Laboratory tests to support the clinical diagnosis of anaphylaxis

INTRODUCTION — Anaphylaxis is a serious systemic allergic reaction that is rapid in onset and may cause death [1]. The diagnosis of anaphylaxis during the acute event is based on the clinical presentation and a history of a recent exposure to an offending agent [1]. There are no laboratory tests available in an emergency department or clinic setting to confirm a diagnosis of anaphylaxis in real time. (See "Anaphylaxis: Emergency treatment".)

However, laboratory tests in serum, plasma, and possibly urine obtained during or shortly after the acute event can help to support the clinical diagnosis of anaphylaxis. These tests can also help identify anaphylaxis in the presence of other disorders that have overlapping clinical presentations, such as severe asthma or myocardial infarction. In addition, these tests may provide evidence for anaphylaxis as a cause of death.

This topic reviews the laboratory tests that can be used to support the clinical diagnosis of anaphylaxis in both adults and children. These tests are different from those that identify sensitization to the inciting allergen, namely measurements of allergen-specific immunoglobulin E (IgE) and those that identify mast cell disorders, which are reviewed separately. (See "Overview of skin testing for IgE-mediated allergic disease" and "Overview of in vitro allergy tests" and "Anaphylaxis: Confirming the diagnosis and determining the cause(s)", section on 'Testing for allergen cause(s)' and "Mastocytosis (cutaneous and systemic) in adults: Epidemiology, pathogenesis, clinical manifestations, and diagnosis".)

OVERVIEW — The principal effector cells of systemic anaphylaxis certainly include mast cells and likely basophils. The various preformed and newly generated mediators secreted by these cells cause many of the signs and symptoms of systemic anaphylaxis. The pathophysiology of anaphylaxis is reviewed in detail separately. (See "Pathophysiology of anaphylaxis".)

Mediators released during anaphylaxis — Mast cells and basophils degranulate upon activation, releasing preformed mediators from intracellular granules into the extracellular environment. Two of the most abundant and best characterized preformed granule mediators are tryptase and histamine. Elevations in tryptase and histamine can sometimes be detected in blood samples obtained shortly after the onset of symptoms. Also, elevated levels of histamine, histamine metabolites (N-methylhistamine and N-methylimidazole acetic acid), the prostaglandin D₂ (PGD₂) metabolites [11-beta-PGF_2-alpha, 2,3-dinor-11-beta-PGF_2-alpha, or 2,3,18,19-tetranor-11-beta-PGF_2-alpha(PGD-M)] and the leukotriene C₄ (LTC₄) metabolite [leukotriene E₄ (LTE₄)], can be measured in urine or in some cases, in serum or plasma after an anaphylactic event.

Available tests — Assays for the following mediators are available commercially in the United States (table 1):

●Tryptase (serum/plasma)

●Histamine (plasma, urine)

●N-methylhistamine (plasma, urine)

●11-beta-PGF_2-alpha (serum/plasma, urine)

●LTE₄ (urine)

The histamine metabolite N-methylimidazole acetic acid (urine) is available in some commercial laboratories.

Platelet-activating factor (PAF) [2], PGD₂ [3], chymase [4], and mast cell carboxypeptidase A3 in serum are also being investigated in research laboratories as biomarkers of anaphylaxis but require further study [5]. (See 'Possible future tests' below.)

Time course of mediator release — Elevations in tryptase and histamine may be transiently detectable following anaphylaxis:

●Total tryptase should ideally be measured 30 to 120 minutes after the onset of anaphylaxis signs or symptoms (table 2), ideally from one to two hours after onset, because most patients have recovered from hypotension by one hour and levels should be at or near peak during the second hour after onset. This level should be compared with one obtained at baseline, which could either be prior to the event (eg, serum sent to the lab for a different test) or at least 24 hours after all signs and symptoms have resolved. During experimental insect sting anaphylaxis, serum tryptase levels peak 30 to 90 minutes after the onset of symptoms and then decline with a half-life of about two hours, so with a very high peak, levels may remain elevated up to six hours [6,7]. The larger the initial elevation of tryptase, the longer the level remains elevated above baseline and may do so for several hours after the event. (See 'Tryptase' below.)

●Histamine elevations are even more fleeting. Plasma histamine levels peak within 5 to 10 minutes after symptom onset and decline with a half-life of 1 to 2 minutes, such that levels typically return to baseline by 15 to 30 minutes [6-10]. Therefore, samples should be drawn as soon as possible, preferably 2 to 15 minutes from the onset of symptoms (table 3). Metabolites of histamine are slightly longer-lasting in blood and may appear in urine made within 30 to 60 minutes of the event. They remain detectable in urine stored in the bladder for hours. (See 'Histamine' below.)

TRYPTASE — Tryptase is produced by both mast cells and basophils, although mast cells contain approximately 500-fold more than basophils [11-13]. The biologic functions of tryptase have not been fully defined. Because tryptase is relatively specific for mast cells, acutely elevated tryptase levels in serum or plasma when compared with a baseline level indicate involvement of these cells in clinical events [14].

Forms — Mast cell tryptase exists in two types (alpha and beta), both of which have mature and immature (pro) forms:

●A portion of the immature form, also known as protryptase, is spontaneously secreted by unstimulated mast cells [15]. Specifically, alpha-protryptase and beta-protryptase account for the vast majority of the tryptase detected in nonanaphylactic serum or plasma. The commercial total tryptase assay measures mature and proforms of both alpha- and beta-tryptases with comparable sensitivities. Any function(s) of immature tryptases, other than serving as precursors of the mature enzymes, are not known [16].

●Another portion of protryptases is proteolytically processed at acidic pH in the presence of heparin to their mature forms, namely beta-tryptase and alpha-tryptase, which form tetramers that are stored inside the secretory granules of mast cells in a macromolecular complex with heparin proteoglycan [17-21]. When mast cells become activated and degranulate, mature tryptase(s) and histamine are released into the extracellular environment. In vitro potential biologic activities of mature beta-tryptase homotetramers include generation of complement anaphylatoxins and kinins, inactivation of fibrinogen, and stimulation of a variety of different cell types, while its activity(ies) in vivo remains uncertain. Unlike beta-tryptase homotetramers, alpha-tryptase homotetramers are nearly inactive as a protease. One function of alpha-tryptase was revealed only recently, when it was found to form heterotetramers with beta tryptase (alpha₂beta₂) [22]. These heterotetramers appear to have protease activities that are distinct from either homotetramer. The portion of tryptase tetramers accounted for by these heterotetramers increases as the ratio of alpha-tryptase to beta-tryptase genes increase and form naturally in anyone who has at least one alpha-tryptase TPSAB1 gene (ie, roughly 75 percent of the United States population). (See 'Hereditary alpha tryptasemia' below.)  

Genetics — There are genetic differences in tryptase genes that have slight effects on total tryptase, but these have been incorporated into the normal laboratory range. The genes for the human alpha- and beta-tryptases reside on chromosome 16. The dimorphic tryptase alpha/beta 1 (TPSAB1) gene can give rise to alpha- or beta-tryptase, and the monomorphic TPSB2 gene gives rise to beta-tryptase only. Humans normally have four functional tryptase genes (two from each parent). In the United States, about 25 percent of people have two alpha and two beta genes, approximately 50 percent have one alpha and three beta genes, and the remaining 25 percent have four beta genes, though there are racial variances in these distributions [23]. The prevalence of alpha-tryptase deficiency varies among subjects with Asian (10 percent), European (23 percent), or African (41 percent) ancestry [24]. In contrast, no subject lacking a gene encoding an active form of beta-tryptase has been reported [24].

The normal tryptase genotypes have small effects on the level of total tryptase (ie, <1 ng/mL difference) [25]. In contrast, having extra copies of alpha-tryptase-encoding TPSAB1 markedly increases serum tryptase levels. (See 'Hereditary alpha tryptasemia' below.)

Assays — Two immunoassays for tryptase have been developed and applied clinically:

●An enzyme-linked immunosorbent assay (ELISA) for "total" tryptase is commercially available as "ImmunoCAP tryptase" through many commercial laboratories [26]. This assay detects all forms of alpha- and beta-tryptases (mature and pro forms) in combination and has been approved by the World Health Organization and the US Food and Drug Administration for use in diagnosing systemic anaphylaxis.

●An ELISA for mature tryptase (alpha and beta) is performed only at the author's laboratory at Virginia Commonwealth University (Richmond, Virginia) [27]. In addition to mature tryptase, this laboratory also routinely measures total tryptase by ImmunoCAP on each sample provided and monitors for the presence of heterophilic antibody. (See 'Causes of false-positive elevations' below.)

Proper collection of samples — Serum or plasma for tryptase should be obtained ideally within the first one to three hours after anaphylaxis (table 1). Printable instructions for proper collection and handling of samples for tryptase measurement are provided (table 3). Measurements of tryptase in serum and plasma are similar [28]. An additional sample should be collected at least 24 hours after all signs and symptoms have resolved to serve as a baseline sample for comparison. Frozen serum or plasma obtained before the event can also be used as a baseline and is often available for hospitalized patients. Tryptase is stable in frozen serum for at least one year.

●Serum and plasma tryptase levels are similar. In a study designed to compare plasma and serum levels, plasma samples obtained from citrated blood were collected from 43 healthy subjects and divided into two portions, one of them was clotted by the addition of calcium chloride (CaCl₂), from which serum was separated from clot, and the other remained as plasma. Total tryptase levels were 4.6±1.5 ng/mL in plasma and 5.1±2.1 ng/mL in serum, which were not statistically different from one another [28].

●Also, if basophils are disrupted or activated during blood collection or clotting, this can theoretically increase the tryptase level in plasma or serum. For example, 100 percent of basophil tryptase (0.3 x 10⁶ basophils/mL x 50 ng/10⁶ basophils) could increase mature tryptase levels to 15 ng/mL, although such release has not been reported and mature tryptase levels in serum are typically <1 ng/mL.

The author's laboratory requires 1 mL of serum (red top tube) or plasma (heparin, citrate, or ethylenediaminetetraacetic acid [EDTA]) [27]. The sample should optimally be stored and shipped frozen by overnight courier, although the enzyme appears to be immunologically stable if refrigerated for more than one week. Samples left at room temperature for more than one or two days might yield falsely low values.

For postmortem samples, collection from the femoral artery or vein rather than the heart is recommended to minimize the contribution of nonspecifically released tryptase from mast cells in disintegrating or traumatized cardiac tissue [29].

Normal levels — Baseline total tryptase levels range from 1 to 11.4 ng/mL with an average of 3 to 5 ng/mL (table 2). Based on limited data, mean levels in infancy may be slightly higher than those in older children and adults, although still within the normal range [30]. The upper limit of 11.4 ng/mL is recommended by the manufacturer, although acute elevations from the baseline within the normal range may also be clinically relevant. A person's baseline total tryptase level shows little variation over time. The primary determinant of that level is genetic, not environmental [31]. Baseline serum levels of mature tryptase are normally undetectable (<1 ng/mL) in healthy individuals who have not experienced anaphylaxis in the preceding few hours.

Elevations in anaphylaxis — Elevated levels of total or mature tryptase in serum or plasma may be useful for distinguishing anaphylaxis from other conditions in the differential diagnosis, such as vasovagal reactions, septic shock, seizures, myocardial shock, benign flushing, or carcinoid syndrome. These other disorders are not normally associated with elevations in serum mature or total tryptase levels (personal experience of author). (See "Differential diagnosis of anaphylaxis in adults and children".)

Tryptase levels in serum or plasma begin to rise as early as 5 minutes after clinical onset of anaphylaxis, reaching maximal levels between 30 and 90 minutes, and then decline with a half-life of about 2 hours. Elevations can range from marginally elevated to levels >100 ng/mL. Dramatic elevations have been recorded following anaphylactic shock induced by medications, for example. The tryptase rise correlates best with the magnitude of hypotension during the anaphylactic event, and not as well with cutaneous, respiratory, or gastrointestinal signs or symptoms [6].

Interpretation of mature and total — An increased level of mature tryptase in serum collected during an acute clinical event indicates that mast cell (and possibly basophil) activation has occurred. There is no other known condition that results in selective elevations of mature tryptase. Such elevation in mature tryptase would lead to an elevation in total tryptase, which may thus also rise during anaphylaxis.

A rise in total tryptase levels above baseline may be more sensitive than a single mature tryptase level [28,32]. The minimal elevation of the acute total tryptase level that is considered to be clinically significant was suggested to be ≥(2 + 1.2 x baseline tryptase levels) in units of ng/mL or mcg/liter [33], allowing mast cell activation to be imputed even when the acute tryptase level is in the normal range [34,35]. The value of the acute versus baseline formula above was shown to have a high positive predictive value and moderate negative predictive value, depending on clinical severity, in perioperative [36] and emergency department [3] settings. If measurement of mature tryptase are also available, the ratio of total to mature tryptase during systemic anaphylaxis is typically <10 and approaches unity at higher levels of mature tryptase (table 2).

Both total and mature tryptase levels should be higher in the acute than in the baseline sample. Comparing acute and baseline levels greatly improves the diagnostic value of total tryptase measurements. If total tryptase is the only test available, then paired levels are essential for interpretation (table 4).

Higher baseline levels of total tryptase initially appeared to increase the risk for more severe anaphylaxis, particularly in insect venom-sensitive subjects, to both field stings and venom immunotherapy [37,38]. However, this risk may reflect the increased likelihood of underlying mastocytosis or an activating c-kit mutation, rather than an effect of the tryptase level per se. Notably, patients with a history of systemic anaphylaxis to insect stings and a baseline tryptase level >5 ng/mL have a high prevalence of underlying clonal mast cell disease (systemic mastocytosis or monoclonal mast cell activation syndrome [MMAS]) [39-41]. This possibility should be considered in any patient with hypotensive reactions to insect stings. (See "Mastocytosis (cutaneous and systemic) in adults: Epidemiology, pathogenesis, clinical manifestations, and diagnosis", section on 'Recurrent severe anaphylactic episodes'.)

Relationship to hypotension — During insect sting-induced anaphylaxis, which has been more extensively studied than other causes of anaphylaxis, the magnitude of the serum tryptase elevation correlates closely with the drop in mean arterial pressure, but not with the severity of gastrointestinal or respiratory symptoms, indicating that the magnitude of mast cell activation is a primary determinant of the severity of this clinical manifestation [8,28]. In the absence of hypotension, there may not be sufficient release of tryptase that enters the circulation to raise serum or plasma levels.

Food-induced anaphylaxis — Elevated levels of total tryptase during acute clinical events implicate mast cell involvement. However, total tryptase levels are less often elevated in food-induced than in insect sting-triggered anaphylaxis [42,43], although elevations are often seen in cases of food-induced anaphylactic fatalities [43]. However, utilizing the acute ≥(2 + 1.2 x baseline tryptase level) formula allows identification of a clinically significant rise in the tryptase level, even when the acute level is <11.4 ng/mL [44]. Several possible explanations for lower or no tryptase elevation in food-induced anaphylaxis have been proposed, including the following:

●Localized rather than systemic release of mast cell mediators may occur in some victims of anaphylaxis (eg, those with laryngeal edema).

●Tryptase released by mast cells at mucosal sites may not diffuse into the circulation as efficiently as that released near blood vessels, and mast cells at mucosal surfaces contain less tryptase than those in the skin and perivascular tissues.

●There may be immunoglobulin E (IgE)-dependent anaphylactic pathways that bypass mast cells and involve basophils and/or other cell types. However, proving basophils are involved in anaphylaxis has been problematic because there is no serum or plasma biomarker for specifically detecting basophil activation.

●Other pathways that could clinically mimic anaphylaxis might include production of nonmast cell vasoactive mediators, such as complement anaphylatoxins (C3a and C5a), kinins, or lipids. (See "Pathophysiology of anaphylaxis".)

Fatal anaphylaxis — Postmortem tryptase levels have been studied in cases of fatal anaphylaxis [43,45]. In one series, mature tryptase was significantly elevated (12 to 150 ng/mL) in nine of nine Hymenoptera sting fatalities, six of eight food-induced fatalities, and two of two reactions to diagnostic therapeutic agents, whereas levels were normal in six postmortem controls [43]. A level of >10 ng/mL for mature tryptase (normal <1 ng/mL) was thought to indicate a magnitude of mast cell activation consistent with anaphylactic shock near the time of death. A study of total tryptase levels in postmortem sera suggests that a total tryptase level >45 ng/mL is consistent with systemic anaphylaxis near the time of death [46], but this might be problematic in the absence of a baseline tryptase level. The etiology and pathogenesis of fatal anaphylaxis are reviewed in more detail separately. (See "Fatal anaphylaxis".)

Diagnostic specificity of postmortem levels — Given the lack of a gold standard for identifying death from anaphylaxis, it is hard to know when an elevated tryptase level indicates mast cell activation near the time of death or is a false positive. The diagnostic specificity of postmortem tryptase levels has been challenged by a study that found elevated levels of mature tryptase in approximately 10 percent (5 of 49) of fatalities believed to be nonanaphylactic [47]. In the five patients with postmortem tryptase levels >10 ng/mL:

●One death was attributed to salicylate overdose, although aspirin sensitivity could have coexisted and caused mast cell activation [47]. Mast cell activation has been well-documented in aspirin-sensitive subjects challenged with aspirin-like drugs [48-51].

●Another death was associated with atherosclerotic coronary vascular disease, although details were not available regarding drugs received near the time of death that could have activated mast cells, such as morphine.

●The remaining three individuals died of trauma, and although there are few studies on serum tryptase elevations in trauma patients, elevated tryptase levels in peritoneal lavage fluids have been noted in patients following intestinal manipulation during abdominal surgery [52].

In other postmortem studies, elevated levels of mature tryptase were reported in the sudden infant death syndrome, heroin-related deaths, and unexplained deaths [29,53,54]. Whether such cases are false positives that do not reflect mast cell activation or true positives that reflect non-IgE-dependent mast cell activation remains to be determined.

In a subsequent postmortem study, total tryptase levels in aortic serum in excess of 110 ng/mL had a sensitivity of 80 percent and specificity of 92 percent for fatal anaphylaxis [55], and in another study, levels above 43 ng/mL had a sensitivity of 90 percent and specificity of 98 percent [56]. However, the cause of an elevated postmortem tryptase is not precisely understood, such as leakage of tryptase from dead or dying mast cells into periaortic tissues and then into blood. Therefore, this remains an area in need of further study.

Conclusions — Collection of blood for tryptase measurements should be considered if the diagnosis of anaphylaxis is being entertained. Elevations in tryptase correlate with hypotension and support the diagnosis of anaphylaxis, although normal levels do not exclude anaphylaxis, particularly in food-induced reactions, samples collected more than four hours after symptom onset, and those without hypotension. The specificity and sensitivity of tryptase elevations have not been precisely determined but increase with clinical severity.

Ideally, samples should be collected between 15 minutes to 3 hours of the event onset or as soon as possible thereafter. Large elevations in tryptase may persist for several hours.

Mature tryptase is more specific for mast cell activation than total tryptase, particularly when only a single acute specimen is available. The predictive value of total tryptase is markedly improved by comparing an acute with a baseline sample, using the formula acute ≥(2 (ng/mL) + 1.2 x baseline (ng/mL)) to calculate the minimal acute tryptase level likely to be clinically significant. Using this formula, the sensitivity of the total tryptase assay appears to be greater than for an acute mature tryptase level alone.  

Detailed descriptions of the clinical event and medications received before and during the event are needed for optimal interpretation of tryptase levels. For example, medications acting as an allergen (eg, penicillin) or nonsteroidal anti-inflammatory drugs (NSAIDs) or opioids may trigger a mast cell activation event. The manner in which blood was obtained can sometimes affect postmortem specimens.

Elevations of tryptase in nonanaphylactic patients — The levels of total tryptase in nonanaphylactic subjects is largely composed of immature protryptases and reflect (in part) the total body burden of mast cells.

Systemic mastocytosis — Total tryptase is elevated (>20 ng/mL) in most patients with systemic mastocytosis, a disorder of clonal mast cell hyperplasia. The total/mature tryptase ratio is usually >20 (table 2). A total tryptase level >20 ng/mL in the absence of another cause for an elevated level is just one of the four World Health Organization's minor criteria for diagnosis [57]. A baseline serum total tryptase level has been approved by the US Food and Drug Administration for use in the evaluation of suspected systemic mastocytosis [58]. Systemic mastocytosis or related monoclonal mast cell activation disorder, both having gain of function mutations in the gene encoding Kit, may present with spontaneous, insect sting-induced, or allergen-induced anaphylaxis [59]. The diagnosis of systemic mastocytosis is reviewed in detail separately. (See "Mastocytosis (cutaneous and systemic) in adults: Epidemiology, pathogenesis, clinical manifestations, and diagnosis" and "Mastocytosis (cutaneous and systemic) in children: Epidemiology, clinical manifestations, evaluation, and diagnosis".)

Hereditary disorders associated with mast cell activation

Hereditary alpha tryptasemia — Hereditary alpha-tryptasemia (HaT) is an autosomal dominant genetic condition, in which affected members have high normal or elevated baseline total tryptase levels (>8 ng/mL) but normal mature tryptase levels (<1 ng/mL) [60]. The prevalence of HaT in the general population may be as high as 6 percent in those with European ancestry, suggesting that it might be the most common reason for an elevation in baseline total tryptase. The genetic defect is a copy number increase in the TPSAB1 gene but only when it encodes alpha tryptase [61]. For example, those with one extra copy of the alpha-tryptase gene have mean ± standard deviation (SD) levels of 15 ± 5 ng/mL, and those with two extra copy numbers have 24 ± 6 ng/mL. Also, a family in which affected members through three generations had elevated baseline total tryptase levels and a primary mast cell activation syndrome were found to have HaT due to quintuplication of TPSAB1 [62,63]. No individuals with extra copies of a beta-tryptase gene have been described to date. Genetic testing for increased copies of the TPSAB1 gene is commercially available, but third party payers may not cover the cost. Testing should be considered on an individual basis. (See 'Genetics' above.)

Based on a relatively limited number of patients, HaT can be asymptomatic or can manifest with one or more of the following conditions [60,64]:

●Severe anaphylaxis, particularly triggered by Hymenoptera insect stings

●Idiopathic anaphylaxis

●Vibratory urticaria

●Musculoskeletal problems (eg, joint hypermobility leading to arthritis)

●Dysautonomia:

•Postural orthostatic tachycardia syndrome

•Gastrointestinal hypomotility associated with irritable bowel syndrome

●Retained primary dentition

Larger studies are needed to fully characterize this disorder, and the associations noted above may be influenced by referral or other biases.

A proposed mechanism underlying some of these signs and symptoms involves the spontaneous formation of alpha/beta-tryptase heterotetramers in mast cells and likely basophils of patients with HaT. These heterotetramers (but neither homotetramer) activate protease-activated receptor-2 (PAR2), which is found on smooth muscle, neurons, epithelial cells, and endothelial cells, and in vitro potentiates acute vascular membrane permeability, possibly increasing the severity of anaphylaxis in vivo [64]. In addition, the heterotetramers cleave the mechanosensory receptor EGF-like module-containing mucin-like hormone receptor-like-2 (EMR2) on mast cells from skin, making these cells susceptible to vibration-triggered degranulation [22]. These heterotetramers also form naturally in people with normal tryptase genotypes, as long as at least one alpha-tryptase-encoding gene is present, and thus may explain similar symptoms that were not previously linked to mast cell or tryptase function.

It is possible that new therapeutic interventions will arise with a more precise understanding of pathogenesis. Moreover, HaT accounts for most cases of an elevated baseline tryptase level in asymptomatic patients [65].

HaT can also coexist with clonal mast cell disease [63]. In patients with systemic mastocytosis, the presence of concomitant HaT is associated with a higher risk of triggered or spontaneous systemic anaphylaxis than observed for either disorder by itself [64].

Other genetic conditions — Other genetic conditions associated with mast cell activation have been reported, although these have not been associated with elevations in baseline tryptase:

●Two families with vibratory urticaria were found to have an autosomal dominant gain-of-function mutation in the gene ADGRE2 which encodes the EMR2 receptor [66]. Mast cells derived from these individuals demonstrated vibration-dependent degranulation in vitro.

●Individuals with familial periodic fever syndromes associated with gain-of-function mutations in cryopyrin (familial cold autoinflammatory syndrome, Muckle-Wells syndrome, and neonatal-onset multisystem inflammatory disorder) had a positive cutaneous ice cube challenge test and mast cells that were activated ex vivo by cold temperatures to secrete interleukin 1-beta [67,68].

●An autosomal dominant primary immunodeficiency caused by a gain-of-function mutation in phospholipase C-gamma-2 results in urticaria during evaporative cooling of the skin and mast cells activated ex vivo by cool temperatures [69].

●Poorly-controlled Gaucher disease [70]. (See "Gaucher disease: Pathogenesis, clinical manifestations, and diagnosis".)

Other conditions — Elevated baseline total tryptase levels are also observed in the following disorders:

●Acute myeloid leukemia [71] (see "Clinical manifestations, pathologic features, and diagnosis of acute myeloid leukemia")

●Myelodysplastic syndromes [72] (see "Clinical manifestations and diagnosis of myelodysplastic syndromes (MDS)")

●The myeloid variants of hypereosinophilic syndrome (particularly those associated with the Fip1-like1-platelet-derived growth factor receptor alpha [FIP1L1-PDGFRA] mutation) [73] (see "Hypereosinophilic syndromes: Clinical manifestations, pathophysiology, and diagnosis")

●Severe renal failure (ie, chronic kidney disease stages 4 and 5 and hemodialysis patients) [74-76]

●GATA-2 haploinsufficiency [77]

●With the treatment of onchocerciasis, though whether this reflected mast cell activation, disruption, or hyperplasia was not clear [78] (see "Onchocerciasis")

●After administration of recombinant stem cell factor due to mast cell hyperplasia [79] (see "Introduction to recombinant hematopoietic growth factors", section on 'Stem cell factor and Flt3 ligand')

Causes of false-positive elevations — False elevations in tryptase levels, though uncommon [80], have been associated with the presence of heterophilic antibodies (ie, usually human antimouse antibodies) in a patient's serum [81-83]. Many laboratories have since added steps to prevent this. Patients who have received any mouse-human chimeric antibody, such as the chimeric (mouse Fab/human Fc) antibody infliximab, or individuals who have extensive exposure to mice, are at increased risk for having such antibodies. The prevalence of false elevations in tryptase is higher in subjects that are rheumatoid factor-positive, although the immunoglobulin G (IgG) or immunoglobulin M (IgM) rheumatoid factors recognizing human IgG may not be the culprits binding to the mouse antitryptase antibodies used in the immunoassay. Instead, these individuals are more likely to have human antimouse antibodies that cross-link the capture and detector mouse antitryptase antibodies, similar to how tryptase cross-links these antibodies.

Phadia began adding a heterophilic antibody suppressor to their tryptase kit in early 2011 and in 2018 altered the detector antitryptase mAb from IgG to F(ab′)₂, which markedly reduce such false-positives, such that this is probably no longer an issue. Nevertheless, the author's diagnostic lab at Virginia Commonwealth University performs a negative control ELISA using a mouse nonimmune detector antibody that appears positive in the presence of heterophilic antibody but not in the presence of tryptase, which serves as a warning of the presence of heterophilic antibody activity.

HISTAMINE — Histamine is a biogenic amine produced by and stored primarily in human mast cells and basophils, which produce comparable amounts of histamine [13]. Histamine may also be produced by neutrophils, monocytes, T lymphocytes, keratinocytes, and enterochromaffin cells that express histidine decarboxylase [84,85]. Histamine is the only preformed granule mediator of mast cells and basophils known to have direct potent vasoactive and smooth muscle spasmogenic activities.

Histamine is generated intracellularly from histidine by histidine decarboxylase. Most of this histamine is then stored in the secretory granules of mast cells and basophils, although a small portion is released constitutively. Histamine binds to carboxyl and possibly sulfate moieties on proteins and proteoglycans in the acidic pH environment of secretory granules. However, in the neutral pH of the extracellular environment, histamine is less positively charged and becomes dissociated from the macromolecular structures to which it had been bound. Free histamine has low molecular mass (111 Da) and high solubility and rapidly diffuses away from its local sites of release. A portion may enter the circulation. A smaller amount of free histamine may pass into the urine unmetabolized.

Elevations during anaphylaxis — Similar to tryptase, any elevation in plasma or urine histamine is consistent with anaphylaxis, although normal levels do not exclude the diagnosis. Ideally, the acute level should be compared with a baseline level.

Histamine elevations correlate well with clinical severity of anaphylaxis. Although histamine levels in properly collected samples of plasma have a greater sensitivity than tryptase, the short plasma half-life of histamine and the difficulties in handling the sample usually limit the utility of this measurement in the clinical setting [86].

Histamine metabolites — Within minutes, the majority of released histamine is sequentially metabolized to N-methylhistamine and then to N-methylimidazole acetic acid by histamine N-methyltransferase and monoamine oxidase or to imidazole acetic acid by diamine oxidase. N-methylhistamine and N-methylimidazole acetic acid are specific metabolites of histamine, while imidazole acetic acid is not. Levels of N-methylhistamine or N-methylimidazole acetic acid, which can be measured in plasma or urine, may also reflect overall levels of released histamine. These metabolites, generated acutely and then stored in the bladder, will be detectable in the urine for several hours after anaphylaxis. Elevated urinary N-methylhistamine was observed in specimens collected six hours after an insect sting challenge in patients experiencing systemic anaphylactic symptoms [87].

Some investigators consider these metabolites to be more reliable indicators of histamine release than histamine itself. N-methylhistamine is longer-lived than histamine in the circulation, accounts for a greater portion of the released histamine that ends up in the urine, and demonstrates less natural diurnal variation [88-90]. However, another study of anaphylaxis during anesthesia found urinary N-methylhistamine to be less sensitive than either plasma tryptase or histamine, so the relative merits of histamine and its metabolites are not fully defined [91].

In summary, urinary histamine, N-methylhistamine, or N-methylimidazole acetic acid levels are helpful when elevated above baseline, but normal levels do not exclude mast cell activation.

Assays and collection of samples — Assays are commercially available to measure histamine and its metabolites in biologic fluids (table 1).

●Blood – Normal levels of histamine in plasma are <617 pg/mL (<5.6 nM, Mayo Clinic Laboratory). Plasma should be used, rather than serum, to avoid the artifactual release of histamine from basophils that can occur during clotting. The blood sample should be collected through a 20-gauge or larger needle and pulled manually under gentle pressure into a syringe containing either citrate or ethylenediaminetetraacetic acid (EDTA). Pulling blood through a narrow-bore needle under vacuum can also cause basophil activation or breakage ex vivo and falsely elevate the levels of histamine. Collected anticoagulated blood should be placed on ice and centrifuged to separate plasma from cells as soon as possible and then frozen until ready to be analyzed. Printable instructions for collection and handling are provided (table 3).

●Urine – Urine for measuring levels of histamine are collected with a standard 24-hour collection. Collection of "acute" urine (ie, urine produced during the acute event and stored in the bladder) should begin as soon as possible after the acute event. A urinary histamine concentration of up to 30 ng/mL is considered normal. Ideally, a second baseline sample should be collected beginning at least 24 hours after all signs and symptoms of anaphylaxis have ceased. Instructions for collecting, storing, and shipping urine specimens can be found on the websites of commercial laboratories [92].

Other causes of elevated histamine — Elevations in plasma or urinary histamine may result from the following:

●Urinary histamine and its metabolites may be elevated due to the presence of histamine-producing bacteria in the urogenital or gastrointestinal tracts, although this has not been extensively studied [93].

●Histamine-rich foods include some fish, aged cheeses, chocolate, red wine, and vegetables, such as eggplant, spinach, and tomatoes, although the relationship between the histamine content in foods and the appearance of that histamine or its metabolites in the circulation or urine is uncertain, in part because the factors affecting absorption of ingested histamine are unclear [94,95].

●Scombroidosis, an illness that results from the ingestion of improperly stored fish that has accumulated high histamine levels, presents with flushing, headache, and widened pulse pressure but not with pruritic urticaria and hypotension as seen in systemic anaphylaxis [96]. Often several people eating the same fish are affected. Urinary and plasma histamine or histamine metabolite levels can be elevated, although tryptase levels are normal. Elevated histamine levels are not found in other forms of food poisoning. The diagnosis of scombroidosis is reviewed in more detail separately. (See "Seafood allergies: Fish and shellfish", section on 'Differential diagnosis'.)

●Constitutive hyperhistaminemia has been considered as a contributing factor to recurrent anaphylaxis, though convincing data have not emerged [97].

●Levels of histamine may be elevated in adults with systemic mastocytosis. (See "Mastocytosis (cutaneous and systemic) in adults: Epidemiology, pathogenesis, clinical manifestations, and diagnosis".)

PROSTAGLANDIN AND METABOLITES — Prostaglandin D₂ (PGD₂) is the principal cyclooxygenase product produced by activated mast cells but is not produced by activated basophils. Antigen-presenting cells, megakaryocytes, T helper type 2 (Th2) lymphocytes, eosinophils, and platelets are also capable of producing this prostaglandin [98]. PGD₂ plays a large role in the hemodynamic changes that accompany episodes of systemic mast cell activation [99,100]. Like histamine, clearance from the systemic circulation is rapid, and elevated serum levels of PGD₂ are generally difficult to detect [7]. Elevations of the major circulating and excreted metabolite (2,3-dinor-11-beta-PGF_2-alpha) can be measured in the urine, where it may be a more sensitive indicator of mast cell activation than N-methylhistamine [101-105]. Urinary prostaglandin is available through a limited number of commercial laboratories [106]. A subsequent study reported that plasma levels of 11-beta-PGF_2-alpha measured within two hours of an anaphylactic event may be more sensitive than tryptase and leukotriene E₄ (LTE₄) [3], though additional studies are warranted.

Measurement of PGD₂ or its metabolites is not useful in patients receiving cyclooxygenase inhibitors, including aspirin and other nonsteroidal anti-inflammatory drugs (NSAIDs), as these medications block the synthesis of this mediator. (See "NSAIDs: Pharmacology and mechanism of action".)

LEUKOTRIENES AND METABOLITES — Leukotriene C₄ (LTC₄) is the principal 5-lipoxygenase product produced by activated mast cells and basophils. Activated eosinophils along with certain phagocytic mononuclear cells, endothelial cells, vascular smooth muscle cells, and platelets also can produce this metabolite. Like histamine and prostaglandin D₂ (PGD₂), it is rapidly metabolized to LTD₄ and then to LTE₄, which can be measured in urine, a test available commercially in the United States [106]. Although the cell source may be uncertain, elevated LTE₄ levels may be seen in the urine after anaphylaxis. In one study, urine LTE₄ appeared to discriminate better between systemic anaphylaxis, bronchial asthma, and healthy controls than either urinary 11-beta-PGF_2-alpha or serum tryptase [107]. LTE₄ also has been measured in plasma, where it seems to be less sensitive than the PGD₂ metabolite or tryptase [3].

POSSIBLE FUTURE TESTS — Chymase, mast cell carboxypeptidase A3, and platelet-activating factor are other products that have been evaluated as possible markers of anaphylaxis. Assays for these mediators are not widely available commercially, although they are performed in research laboratories.

Chymase and carboxypeptidase A3 — Chymase and carboxypeptidase A3 are mast cell-derived proteases that are expressed by a subset of mast cells called MC_TC (mast cells containing tryptase and chymase), which are the predominant type of mast cells in skin, perivascular tissue, heart, conjunctiva, and bowel submucosa [108]. Chymase and carboxypeptidase A3 are stored in secretory granules and are released from mast cells as a macromolecular protease:proteoglycan complex [21]. Subtypes of mast cells are reviewed in more detail separately. (See "Mast cells: Development, identification, and physiologic roles".)

These proteases are not expressed by basophils:

●Chymase levels measured by enzyme-linked immunosorbent assay (ELISA) in postmortem serum were elevated (>3 ng/mL) in fatal anaphylaxis, correlated with elevated levels of tryptase, and were undetectable in most cases of nonanaphylactic deaths [4]. Further evaluation of this marker of anaphylaxis is needed.

●Carboxypeptidase A3 levels measured by an ELISA were elevated (>14 ng/mL) in serum obtained from individuals with a clinical diagnosis of anaphylaxis but not in healthy controls or those with asthma or atopy [109,110]. However, carboxypeptidase A3 and total tryptase levels did not correlate with one another, perhaps because of delayed diffusion of carboxypeptidase A3 into the circulation. Further studies of this potential biomarker are needed.

PLATELET-ACTIVATING FACTOR — Platelet-activating factor (PAF) is generated during anaphylactic reactions to foods [111,112] and perhaps other types of anaphylaxis, but also during stroke, sepsis, myocardial infarction, and colitis, limiting its discriminatory ability as a biomarker. Although PAF undergoes rapid metabolism, plasma levels measured after food-triggered anaphylaxis correlate with clinical severity. Also, low levels of serum PAF acetylhydrolase, the enzyme that metabolizes and inactivates PAF, appear to increase risk for more severe anaphylaxis. The enzyme resides on lipid particles in the circulation, and its concentration also relates directly to the concentration of these lipid particles, which decreases in response to statins [112].

TESTS FOR CONDITIONS MIMICKING ANAPHYLAXIS — Anaphylaxis can be difficult to distinguish from other medical conditions that mimic signs or symptoms of anaphylaxis, particularly in the absence of an inciting allergen. For example, flushing occurs with carcinoid syndrome, tumors secreting vasoactive intestinal protein (VIPomas), pheochromocytomas, and with activation of the complement or kinin pathways, leading to production of complement C3a, C5a, anaphylatoxins, or bradykinin. To help distinguish these entities from anaphylaxis, several laboratory tests should be considered when clinically appropriate (table 5):

●Plasma-free metanephrine and urinary vanillylmandelic acid are elevated in pheochromocytoma. (See "Clinical presentation and diagnosis of pheochromocytoma".)

●Serum serotonin and urinary-5-hydroxyindoleacetic acid may be elevated in carcinoid syndrome. (See "Diagnosis of carcinoid syndrome and tumor localization".)

●Pancreastatin, vasoactive intestinal peptide, substance P, neurokinin, and/or calcitonin may be elevated in gastrointestinal VIPomas or medullary carcinoma of the thyroid. (See "VIPoma: Clinical manifestations, diagnosis, and management" and "Medullary thyroid cancer: Clinical manifestations, diagnosis, and staging".)

●Plasma kallikrein is activated in plasma with activation of the contact pathway (eg, as occurred with heparin preparations contaminated with over-sulfated chondroitin sulfate) [113].

●Complement activation with production of anaphylatoxins C3a and C5a may occur after exposure to polyethylene glycol, snake venoms, older dialysis membranes, and nanoparticles [114-117].

The differential diagnosis of anaphylaxis is reviewed separately. (See "Differential diagnosis of anaphylaxis in adults and children".)

SOCIETY GUIDELINE LINKS — Links to society and government-sponsored guidelines from selected countries and regions around the world are provided separately. (See "Society guideline links: Anaphylaxis".)

INFORMATION FOR PATIENTS — UpToDate offers two types of patient education materials, "The Basics" and "Beyond the Basics." The Basics patient education pieces are written in plain language, at the 5^th to 6^th grade reading level, and they answer the four or five key questions a patient might have about a given condition. These articles are best for patients who want a general overview and who prefer short, easy-to-read materials. Beyond the Basics patient education pieces are longer, more sophisticated, and more detailed. These articles are written at the 10^th to 12^th grade reading level and are best for patients who want in-depth information and are comfortable with some medical jargon.

Here are the patient education articles that are relevant to this topic. We encourage you to print or e-mail these topics to your patients. (You can also locate patient education articles on a variety of subjects by searching on "patient info" and the keyword(s) of interest.)

●Beyond the Basics topics (see "Patient education: Anaphylaxis symptoms and diagnosis (Beyond the Basics)" and "Patient education: Anaphylaxis treatment and prevention of recurrences (Beyond the Basics)")

SUMMARY AND RECOMMENDATIONS

●The diagnosis of anaphylaxis is made clinically, and there are no laboratory tests available in an emergency department or clinic setting to verify a diagnosis of anaphylaxis in real time. However, serum, plasma, and urine obtained during or shortly after the acute event can support the clinical diagnosis of anaphylaxis.

●Mast cells and basophils are the principal effector cells of systemic anaphylaxis and release a variety of mediators upon activation. Assays for tryptase (in serum or plasma), histamine (plasma), histamine metabolites (urine), prostaglandin D₂ (PGD₂) or its metabolite prostaglandin F_2-alpha (PGF_2-alpha) (urine or plasma), and leukotriene E₄ (LTE₄) (urine or plasma) are available commercially (table 1). (See 'Overview' above.)

●Collection of blood for tryptase measurements should be considered if the diagnosis of anaphylaxis is being entertained. Elevations in tryptase correlate with hypotension and support the diagnosis of anaphylaxis, although normal levels do not exclude anaphylaxis. Tryptase should ideally be measured within 30 to 120 minutes after onset of anaphylaxis symptoms, but elevations may still be detectable several hours after a severe reaction (table 3). Plasma histamine levels should be measured preferably less than 30 minutes after symptom onset. Metabolites of histamine in urine accumulating over hours may remain elevated for several hours after an anaphylactic event. (See 'Time course of mediator release' above.)

●Tryptase exists in mature and immature (precursor) pro forms. A portion of the pro forms are constitutively released and reflect genetics and total body mast cell content, while mature tryptase is released from mast cells upon degranulation (eg, in anaphylaxis). Assays for total tryptase, which are available commercially, measure both immature and mature forms of tryptase. The only assay available for mature tryptase is performed in the author's laboratory (table 1). Both mature and total tryptase can be useful in the evaluation of anaphylaxis, although levels of mature tryptase are more specific (table 2). (See 'Tryptase' above.)

•Any elevation in mature tryptase in serum or plasma is suggestive of anaphylaxis.

•Total tryptase will rise after anaphylaxis, but a follow-up baseline sample is required to interpret total tryptase accurately (table 4). The minimal elevation of an acute total tryptase level that is considered to be clinically significant (ie, indicative of mast cell activation) is ≥(2 + 1.2 × baseline tryptase levels) in ng/mL.

•The ratio of total to mature tryptase during systemic anaphylaxis is typically <10 (table 2). (See 'Elevations in anaphylaxis' above.)

●Conditions that cause elevated total tryptase, with minimal or no elevations in mature tryptase, include hereditary alpha tryptasemia, systemic mastocytosis, acute myeloid leukemia, myelodysplastic syndromes, hypereosinophilic syndrome (myeloid variant), Gaucher disease, renal failure, and onchocerciasis (during active treatment). (See 'Elevations of tryptase in nonanaphylactic patients' above.)

●Plasma histamine elevations correlate well with clinical severity of anaphylaxis, although elevations are fleeting, and blood samples must be handled with great care, limiting the practical utility of this test (table 3). Urinary metabolites of histamine, such as N-methylhistamine, are longer-lasting and can be assayed in a routine 24-hour urine sample. Like tryptase, any elevations of these mediators collected during the acute over baseline levels support the diagnosis of anaphylaxis, but normal levels do not exclude the possibility of anaphylaxis. (See 'Histamine' above.)

●Urinary PGF_2-alpha, a metabolite of PGD₂, is elevated after mast cell-mediated anaphylaxis, unless the patient has taken a cyclooxygenase inhibitor, such as aspirin, and can be measured in the same urine specimen as N-methylhistamine. Assays for urinary LTE₄ are available from commercial laboratories. (See 'Prostaglandin and metabolites' above and 'Leukotrienes and metabolites' above.)

●Chymase, mast cell carboxypeptidase A3, or platelet-activating factor (PAF) are other products that may be useful in the future for diagnosing anaphylaxis. Assays for these proteases are available only in research settings. (See 'Possible future tests' above.)

●Several other laboratory tests are useful in excluding rare disorders that can mimic anaphylaxis (table 5). (See 'Tests for conditions mimicking anaphylaxis' above.)