Hymenoptera Venom Allergy (HVA) With Components Profile

Serum

2 mL

1.0 mL (Note: This volume does not allow for repeat testing.)

Gel-barrier tube

Support the diagnosis of hymenoptera venom allergy (HVA) by detection of sIgE antibodies to whole venom extracts and individual allergenic venom proteins. Identifying sIgE responses to specific molecular targets with component resolved diagnostics (CRD) helps fine-tune the diagnosis by distinguishing species-specific, co-reactive, or cross-reactive sensitizations. An accurate diagnosis, in turn, facilitates treatment, including prescription of venom immunotherapy.¹

The high rate of asymptomatic sensitization to hymenoptera venom makes an accurate diagnosis of Hymenoptera venom allergy challenging.^2,3,15,17

There is no correlation between the severity of sting reactions and the concentration of venom sIgE to whole venom extracts.^4-7,19 or individual components,⁷¹ and some patients with minimal level or absent specific IgE to these entities can develop severe anaphylaxis.

An increase in serum specific IgE levels after a sting is not an indicator for conversion into a clinically relevant hypersensitivity in patients with no history of severe systemic reaction to hymenoptera sting.¹⁷

sIgE to CCDs does not exclude clinical relevant sensitization to different venoms.¹¹

Negative allergy test results can occur in patients with convincing history of sting-induced systemic reactions due to a number of causes including:

1. Testing performed in the refractory period following the sting reaction;

2. Loss of sensitization over time;

3. Sensitizing allergen component(s) under-represented in diagnostic test reagent;

4. The presence of systemic mastocytosis, as negative results occur in up to 15% of these patients.⁸

Thermo Fisher ImmunoCAP® Allergen-specific IgE

Hymenoptera venom allergy (HVA) is a potentially life-threatening allergic reaction that occurs following a honeybee, vespid, or ant sting. Most hymenoptera stings produce a transient local reaction that can last up to several days and generally resolves without treatment. More marked swelling extending from the sting site can occur in sensitized individuals as the result of an IgE-mediated late-phase reaction.⁹ Systemic reactions to the venoms of stinging Hymenoptera may be restricted to generalized symptoms of the skin, but can also affect the respiratory and vascular system and lead to multi-organ failure. Fatal anaphylaxis after Hymenoptera stings is a rare but well-recognized cause of sudden death.^10-12 The risk of a future systemic reaction on hymenoptera sting is significantly increased in patients who have historically experienced large local reactions after a sting.¹³

Identification of the species of insect responsible for the sting reaction is useful in establishing the diagnosis, prescribing treatment, and educating patients in avoidance measures. The most prevalent hymenoptera causing allergic reactions in the US belong to the Apidae and Vespidae families.^14-16 The Apidae family includes the subfamilies Apinae (honeybees; Apis mellifera) and Bombinae (bumblebees; Bombus terrestris), while the Vespidae family is composed of the Vespinae subfamily, including the genera Vespula (yellow jacket; Vespula vulgaris), and the Polistinae subfamily, which includes the genus Polistes (paper wasp; Polistes dominula).

The diagnosis of HVA is based on the clinical history of a systemic/anaphylactic sting reaction and the detection of IgE sensitization to relevant insect venoms.^9,15 Patient work-ups have historically included tests using whole venom preparations, either “in vivo” via skin testing or “in vitro” by measuring serum levels of venom specific IgE (sIgE) antibody.¹¹ Specific IgE antibodies are produced after the very first sensitizing event and can be detected immediately after the first allergic reaction, although it is recommended to perform the testing 1-4 weeks after the last sting.¹² A clear documented history of a sting-associated reaction is a prerequisite to a patient work-up for HVA because many patients express positive test results without a history of clinically significant sting-associated symptoms.^17,18 Venom-specific immunotherapy (VIT) is currently the only known curative therapy, but its efficacy greatly depends on the correct identification of the culprit insect species.^19-22

Hymenoptera venoms contain complex mixtures of glycosylated and non-glycosylated proteins and peptides.^23,24 While the venom from each species of hymenoptera is unique, the various venoms contain multiple homologous protein components. One of the main obstacles to identification of the correct allergy-eliciting insect is immunological cross reactivity, due to the significant redundancy of the amino acid sequences of their allergenic proteins. Differentiation between primary sensitization and cross-reactivity-driven responses using whole venom extract based tests can be challenging, especially in the cases where tests with multiple extracts are positive.^11,15 Dual positivity complicates the process of selecting the appropriate venom for immunotherapy in cases where the culprit insect is unknown.¹⁵ As many as half of HVA allergic patients have positive results for both honeybee venom (HBV) and yellow jacket venom (YJV) using whole venom extract-based diagnostic tests.^{12,15,18,25,26,27} Dual positivity for YJV and paper wasp venom (PWV)is even more common.^{12,15,18,23,25,26} While some duel positive patients may have been stung by multiple types of hymenoptera engendering multiple primary sensitizations, a single, primary sensitizer is a more common cause of HVA.

Component Resolved Diagnostics (CRD) refers to the use of individual, purified, or recombinant, allergens for in vitro detection of allergen sIgE.^{11,12,15,21,28,29,32} CRD can be used to discriminate between primary sensitization and cross-reactivity in patients with double-positive results by whole venom-based diagnostic tests.¹² Some allergen components present in a given whole venom preparation are unique to that venom and not present in other potential venom sensitizers. These species-specific components are referred to as “marker allergens” as they serve as a marker of genuine sensitization to a specific venom source.¹⁵ Thus, CRD can often distinguish true dual- from single-species sensitization.^{11,15,28,30,31} CRD can also be valuable in patients with a proven history of a venom-induced systemic reaction but negative whole venom extract-based allergy tests, as it allows for the detection of sensitization to potent individual venom allergens that are underrepresented in whole venom extracts.^{11,12,15,21,28,32,33} Studies have also found a correlation between the pattern of CRD sensitization and VIT efficacy,^34,35 as well as VIT side effects.³⁶

HBV: Honeybee (Apis mellifera) Venom

HBV is the best characterized of the hymenoptera venoms due to the prominence of beekeeping and the associated high prevalence of allergic reactions associated with honeybee stings.^37,38 The venom allergens of different honeybee species are similar and are reported to be highly cross-reactive.¹¹ Twelve key proteins designated Api m 1-12 are currently included in the official allergen nomenclature database (www.allergen.org).^21,39 Among them, Api m 1, Api m 2, Api m 3, Api m 5 and Api m 10 are referred to as major allergens in that more than 50% of confirmed honeybee-sensitized patients show IgE reactivity to these components.^40,41 The combined testing for sIgE to these five HBV components has a diagnostic sensitivity of approximately 95% for identifying HBV-allergic patients.^21,40 Of these five HBV components, three (Api m 1, Api m 3, and Api m 10) are unique to the Apidae family and not present in the venom of yellow jacket or other Vespidae,^15,24 and thus serve as a markers of primary HBV sensitization. In contrast, Api m 2 and Api m 5 are cross-reactive with venom proteins of hymenoptera of the Vespidae family.^15,24

Api m 1, referred to as phospholipase A2, is a neurotoxin that effects the peripheral neuromuscular system.²⁴ The prevalence of Api m 1 sensitization is reported to range between 57% and 97% among HBV-allergic patients.^{27,33,34,40,42-46,54} While detection of Api m 1 sIgE indicates likely primary HBV sensitization, a lack of sensitization to Api m 1 in patients suspected of having HBV allergy is insufficient to rule out genuine HBV sensitization.¹⁵

Api m 2 is an enzyme that breaks down hyaluronic acid, a polysaccharide of high molecular mass that maintains cell adhesion.⁴⁷ This facilitates toxin diffusion beyond the site of the sting.^24,47 Api m 2 is structurally similar to the Ves v 2, a hyaluronidase from yellow jacket venom (YJV),^48,50,51 and thus positive Api m 2 sIgE results can occur as the result of exposure either to HBV to YJV.¹⁵ Ves v 2 represents only a minor allergen of YJV, while Api m 2 is an important major allergen of HBV.^34,40,49,51 Assessment of sensitization to venom marker components (from both HBV and YJV) is required to ascertain the primary sensitizer responsible for Api m 2 sIgE positivity.^11,15

Api m 3 is a marker allergen for primary HBV sensitization since it is not found in the venoms of other hymenoptera. Api m 3 is present in relatively low concentration in HBV and is poorly represented in several of the licensed preparations routinely used for honeybee venom immunotherapy and diagnostics, so Api m 3 can be positive in whole venom extract test-negative patients.^11,50,54

Api m 5 is a dipeptidyl peptidase and plays important role in venom toxicity by activating proteins by cleaving amino acids from their n-terminals. Api m 5 is structurally similar to dipeptidyl peptidase proteins from other hymenoptera, including Ves v 3 from YJV^50,51 and a dipeptidyl peptidase from Polistes dominula venom.⁵² Due to high inter-species cross-reactivity, positive Api m 5 results are not specific for HBV exposure as they can reflect sensitization to other hymenoptera venom.¹⁵

Api m 10 is a complex protein that is also referred to as icarapin.^15,34,53-56 It is an unstable protein of unknown function that has homologs in other insect species.⁵⁶ This HBV marker allergen is present in relatively low abundance in HBV and is poorly represented or missing in several of the licensed preparations routinely used for honeybee venom immunotherapy. A retrospective multi-center study found that a predominant sensitization to Api m 10 represents a relevant risk factor for treatment failure of VIT,⁵⁴ ostensibly due to its low relative concentration in certain immunotherapy extracts.^54,57

YJV: Yellow Jacket (Vespinae Vespula) Venom

Yellow jacket is the common American name for predatory social wasps of the genera vespula, dolichovespula and vespa.^11,58 Members of these genera are known simply as "wasps" in other English-speaking countries. YJV proteins from the various species are closely related^21,25 and include the prominent allergens Ves v 1 and Ves v 5.^59-61 Ves v 1 and Ves v 5 are marker allergens, and the presence of serum sIgE to these proteins indicates primary yellow jacket sensitization.¹⁵

Ves v 1, also referred to as phospholipase A1, is responsible for the hydrolysis of the plasma membrane phospholipids, allowing the diffusion of toxins into adjacent cells and producing edema.⁴⁷ The prevalence of Ves v 1 sensitization in YJV-allergic patients is reported to range between 33% and 79%.^{9,11,12,41,62-65}

Ves v 5 is the “Antigen 5” protein from vespids.⁶¹ Antigen 5 proteins are recognized as the major and most potent allergen in venoms of the Vespidae family and are found in the venom of all Vespidae species, including YJV. The biological function of these proteins is as of yet unknown. Antigen 5 proteins are not found in HBV. Antigen 5 proteins are related by high amino acid sequence identity to pathogenesis-related proteins from mammals, reptiles, insects, fungi, and plants.⁶⁶ Homologous Antigen 5's from a large number of different yellow jackets, hornets, and paper wasps are known and patients show varying extents of cross-reactivity to these proteins.^47,66 Sensitization rates to Ves v 5 in YJV-allergic patients are reported to range between 85% and 98%.^{27,41,51,62-64,67,68}

Testing for sIgE to both Ves v 1 and Ves v 5 results in a diagnostic sensitivity as high as 92% to 98% for the detection of YJV allergic patients.^{27,28,42,43,46,48,51,63,64,69-71} Patients that test negative for sIgE to both Ves v 1 and Ves v 5 are very unlikely to have been sensitized by YJV exposure.^11,12,18

PWV: Paper Wasp (Polistes dominula) Venom

Polistes dominula is an invasive species that originated in Europe and is now found in the northeastern United States as well as in Europe and Australia. The related species Polistes exclamans, Polistes annularis, and Polistes fuscatus are indigenous to North America.^11,58 There are no marker allergens specific to Polistes, and patients primarily sensitized to PWV are often misdiagnosed as YJV allergic and at risk for being inadequately protected by yellow jacket VIT.^15,72

Pol d 5 is the “Antigen 5” protein from Polistes dominula and exhibits significant cross-reactivity with Ves v 5 of YJV. Antigen 5 proteins from Vespula and Polistes have sequence identity of approximately 57%.^47,73

Cross-reactive Carbohydrate Determinants (CCDs)

One of the main obstacles to determining the species responsible for HVA in a given individual using whole venom extract-based diagnostics is the occurrence of specific N-linked glycosylation of some Hymenoptera venom allergens. Many relevant venom allergen proteins are post-translationally glycosylated with unique alpha 1,3-linked fucose residues.¹⁵ While many mammalian proteins are glycosylated, only plants and insect proteins contain this specific glycan residue. Since this specific glycosylation does not occur in humans, exposure to proteins that are glycosylated with alpha 1,3-linked fucose residues often induces allergic sensitization and IgE production. This glycan sIgE does not reflect exposure to any specific allergen but represents a pan-sensitization to “Cross-reactive Carbohydrate Determinants” or “CCD” sensitization.¹⁵ IgE antibodies directed against CCD confound diagnostic tests using whole venom extracts.¹¹ CCD-IgE produces positive test results using multiple venom extracts that doesn’t reflect specific venom exposure.¹¹ CCD-sIgE accounts for up to 50% of double positivity to HBV and YJV.⁷² Glycosylation with CCD sugars does not occur in the manufacture of recombinant allergen components. As a consequence, tests using component proteins can be used to differentiate true venom sensitization from CCD sensitization in venom-extract positive patients.^{9,11,15,48,69,75} Unlike YJV and HBV, the venom of Polistes species are devoid of any a 1,3-core-fucosylation and hence are not confounded by CCD interference.⁷⁶

The measurement of CCD-specific IgE can be of value in cases where venom component testing is not definitive, especially where a venom extract IgE is positive but all venom components for that species is negative.^21,28

Resolving Double Positivity

Positivity to more than one venom extract can occur due to:

- True double-sensitization to multiple species of hymenoptera;

- Sensitization to cross-reacting allergen proteins present in venoms of different species;

- Sensitization to CCD (carbohydrate determinants) present in venoms of different species.

CRD can help resolve the cause of double positivity.

Apidae vs Vespula. It has been demonstrated that the use of Api m 1 on its own is insufficient to detect all HBV allergic individuals and therefore to distinguish between primary bee and wasp venom sensitization.^46,77 The use of other HBV marker allergens Api m 3 and Api m 10 and YJV marker allergens Ves v 1 and rVes v 5 improves the accuracy of determination of primary allergen sensitization.^{19,45,46,54,62}

Vespula vs Polistes. The discrimination between Vespula spp. and Polistes spp. sensitization is challenging due to high phylogenetic overlap between the two species.^12,24,61,73 Measurement of Ves v 5 and Pol d 5 can suggest the primary sensitizer in cases where the difference in specific IgE levels between the two molecules is particularly significant, with at least double values of one recombinant over the other.^70,78-80 However, a recent study showed that such proposed ratio was less accurate than CAP-inhibition results.⁸¹

Venom Allergy in patients with Mast Cell Disease

Patients suffering from mastocytosis and/or elevated baseline serum tryptase are more likely than others to go into anaphylaxis when experiencing an insect sting.^{8,9,15,31,38,63,82-84} The Stinging Insect Hypersensitivity Practice Parameter Update 2016 workgroup has recommended that venom allergy testing is appropriate in patients with indolent systemic mastocytosis, even with no history of allergic reaction to a sting, and that the clinician should consider the possibility of testing (and treating) such patients.⁹ Patients with mastocytosis frequently have negative allergy test results despite a clear history of hymenoptera sting-induced systemic allergic reaction.^8,9,15,31,85 Basal serum tryptase should be measured when there is a history of severe insect sting anaphylaxis (especially with hypotension or the absence of urticaria) and when skin and serum test results for venom-specific IgE are negative.^9,86 Adult patients with mastocytosis and/or elevated baseline serum tryptase are at risk for more severe reactions following stings.^11,87 A serum tryptase level within the upper normal range (8-13.2 ug/L) in patients with a history of venom-induced anaphylaxis have an increased risk of severe symptoms on re-exposure to hymenoptera venom.^88,89