im-mediate hypersensitivity reaction, i.e. a type I re-action according to the Coombs and Gell classi-

fication, where it plays a key role [7]. Tryptase isone of many mediators released from mast cellsinvolved in immediate-type reactions. [8, 9]. Othersubstances stored in granules (preformed [includ-ing tryptase]), exhibit a pharmacological effect onblood vessels of the respiratory tract and the respi-

ratory tract itself, resulting in clinical symptoms ofhay fever, bronchial asthma, urticaria, or anaphy-laxis.

Data presented in the literature also suggeststhat mast cells are involved in inflammatory pro-cesses in the course of other diseases. Attention isdrawn to the role of mast cells in autoimmune dis-eases such as: rheumatoid arthritis, scleroderma orpemphigoid.

The process of releasing substances storedin granules is referred to as degranulation. De-granulation of mast cells is an active process of

secretion whose essential elements are energy andcalcium. A mastocyte can be activated both immu-nologically and non-immunologically. The formercan occur in two ways: in the mechanism depen-dent and independent of IgE antibodies. Mast cellshave membrane receptors for these antibodies.When a mutually complementary antibody joinsthe membrane receptor and then the mastocytecontacts an allergen, the cell is degranulated [10,11]. Many non-specific stimuli may, however, re-lease its mediators from the cell. Among them aretrauma, high temperature and toxins.

The cytoplasm of a mastocyte contains numer-ous basophil granules which consist of such sub-stances as histamine, serotonin, glycosaminogly-cans, peroxidase, superoxide dismutase, eosinophilchemotactic factors (ECF), neutrophil chemotacticfactors (NCF), tryptase, chymase, heparin and nu-merous cytokines.

The role of cytokines is associated with the ini-tiation of the late allergic response (LAR), or thechronic phase of allergic inflammation (chemot-axis, infiltration and accumulation of inflamma-tory cells). Clinically, the late allergic response

occurs analogically to the early symptoms. It canbegin 24 hours after the immediate allergic reac-tion. It peaks after about 812 hours and lasts upto 24 hours.

Tryptase

Tryptase is an enzyme specific to mast cellgranules. The enzyme is present in complexes withheparin and has a high molecular weight comparedto histamine (approximately 140 kDa). The genes

responsible for tryptase development and synthe-sis are located on the short arm of chromosome16. In the human body, depending on the ami-


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no acid composition, it is present as: -tryptase,

-tryptase, -tryptase and -tryptase. Each of themhas subtypes. Table 1 presents the characteristicsof each type.

Resistance to protease inhibitors characteristicof tryptase results from an unprecedented abilityof this protein to create molecules with a tetramerstructure and the active center facing the insideand unreachable to proteins with inhibiting prop-erties [14]. The regulation of tryptase activity dueto the specific protection mechanism is not subjectto protease-protease inhibitor dependence. Theonly suggested regulatory mechanism, based on

observations in vitro, is the pH-dependent abilityof tryptase to bind to the heparin molecule, whichfacilitates the formation of active tetrameres. Atneutral pH, this ability disappears, and tetramersdisintegrate. Acidic pH is conducive to formingtetramers [15].

Studies describe the multidirectional effectsof the biological activity of tryptase. It causes thedisintegration of bronchodilator mediators, whichmay result in a smooth muscle spasm in the re-spiratory track. In the endothelium of blood ves-sels, their nitric-oxide-dependent vasodilation

and increased permeability with the characteris-tics of hypotension have been observed. Tryptasedegrades the vasoactive intestinal peptide (VIP)and calcitonin gene-related peptide (CGRP). Italso exhibits mitogenic activity for fibroblasts andincreases expression of the first vascular cellularadhesion molecule (VCAM-1). Tryptase showsactivity against plasma coagulation factors (fibrin-ogen degradation, prekallikrein activation, kinino-gen distribution, activation of the pro-urokinase-type plasminogen activator (pro-uPA)), whereasthe end result is the anticoagulant activity [16].

Tryptase secretion from mast cells depends onthe availability of intra- and extracellular calciumand magnesium resources, as well as the presence

of phospholipase A2 (PLA2) and phospholipase

C (PLC) inhibitors [17, 18].Determination of tryptase concentration is

used as a marker of mast cell activation in inflam-matory diseases as the enzyme can be described astypical of mast cells. Available in the pharmacolog-ical market, the reference set (UniCAP 100 Phadia,Pharmacia, Uppsala, Sweden) makes it possible todetermine the total tryptase concentration. It isa fluorescence immunoassay [19]. Antitryptase re-acts with tryptase in the patient serum specimen.After washing, enzyme-labeled antibodies against-tryptase are added to form a complex. After in-

cubation, unbound enzyme-antitryptase is washedaway and the bound complex is incubated witha developing agent. At the end of the reaction thefluorescence is measured, and the degree of fluo-rescence correlates with the amount of tryptasepresent.

An increase in serum tryptase levels is ob-served between 36 hours after the onset of ananaphylactic reaction. It returns to normal with-in 1214 hours [20]. The practical significanceof this is the window of opportunity for testingfor systemic anaphylaxis. Tryptase testing can be

performed on blood samples obtained 16 hoursafter onset of the reaction. The working party ofthe Association of Anaesthetists of Great Britainand Ireland on suspected anaphylactic reactionsassociated with anaesthesia advises taking threesamples [21]. One immediately after the initial re-suscitation, another 1 hour after the start of thereaction at the moment the MCT concentrationnormally peaks and the last simple 24 hours afterthe reaction to get a baseline value. The sampletaken 1 hour after the start of the reaction is obvi-ously the most important.

Table 1.Types of human tryptase [12, 13]

Tabela 1.Typy ludzkiej tryptazy [12, 13]

Type (Typ) Subtype (Podtyp) Characteristics (Charakterystyka)

-tryptase I II

exhibits little biological activity, present in low concentrations in blood serum even in the absence of mast

cell degranulation

-tryptase 1 2 3

the main form stored in granules of mast cells, released during severe inflammation, such as anaphylactic shock. Absent in

serum

-tryptase I II

a protein embedded in the cell membrane of both mast cell granules and itsouter membrane, its function is unknown

-tryptase I II

the type has a significantly shorter chain and different affinity for peptides


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The Application of TryptaseLevel Determination

The longer half-life of tryptase in serum makesit a more convenient diagnostic marker than his-

tamine. Histamine can also be released by othercells such as basophils, antigen-presenting cellsand bacteria. The optimum time for tryptase de-termination is between 16 hours after the expo-sure to an allergen (peak secretion between thefifteenth minute and the second hour, with a half-life of 1.52.5 hours). It should be remembered,however, that a higher level of tryptase may beobserved in diseases and conditions other thananaphylactic allergy. The examples can be woundhealing, scar formation involving the mast cell,multiorgan trauma, hypoxia, myocardial infarc-

tion, heroin intoxication, mastocytosis. An in-crease in tryptase levels beyond systemic mastocy-tosis is also diagnosed in acute myeloid leukemia,myelodysplastic syndromes, hypereosinophilicsyndrome related to FIP1L1-PDGFRA mutation,the exogenous administration of SCF (stem cellfactor) and end-stage renal insufficiency with in-creased endogenous SCF levels. Systemic masto-cytosis is characterized by mast cell hyperplasia inbone marrow, skin, liver, spleen and gastrointesti-nal mucosa [22]. A study of tryptase levels in pa-tients with biopsy-proven mastocytosis reported

concentrations of total tryptase > 20 ng.mL andrations of total tryptase to -tryptase > 20, whereasnormal patients had total tryptase levels < 14 ng.mL [23]. The sensitivity of the total mast celltryptase concentration as a diagnostic test is 83%,with a specificity of > 98%. Total serum tryptaseconcentrations may be useful in evaluating treat-ment aimed to reduce mast cell numbers in pa-tients with systemic mastocytosis.

Myelodysplastic syndrome (MDS) is a largegroup of acquired neoplastic disorders of the bonemarrow most common in the elderly and is caused

by an abnormal differentiation and maturation ofhaemopoetic cells. Serum tryptase levels can beused to differentiate the MDS variants. Follow-upstudies should clarify whether an elevated serumtryptase concentration in MDS is of prognosticsignificance.

Serum tryptase determination can be used toevaluate the allergenic activity of drugs taken dur-ing anesthesia and the perioperative period. A sin-gle tryptase level test can be insufficient, therefore,in order to improve the specificity and sensitivityof determination, a serial test and its comparison

with the basic concentration is suggested. It wasshown that suxamethonium did not cause the re-lease of this substance from both the basophils of

peripheral blood and mast cells of the skin andlungs. On the contrary, after a dose of atracuriumand vecuronium the effect of tryptase releasewas directly proportional to the dose used, and thedynamics of concentrations stimulated by aminos-teroid and benzylisoquinoline drugs was similar.

High intersubject variability was observed in atra-curium activity in response to mast cell tryptasesecretion from the lungs. Among the analyzeddrugs, only atracurium resulted in the release ofhistamine from mast cells obtained from the atriaof the heart. Intradermal stimulation of mast cellswith aminosteroid and benzylisoquinoline relax-ants caused a dose-dependent biochemical effect(tryptase release) and increase in objective (erythe-ma) and subjective (pain, itching) symptoms. Inthe case of suxamethonium and benzylisoquino-lines, the occurrence and intensity of intradermal

stimulation subjective symptoms (itching, erythe-ma and pain) corresponded with increasing con-centrations of tryptase, which was not observed inrelation to aminosteroids [18, 24].

Tryptase was also used in the diagnosis of fatalanaphylactic reactions. It is suggested that an el-evated level of tryptase collected post-mortem maypersist up to the third day after death from anaphy-laxis [25]. Such a determination of serum tryptasein the event of a suspected anaphylactic reactionis useful in most cases of parenteral penetration ofallergens. This was proved in post-mortem exami-

nations carried out up to 24 hours after the deathof 19 victims. A tryptase level above 10 ng/ml wasfound in nine out of nine victims of anaphylaxiswho were bitten by insects, two of two cases ofanaphylaxis after drugs given parenterally, and sixof eight cases of food allergy. At the same time,a level below 5 ng.ml was observed in 57 cases ofpost-mortem serum analysis in persons who diedof other reasons than anaphylaxis [25].

Determination of serum tryptase levels waswidely used in the diagnosis of mastocytosis.A tryptase level above 25 ng.mL is one of the di-

agnostic criteria for the disease, although it is nota specific marker of mastocytosis. The diagnosis ofthis disease is also affected by clinical features anda histopathological picture of the bone marrow.

A number of laboratory methods for determin-ing tryptase levels in bodily fluids have been devel-oped. The radioimmunoenzymatic method is themost useful and the most common method whichmakes it possible to detect II-tryptase, specific toallergic processes, at concentrations of less than1 ng.mL, and the range of determinability on thecalibration graph is 1200 ng.mL [11]. It has been

shown that in the time interval up to 6 hours afterthe first symptoms of anaphylaxis, there is no cor-relation between the time of blood collection and


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Tryptase in Anaphylactic Reactions 407

the recorded tryptase levels [11, 19]. Kinetic pa-rameters and enzyme stability under different ex-ternal environmental conditions make it possibleto collect biological material, and then to conductquantification of tryptase not only at the time ofthe onset of allergic symptoms. It is recommended

to collect blood for clot in the amount of 510 ml,within 14 hours from the onset of allergic symp-toms [4, 18, 19, 26]. It is recommended to comparethe resultant level of tryptase with a control value,obtained at least 24 hours after an anaphylacticreaction. Its elevated level in comparison with thecontrol value is a highly sensitive indicator of ana-phylactic reaction in the perioperative period [4].

However, there is a group of patients withclinical symptoms of severe anaphylactic reaction,but their tryptase is maintained at normal levels.They require further diagnostic tests. Namely, an

elevated tryptase level does not always correlatewith severe anaphylaxis. A negative test result ofa tryptase level does not justify a withdrawal fromfurther examination. No increase in tryptase con-centration in each case of anaphylaxis is attributedto its local release (e.g. in laryngeal edema) whichis not sufficient to increase the total serum concen-tration, or a greater participation of basophils thanmast cells in the pathomechanism of anaphylaxisin a given patient.

Perioperative anaphylactic reactions are a veryimportant anesthesiological problem, because most

often they result from the effects of drugs used forgeneral anesthesia. The greatest risk of allergic re-actions is associated with the anesthesia inductionphase, and skeletal muscle relaxants are one of theallergenic factors. Aminosteroids trigger allergicreactions more often than benzylisoquinolines constituting about 50% of cases, among them ro-curonium (30%), vecuronium (17%) and pancuro-nium (6%). Among benzylisoquinolines, allergicreactions most often occur after atracurium and

mivacurium, 20% and 6% respectively. The fewestallergic reactions are observed after cisatracuium 0.3% of cases. Depolarizing relaxants (suxametho-nium) cause anaphylaxis as often as drugs from thegroup of non-depolarizing aminosteroids.

In a case of suspected anaphylaxis during anes-

thesia, it is important to conduct three basic diag-nostic tests: biochemical determination, serologicaldetermination and skin tests. Tryptase concentra-tion determination is a reliable and effective meth-od of confirming the incidence of anaphylaxis.

The multidirectional effects of drugs used inthe perioperative period makes anaphylaxis a verycomplex problem. This is a very topical issue, andepidemiological data proves the problem is reallyserious. The severity of an allergic reaction in thefinal assessment of the suitability of measuringtryptase level as an important element in the di-

agnosis of anaphylaxis in the perioperative periodshould undoubtedly be taken into account. How-ever, determination of tryptase levels can not bethe only diagnostic tool in perioperative anaphy-laxis. A detailed medical history to define the caus-ative agent and clarify the accompanying clinicalsymptoms should play the primary role. The diag-nosis of anaphylaxis is particularly difficult or evenimpossible in the perioperative period due to thelack of cooperation with the patient (influenced bysoporific drugs) and because of the large number ofmedicines used both for anesthesia and periopera-

tive care. The diagnosis of anaphylaxis in the pe-rioperative period, despite the constant expansionof knowledge, is still a difficult challenge in dailyclinical practice. Difficulties in assessing individualrisk of anaphylaxis result from a lack of sensitive,widely available diagnostic methods confirmingan anaphylaxis episode, and the inability to dis-tinguish between people who are allergic to an al-lergen and those in whom the exposure to a givenfactor may cause a life-threatening reaction.

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Address for correspondence:

Grayna Michalska-KrzanowskaStroma 4c70-004 SzczecinPolandE-mail: [email protected]

Conflict of interest: None declared

Received: 16.09.2011Revised: 2.01.2012Accepted: 6.06.2012

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Tryptase in Diagnosing Adverse Suspected Anaphylactic Reaction