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A WAO — ARIA — GA2LEN consensus document on molecular-based allergy diagnosis ([email protected]): Update 2020

Open AccessPublished:March 07, 2020DOI:https://doi.org/10.1016/j.waojou.2019.100091

      Abstract

      Precision allergy molecular diagnostic applications ([email protected]) is increasingly entering routine care. Currently, more than 130 allergenic molecules from more than 50 allergy sources are commercially available for in vitro specific immunoglobulin E (sIgE) testing. Since the last publication of this consensus document, a great deal of new information has become available regarding this topic, with over 100 publications in the last year alone. It thus seems quite reasonable to publish an update. It is imperative that clinicians and immunologists specifically trained in allergology keep abreast of the new and rapidly evolving evidence available for [email protected]
      [email protected] may initially appear complex to interpret; however, with increasing experience, the information gained provides relevant information for the allergist. This is especially true for food allergy, Hymenoptera allergy, and for the selection of allergen immunotherapy. Nevertheless, all sIgE tests, including [email protected], should be evaluated within the framework of a patient's clinical history, because allergen sensitization does not necessarily imply clinical relevant allergies.

      Keywords

      Introduction

      In the late 1960s, the discovery of the immunoglobulin (IgE) antibody provided a specific biomarker that could be used to identify allergic diseases triggered by environmental allergens (generally proteins). Traditional IgE antibody tests such as skin prick tests (SPTs) and in vitro specific IgE (sIgE) tests are based on crude extracts composed of allergenic and non-allergenic molecules obtained from an allergenic source. With the application of DNA technology in the late 1980s, allergenic molecules were characterized and cloned to resolve the determinants of various allergic diseases.
      • Fiocchi A.
      Setting the stage of innovations in allergy globally.
      • Canonica G.W.
      • Ansotegui I.J.
      • Pawankar R.
      • et al.
      Wao - ARIA - GA(2)LEN consensus document on molecular-based allergy diagnostics.
      • Luengo O.
      • Cardona V.
      Component resolved diagnosis: when should it be used?.
      • Tallar M.T.
      • Grayson M.H.
      Component-resolved allergen testing: the new frontier.
      The availability of allergenic molecules in the last decade has ushered in a new phase of diagnostics,
      • Kleine-Tebbe J.
      • Jakob T.
      Molecular Allergy Diagnostics.
      now termed precision allergy molecular diagnostic applications ([email protected]), allowing improved management of allergic diseases.
      • Van Gasse A.L.
      • Mangodt E.A.
      • Faber M.
      • Sabato V.
      • Bridts C.H.
      • Ebo D.G.
      Molecular allergy diagnosis: status anno 2015.
      In previous years, this diagnostic strategy has been termed component-resolved diagnostics (CRD), molecular-based allergy diagnostics (MBAD), or molecular allergy diagnostics (MAD). A multitude of articles regarding [email protected] have been published that reinforce the utility of adding this testing method to the care of the allergic patient.
      • Fiocchi A.
      Setting the stage of innovations in allergy globally.
      Thus, it appears useful to provide an update to the WAO — ARIA — GA2LEN consensus document on [email protected] which was published in 2013.
      • Canonica G.W.
      • Ansotegui I.J.
      • Pawankar R.
      • et al.
      Wao - ARIA - GA(2)LEN consensus document on molecular-based allergy diagnostics.

      Nomenclature

      Today, many of the most common allergenic molecules have been cloned or purified, have had their 3-dimensional structures elucidated, and can be consistently produced.
      • Maruyama N.
      • Nakagawa T.
      • Ito K.
      • et al.
      Measurement of specific IgE antibodies to Ses i 1 improves the diagnosis of sesame allergy.
      Because of the growing number of allergens identified, a systematic allergen nomenclature, approved by the World Health Organization and International Union of Immunological Species (WHO/IUIS) Allergen Nomenclature Subcommittee, has been established.
      • Pomes A.
      • Davies J.M.
      • Gadermaier G.
      • et al.
      WHO/IUIS allergen nomenclature: providing a common language.
      The subcommittee has been charged with developing and maintaining not only the systematic nomenclature developed for allergenic molecules but also a comprehensive database of known allergenic proteins, which can be accessed at http://www.allergen.org. Allergenic molecules are named using their Latin binomial name (genus and species). A detailed description of the terminology has been recently published.
      For example, allergens that begin with Phl p are from Phleum pratense (Timothy grass). A number is added to the name to distinguish the various allergens from the same species (e.g., Phl p 1, Phl p 2). The numbers are assigned to the allergens in the order of their identification. Allergenic molecules are classified into protein families according to their structure and biological function.
      • Letran A.
      • Espinazo M.
      • Moreno F.
      Measurement of IgE to pollen allergen components is helpful in selecting patients for immunotherapy.
      • Hamilton R.G.
      • Oppenheimer J.
      Serological IgE analyses in the diagnostic algorithm for allergic disease.
      • Jakob T.
      • Forstenlechner P.
      • Matricardi P.
      • Kleine-Tebbe J.
      Molecular allergy diagnostics using multiplex assays: methodological and practical considerations for use in research and clinical routine: Part 21 of the Series Molecular Allergology.
      • Woodfolk J.A.
      • Commins S.P.
      • Schuyler A.J.
      • Erwin E.A.
      • Platts-Mills T.A.
      Allergens, sources, particles, and molecules: why do we make IgE responses?.
      Many different molecules share common epitopes (antibody binding sites), and the same IgE antibody can bind and induce an immune response to allergenic molecules with similar structures from various allergen sources. These cross-reactive allergens give valuable information regarding sensitization to several different sources. In contrast, some molecules are unique markers for specific allergen sources, allowing for the identification of the primary sensitizer. Table 1 lists the components belonging to the most frequent allergen families and their availability on 3 different multiplex tests.
      Table 1Components belonging to the 8 most common allergen families in ISAC, ALEX, and Euroline
      • Heffler E.
      • Puggioni F.
      • Descalzi D.
      • Racca F.
      • Canonica G.W.
      • Melioli G.
      Microarray immunodiagnostics for aeroallergens.
      FamilySpeciesAllergenMultiplex text
      ISACALEXEuroline
      Bet v 1–related protein (PR-10)Actinidia deliciosa (green kiwi)Act d 8X
      Alnus glutinosa (alder)Aln g 1XX
      Apium graveolens (celery)Api g 1XX
      Arachis hypogaea (peanut)Ara h 8XX
      Betula pendula (white birch)Bet v 1XXX
      Corylus avellana (hazel)Cor a 1XX
      Glycine max (soybean)Gly m 4XX
      Daucus carota (carrot)Dau c 1X
      Malus domestica (apple)Mal d 1XX
      Prunus persica (peach)Pru p 1X
      Venom group 5 allergen familyPolistes dominulus (European paper wasp)Pol d 5XXX
      Vespula vulgaris (yellow jacket)Ves v 5XXX
      Cupin SuperfamilyAnacardium occidentale (cashew)Ana o 2X
      A. hypogaea (peanut)Ara h 1XXX
      Ara h 3XXX
      Corylus avellana (hazel)Cor a 9XX
      Cor a 11X
      G. max (soybean)Gly m 5XX
      Gly m 6XX
      Juglans regia (English walnut)Jug r 2XX
      EF hand domain (Ca++ binding proteins)B. pendula (white birch)Bet v 4X
      A. glutinosa (alder)Aln g 4X
      Gadus callarias (Baltic cod)Gad c 1X
      Gadus morhua (Baltic cod)Gad m 1X
      Penaeus monodon (black tiger shrimp)Pen m 4X
      Cyprinus carpio (European carp)Cyp c 1XX
      Phleum pratense (Timothy grass)Phl p 7XXX
      Expansin, C-terminal domainCynodon dactylon (Bermuda grass)Cyn d 1X
      P. pratense (Timothy grass)Phl p 1XXX
      Phl p 2XX
      LipocalinBlatella germanica (cockroach)Bla g 4X
      Bos domesticus (domestic cattle)Bos d 2X
      Bos d 5XXX
      Canis familiaris (domestic dog)Can f 1XX
      Can f 2XX
      Equus caballus (horse)Equ c 1XX
      Felis domesticus (domestic cat)Fel d 4XX
      Mus musculus (mouse)Mus m 1XX
      ProfilinB. pendula (white birch)Bet v 2XX
      Hevea brasiliensis (Para rubber tree)Hev b 8XX
      Mercurialis annua (annual mercury)Mer a 1X
      P. pratense (Timothy grass)Phl p 12XXX
      Phoenix dactylifera (date palm)Pho d 2X
      Olea europaea (olive)Ole e 2X
      Prolamin superfamilyA. occidentale (cashew)Ana o 3X
      A. hypogaea (peanut)Ara h 2XXX
      Ara h 6XXX
      Ara h 9XXX
      Artemisia vulgaris (mugwort)Art v 3XX
      Bertholletia excelsa (Brazil nut)Ber e 1XX
      Brassica /Sinapis spp.Sin a 1X
      Corylus avellana (hazel)Cor a 8XX
      Cor a 14X
      Fagopyrum esculentum (buckwheat)Fag e 2XX
      G. max (soybean)Gly m 8X
      J. regia (Englich walnut)Jug r 1XX
      Macadamia integrifolia (macadamia)Mac i 2SX
      Papaver somniferum (poppy)Pap s 2SX
      Sesamum indicum (sesame)Ses i 1X

      New concepts regarding the mechanisms of action of allergens

      Allergens induce sIgE sensitization of mast cells and trigger allergic inflammation upon re-exposure. The availability of natural purified (n) or recombinant (r) allergens has helped to improve our understanding of the mechanisms leading to this phenomenon, which vary depending on several ecological, biological, and structural characteristics of the allergenic molecules.
      • Valenta R.
      • Karaulov A.
      • Niederberger V.
      • et al.
      Molecular aspects of allergens and allergy.
      In addition to the production of sIgE and the IgE binding associated with Th2 immunity, allergens may also act by promoting tissue inflammation directly because of their enzymatic or other (still unknown) biological properties.
      The induction of sIgE and sensitization are not straightforward processes. For example, because aeroallergens are transported in particles (e.g., the feces of mites) and mostly interact with respiratory mucosae, the immune response results from stimulation by several components in addition to allergens.
      • Woodfolk J.A.
      • Commins S.P.
      • Schuyler A.J.
      • Erwin E.A.
      • Platts-Mills T.A.
      Allergens, sources, particles, and molecules: why do we make IgE responses?.
      This means that natural exposure sometimes does not result in important sensitization (and probably a greater IgG response than IgE response) if Th1-promoting components are inhaled simultaneously and overcome the effect of allergenic molecules, or if interleukin (IL)-10–mediated tolerance is induced by the presence of bacteria.
      • Schulke S.
      Induction of interleukin-10 producing dendritic cells as a tool to suppress allergen-specific T helper 2 responses.
      In addition, the immune response to allergens starts with the activation of innate immune receptors, which can also modulate the strength of the Th2 response.
      • Wang J.Y.
      The innate immune response in house dust mite-induced allergic inflammation.
      • Jacquet A.
      Innate immune responses in house dust mite allergy.
      • Satitsuksanoa P.
      • Kennedy M.
      • Gilis D.
      • et al.
      The minor house dust mite allergen Der p 13 is a fatty acid-binding protein and an activator of a TLR2-mediated innate immune response.
      Here again, we can infer that there are several pathways, some of them antagonistic, to the production of sIgE and clinically relevant sensitization.
      These pathways involve various types of cells, including the recently discovered innate lymphoid cells (ILC). Because several members of this cell population have been described, more data have to be obtained before a clear role for each member during the allergic response can be defined. The participation of ILC type 2 (ILC2) seems to be influenced by the type of allergen; for example, ILC2 cells are expanded more in allergic rhinitis induced by house dust mite (HDM) than in that induced by mugwort.
      • Fan D.
      • Wang X.
      • Wang M.
      • et al.
      Allergen-dependent differences in ILC2s frequencies in patients with allergic rhinitis.
      Also, in parasite-infected mice, HDM can induce the production of IL-13 and mediation of conventional type 2 inflammatory responses independently of T-cell receptor stimulation.
      • Guo L.
      • Huang Y.
      • Chen X.
      • Hu-Li J.
      • Urban Jr., J.F.
      • Paul W.E.
      Innate immunological function of TH2 cells in vivo.
      ILC2 may act to intensify the specific Th2 response or to provide, together with other ILC types, pro-inflammatory cytokines in reactions not mediated by IgE. Activation of ILC2 is mediated by IL-25, thymic stromal lymphopoietin, and particularly IL-33 (alarmin), which is produced by stimulated epithelial cells after exposure to allergens, infectious organisms, and pollutants.
      • Peebles Jr., R.S.
      At the bedside: the emergence of group 2 innate lymphoid cells in human disease.
      An example of the pro-inflammatory properties of allergens that are not mediated by IgE includes the proteolytic activity of Der p 1, which directly activates epithelial cells to induce the production of pro-inflammatory cytokines.
      • Lambrecht B.N.
      • Hammad H.
      Allergens and the airway epithelium response: gateway to allergic sensitization.
      Also, the non-proteolytic components of Der p 2 induce inflammation by other IgE-independent mechanisms.
      • Osterlund C.
      • Gronlund H.
      • Polovic N.
      • Sundstrom S.
      • Gafvelin G.
      • Bucht A.
      The non-proteolytic house dust mite allergen Der p 2 induce NF-kappaB and MAPK dependent activation of bronchial epithelial cells.
      • Chiou Y.L.
      • Lin C.Y.
      Der p2 activates airway smooth muscle cells in a TLR2/MyD88-dependent manner to induce an inflammatory response.
      • Ye Y.L.
      • Wu H.T.
      • Lin C.F.
      • et al.
      Dermatophagoides pteronyssinus 2 regulates nerve growth factor release to induce airway inflammation via a reactive oxygen species-dependent pathway.
      • Yu S.J.
      • Liao E.C.
      • Sheu M.L.
      • Chang D.T.
      • Tsai J.J.
      Cell-penetrating peptide derived from human eosinophil cationic protein inhibits mite allergen Der p 2 induced inflammasome activation.
      • Yin S.C.
      • Liao E.C.
      • Chiu C.L.
      • Chang C.Y.
      • Tsai J.J.
      Der p2 internalization by epithelium synergistically augments toll-like receptor-mediated proinflammatory signaling.
      • Trompette A.
      • Divanovic S.
      • Visintin A.
      • et al.
      Allergenicity resulting from functional mimicry of a Toll-like receptor complex protein.
      • Wang W.C.
      • Tsai J.J.
      • Kuo C.Y.
      • Chen H.M.
      • Kao S.H.
      Non-proteolytic house dust mite allergen, Der p 2, upregulated expression of tight junction molecule claudin-2 associated with Akt/GSK-3beta/beta-catenin signaling pathway.
      These characteristics could influence the clinical impact of each allergen but may not correlate with the frequency of IgE binding. This could be the case for Der p 13
      • Satitsuksanoa P.
      • Kennedy M.
      • Gilis D.
      • et al.
      The minor house dust mite allergen Der p 13 is a fatty acid-binding protein and an activator of a TLR2-mediated innate immune response.
      ; Der p 18
      • Resch Y.
      • Blatt K.
      • Malkus U.
      • et al.
      Molecular, structural and immunological characterization of Der p 18, a chitinase-like house dust mite allergen.
      ,
      • Mueller G.A.
      • Edwards L.L.
      • Aloor J.J.
      • et al.
      The structure of the dust mite allergen Der p 7 reveals similarities to innate immune proteins.
      ; Der p 7
      • Mueller G.A.
      • Edwards L.L.
      • Aloor J.J.
      • et al.
      The structure of the dust mite allergen Der p 7 reveals similarities to innate immune proteins.
      ; Der p 5
      • Pulsawat P.
      • Soongrung T.
      • Satitsuksanoa P.
      • et al.
      The house dust mite allergen Der p 5 binds lipid ligands and stimulates airway epithelial cells through a TLR2-dependent pathway.
      ; other proteases such as Der p 3, Der p 6, and Der p 9
      • Chevigne A.
      • Jacquet A.
      Emerging roles of the protease allergen Der p 1 in house dust mite-induced airway inflammation.
      ; and other lipid-binding proteins such as Der p 21 and Blo t 13. Therefore, in some cases, the IgE binding detected by [email protected] might be considered a proxy for more crucial allergenic properties.
      An interesting mechanism of action of HDM is that it induces epigenetic changes in immune cells and epithelial/bronchial muscle cells. Recent studies revealed that modifications to DNA methylation in B cells might influence the susceptibility to mite sensitization,
      • Pascual M.
      • Suzuki M.
      • Isidoro-Garcia M.
      • et al.
      Epigenetic changes in B lymphocytes associated with house dust mite allergic asthma.
      and hypomethylation of the IL-13 gene is associated with an increased risk of allergic rhinitis due to HDM sensitization.
      • Li J.Y.
      • Zhang Y.
      • Lin X.P.
      • et al.
      Association between DNA hypomethylation at IL13 gene and allergic rhinitis in house dust mite-sensitized subjects.
      Also, HDM can induce epigenetic modifications in experimental airway inflammation in mice, changing the methylation pattern of important genes such as pde4 d
      • Shang Y.
      • Das S.
      • Rabold R.
      • Sham J.S.
      • Mitzner W.
      • Tang W.Y.
      Epigenetic alterations by DNA methylation in house dust mite-induced airway hyperresponsiveness.
      and tgfb1.
      • Cheng R.Y.
      • Shang Y.
      • Limjunyawong N.
      • et al.
      Alterations of the lung methylome in allergic airway hyper-responsiveness.
      Also, HDM induces the same epigenetic modifications as diesel exhaust in an ex vivo model of inflammation in human bronchial epithelial cells.
      • Zhang X.
      • Chen X.
      • Weirauch M.T.
      • et al.
      Diesel exhaust and house dust mite allergen lead to common changes in the airway methylome and hydroxymethylome.
      These studies suggest that HDM also induces IgE-mediated bronchial inflammation by altering the epigenetic patterns of cells involved in bronchial homeostasis.
      Another important point on the mechanisms of action of allergens is the role of IgG and its subclasses, which in turn also has 2 aspects. One is the involvement of IgG as an effector mechanism in the pathogenesis of some allergic reactions, such as food allergy. Although there is some evidence that IgG participates in food allergy not mediated by IgE,
      • Liu F.
      • Lin L.R.
      • Zhang H.L.
      • et al.
      Laboratorial characteristics of patients with diarrhoea suffering from egg white allergy.
      the evaluation of serum-specific IgG and IgG4 has no proven predictive value in food allergy diagnosis.
      • Stapel S.O.
      • Asero R.
      • Ballmer-Weber B.K.
      • et al.
      Testing for IgG4 against foods is not recommended as a diagnostic tool: EAACI Task Force Report.
      ,
      • Werfel T.
      • Asero R.
      • Ballmer-Weber B.K.
      • et al.
      Position paper of the EAACI: food allergy due to immunological cross-reactions with common inhalant allergens.
      More studies are therefore needed to define specific IgG as a marker of food allergy.
      • Antico A.
      • Pagani M.
      • Vescovi P.P.
      • Bonadonna P.
      • Senna G.
      Food-specific IgG4 lack diagnostic value in adult patients with chronic urticaria and other suspected allergy skin symptoms.
      ,
      • Lucas Moreno J.M.
      Diarrhoea due to allergy to egg: is there a role for specific IgG?.
      The second aspect is the potential clinical impact of the IgG/IgE ratio, an interesting and traditional theme that has been revived in recent years, probably because of the availability of purified components for [email protected] There is important evidence suggesting that, in addition to what is observed during allergen immunotherapy (AIT), a high IgG/IgE ratio is associated with fewer allergic symptoms.
      • Santos A.F.
      • James L.K.
      • Bahnson H.T.
      • et al.
      IgG4 inhibits peanut-induced basophil and mast cell activation in peanut-tolerant children sensitized to peanut major allergens.
      • Hofmaier S.
      • Hatzler L.
      • Rohrbach A.
      • et al.
      Default" versus "pre-atopic" IgG responses to foodborne and airborne pathogenesis-related group 10 protein molecules in birch-sensitized and nonatopic children.
      • Holt P.G.
      • Strickland D.
      • Bosco A.
      • et al.
      Distinguishing benign from pathologic TH2 immunity in atopic children.
      In particular, IgG4 seems to have a direct role in the induction of tolerance.
      • Bianchini R.
      • Roth-Walter F.
      • Ohradanova-Repic A.
      • et al.
      IgG4 drives M2a macrophages to a regulatory M2b-like phenotype: potential implication in immune tolerance.
      This area of research should provide useful information regarding the inception, evolution, and diagnosis of allergic diseases, but there is currently no standardized way to apply these findings to [email protected]

      The usefulness of [email protected] for management of allergic diseases: a bird's eye view

      [email protected] is increasingly entering routine care and improves management of allergy. This is particularly evident for food allergy.
      • Luengo O.
      • Cardona V.
      Component resolved diagnosis: when should it be used?.
      ,
      • Tallar M.T.
      • Grayson M.H.
      Component-resolved allergen testing: the new frontier.
      ,
      • Van Gasse A.L.
      • Mangodt E.A.
      • Faber M.
      • Sabato V.
      • Bridts C.H.
      • Ebo D.G.
      Molecular allergy diagnosis: status anno 2015.
      ,
      • Maruyama N.
      • Nakagawa T.
      • Ito K.
      • et al.
      Measurement of specific IgE antibodies to Ses i 1 improves the diagnosis of sesame allergy.
      ,
      • Hamilton R.G.
      • Oppenheimer J.
      Serological IgE analyses in the diagnostic algorithm for allergic disease.
      • Jakob T.
      • Forstenlechner P.
      • Matricardi P.
      • Kleine-Tebbe J.
      Molecular allergy diagnostics using multiplex assays: methodological and practical considerations for use in research and clinical routine: Part 21 of the Series Molecular Allergology.
      • Woodfolk J.A.
      • Commins S.P.
      • Schuyler A.J.
      • Erwin E.A.
      • Platts-Mills T.A.
      Allergens, sources, particles, and molecules: why do we make IgE responses?.
      Knowing which allergenic molecules the patient is sensitized to can help to determine the likelihood of local versus systemic reactions and to predict the persistence of clinical symptoms. For example, some allergens, such as storage proteins in peanuts (e.g., Ara h 2) and nuts (e.g., Cor a 9), are associated with severe reactions, whereas sensitization to other allergens is usually associated with less severe reactions. Another critical aspect, difficult to elucidate using traditional tests, is the stability of the allergen. Allergens that are stable to heat and digestion (e.g., Ara h 2 from peanut) are more likely to cause severe clinical reactions, whereas heat- and digestion-labile molecules (e.g., Ara h 8 from peanut) are more likely to cause milder, local reactions (such as pruritus) or to be tolerated. Similarly, identifying whether the sensitization is primary and likely to be clinically relevant or due to clinically irrelevant cross-sensitization helps to evaluate the likelihood of reaction on exposure to different allergen sources. A recent meta-analysis determined that molecular diagnostics are particularly useful for food allergy.
      • Flores Kim J.
      • McCleary N.
      • Nwaru B.I.
      • Stoddart A.
      • Sheikh A.
      Diagnostic accuracy, risk assessment, and cost-effectiveness of component-resolved diagnostics for food allergy: a systematic review.
      [email protected] may also be helpful in the assessment of idiopathic anaphylaxis. If positive, it may orient the allergist to the triggering allergen; if negative, a non-IgE-mediated mechanism underlying the anaphylaxis (such as mast cell disorders) should be considered.
      • Luengo O.
      • Cardona V.
      Component resolved diagnosis: when should it be used?.
      ,
      • Cardona V.
      • Ansotegui I.J.
      Component-resolved diagnosis in anaphylaxis.
      It is also useful in cases of Hymenoptera venom allergy, with a recent study demonstrating that [email protected] can identify a subgroup of patients likely to fail on honey bee venom (HBV) immunotherapy.
      • Satitsuksanoa P.
      • Kennedy M.
      • Gilis D.
      • et al.
      The minor house dust mite allergen Der p 13 is a fatty acid-binding protein and an activator of a TLR2-mediated innate immune response.
      ,
      • Fan D.
      • Wang X.
      • Wang M.
      • et al.
      Allergen-dependent differences in ILC2s frequencies in patients with allergic rhinitis.
      ,
      • Frick M.
      • Fischer J.
      • Helbling A.
      • et al.
      Predominant Api m 10 sensitization as risk factor for treatment failure in honey bee venom immunotherapy.
      [email protected] may also improve the selection of both patients and specific allergens for AIT for inhalant allergies (e.g., for pollen),
      • Letran A.
      • Espinazo M.
      • Moreno F.
      Measurement of IgE to pollen allergen components is helpful in selecting patients for immunotherapy.
      ,
      • Wang J.Y.
      The innate immune response in house dust mite-induced allergic inflammation.
      ,
      • Jacquet A.
      Innate immune responses in house dust mite allergy.
      with a recent study demonstrating that the added precision [email protected] brought to the prescribing of immunotherapy reduced overall costs.
      • Saltabayeva U.
      • Garib V.
      • Morenko M.
      • et al.
      Greater real-life diagnostic efficacy of allergen molecule-based diagnosis for prescription of immunotherapy in an area with multiple pollen exposure.
      All of these topics are described in greater detail in this revised WAO - ARIA - GA2LEN consensus document.

      In vitro diagnostics

      Singleplex and multiplex measurement arrays

      An ever-increasing number of studies focusing on different allergenic molecules or allergic diseases are rapidly being published. However, the search for additional clinically relevant molecules is ongoing and needed to improve the positive predictive value of allergy diagnostic testing. Both the scientific and industrial communities have been involved in developing new reagents and new diagnostic tools in the past few years. At present, the presence of IgE antibodies against allergenic molecules may be determined using a singleplex (one assay per sample) or multiplex (multiple assays per sample) measurement platform. A singleplex platform allows the clinician to select those allergenic molecules necessary for an accurate diagnosis as determined by the clinical history of the patient.
      • van Hage M.
      • Hamsten C.
      • Valenta R.
      ImmunoCAP assays: pros and cons in allergology.
      The multiplex approach allows for the IgE response to a broad array of preselected allergens on a chip to be characterized independently of the clinical history. Notably, analysis by multiplex assay may be even possible with a dried blood spot, which can be easily transported.
      • Garib V.
      • Rigler E.
      • Gastager F.
      • et al.
      Determination of IgE and IgG reactivity to more than 170 allergen molecules in paper-dried blood spots.
      There are several commercially available immuno-solid-phase multiplex allergen arrays: the Thermo Fisher ImmunoCAP ISAC (Immuno-solid-phase Allergen Chip), which contains 112 allergens from 51 allergen sources
      • Jakob T.
      • Forstenlechner P.
      • Matricardi P.
      • Kleine-Tebbe J.
      Molecular allergy diagnostics using multiplex assays: methodological and practical considerations for use in research and clinical routine: Part 21 of the Series Molecular Allergology.
      ; the new ImmunoCAP ISAC 112i, with 112 components from 48 allergen sources, where some components have been removed (Hymenoptera components, Pla a 2, Jug r 2) and some others were added (Ana o 3, Can f 4, Can f 6, Cor a 14, Der p 23 and alpha-Gal); the MADx Allergen Explorer (ALEX; containing 282 allergens: 156 extracts and 126 components)
      • Heffler E.
      • Puggioni F.
      • Peveri S.
      • Montagni M.
      • Canonica G.W.
      • Melioli G.
      Extended IgE profile based on an allergen macroarray: a novel tool for precision medicine in allergy diagnosis.
      ; and the Euroline microstrips.
      • Di Fraia M.
      • Arasi S.
      • Castelli S.
      • et al.
      A new molecular multiplex IgE assay for the diagnosis of pollen allergy in Mediterranean countries: a validation study.
      The large number of extracts/allergens from multiple classes of allergenic sources (Table 2) provides extensive and detailed information about a patient's sensitization profile.
      • Heffler E.
      • Puggioni F.
      • Peveri S.
      • Montagni M.
      • Canonica G.W.
      • Melioli G.
      Extended IgE profile based on an allergen macroarray: a novel tool for precision medicine in allergy diagnosis.
      ,
      • Melioli G.
      • Bonifazi F.
      • Bonini S.
      • et al.
      The ImmunoCAP ISAC molecular allergology approach in adult multi-sensitized Italian patients with respiratory symptoms.
      Other diagnostic tools based on allergen arrays are under development, and new tools will likely be available in the near future.
      Table 2List of Components available for the Molecular Allergy Diagnosis
      Common components available on the market. Components are in in alphabetic order, as of June 2019, from different sources (Thermofisher, MADx, Hycor, Euroline, Siemens)
      Act d 1, Act d 2, Act d 5, Act d 8, Alkalase, Alt a 1, Alt a 1, Alt a 6, Amb a 1, Amb a 4, Ana c 2, Ana o 2, Ana o 3, Ani s 1, Ani s 3, Api g 1., Api g 2, Api g 6, Api m 1, Api m 4, Api m 10, Api m 2, Api m 3, Api m 5, Ara h 1, Ara h 2, Ara h 3, Ara h 6, Ara h 8, Ara h 9, Art v 1, Art v 3, Asp f 1, Asp f 2, Asp f 3, Asp f 4, Asp f 6, Asp o 21, Asp r 1, Ber e 1, Bet v 1, Bet v 2, Bet v 4, Bet v 6, Bla g 1, Bla g 2, Bla g 4, Bla g 5, Bla g 7, Blo t 5, Bos d 2, Bos d 4, Bos d 5, Bos d 6, Bos d 8, Bos d Lactoferrin, Can f 1, Can f 2, Can f 3, Can f 4, Can f 5, Can f 6, Car p 1, Che a 1, Cla h 8, Cor a 1, Cor a 11, Cor a 14, Cor a 8, Cor a 9, Cry j 1, Cup a 1, Cyn d 1, Cyp c 1, Dau c 1, Der f 1, Der f 2, Der p 1, Der p 10, Der p 11, Der p 2, Der p 23, Der p 5, Der p 7, Equ c 1, Equ c 3, Fag e 2, Fel d 1, Fel d 2, Fel d 4, Fel d 7, Fra e 1, Gad m 1, Gad c 1, Gal d 1, Gal d 2, Gal d 3, Gal d 4, Gal d 5, Gal-alpha, gliadin, Gly d 2, Gly m 4, Gly m 5, Gly m 6, Gly m 8, Hev b 1, Hev b 11, Hev b 3, Hev b 5, Hev b 6., Hev b 8, Hom s LF, Jug r 1, Jug r 2, Jug r 3, Lep d 2, Lol p 1, Mac i 2S Albumin, Mal d 1, Mal d 2, Mal d 3, Mal d 4, Maxatase, Mala s 1, Mala s 11, Mala s 5, Mala s 6, Mala s 9, Mer a 1, Mus m 1, MUXF3, Ole e 1, Ole e 2, Ole e 7, Ole e 9, Pap s 2S Albumin, Par j 2, Pen a 1, Pen m 1, Pen m 2, Pen m 4, Per a 7, Phl p 1, Phl p 11, Phl p 12, Phl p 2, Phl p 4, Phl p 5, Phl p 6, Phl p 7, Pho d 2, Pla a 1, Pla a 2, Pla a 3, Pla l 1, Pol d 5, Pru av 1, Pru av 3, Pru av 4, Pru p 1, Pru p 3, Pru p 4, Sal k 1, Savinase, Ses i 1, Sin a 1, Sola l 6, Sus s Pepsin, Sus s PSA, Tri a gliadin, Tri a 14, Tri a 19, Tri a aA_TI, Ves v 1, Ves v 5, Vit v 1.
      Components only available in the Thermofisher ImmunoCAP system (including ISAC)
      Act d 8, Alkalase, Ana o 2, Api m 3, Api m 4, Api m 5, Asp f 1, Asp f 2, Asp o 21, Bet v 4, Bla g 7, Blo t 5, Bos d lactoferrin, Can f 4, Can f 5, Can f 6, Car p 1, Cry j 1, Cyn d 1, Equ c 3, Fel d 7, Gad c 1, Gal-alpha, gliadin f9, Jug r 3, Lep d 2, Maxatase, Mer a 1, MUXF3, Ole e 7, Ole e 9, Pen a 1, Pen m 2, Pen m 4, Phl p 11, Phl p 4, Pla a 2, Pla a 3, Pru p 1, Pru p 4, Sal k 1, Savinase, Sus s Pepsin, Sus s PSA, Tri a 14, Tri a 19.0101, Tri a aA_TI, Ves v 1
      Components only available in the MADx ALEX
      Act d 10, Amb a 4, Api g 2, Api g 6, Bla g 4, Bos d 2, Cor a 11, Dau c 1, Der p 11, Der p 5, Der p 7, Fra e 1, Gad m 1, Gly d 2, Gly m 8, Hom s LF, Lol p 1, Mac i 2S Albumin, Mal d 2, Mala s 1, Mala s 11, Mala s 5, Mala s 6, Mala s 9, Ole e 2, Pap s 2S Albumin, Per a 7, Pho d 2, Sin a 1, Sola l 6, Tri a gliadin, Vit v 1
      Multiplex assays are especially suited for use in patients with complex sensitization patterns or symptoms. The multiplex technology is a consolidated [email protected] approach for improved diagnosis, prognosis, and selection of patients for AIT. Although they are commercial products, they have been the mainstay of many studies. In recent years, an increasing number of reagents have been made available for singleplex assays, but multiplex assays have been the object of both research and development by the diagnostic industry, and they contain many more components than are available for singleplex assays. These new features are discussed in more detail in the sections below, together with the available informatics support and criteria for interpreting allergen arrays and supporting the diagnosis.
      Precision medicine was launched at a worldwide level by the initiative of then US President Barack Obama in 2015. Before that, precision (or personalized) medicine was more a dream than a reality. Precision or personalized medicine is a discipline that identifies specific biomarkers of a given disease in a given patient (the so-called “endotype”) that are based on the patient's characteristics, evaluated in real time, and may impact the therapeutic approach. Precision or personalized medicine is expected to deeply affect all medical procedures in the near future,
      • Hamburg M.A.
      • Collins F.S.
      The path to personalized medicine.
      including more-appropriate selection of patients and treatments and more-appropriate allocation of resources and interventions in general. The increasing availability of biological drugs, bio-engineering, and genetic and “omics” tools have already started to affect the decisional processes in medicine.
      Allergy represents an excellent paradigm for precision medicine because, in many cases, the patient can be well characterized by available clinical features, diagnostic tests, and biomarkers. In addition, many of the underlying mechanisms are known in detail even if others are still being investigated. In this context, the introduction and availability of [email protected] represent a real advance in the description of the patient's IgE repertoire. At present, the therapeutic strategy based on standard drugs (such as inhaled corticosteroids, bronchodilators, and antihistamines) has not substantially changed, but the detailed definition of the sensitization profile has allowed the use of AIT to be refined.
      • Passalacqua G.
      • Canonica G.W.
      AIT (allergen immunotherapy): a model for the "precision medicine.
      • Riccio A.M.
      • De Ferrari L.
      • Chiappori A.
      • et al.
      Molecular diagnosis and precision medicine in allergy management.
      • Kleine-Tebbe J.
      • Matricardi P.M.
      • Hamilton R.G.
      Allergy work-up including component-resolved diagnosis: how to make allergen-specific immunotherapy more specific.
      In this context, AIT still represents an etiology-based treatment when the clinical aspects and the diagnostic procedures are well standardized, but [email protected] allows its prescription to be better focused, and to achieve the best results, reducing costs. The impact of [email protected] on the use and prescription of AIT will be discussed below.
      In summary, with increasing experience, [email protected] is generally straightforward to interpret and can provide relevant, additional information for the allergist. However, the clinical utility of many of the allergenic molecules needs further investigation. Because of the speed at which new [email protected] data are becoming available, clinicians are required to keep pace with a large amount of novel information. This WAO - ARIA - GA2LEN consensus update document on [email protected] provides a practical guide to the indication, determination, and interpretation of [email protected] It is designed for clinicians specifically trained in allergology but can also be a good starting point for new users.

      Available diagnostic tools

      In the last year, the multiplex approach to in vitro [email protected] has greatly improved. The ISAC 103 version was described in the original consensus statement,
      • Canonica G.W.
      • Ansotegui I.J.
      • Pawankar R.
      • et al.
      Wao - ARIA - GA(2)LEN consensus document on molecular-based allergy diagnostics.
      and an improved version of ISAC, based on 112 different components from 51 allergen sources, was released in 2011. The new assay has been shown to be repeatable (intra-assay assessment), reproducible (interassay assessment), and suitable for supporting a multiplex allergy diagnosis.
      • Martinez-Aranguren R.
      • Lizaso M.T.
      • Goikoetxea M.J.
      • et al.
      Is the determination of specific IgE against components using ISAC 112 a reproducible technique?.
      A deeper analysis of ISAC 112 characteristics also demonstrated that it is a highly reproducible and accurate method that may be considered as a single analyte assay given the EN ISO 15189 accreditation procedure.
      • Lambert C.
      • Sarrat A.
      • Bienvenu F.
      • et al.
      The importance of EN ISO 15189 accreditation of allergen-specific IgE determination for reliable in vitro allergy diagnosis.
      Results obtained by ISAC 112 also correlate well with singleplex ImmunoCAP results.
      • Huss-Marp J.
      • Gutermuth J.
      • Schaffner I.
      • et al.
      Comparison of molecular and extract-based allergy diagnostics with multiplex and singleplex analysis.
      Recently, the new ISAC 112i was introduced.
      Despite the success of the ISAC assays, some partially unexpected cross-binding between its components have been identified.
      • Villalta D.
      • Conte M.
      • Asero R.
      • Da Re M.
      • Stella S.
      • Martelli P.
      Isolated IgE reactivity to native walnut vicilin-like protein (nJug r 2) on ISAC microarray is due to cross-reactive carbohydrate epitopes.
      For example, nPhl p 4, a highly glycosylated protein, can bind to IgE specific for cross-reactive carbohydrate determinants (CCDs), and IgE to the native walnut vicilin-like protein nJug r 2 can also be raised in patients sensitized to CCDs.
      • Villalta D.
      • Conte M.
      • Asero R.
      • Da Re M.
      • Stella S.
      • Martelli P.
      Isolated IgE reactivity to native walnut vicilin-like protein (nJug r 2) on ISAC microarray is due to cross-reactive carbohydrate epitopes.
      For this reason, the real clinical significance of a positive nJug r 2 result must be carefully evaluated in the context of the results of other components and clinical findings.
      Allergen microarrays have also been used to evaluate the presence of sIgE in fluids other than serum or plasma. Leonardi et al.
      • Leonardi A.
      • Borghesan F.
      • Faggian D.
      • Plebani M.
      Microarray-based IgE detection in tears of patients with vernal keratoconjunctivitis.
      recently showed that in vernal keratoconjunctivitis, ISAC can detect the presence of sIgE to grass, tree, mites, animals, and food allergens in tears. What was particularly interesting was that, in some patients, sIgE were absent in serum but detectable in tears. The presence of sIgE only in tears of patients with symptoms only in the eyes supports the idea that tissues can be locally sensitized. Using an innovative approach,
      • Hochwallner H.
      • Alm J.
      • Lupinek C.
      • et al.
      Transmission of allergen-specific IgG and IgE from maternal blood into breast milk visualized with microarray technology.
      Valenta and co-workers analyzed the presence of IgE in samples of breast milk. This use of microarray technology for 2 alternative specimen types, tears and breast milk, opens new fields for research and in clinics.
      [email protected] is useful for identifying sensitivities to many, but not all, allergens. For example, D'Amelio et al.
      • D'Amelio C.M.
      • Goikoetxea M.J.
      • Martinez-Aranguren R.
      • et al.
      Is the performance of ImmunoCAP ISAC 112 sufficient to diagnose peach and apple allergies?.
      examined whether the performance of ImmunoCAP ISAC 112 is sufficient to diagnose peach and apple allergies. They conclude that although the sensitivity of the peach components in ISAC could be improved, it is adequate in Italy. The same authors
      • Goikoetxea M.J.
      • D'Amelio C.M.
      • Martinez-Aranguren R.
      • et al.
      Is microarray analysis really useful and sufficient to diagnose nut allergy in the mediterranean area?.
      concluded that the diagnostic performance of ISAC was adequate for hazelnut and walnut allergy but not for peanut allergy. Finally, in a different situation,
      • Javaloyes G.
      • Goikoetxea M.J.
      • Garcia Nunez I.
      • et al.
      Pru p 3 acts as a strong sensitizer for peanut allergy in Spain.
      even if standard ImmunoCAP have, for apple and peach, a wider number of available components (in particular Mad d 3, a lipid transfer protein [LTP]), the evaluation of Pru p 3 (largely homologous to Mal d 3) may support the identification of an apple sensitivity even if “the presence of sIgE against Pru p 3 in LTP sensitized patients can be due to cross-sensitization and should therefore not be used to predict clinical symptoms".
      In the period that ISAC 103 and ISAC 112 have been used in clinics, other strategies for multiplex molecular diagnostics have been developed. The MeDALL group developed a novel microarray adding more than 70 new components to the standard panel of ISAC 112.
      • Lupinek C.
      • Wollmann E.
      • Baar A.
      • et al.
      Advances in allergen-microarray technology for diagnosis and monitoring of allergy: the MeDALL allergen-chip.
      These new components are allergens from peanut, nuts (almond, cashew, and pistachio), cow's milk, wheat, olive pollen, mites, dogs, insect venom, Staphylococcus aureus toxins, and maltose binding protein. The clinical features of the MeDALL microarray were evaluated during the so-called allergen march from childhood to adolescence.
      • Skrindo I.
      • Lupinek C.
      • Valenta R.
      • et al.
      The use of the MeDALL-chip to assess IgE sensitization: a new diagnostic tool for allergic disease?.
      The prevalence of allergic sensitization increased from age 10–16 years and was similar by SPT and ImmunoCAP and significantly higher with the MeDALL chip at age 10. All 3 tests were comparable for identification of allergic sensitization among children with current rhinitis or asthma.
      A different approach has been developed by an English/Swedish company that designed and further implemented a microarray, the Microtest system, where whole extracts are spotted in addition to single-allergen components.
      • Williams P.
      • Onell A.
      • Baldracchini F.
      • Hui V.
      • Jolles S.
      • El-Shanawany T.
      Evaluation of a novel automated allergy microarray platform compared with three other allergy test methods.
      This combination of extracted allergens and recombinant components was tested against 3 other allergy test methods (SPT, ImmunoCAP, and ISAC 112) in a pairwise fashion for each component. The methods produced concordant results 81%–88% of the time, with correlation coefficients for the most prevalent allergens (cat, dog, mite, Timothy, birch, and peanut) ranging from 0.73 to 0.95, although results of the different tests were discordant in some patients. Thus, the Microtest system provides another alternative for testing common allergens.
      More recently, ALEX (Allergen Explorer) was developed by MADx in Vienna, Austria. ALEX is an array of allergens spotted on a solid phase by the use of nanoparticles. ALEX contains 282 reagents (156 allergen extracts and 126 recombinant or highly purified molecules). Thus, this chip, like the Microtest, contains second-level diagnostics (represented by extract allergens) and third-level diagnostics (represented by single molecules) are available, which is remarkable, and the results from ALEX correlate well with those from ISAC.
      • Heffler E.
      • Puggioni F.
      • Peveri S.
      • Montagni M.
      • Canonica G.W.
      • Melioli G.
      Extended IgE profile based on an allergen macroarray: a novel tool for precision medicine in allergy diagnosis.
      ,
      • Buzzulini F.
      • Da Re M.
      • Scala E.
      • et al.
      Evaluation of a new multiplex assay for allergy diagnosis.
      This microarray allows the measurement of an IgE profile including “whole” allergens and recombinant or purified allergen proteins in a single chip. Thus, 2 characteristics are specific of this assay: the first is that it can be a suitable method for the bottom-up strategy of allergy diagnosis, which tests isolated allergens before whole extracts.
      • Matricardi P.M.
      • Kleine-Tebbe J.
      • Hoffmann H.J.
      • et al.
      EAACI molecular allergology user's guide.
      The second is that it provides an extended IgE profile in line with the indications of a precision medicine approach.
      • Riccio A.M.
      • De Ferrari L.
      • Chiappori A.
      • et al.
      Molecular diagnosis and precision medicine in allergy management.
      This approach requires the most accurate definition of the patient's phenotype to identify the patient's endotype
      • Canonica G.W.
      • Ferrando M.
      • Baiardini I.
      • et al.
      Asthma: personalized and precision medicine.
      and to provide an accurate diagnosis and appropriate treatment. In consideration of the very large number of allergens and components and the significant complexity of the interpretation of the results (at least for non-professional molecular allergists), ALEX has been associated with a new version of the expert system Allergenius, originally developed for the interpretation of ISAC results.
      • Matricardi P.M.
      • Kleine-Tebbe J.
      • Hoffmann H.J.
      • et al.
      EAACI molecular allergology user's guide.
      ,
      • Melioli G.
      • Spenser C.
      • Reggiardo G.
      • et al.
      Allergenius, an expert system for the interpretation of allergen microarray results.
      Perhaps the greatest value of ALEX is the provision of a CCD inhibitor, which reduces clinically irrelevant cross-sensitization and has a direct impact on primary sensitization information.
      • Heffler E.
      • Puggioni F.
      • Peveri S.
      • Montagni M.
      • Canonica G.W.
      • Melioli G.
      Extended IgE profile based on an allergen macroarray: a novel tool for precision medicine in allergy diagnosis.
      Another group of tests are represented by the multiple allergen simultaneous tests (MAST) immunoblots, such as Euroline, which is a commercially available assay for [email protected] based on the immunoblot technique. A sensitization profile can be generated from the simultaneous determination of sIgE to different allergen components and extracts. Notably, different IgE profiles are available. Substantial agreement between MAST and ImmunoCAP was found for inhalant, food, and venom allergens, and it thus represents a valid diagnostic alternative.
      • Di Fraia M.
      • Arasi S.
      • Castelli S.
      • et al.
      A new molecular multiplex IgE assay for the diagnosis of pollen allergy in Mediterranean countries: a validation study.
      Finally, epitope mapping will be the real future, provided that a large number of clinical and experimental data become available. Indeed, peptides are recognized by humans in an HLA-restricted manner. For this reason, “dominant peptides” can be identified, but many other “individual peptides” should also be considered. A panel of partially overlapping peptides fully covering the whole allergenic protein could be used for highly represented allergens and has meaning only if sIgE have been previously observed in a more classic allergen-component assay. Keeping this consideration in mind, many groups have published extremely exciting results that clearly show that both diagnostic and prognostic indications can be derived from the analysis of the IgE profile directed to peptides of different allergen sources.
      • Kuhne Y.
      • Reese G.
      • Ballmer-Weber B.K.
      • et al.
      A novel multipeptide microarray for the specific and sensitive mapping of linear IgE-binding epitopes of food allergens.
      • Savilahti E.M.
      • Kuitunen M.
      • Valori M.
      • et al.
      Use of IgE and IgG4 epitope binding to predict the outcome of oral immunotherapy in cow's milk allergy.
      • Lisson M.
      • Erhardt G.
      Mapping of epitopes occurring in bovine alpha(s1)-Casein variants by peptide microarray immunoassay.
      • De-Simone S.G.
      • Napoleao-Pego P.
      • De-Simone T.S.
      Spot synthesis: an optimized microarray to detect IgE epitopes.
      • Perez-Gordo M.
      • Pastor-Vargas C.
      • Lin J.
      • et al.
      Epitope mapping of the major allergen from Atlantic cod in Spanish population reveals different IgE-binding patterns.
      • Martinez-Botas J.
      • Cerecedo I.
      • Zamora J.
      • et al.
      Mapping of the IgE and IgG4 sequential epitopes of ovomucoid with a peptide microarray immunoassay.
      • Gimenez G.
      • Benede S.
      • Lin J.
      IgE epitope mapping using peptide microarray immunoassay.
      Of course, the need for large epidemiological studies will be necessary to define rules that can be applied to different populations of patients. However, the possibility of dissecting the immune response mediated by IgE will be included in the fourth diagnostic level of allergic diseases, such as the basophil activation test, in the near future. In this context, it is clear that the diagnostic process in complex or highly complex patients will need to be managed by highly skilled groups with a specialized laboratory and advanced informatics tools.

      Patients most likely to benefit from [email protected]

      There is a general consensus that patients with multiple sensitizations will likely benefit from [email protected] This includes patients with respiratory sensitization to a large number of allergen families and patients with pollen-food or inhalant food syndromes. Another relevant application of [email protected] is food allergy and the food protein-induced enterocolitis syndrome,
      • Mehr S.
      • Lee E.
      • Hsu P.
      • et al.
      Innate immune activation occurs in acute food protein-induced enterocolitis syndrome reactions.
      • Nowak-Wegrzyn A.
      • Warren C.M.
      • Brown-Whitehorn T.
      • Cianferoni A.
      • Schultz-Matney F.
      • Gupta R.S.
      Food protein-induced enterocolitis syndrome in the US population-based study.
      • Feuille E.
      • Nowak-Wegrzyn A.
      Recognizing and Treating Food Protein-Induced Enterocolitis Syndrome.
      because it is now possible to determine the individual pattern of IgE sensitization by analyzing single allergenic molecules instead of complex allergenic extracts.
      Allergens commonly used for [email protected] can be either recombinant or purified native, and serum sIgE levels can be detected through singleplex assays or in multiplex arrays. In order to choose the test correctly, the specialist should take several factors into consideration: the target of the [email protected] (e.g., immunotherapy for respiratory diseases, latex allergy, food allergy, cross-reactions between food and inhalant allergens), the complexity of the clinical case, and the availability in each country of the molecular diagnostic tools. Complex cases, such as multiple sensitizations to respiratory and food allergens and idiopathic anaphylaxis, are preferably studied with multiplex assays.
      • Fedenko E.
      • Elisyutina O.
      • Shtyrbul O.
      • et al.
      Microarray-based IgE serology improves management of severe atopic dermatitis in two children.
      Multiplex assays performed early in life seem to be also useful for predicting the risk of developing allergic symptoms in later life.
      • Westman M.
      • Lupinek C.
      • Bousquet J.
      • et al.
      Early childhood IgE reactivity to pathogenesis-related class 10 proteins predicts allergic rhinitis in adolescence.
      • Posa D.
      • Perna S.
      • Resch Y.
      • et al.
      Evolution and predictive value of IgE responses toward a comprehensive panel of house dust mite allergens during the first 2 decades of life.
      • Wickman M.
      • Lupinek C.
      • Andersson N.
      • et al.
      Detection of IgE reactivity to a handful of allergen molecules in early childhood predicts respiratory allergy in adolescence.
      Clinicians should always be adequately trained, familiar with the main allergen protein families, and aware of the characteristics of the [email protected] assays. In particular, the multiplex tests can produce complex results, and the clinician should therefore be familiar with the results each type of assay might produce. In general, educational programs for both the use and interpretation of [email protected] tests should be implemented.
      • Melioli G.
      • Spenser C.
      • Reggiardo G.
      • et al.
      Allergenius, an expert system for the interpretation of allergen microarray results.
      Multiplex platforms allow for testing of more than 100 molecules simultaneously (Table 2) in a very low quantity of serum and without interference from high total IgE levels, but they are less sensitive and less appropriate for monitoring sensitization than their singleplex counterparts.
      • Canonica G.W.
      • Ansotegui I.J.
      • Pawankar R.
      • et al.
      Wao - ARIA - GA(2)LEN consensus document on molecular-based allergy diagnostics.
      However, when the detection of more than 12 or 13 sIgE is needed, it has been suggested that the multiplex assay is more cost-effective than the singleplex diagnostic approach and is therefore preferred.
      • Passalacqua G.
      • Melioli G.
      • Bonifazi F.
      • et al.
      The additional values of microarray allergen assay in the management of polysensitized patients with respiratory allergy.

      Risk assessment and [email protected]

      [email protected] can increase the accuracy of an allergy diagnosis in certain circumstances.
      • Luengo O.
      • Cardona V.
      Component resolved diagnosis: when should it be used?.
      In allergic patients, a molecular approach is suitable for the following:
      • assessing the risk of potential allergic reactions, which depend on the individual allergic (clinical) sensitization profile;
      • evaluating whether unknown potential triggering factors are present (i.e., the presence of sIgE versus allergenic molecules correlated with high risk for allergic reactions).
      In particular, in the case of polysensitization, [email protected] makes it possible to distinguish between primary and cross-sensitization, leading to a significant improvement in patient management. For example, appropriate avoidance diets can be recommended when the correct food allergen is identified; conversely, by identifying cross-sensitivities [email protected] can prevent needless food avoidance or prescriptions for self-injectable adrenaline. Cross-sensitization between aeroallergens and food allergens is very common. In some cases, the presence of a respiratory allergy to an allergen with a shared epitope to food may lead to a clinically relevant cross-reactivities. Pollen sensitization may lead to “pollen-food syndromes”, such as birch-apple or celery-mugwort-spice syndrome. Nonpollen aeroallergens cross-reacting with foods include mite-shrimp syndrome. This topic has been covered in an extensive review
      • Popescu F.D.
      Cross-reactivity between aeroallergens and food allergens.
      that describes cross-reactivities between different combinations of food and inhalant allergens and which could be an important tool for the interpretation of multiple sensitivities detected with [email protected] (either multiplex or singleplex). The fundamental work of Valenta et al. is another useful guide for understanding the molecular mechanisms underlying allergic sensitization and cross-reactivities of food allergens.
      • Valenta R.
      • Hochwallner H.
      • Linhart B.
      • Pahr S.
      Food allergies: the basics.
      If patients have sIgE to natural allergens but no clinical manifestations after exposure to the allergen, the presence of IgE directed to CCDs should be investigated; in 10–20% of patients with pollen sensitivity, sIgE against carbohydrate epitopes called N-glycans can be detected.
      • Fotisch K.
      • Vieths S.
      N- and O-linked oligosaccharides of allergenic glycoproteins.
      The presence of serum sIgE directed towards proteins that are stable to heat and acidic conditions (such as LTPs, storage proteins, gliadins, tropomyosins, parvalbumins, caseins, and ovomucoid) is generally associated with a higher risk of systemic or severe reactions. In contrast, a pattern of sensitization to gastro- and heat-labile proteins (families such as profilins, PR-10, thaumatins, ovalbumin, and lactalbumin) is generally correlated with a lower risk of severe allergic manifestations; an exception is Gly m 4, a member of the PR-10 family, which is a potential marker for severe reactions in patients consuming large quantities of soy drinks and sensitized to Bet v1, the major allergen of birch.
      • Berneder M.
      • Bublin M.
      • Hoffmann-Sommergruber K.
      • Hawranek T.
      • Lang R.
      Allergen chip diagnosis for soy-allergic patients: Gly m 4 as a marker for severe food-allergic reactions to soy.
      There are other exceptions to the classical rule of “stable allergens–severe reactions and labile allergens–mild reactions”. Indeed, anaphylactic reactions to foods have occurred in patients monosensitized to PR-10 allergens
      • Roseler S.
      • Balakirski G.
      • Plange J.
      • et al.
      [Anaphylaxis to PR-10 proteins (Bet v1 homologues)].
      or profilins,
      • Alvarado M.I.
      • Jimeno L.
      • De La Torre F.
      • et al.
      Profilin as a severe food allergen in allergic patients overexposed to grass pollen.
      and it is well known that clinical expression of LTPs varies from asymptomatic to anaphylactic reactions.
      A molecular approach allows patients (e.g., those with occupational latex sensitization) to be stratified according to the risk of reactions as a secondary prevention strategy, but it is important to remember that the severity of allergic reactions also depends on other factors, such as the route of allergen exposure and the presence of cofactors, such as exercise or concomitant drug consumption. Interestingly, the geographic area can also influence the risk of allergic manifestations: in the Mediterranean area, for instance, Pru p 3 is one of the most common triggers for anaphylaxis and severe reactions. A cohort of 133 Italian patients allergic to peach with positive SPT to whole peach extract and sIgE against Pru p 3 has been studied.
      • Uasuf C.G.
      • Villalta D.
      • Conte M.E.
      • et al.
      Different co-sensitizations could determine different risk assessment in peach allergy? Evaluation of an anaphylactic biomarker in Pru p 3 positive patients.
      The population recruited from the northeastern part of the country, where sensitization to PR-10 and profilin is more common than in the south, also had sIgE to Pru p 1 (42.8%) and Pru p 4 (12.7%), whereas no patients from the south were sensitized to PR-10 or profilin. In the southern population, the levels of sIgE to Pru p 3 were significantly different in symptomatic patients and asymptomatic patients, and in subjects with mild systemic reactions and subjects with severe systemic reactions. In contrast, in the northeastern population, no differences were found in the levels of sIgE to Pru p 3 between these groups. Also, in the southern population, the severity scores of the clinical reactions and the levels of sIgE to Pru p 3 were statistically correlated, whereas in the northeastern population, the correlation was not statistically significant due to the low number of patients with severe allergic reactions. Another important finding of this study was the determination of cutoff levels for sIgE to Pru p 3 that allowed discriminating asymptomatic from symptomatic patients (2.87 kUA/L for the southern Italian population and 2.69 kUA/L for the northeastern population). Lower levels of sIgE to Pru p 3 and the co-sensitization to Pru p1 and/or Pru p 4 could explain the lower risk of severe reactions in the northeastern patients than in the southern patients, who were monosensitized and presented with higher levels of sIgE to Pru p 3, as if the simultaneous positivity to Pru p 1 and/or Pru p 4 can play a sort of ‘protective’ role against the development of severe symptoms induced by Pru p 3.
      • Pastorello E.A.
      • Farioli L.
      • Pravettoni V.
      • et al.
      Pru p 3-sensitised Italian peach-allergic patients are less likely to develop severe symptoms when also presenting IgE antibodies to Pru p 1 and Pru p 4.
      Geographic differences are extremely relevant, and the future work of the [email protected] committee will attempt to define a geographic map of allergen sensitization for the whole world.
      The role of [email protected] in food allergy risk assessment is relevant in pediatric patients, where it is now possible to map the sensitization profile in a low quantity of serum and, in some cases, to avoid oral food challenges that are costly, time-consuming, and not free from risk.
      • Van Hoeyveld E.D.S.L.
      Diagnosis of food allergy Part 2. The use of allergen components for in vitro diagnosis of food allergy in children.
      An interesting review recently reported the cutoff values of sIgE levels for stratifying the risk of reactions to food (peanuts and tree nuts, cow's milk, egg, wheat, fish and seafood, fruits, and vegetables) in pediatric populations.
      • Borres M.P.
      • Maruyama N.
      • Sato S.
      • Ebisawa M.
      Recent advances in component resolved diagnosis in food allergy.
      Nevertheless, the results probably depend on the in vitro method used and comorbidities, and further large-scale studies are required before these cutoff values can be officially validated.
      • Borres M.P.
      • Maruyama N.
      • Sato S.
      • Ebisawa M.
      Recent advances in component resolved diagnosis in food allergy.
      Another paper extensively discussed the role of specific allergenic molecules in the development and persistence of egg allergy; for instance, the presence of elevated sIgE levels against ovomucoid (Gal d 1) seems to predict the persistence of allergy over time
      • Ando H.
      • Moverare R.
      • Kondo Y.
      • et al.
      Utility of ovomucoid-specific IgE concentrations in predicting symptomatic egg allergy.
      ,
      • Ohtani K.
      • Sato S.
      • Syukuya A.
      • et al.
      Natural history of immediate-type hen's egg allergy in Japanese children.
      and the inability to tolerate even extensively cooked eggs. Also, the evaluation of protein-specific sIgE levels and IgE/IgG4 ratios seems to be more helpful than the SPT in predicting tolerance to baked egg. In general, children sensitized to sequential epitopes are less likely to resolve their egg allergy than those presenting with sIgE to conformational epitopes. Nevertheless, even though [email protected] represents a promising diagnostic tool, larger studies (including challenges with cooked egg) are needed to confirm its clinical utility; furthermore, the possibility of performing [email protected] does not exclude the need for oral provocation tests in many patients
      • Chokshi N.Y.
      • Sicherer S.H.
      Molecular diagnosis of egg allergy: an update.
      because sIgE levels do not significantly predict the severity of allergic reactions or the outcome of oral provocation tests with the culprit food; that is, sIgE represents sensitization but not necessarily allergy. The presence of serum sIgE against food allergens in the absence of a significant clinical history is a common finding. In this case, the patient should be followed carefully for possible allergic manifestations, including reactions to other cross-reacting foods, but avoiding the suspected trigger only because of a positive sIgE result is not recommended, particularly during childhood when avoidance can lead to failure to thrive. An elimination diet, especially when the suspected food allergy could be due to cross-reactions, should be required only when a clear history is present or an oral food challenge is positive.
      [email protected] is also useful in cases of anaphylaxis, especially idiopathic anaphylaxis, food-dependent exercise-induced anaphylaxis, and red-meat anaphylaxis.
      • Pattanaik D.
      • Lieberman P.
      • Lieberman J.
      • Pongdee T.
      • Keene A.T.
      The changing face of anaphylaxis in adults and adolescents.
      Multiplex testing was used to identify potential causes of idiopathic anaphylaxis.
      • Heaps A.
      • Carter S.
      • Selwood C.
      • et al.
      The utility of the ISAC allergen array in the investigation of idiopathic anaphylaxis.
      The test identified 203 sensitizations in 22 (20%) of the 110 patients examined. Of these, 35 were considered to be plausible causes of the anaphylaxis, and the newly identified triggers were confirmed in 11 of the 22 patients. Omega-5-gliadin and shrimp allergens represented 45% of the previously unrecognized sensitizations, followed by seed storage proteins, nonspecific LTP (nsLTP), and latex allergens.
      Specialists should keep in mind that some allergenic molecules are poorly represented in allergen extracts, and the predictive value of first-level diagnostic tests (SPT, sIgE detection) may not allow a proper diagnosis. For example, a proper investigation of serum sIgE directed against proteins belonging to the 2S-albumin family of seed storage proteins is quite necessary for risk assessment.
      • Borres M.P.
      • Maruyama N.
      • Sato S.
      • Ebisawa M.
      Recent advances in component resolved diagnosis in food allergy.
      Molecules such as Ara h 2 and 6, Cor a 14, Ana o 3, Ber e 1, and Jug r 1 are significantly predictive of allergic reactions to peanut, hazel, cashew, Brazil nut, and walnut, respectively. Co-sensitization to Ara h 2 and Ara h 6 is associated with severe reactions to peanut. Recent data indicate that Ses i 1 may represent the best marker for sesame allergy.
      • Maruyama N.
      • Nakagawa T.
      • Ito K.
      • et al.
      Measurement of specific IgE antibodies to Ses i 1 improves the diagnosis of sesame allergy.
      A recent review has been published regarding the relevance of lipophilic molecular allergens in diagnosis of food allergy.
      • Jappe U.
      • Schwager C.
      Relevance of lipophilic allergens in food allergy diagnosis.
      In fact, some severe allergic reactions are caused by lipophilic molecules that are not contained in the allergenic extracts for in vivo tests; among them are the oleosins, which are insoluble in saline or aqueous solutions. At the time this guideline was written, only oleosins of peanut (Ara h 10, Ara h 11), sesame (Ses i 4 and Ses i 5), and hazelnut (Cor a 12 and Cor a 13) had been registered as allergens. Furthermore, serum IgE specific for a group of molecular allergens, such as Jug r 2 (vicilin-like protein), Ana o 2 (legumin-like protein), Ses i 1 (2S albumin storage protein), Pen m 2 (arginine kinase), and Pen m 4 (sarcoplasmic calcium-binding protein), are identifiable only through multiplex arrays and are not available in singleplex.
      In the case of food-dependent exercise-induced anaphylaxis, [email protected] makes it possible to identify the pattern of allergic sensitization and then to avoid the trigger allergen(s) and other possible cofactors (such as nonsteroidal anti-inflammatory drugs or alcohol). Wheat-dependent exercise-induced anaphylaxis (WDEIA), for instance, is classically associated with allergic sensitization to omega-5-gliadin (Tri a 19)
      • Matsuo H.
      • Dahlstrom J.
      • Tanaka A.
      • et al.
      Sensitivity and specificity of recombinant omega-5 gliadin-specific IgE measurement for the diagnosis of wheat-dependent exercise-induced anaphylaxis.
      ; nevertheless, patients reporting symptoms suggestive for WDEIA should be tested also for nsLTP, such as Pru p 3 and Tri a 14, because sensitization to the LTP family has been reported to be very common in cofactor-exacerbated allergic reactions in the Mediterranean basin.
      • Pastorello E.A.
      • Farioli L.
      • Stafylaraki C.
      • et al.
      Wheat-dependent exercise-induced anaphylaxis caused by a lipid transfer protein and not by omega-5 gliadin.
      ,
      • Cardona V.
      • Luengo O.
      • Garriga T.
      • et al.
      Co-factor-enhanced food allergy.
      In another study, 64 (78%) of 82 patients with food-dependent exercise-induced anaphylaxis were positive to Pru p 3, indicating that LTPs are the most frequent sensitizer in Italian subjects.
      • Romano A.
      • Scala E.
      • Rumi G.
      • et al.
      Lipid transfer proteins: the most frequent sensitizer in Italian subjects with food-dependent exercise-induced anaphylaxis.
      Of recent interest is the use of [email protected] in the diagnosis of anaphylaxis occurring after ingestion of red meat (e.g., beef, pork, and lamb), despite tolerance to other meats like chicken or turkey. Tick bites from Ixodes ricinus in Europe and Amblyomma americanum in the US are considered the main determinants for the IgE responses against a mammalian oligosaccharide epitope called galactose-α-1,3-galactose (alpha-gal).
      • Apostolovic D.
      • Mihailovic J.
      • Commins S.P.
      • et al.
      Allergenomics of the tick Ixodes ricinus reveals important alpha-Gal-carrying IgE-binding proteins in red meat allergy.
      IgE to alpha-gal has been associated with 2 distinct forms of anaphylaxis: i) delayed-onset anaphylaxis, which occurs 3–6 h after ingestion of mammalian food products (e.g., beef or pork); and ii) immediate-onset anaphylaxis during the first exposure to intravenous cetuximab, a monoclonal antibody for the treatment of metastatic colorectal cancer.
      • Levin M.
      • Apostolovic D.
      • Biedermann T.
      • et al.
      Galactose alpha-1,3-galactose phenotypes: lessons from various patient populations.
      sIgE levels seem to decrease over time, and some patients can tolerate red meat again after about 1–2 years if no additional tick bites occur.
      • Wilson J.M.
      • Platts-Mills T.A.E.
      Meat allergy and allergens.

      [email protected] in selected sensitizations

      Respiratory allergens

      Data have been published recently related to the molecular profiles associated with the development of allergic asthma.
      • Wickman M.
      • Lupinek C.
      • Andersson N.
      • et al.
      Detection of IgE reactivity to a handful of allergen molecules in early childhood predicts respiratory allergy in adolescence.
      ,
      • Karaulov A.V.
      • Garib V.
      • Garib F.
      • Valenta R.
      Protein biomarkers in asthma.
      ,
      • Simpson A.
      • Lazic N.
      • Belgrave D.C.
      • et al.
      Patterns of IgE responses to multiple allergen components and clinical symptoms at age 11 years.
      In a Korean study of 168 patients with allergic rhinitis, sIgE to molecular HDM allergens obtained with ISAC correlated with the clinical diagnosis and was 78.9% comparable to ImmunoCAP specificity, although the concordance was lower for Alternaria, birch, and mugwort.
      • Jung J.H.
      • Kang I.G.
      • Kim S.T.
      Comparison of component-resolved diagnosis by using allergen microarray with the conventional tests in allergic rhinitis patients: the first using in korea.
      In a study from Singapore, 135 atopic subjects presenting mostly with allergic rhinitis underwent multiplex testing. A strong association was found between allergic rhinitis and HDM sensitization, whereas asthma and atopic dermatitis were not correlated with sensitization to any allergen, prompting the conclusion that the microarray should probably be adjusted for regional allergens.
      • Santosa A.
      • Andiappan A.K.
      • Rotzschke O.
      • et al.
      Evaluation of the applicability of the Immuno-solid-phase allergen chip (ISAC) assay in atopic patients in Singapore.
      A Bavarian study with 86 patients showed that the IgE reactivities to major allergens had direct clinical relevance, whereas pan-allergens alone did not lead to clinical symptoms.
      • San Nicolo M.
      • Braun T.
      • Eder K.
      • Berghaus A.
      • Groger M.
      Clinical relevance of IgE to profilin and/or polcalcin in pollen-sensitized patients.
      The often complex patterns of multisensitizations, which include IgE to cross-binding epitopes, may prompt the development of more sophisticated analysis tools. A UK-based group used latent variable modeling to study the patterns of sensitivities on ISAC in 461 children.
      • Simpson A.
      • Lazic N.
      • Belgrave D.C.
      • et al.
      Patterns of IgE responses to multiple allergen components and clinical symptoms at age 11 years.
      Allergens clustered into 3 groups of allergenic components, which allowed the authors to correlate different clinical patterns with these clusters of IgE sensitivity. Because multiplex diagnostics are complex, it is likely that such models will be needed to understand the clinical implications of the results.
      • Simpson A.
      • Lazic N.
      • Belgrave D.C.
      • et al.
      Patterns of IgE responses to multiple allergen components and clinical symptoms at age 11 years.
      The specific molecular sensitization pattern of IgE, combined with the semiquantitative level determination, may predict the risk for allergic rhinitis and asthma, as well as the risk of accelerated and severe clinical reactivity for Hymenoptera venom,
      • Jakob T.
      • Rafei-Shamsabadi D.
      • Spillner E.
      • Muller S.
      Diagnostics in Hymenoptera venom allergy: current concepts and developments with special focus on molecular allergy diagnostics.
      food allergy, exercise-induced food allergy, red-meat delayed anaphylaxis, latex allergy, or Anisakis allergy in the individual patient.
      • Cardona V.
      • Ansotegui I.J.
      Component-resolved diagnosis in anaphylaxis.
      Equally important is the fact that clinically relevant sensitizations can be differentiated from non-relevant sensitizations to CCDs and profilins, which translate to practical, relevant recommendations for patients.
      • Larenas-Linnemann D.
      • Luna-Pech J.A.
      • Mosges R.
      Debates in Allergy Medicine: allergy skin testing cannot be replaced by molecular diagnosis in the near future.
      ,
      • Jensen-Jarolim E.
      • Jensen A.N.
      • Canonica G.W.
      Debates in allergy medicine: molecular allergy diagnosis with ISAC will replace screenings by skin prick test in the future.
      Along these lines, it has been observed that co-sensitization to profilins is associated with a lower occurrence of systemic reactions to nsLTP or storage proteins.
      • Scala E.
      • Abeni D.
      • Guerra E.C.
      • et al.
      Cosensitization to profilin is associated with less severe reactions to foods in nsLTPs and storage proteins reactors and with less severe respiratory allergy.

      House dust mites

      The presence of IgE to both Der p 2 and Der p 1 has highly significant predictive value for immediate-type asthma.
      • Minami T.
      • Fukutomi Y.
      • Lidholm J.
      • et al.
      IgE Abs to Der p 1 and Der p 2 as diagnostic markers of house dust mite allergy as defined by a bronchoprovocation test.
      In a birth cohort study of 1184 subjects in Italy,
      • Custovic A.
      • Sonntag H.J.
      • Buchan I.E.
      • Belgrave D.
      • Simpson A.
      • Prosperi M.C.
      Evolution pathways of IgE responses to grass and mite allergens throughout childhood.
      a combined sensitization to Der p 1 and Der p 2 represented the highest risk factor for asthma development, independent of age. Others have reported the specific impact of Der p 2 and Der f 2 allergens on severe asthma.
      • Sylvestre L.
      • Jegu J.
      • Metz-Favre C.
      • Barnig C.
      • Qi S.
      • de Blay F.
      Component-based allergen-microarray: der p 2 and der f 2 dust mite sensitization is more common in patients with severe asthma.
      Cysteine proteases, such as Der p 1 and papain, may have a percutaneous sensitization capacity, in addition to the described capacity to disrupt bronchial epithelial barriers.
      • Stremnitzer C.
      • Manzano-Szalai K.
      • Willensdorfer A.
      • et al.
      Papain degrades tight junction proteins of human keratinocytes in vitro and sensitizes C57bl/6 mice via the skin independent of its enzymatic activity or TLR4 activation.
      Der p 23 is a major allergen associated with asthma in both pediatric and adult populations.
      • Posa D.
      • Perna S.
      • Resch Y.
      • et al.
      Evolution and predictive value of IgE responses toward a comprehensive panel of house dust mite allergens during the first 2 decades of life.
      ,
      • Celi G.
      • Brusca I.
      • Scala E.
      • et al.
      House dust mite allergy in Italy-Diagnostic and clinical relevance of Der p 23 (and of minor allergens): a real-life, multicenter study.
      The recently identified Der p 11 (a non-fecal allergen from Dermatophagoides pteronyssinus) seems to be a useful serological marker for the identification of a subgroup of HDM-allergic patients suffering from atopic dermatitis.
      • Banerjee S.
      • Resch Y.
      • Chen K.W.
      • et al.
      Der p 11 is a major allergen for house dust mite-allergic patients suffering from atopic dermatitis.
      The development of HDM allergen sensitization during life (the so-called allergen march) has been studied at a molecular level with their relationships with symptoms.
      • Posa D.
      • Perna S.
      • Resch Y.
      • et al.
      Evolution and predictive value of IgE responses toward a comprehensive panel of house dust mite allergens during the first 2 decades of life.
      Dermatophagoides components cross-react with components of other HDM, in particular those from Blomia tropicalis. In this context, a positive IgE to Blomia components is specific in tropical environments, whereas it is related to a cross-reaction in temperate climates.
      The cross-reactive capacity of mite allergens was analyzed in 200 Chinese patients with HDM allergy by [email protected], showing that IgE to Der p 10 correlated with cross-sensitization to shrimp, moth, and cockroach allergens (Pen m 1, Ani s 3, and Bla g 7).
      • Zeng G.
      • Luo W.
      • Zheng P.
      • et al.
      Component-resolved diagnostic study of Dermatophagoides pteronyssinus major allergen molecules in a southern Chinese cohort.
      A clinical risk assessment of the apparent cross-reactivity revealed that higher levels of IgE to rPen a 1 and rDer p 10 correlated with positive responses in double-blind placebo-controlled food challenges (DBPCFC) with shrimp. Furthermore, diagnosed asthma to HDM and IgE to nDer p 1, 2, and 10 predicted a higher risk for clinically relevant shrimp allergy.
      • Farioli L.
      • Losappio L.M.
      • Giuffrida M.G.
      • et al.
      Mite-induced asthma and IgE levels to shrimp, mite, tropomyosin, arginine kinase, and der p 10 are the most relevant risk factors for challenge-proven shrimp allergy.
      Tropical climates, although scarcely studied, are a suitable environment for the development of mite sensitization; indeed, mites are the most prevalent allergen source in the tropics, followed by pets and cockroaches,
      • Sanchez-Borges M.
      • Capriles-Hulett A.
      • Torres J.
      • et al.
      Diagnosis of allergic sensitization in patients with allergic rhinitis and asthma in a tropical environment.
      whereas pollen and molds are less relevant than in North America and Europe. In tropical places where helminth infections are prevalent, cross-reactivity between mites and tropomyosins from the nematode Ascaris lumbricoides are associated with asthma symptoms.
      • Celi G.
      • Brusca I.
      • Scala E.
      • et al.
      House dust mite allergy in Italy-Diagnostic and clinical relevance of Der p 23 (and of minor allergens): a real-life, multicenter study.
      ,
      • Ahumada V.
      • Garcia E.
      • Dennis R.
      • et al.
      IgE responses to Ascaris and mite tropomyosins are risk factors for asthma.
      ,
      • Buendia E.
      • Zakzuk J.
      • Mercado D.
      • Alvarez A.
      • Caraballo L.
      The IgE response to Ascaris molecular components is associated with clinical indicators of asthma severity.
      In tropical climates, Blo t 5 from B. tropicalis has been associated with severe allergic dermatitis.
      • Camargo Lopes de Oliveira L.
      • Pierotti F.F.
      • Mallozi M.
      • et al.
      rBlo t 5 is a potential contributor to the severity of atopic dermatitis in a Brazilian population.
      Recently, a clear relationship has been shown between sensitization to HDM components and the outcome of AIT with the relevant components.
      • Chen K.W.
      • Zieglmayer P.
      • Zieglmayer R.
      • et al.
      Selection of house dust mite-allergic patients by molecular diagnosis may enhance success of specific immunotherapy.
      Several cases of anaphylaxis after oral ingestion of mites have been reported in patients sensitized to mites who ingested contaminated food (pancake syndrome). The species identified were Suidasia spp., D. pteronyssinus, Aleuroglyphus ovatus, Lepidoglyphus destructor, and B. tropicalis.
      • Sanchez-Borges M.
      • Fernandez-Caldas E.
      Hidden allergens and oral mite anaphylaxis: the pancake syndrome revisited.
      Of note, group 2 mite allergens (NPC2), which are heat resistant, have been suggested to be responsible for (severe) symptoms of oral mite anaphylaxis.
      • Sanchez-Borges M.
      • Fernandez-Caldas E.
      Hidden allergens and oral mite anaphylaxis: the pancake syndrome revisited.

      Pollens

      Regional and climate differences determine pollen counts and species. Birch pollen is a dominant pollen source in much of Europe, with Bet v 1 as the single major allergen that is responsible for cross-sensitizations and cross-reactivities with pollen from other species and plant-derived food. The higher the IgE levels to Bet v 1, the higher the risk for cross-reactivities in a hierarchical clustering of Bet v 1 > Mal d 1 > Cor a 1.04 > Ara h 8 > Pru p 1 > Aln g 1 > Api g 1 > Act d 8 > Gly m 4, such that if IgE was present to the allergens lower in the hierarchy it was also present to those allergens higher in the list.
      • Westman M.
      • Lupinek C.
      • Bousquet J.
      • et al.
      Early childhood IgE reactivity to pathogenesis-related class 10 proteins predicts allergic rhinitis in adolescence.
      Additionally, high levels of IgE to Bet v 1 correlate with the likelihood of allergic rhinitis persisting beyond the age of 16 years. Interesting associations between clinical presentation and specific PR-10 sensitization profiles were observed in a birch-free Mediterranean area: Bet v 1, Cor a 1.0101, and Aln g 1 reactivity are associated with respiratory symptoms, whereas Ara h 8, Cor a 1.0401, Gly m 4, Mal d 1, and Pru p 1 are selectively linked to the occurrence of oral allergic syndrome.
      • Scala E.
      • Abeni D.
      • Cecchi L.
      • et al.
      Molecular recognition profiles and clinical patterns of PR-10 sensitization in a birch-free mediterranean area.
      In Brazil, IgE profiles typical of grass pollen sensitization were found.
      • Moreira P.F.
      • Gangl K.
      • Vieira Fde A.
      • et al.
      Allergen microarray indicates pooideae sensitization in Brazilian grass pollen allergic patients.
      In Iran, IgE in 202 adult asthmatic patients was directed against the grass pollen allergens Phl p 1 and 5, but even more to Salsola kali (Sal k 1), which is an important pollen source in this area.
      • Teifoori F.
      • Shams-Ghahfarokhi M.
      • Postigo I.
      • et al.
      Identification of the main allergen sensitizers in an Iran asthmatic population by molecular diagnosis.
      However, the authors did not find a correlation between IgE levels and asthma symptoms.
      The development of IgE to grass pollen and mite during childhood was studied in a component-based fashion in the birth cohort of 1184 subjects in Italy mentioned above. Whereas an early onset of sensitization was associated with decreased lung function and asthma, the late-onset type was predictive for allergic rhinitis.
      • Custovic A.
      • Sonntag H.J.
      • Buchan I.E.
      • Belgrave D.
      • Simpson A.
      • Prosperi M.C.
      Evolution pathways of IgE responses to grass and mite allergens throughout childhood.
      In the case of grass pollen sensitization, the timing of IgE development (early vs. late) was rather decisive for the clinical course, whereas in mite allergy, the presence of IgE to Der p 1 or 2, or combined sensitization, had a higher predictive value. Another extensive study on the development of grass pollen allergy has been published in the past.
      • Hatzler L.
      • Panetta V.
      • Lau S.
      • et al.
      Molecular spreading and predictive value of preclinical IgE response to Phleum pratense in children with hay fever.
      Savi et al. found that in 140 patients with allergic rhinitis or asthma caused by sensitization to grass pollen, the only correlation between allergens and symptoms was that low levels of Phl p 5 IgE were correlated with an absence of asthma.
      • Savi E.
      • Peveri S.
      • Incorvaia C.
      • et al.
      Association between a low IgE response to Phl p 5 and absence of asthma in patients with grass pollen allergy.
      Similarly, Bokanovic et al. did not find a correlation of IgE levels to any of the tested grass pollen molecules, nor to symptom severity, and suggested that Phl p 1 is sufficient as a marker for sensitization without clinical relevance.
      • Bokanovic D.
      • Aberer W.
      • Hemmer W.
      • et al.
      Determination of sIgE to rPhl p 1 is sufficient to diagnose grass pollen allergy.
      Interesting work on mugwort sensitization comes from China: Art v 1 and Art an 7 were predominant in a patient cohort in the northern part of the country that suffered more often from asthma than a cohort in the Southwest, where Art v 3 was prominent in addition to Art v 1 and Art an 7. However, potential confounding environmental factors were not addressed.
      • Guo L.
      • Huang Y.
      • Chen X.
      • Hu-Li J.
      • Urban Jr., J.F.
      • Paul W.E.
      Innate immunological function of TH2 cells in vivo.
      ,
      • Gao Z.
      • Fu W.Y.
      • Sun Y.
      • et al.
      Artemisia pollen allergy in China: component-resolved diagnosis reveals allergic asthma patients have significant multiple allergen sensitization.
      Vernal conjunctivitis usually occurs on a background of a family history of allergies, most often in young boys. Armentia et al.
      • Armentia A.
      • Iglesias B.
      • Iglesias D.
      • et al.
      Component-resolved diagnostics in vernal conjunctivitis.
      showed that [email protected] could be used to diagnose allergy and to introduce precise AIT, which was successful in reducing eye diseases after 1 year in 13 of the 25 patients examined.

      Pet dander

      Animals are the leading cause of allergic asthma after mites and pollens,
      • Quirce S.
      Asthma in alergologica-2005.
      with some geographic exceptions. In Europe, about 26% of adults evaluated for suspected allergy to inhalant allergens are sensitized to cats and about 27% are sensitized to dogs.
      • Heinzerling L.M.
      • Burbach G.J.
      • Edenharter G.
      • et al.
      GA(2)LEN skin test study I: GA(2)LEN harmonization of skin prick testing: novel sensitization patterns for inhalant allergens in Europe.
      In the US, 12.1% are sensitized to cats and 11.8% are sensitized to dogs.
      • Konradsen J.R.
      • Fujisawa T.
      • van Hage M.
      • et al.
      Allergy to furry animals: new insights, diagnostic approaches, and challenges.
      Although there are possible differences between breeds, all dogs produce allergenic proteins found in the epithelium, dander, lingual glands, prostate, and parotid glands.
      • Smith D.M.
      • Coop C.A.
      Dog allergen immunotherapy: past, present, and future.
      The major dog allergens are Can f 1 and Can f5 149; Can f 5, a prostatic kallikrein, is present only in male dogs that have not been neutered.
      • Portnoy J.
      • Kennedy K.
      • Sublett J.
      • et al.
      Environmental assessment and exposure control: a practice parameter--furry animals.
      IgE to Can f1 and Can f 5 are highly predictive of dog allergy, although other allergens such as Can f 4 and Can f 6 may be clinically significant. The major cat allergens are Fel d 1 and Fel d 4; the sources are salivary, sebaceous, and perianal glands.
      • Portnoy J.
      • Kennedy K.
      • Sublett J.
      • et al.
      Environmental assessment and exposure control: a practice parameter--furry animals.
      Fel d 1 is associated with hormone production and acts as a uteroglobin; it is found mainly in saliva, sebaceous glands of the skin, and the urine of male cats. Fel d 1 is carried in very small airborne particles <5 μm in diameter; this likely renders it able to reach small bronchioles and can explain why it is difficult to remove from the house.
      The homology between different dog and cat allergens such as albumins and lipocalins explains the cross-sensitization between them and allergens from other mammals and the presence of simultaneous sensitization to dogs, cats, and other mammals regardless of whether there is or is not direct exposure to all of them.
      • Cabanas R.
      • Lopez-Serrano M.C.
      • Carreira J.
      • et al.
      Importance of albumin in cross-reactivity among cat, dog and horse allergens.
      Some of these antigens cause clinically relevant cross-reactivity between different animals (Fig. 1). The most significant cross-binding patterns between allergens of cats, dogs, and other mammals are with lipocalins and Can f 5. Lipocalins have amino acid sequences with up to 60% identity with Can f 6 (dog), Equ c 1 (horse), Fel d 4 (cat), Ory c 4 (rabbit), and Mus m 1 (mouse). Can f 5 shows a certain homology with prostate-specific antigen, which is also in the kallikrein family. It has been speculated that prior sensitization to Can f 5 from dogs could be associated with a greater propensity for developing allergic reactions to human seminal fluid.
      • Basagana M.
      • Bartolome B.
      • Pastor C.
      • et al.
      Allergy to human seminal fluid: cross-reactivity with dog dander.
      Finally, Can f 7, an NPC2-like protein that is homologous with the NPC2 components of HDM, has been identified in dogs.
      • Khurana T.
      • Newman-Lindsay S.
      • Young P.R.
      • Slater J.E.
      The NPC2 protein: a novel dog allergen.
      Fig. 1
      Fig. 1Relevant molecules involved in the identification of cat, dog and horse sensitization
      Exposure before and around birth to dog dander or to dust from cow barns is regarded as protective against allergies and asthma. However, if sensitization does occur, multiplexing could be helpful to identify the extent of risk for the individual patient to define avoidance or AIT strategies,
      • Liccardi G.
      • Bilo M.B.
      • Manzi F.
      • Piccolo A.
      • Di Maro E.
      • Salzillo A.
      What could be the role of molecular-based allergy diagnostics in detecting the risk of developing allergic sensitization to furry animals?.
      and to identify other animals that may be a source of clinically relevant cross-reacting allergens despite no prior exposure. Sensitization to Can f 1 and Fel d1 and polysensitization to cat and dog allergens during childhood have been associated with the development of subsequent allergy to cats and dogs.
      • Asarnoj A.
      • Hamsten C.
      • Waden K.
      • et al.
      Sensitization to cat and dog allergen molecules in childhood and prediction of symptoms of cat and dog allergy in adolescence: a BAMSE/MeDALL study.
      In 259 children sensitized to cats, co-sensitization to Fel d 1 and Fel d 4 was associated with a risk for asthma; for dog sensitization, IgE to Can f 5, 1, and 2 were the most significant risk factors.
      • Bjerg A.
      • Winberg A.
      • Berthold M.
      • Mattsson L.
      • Borres M.P.
      • Ronmark E.
      A population-based study of animal component sensitization, asthma, and rhinitis in schoolchildren.
      In contrast, IgE to serum albumins was rare and not clinically relevant. In 304 children, early sensitization to pet allergens (lipocalins, secretoglobins) preceded a risk for respiratory sensitization, followed by asthma and by meat and wheat allergy.
      • Nagao M.
      • Borres M.P.
      • Sugimoto M.
      • et al.
      Sensitization to secretoglobin and lipocalins in a group of young children with risk of developing respiratory allergy.
      Sensitivity to the dog allergens Can f 4 and Can f 6 were associated with positive nasal provocation in children with allergic rhinitis.
      • Kack U.
      • Asarnoj A.
      • Gronlund H.
      • et al.
      Molecular allergy diagnostics refine characterization of children sensitized to dog dander.
      The authors did not, however, regard monosensitization to Can f 5 as an indicator of a dog allergy.
      In contrast to its usefulness in determining dog allergy, [email protected] has limited effectiveness for cat allergy. Its perfomance in determining sIgE is similar for complete cat extract and Fel d 1,159 a predictive marker of allergy to cats.
      Sensitization to certain allergens seems to be associated with the severity and persistence of clinical symptoms, and sensitization to more than 1 allergen or sensitization to albumins seems to be associated with more-severe respiratory symptoms.
      • Konradsen J.R.
      • Fujisawa T.
      • van Hage M.
      • et al.
      Allergy to furry animals: new insights, diagnostic approaches, and challenges.
      ,
      • Uriarte S.A.
      • Sastre J.
      Clinical relevance of molecular diagnosis in pet allergy.
      In patients allergic to cats, the main cross-reacting food allergy syndrome is the pork-cat syndrome due the cross-binding of Fel d 2 with other albumins from mammals; it can lead to immediate anaphylactic reactions after consuming raw or undercooked pork. As noted above in the section on meat allergy, alpha-gal is also present on cat IgA Fel d 5 and IgM Fel d 6.
      • Adedoyin J.
      • Gronlund H.
      • Oman H.
      • Johansson S.G.
      • van Hage M.
      Cat IgA, representative of new carbohydrate cross-reactive allergens.

      Cockroach

      SIgE to Per a 2 has been found in patients with persistent asthma and is a potential marker for more-severe airway disease. [email protected] could help to detect genuine sensitization to mite and cockroach allergens.
      • Barbosa M.C.
      • Santos A.B.
      • Ferriani V.P.
      • Pomes A.
      • Chapman M.D.
      • Arruda L.K.
      Efficacy of recombinant allergens for diagnosis of cockroach allergy in patients with asthma and/or rhinitis.
      Two cockroach allergens, Per a 11 (alpha-amylase) and Per a 12 (chitinase), have been identified by serological and in vitro investigations and SPT in 47 cockroach-sensitized patients.
      • Fang Y.
      • Long C.
      • Bai X.
      • et al.
      Two new types of allergens from the cockroach, Periplaneta americana.

      Fungal allergens

      Tanimoto et al. aimed to differentiate between allergic bronchopulmonary aspergillosis (ABPA) and Aspergillus fumigatus–sensitized asthmatic patients without ABPA.
      • Tanimoto H.
      • Fukutomi Y.
      • Yasueda H.
      • et al.
      Molecular-based allergy diagnosis of allergic bronchopulmonary aspergillosis in Aspergillus fumigatus-sensitized Japanese patients.
      ImmunoCAP showed that ABPA patients (n = 53) had significantly higher levels of IgE to Asp f 1 and Asp f 2 than patients with asthma (n = 253). The result was complicated by comorbid atopic dermatitis, where high levels of anti–Asp f 6 IgE levels were found. Interestingly, Asp f 13, a serine protease, exerts innate adjuvant effects by disrupting fixation of smooth muscles to the extracellular matrix in the bronchi,
      • Balenga N.A.
      • Klichinsky M.
      • Xie Z.
      • et al.
      A fungal protease allergen provokes airway hyper-responsiveness in asthma.
      but the risk of a clinical reaction has not yet been defined. In a meta-analysis of 26 studies on Aspergillus, IgE to Asp f 1 and Asp f 3 was most specific for ABPA.
      • Muthu V.
      • Sehgal I.S.
      • Dhooria S.
      • Aggarwal A.N.
      • Agarwal R.
      Utility of recombinant Aspergillus fumigatus antigens in the diagnosis of allergic bronchopulmonary aspergillosis: a systematic review and diagnostic test accuracy meta-analysis.
      A combination of several molecules was suggested to help with diagnosis, but the clinical utility was not assessed.

      Food allergens

      Multiplex testing provides a major advantage for diagnosing food allergy, whereas IgE binding to single molecules may help to predict the severity of symptoms upon allergy exposure according to the stability of the allergen. This prediction is based on the classification of food allergens according to whether they only elicit local symptoms in the mouth and are then digested, or they survive gastrointestinal digestion and, via effective mucosal adsorption, lead to systemic symptoms.
      • Pali-Scholl I.
      • Untersmayr E.
      • Klems M.
      • Jensen-Jarolim E.
      The effect of digestion and digestibility on allergenicity of food.
      This classification, however, is only a prediction, because cofactors like age, hormonal status, the capacity for gastric digestion, drugs, infections, menses, alcohol, and exercise may accelerate the allergen entry into the body, thereby lowering the threshold for allergic reactivity and increasing the likelihood of systemic reactions.
      • Simons F.E.
      • Ardusso L.R.
      • Bilo M.B.
      • et al.
      2012 update: world allergy organization guidelines for the assessment and management of anaphylaxis.
      In 86 patients with food allergies, a prescription for epinephrine was taken as a measure of the clinically predicted risk for severe reactions.
      • Antonicelli L.
      • Massaccesi C.
      • Braschi M.C.
      • Cinti B.
      • Bilo M.B.
      • Bonifazi F.
      Component resolved diagnosis in real life: the risk assessment of food allergy using microarray-based immunoassay.
      The presence of a prescription was in fair agreement with the predictive value of multiplex testing. However, the authors found 3 problems with the microarray that need to be addressed: poor sensitivity, some discrepancies between the clinical and serological risk assessments, and the absence of some causative allergens from the microarray. For instance, LTPs from tomato are not yet included in the multiplex test, but studies indicate their importance in clinically relevant sensitization, particularly in the Mediterranean area.
      • Lopez-Matas M.A.
      • Larramendi C.H.
      • Huertas A.J.
      • et al.
      Tomato nsLTP as an “in vivo" diagnostic tool: sensitization in a Mediterranean population.
      A European consensus paper focused on food-inhalant cross-reactivity noted that respiratory sensitizations may lead to clinically relevant, even severe, reactions to cross-reactive food.
      • Werfel T.
      • Asero R.
      • Ballmer-Weber B.K.
      • et al.
      Position paper of the EAACI: food allergy due to immunological cross-reactions with common inhalant allergens.
      This is a noncanonical view, because pollen-associated foods are usually regarded as labile and less potent, despite reports of anaphylactic reactions to foods in patients monosensitized to PR-10 allergens
      • Roseler S.
      • Balakirski G.
      • Plange J.
      • et al.
      [Anaphylaxis to PR-10 proteins (Bet v1 homologues)].
      or profilins.
      • Alvarado M.I.
      • Jimeno L.
      • De La Torre F.
      • et al.
      Profilin as a severe food allergen in allergic patients overexposed to grass pollen.
      There is accumulating evidence that pollen also contains LTPs and nsLTPs that may elicit cross-reactivity to foods that are associated with a higher anaphylaxis risk.
      • Scala E.
      • Till S.J.
      • Asero R.
      • et al.
      Lipid transfer protein sensitization: reactivity profiles and clinical risk assessment in an Italian cohort.
      An Italian study of 568 patients
      • Scala E.
      • Till S.J.
      • Asero R.
      • et al.
      Lipid transfer protein sensitization: reactivity profiles and clinical risk assessment in an Italian cohort.
      found that hierarchical patterns and cluster relationships predicted a higher risk for systemic food-associated reactions when a subject had IgE directed against more than 5 different nsLTPs, including peach Pru p 3 and walnut Jug r 3. Sensitization to mugwort Art v 3 and plantain Pla a 3 was associated with an increased likelihood of respiratory symptoms. Finally, Ole e 7 reactivity in olive tree–sensitized subjects is associated with the presence of adverse reactions to food and not with respiratory symptoms.
      • Scala E.
      • Abeni D.
      • Pomponi D.
      • et al.
      Ole e 1, Ole e 7, and Ole e 9: identifying distinct clinical subsets of olive tree-allergic patients.
      IgE to LTPs or storage proteins of a specific plant food did not correlate with clinical reactivity in the majority of 130 Spanish children with food-associated anaphylaxis to mostly peach, walnut, peanut, and hazelnut,
      • Pascal M.
      • Vazquez-Ortiz M.
      • Folque M.M.
      • et al.
      Asymptomatic LTP sensitisation is common in plant-food allergic children from the Northeast of Spain.
      calling into question the predictive value of [email protected] for food allergy to LTPs and seed storage proteins. These data were in line with an earlier study
      • Vieira T.
      • Cunha L.
      • Neves E.
      • Falcao H.
      Diagnostic usefulness of component-resolved diagnosis by skin prick tests and specific IgE to single allergen components in children with allergy to fruits and vegetables.
      that found no correlation between sensitization to the LTP Pru p 3, as measured with component-resolved SPT, and the severity of clinical reactivity. Therefore, more studies need to be performed that examine the correlation between the multiplex IgE results, the level of sensitization, and the clinical picture. However, considering the risk of in vivo tests for diagnosing patients with a previous systemic reaction, multiplex IgE testing is an interesting alternative tool to examine the IgE profile and level of sensitization in a semiquantitative manner in settings when SPT cannot be applied.
      • Jensen-Jarolim E.
      • Jensen A.N.
      • Canonica G.W.
      Debates in allergy medicine: molecular allergy diagnosis with ISAC will replace screenings by skin prick test in the future.
      Of course, cofactors cannot be considered or predicted. Likely, clustering algorithms like those recently developed for allergic rhinitis and asthma
      • Simpson A.
      • Lazic N.
      • Belgrave D.C.
      • et al.
      Patterns of IgE responses to multiple allergen components and clinical symptoms at age 11 years.
      are needed for improving the accuracy of risk evaluation in food allergy by [email protected], but overall, clinical studies on multiplex IgE testing indicate good reliability for risk assessment.
      • Patelis A.
      • Borres M.P.
      • Kober A.
      • Berthold M.
      Multiplex component-based allergen microarray in recent clinical studies.
      The highest diagnostic accuracy is achieved with Bos d 4 for cow's milk, Gal d 1 for heated or raw hen's egg, Ara h 6 for peanut, Cor a 14 for hazelnut, and Lit v 1 for shrimp.
      • Flores Kim J.
      • McCleary N.
      • Nwaru B.I.
      • Stoddart A.
      • Sheikh A.
      Diagnostic accuracy, risk assessment, and cost-effectiveness of component-resolved diagnostics for food allergy: a systematic review.
      In addition [email protected] may provide an economic benefit, especially in multisensitized patients.
      • Peveri S.
      • Pattini S.
      • Costantino M.T.
      • et al.
      Molecular diagnostics improves diagnosis and treatment of respiratory allergy and food allergy with economic optimization and cost saving.

      Milk

      In milk allergy, desensitization strategies are important both for the management of the disease and for nutrition in children. Kuitunen et al. treated 76 milk-allergic children with oral immune therapy (OIT). Children with high levels of IgE to α-lactalbumin, β-lactoglobulin, and casein reached a lower maintenance dose than the target dose.
      • Kuitunen M.
      • Englund H.
      • Remes S.
      • et al.
      High IgE levels to alpha-lactalbumin, beta-lactoglobulin and casein predict less successful cow's milk oral immunotherapy.
      In patients who did respond, IgG4 was produced against these same molecules and lactoferrin during OIT. The authors suggested that the use of [email protected] before OIT could improve the selection of children for whom OIT would probably succeed
      • Kuitunen M.
      • Englund H.
      • Remes S.
      • et al.
      High IgE levels to alpha-lactalbumin, beta-lactoglobulin and casein predict less successful cow's milk oral immunotherapy.
      ; that is, these molecules represent biomarkers.
      • Sato S.
      • Yanagida N.
      • Ohtani K.
      • Koike Y.
      • Ebisawa M.
      A review of biomarkers for predicting clinical reactivity to foods with a focus on specific immunoglobulin E antibodies.
      On the other hand, those children with the highest IgE have the highest need for desensitization. For example, children with higher levels of IgE to cow's milk allergens in [email protected] had a higher risk for persisting milk allergy.
      • Petersen T.H.
      • Mortz C.G.
      • Bindslev-Jensen C.
      • Eller E.
      Cow's milk allergic children-Can component-resolved diagnostics predict duration and severity?.
      Using [email protected], the IgE/IgG4 ratio and the sIgE sensitization pattern allowed the discrimination of patients with cow's milk allergy who tolerate cooked milk.
      • Bartuzi Z.
      • Cocco R.R.
      • Muraro A.
      • Nowak-Wegrzyn A.
      Contribution of molecular allergen analysis in diagnosis of milk allergy.
      However, further [email protected] studies are needed to predict tolerance, with the hope of being able to reduce the need for food challenges.
      • Agyemang A.
      • Saf S.
      • Sifers T.
      • et al.
      Utilizing boiled milk sIgE as a predictor of baked milk tolerance in cow's milk allergic children.

      Egg

      Sato et al. reported that ovomucoid is a diagnostic marker for egg allergy.
      • Sato S.
      • Yanagida N.
      • Ohtani K.
      • Koike Y.
      • Ebisawa M.
      A review of biomarkers for predicting clinical reactivity to foods with a focus on specific immunoglobulin E antibodies.
      However, this may differ with age: Kim et al. divided 27 children with convincing egg-related, immediate-type symptoms into 3 age groups and analyzed their IgE reactivity to ovomucoid (Gal d 1), ovalbumin (Gal d 2), and ovotransferrin (Gal d 3) by enzyme-linked immunosorbent assay or immunoblot.
      • Kim J.
      • Lee J.
      • Park M.R.
      • Han Y.
      • Shin M.
      • Ahn K.
      Special consideration is required for the component-resolved diagnosis of egg allergy in infants.
      The study showed that the IgE against Gal d 2 developed in the first year of life, in the second year the IgE changed to be predominantly against Gal d 1, and after 24 months an additional Gal d 3 response developed. Hence, there appears to be an age-dependent effect on IgE evolution to egg allergens in small children. Dang et al. calculated the risk for clinically relevant, persistent egg allergy in the HealthNuts cohort: IgE to Gal d 1 elevated the risk 2- to 3-fold, and IgE to Gal d 1, 2, 3, or 5 elevated the risk up to 4-fold.
      • Dang T.D.
      • Peters R.L.
      • Koplin J.J.
      • et al.
      Egg allergen specific IgE diversity predicts resolution of egg allergy in the population cohort HealthNuts.
      ,
      • Petrosino M.I.
      • Scaparrotta A.
      • Marcovecchio M.L.
      • et al.
      Usefulness of molecular diagnosis in egg allergic children.
      Accordingly, Gal d 1 and 2 are associated with a higher risk for egg anaphylaxis.
      • Petrosino M.I.
      • Scaparrotta A.
      • Marcovecchio M.L.
      • et al.
      Usefulness of molecular diagnosis in egg allergic children.

      Peanut

      Among the food allergies, multiplex testing is probably the most advanced for peanut allergy, where it has found its place in risk assessment. Based on a retrospective serological examination of 89 Belgian patients with peanut allergy, Ara h 1, 2, and 3 were proposed as markers for sensitization and were associated with clinical reports of more-severe reactions, whereas IgE to Ara h 8 was a sign of cross-sensitization by pollen and was associated with oral allergy syndrome rather than severe reactivity.
      • Faber M.A.
      • Donne I.
      • Herrebosch E.
      • et al.
      Sensitization profiles to peanut allergens in Belgium; cracking the code in infants, children and adults.
      This was supported by Giovanni et al., who found that IgE to Ara h 1 and 2 was associated with anaphylaxis, whereas Ara h 8 produced only weak clinical reactivity.
      • Giovannini M.
      • Comberiati P.
      • Piazza M.
      • et al.
      Retrospective definition of reaction risk in Italian children with peanut, hazelnut and walnut allergy through component-resolved diagnosis.
      In Austria, sensitization to Ara h 2 and 6 was more common than to Ara h 8, 3, and 9,
      • Ackerbauer D.
      • Bublin M.
      • Radauer C.
      • et al.
      Component-resolved IgE profiles in Austrian patients with a convincing history of peanut allergy.
      although the diagnostic sensitivity was good for each.
      A double-blind, placebo-controlled study in 102 patients provided evidence that the storage proteins Ara h 2 and Ara h 6 are the best predictors of severe systemic reactions to peanuts that require treatment with epinephrine.
      • Kukkonen A.K.
      • Pelkonen A.S.
      • Makinen-Kiljunen S.
      • Voutilainen H.
      • Makela M.J.
      Ara h 2 and Ara 6 are the best predictors of severe peanut allergy: a double-blind placebo-controlled study.
      The authors concluded that multiplex IgE testing could replace provocation for diagnosis of severe peanut allergy. The EuroPrevall team stated that Ara h 2 might be regarded as the single major allergen,
      • Ballmer-Weber B.K.
      • Lidholm J.
      • Fernandez-Rivas M.
      • et al.
      IgE recognition patterns in peanut allergy are age dependent: perspectives of the EuroPrevall study.
      whereas peanut-tolerant individuals had more IgE to Ara h 8 and 9. However, in an Asian cohort of 40 patients tolerant patients had IgE to neither Ara h 2 nor Ara h 9. A ratio of Ara h 2 IgE to peanut IgE above 0.6 was suggested to be predictive of severe reactions,
      • Suratannon N.
      • Ngamphaiboon J.
      • Wongpiyabovorn J.
      • Puripokai P.
      • Chatchatee P.
      Component-resolved diagnostics for the evaluation of peanut allergy in a low-prevalence area.
      although different methods can result in different cut-off values.
      Interestingly, IgE to CCDs was exclusively found in tolerant patients.
      • Suratannon N.
      • Ngamphaiboon J.
      • Wongpiyabovorn J.
      • Puripokai P.
      • Chatchatee P.
      Component-resolved diagnostics for the evaluation of peanut allergy in a low-prevalence area.
      This phenomenon was also seen in a Ghanaian cross-sectional study of 1604 children, with adverse reactions in 17% of them, mostly associated with a sIgE response. A co-infection with Schistosoma haematobium was revealed as the sensitizer to CCDs, resulting in peanut-cross-reactive IgE without clinical implications.
      • Amoah A.S.
      • Obeng B.B.
      • Larbi I.A.
      • et al.
      Peanut-specific IgE antibodies in asymptomatic Ghanaian children possibly caused by carbohydrate determinant cross-reactivity.
      A [email protected] result including CCDs may thus be an indicator of tolerance. In contrast, sensitization to the defensins Ara h 12 and Ara h 13 in the lipophilic peanut fraction was associated with more-severe reactions.
      • Petersen T.H.
      • Mortz C.G.
      • Bindslev-Jensen C.
      • Eller E.
      Cow's milk allergic children-Can component-resolved diagnostics predict duration and severity?.
      Alternative efforts include determining IgE, IgG, and IgG4 in the same sample. Lower levels of IgE to Ara h 2 predicted clinically relevant tolerance, in contrast to IgG and IgG4, which failed to discriminate.
      • Lopes de Oliveira L.C.
      • Aderhold M.
      • Brill M.
      • et al.
      The value of specific IgE to peanut and its component Ara h 2 in the diagnosis of peanut allergy.

      Soy

      Because soy is related to peanut, it also represents a source of dangerous allergens. Notably, in addition to Gly m 5 and 6, the Bet v 1 homolog Gly m 4 harbors high allergenic potency due to its heat stability. In a multiplex study with sera from 20 soy-allergic patients, IgE to Gly m 4 predicted severe systemic reactions.
      • Berneder M.
      • Bublin M.
      • Hoffmann-Sommergruber K.
      • Hawranek T.
      • Lang R.
      Allergen chip diagnosis for soy-allergic patients: Gly m 4 as a marker for severe food-allergic reactions to soy.
      Soy is important in global nutrition, and its allergenicity is best evaluated at a molecular level. Lu et al.
      • Lu M.
      • Jin Y.
      • Cerny R.
      • Ballmer-Weber B.
      • Goodman R.E.
      Combining 2-DE immunoblots and mass spectrometry to identify putative soybean (Glycine max) allergens.
      identified β-conglycinin (Gly m 5), glycinin (Gly m 6), and soybean Kunitz trypsin inhibitor as major IgE binders in the sera of patients with clinical symptoms of soy allergy; in contrast, in sensitized-only subjects, IgE was bound by soybean agglutinin, seed biotinylated protein, late embryogenesis protein, and sucrose-binding protein. Gly m 8 (a 2S albumin) is also a relevant allergen in both children and adults.
      • Ebisawa M.
      • Brostedt P.
      • Sjolander S.
      • Sato S.
      • Borres M.P.
      • Ito K.
      Gly m 2S albumin is a major allergen with a high diagnostic value in soybean-allergic children.
      ,
      • Klemans R.J.
      • Knol E.F.
      • Michelsen-Huisman A.
      • et al.
      Components in soy allergy diagnostics: Gly m 2S albumin has the best diagnostic value in adults.

      Hazelnut

      In the EuroPrevall setting, hazelnut sensitization across Europe is dominated by IgE to Cor a 1,
      • Datema M.R.
      • Zuidmeer-Jongejan L.
      • Asero R.
      • et al.
      Hazelnut allergy across Europe dissected molecularly: a EuroPrevall outpatient clinic survey.
      a labile Bet v 1 homolog that cross-reacts with birch pollen but is less dangerous. There were, however, a few geographic exceptions. For example, in Athens the storage protein Cor a 8 was a frequent sensitizer, and this sensitization was associated with food allergy to other nuts and rosacea fruits, and with an allergy to pollen from the goosefoot weed, plane tree, and mugwort. Increasing evidence supports the LTP Cor a 9 and the storage proteins Cor a 14 as causing severe hazelnut-associated symptoms and the more-severe type of cross-reactivity symptoms. In the Eastern Mediterranean area, IgE to Cor a 14 was identified in 56 children with clinically relevant sensitization.
      • Buyuktiryaki B.
      • Cavkaytar O.
      • Sahiner U.M.
      • et al.
      Cor a 14, hazelnut-specific IgE, and SPT as a reliable tool in hazelnut allergy diagnosis in eastern mediterranean children.
      This is in agreement with a Dutch study of 161 hazelnut-sensitized patients that confirmed the predictive value of Cor a 9 and 14 sensitization by DBPCFC, where 13% of children and 49% of adults could be identified objectively.
      • Masthoff L.J.
      • Mattsson L.
      • Zuidmeer-Jongejan L.
      • et al.
      Sensitization to Cor a 9 and Cor a 14 is highly specific for a hazelnut allergy with objective symptoms in Dutch children and adults.
      Importantly, the levels of IgE to Cor a 9 and 14 directly correlated with the reactivity level of 40 hazelnut-allergic children in DBPCFC.
      • Brandstrom J.
      • Nopp A.
      • Johansson S.G.
      • et al.
      Basophil allergen threshold sensitivity and component-resolved diagnostics improve hazelnut allergy diagnosis.
      Moreover, IgE cross-binding between rCor a 9, rCor a 14, and rJug r 1 indicated the potential clinical implications for patients sensitized to one of these nuts.
      • Villalta D.
      • Scala E.
      • Mistrello G.
      • Amato S.
      • Asero R.
      Evidence of cross-reactivity between different seed storage proteins from hazelnut (corylus avellana) and walnut (juglans regia) using recombinant allergen proteins.
      Two studies using [email protected] identified sIgE to Cor a 9 and Cor a 14 as being associated with a history of anaphylaxis in response to hazelnut.
      • Giovannini M.
      • Comberiati P.
      • Piazza M.
      • et al.
      Retrospective definition of reaction risk in Italian children with peanut, hazelnut and walnut allergy through component-resolved diagnosis.
      ,
      • Datema M.R.
      • van Ree R.
      • Asero R.
      • et al.
      Component-resolved diagnosis and beyond: multivariable regression models to predict severity of hazelnut allergy.
      In contrast, Cor a 1 was associated with mild reactions. When symptoms like atopic dermatitis and pollen allergy were combined with [email protected], the highest risks for symptoms were found when IgE to both Cor a 14 and walnut were present. Another study found that [email protected] allowed for more accuracy in predicting severe hazelnut-associated symptoms in patients with the pollen-food syndrome, but was poor in peanut allergy diagnosis.
      • Beck S.C.
      • Huissoon A.P.
      • Collins D.
      • Richter A.G.
      • Krishna M.T.
      The concordance between component tests and clinical history in British adults with suspected pollen-food syndrome to peanut and hazelnut.

      Walnut

      As noted above, 2S seed storage proteins were recently identified as eliciting cross-reactivity between walnut and hazelnut.
      • Villalta D.
      • Scala E.
      • Mistrello G.
      • Amato S.
      • Asero R.
      Evidence of cross-reactivity between different seed storage proteins from hazelnut (corylus avellana) and walnut (juglans regia) using recombinant allergen proteins.
      Giovannini et al. showed that the presence of Jug r 1, Jug r 2, or Jug r 3 sIgE was associated with preceding anaphylaxis,
      • Giovannini M.
      • Comberiati P.
      • Piazza M.
      • et al.
      Retrospective definition of reaction risk in Italian children with peanut, hazelnut and walnut allergy through component-resolved diagnosis.
      indicating that sensitivity to these allergens should be considered a risk factor for anaphylaxis.

      Wheat

      ImmunoCAP and immunoblot were used for [email protected] in sera from children with a suspected wheat allergy.
      • Sievers S.
      • Rawel H.M.
      • Ringel K.P.
      • Niggemann B.
      • Beyer K.
      Wheat protein recognition pattern in tolerant and allergic children.
      Of these children, 44 were reactive and 62 nonreactive to the food challenge. Although the authors confirmed α-, β-, γ-, and ω-gliadins and low-molecular-weight glutenin as major allergens in terms of IgE binding, these allergens were not able to discriminate the tolerant from the challenge-positive children. In contrast, a Scandinavian study found that these same compounds, and the dimeric alpha-amylase inhibitor AAI 0.19 (Tri a 28), were clinically relevant antigens that helped to identify reactive patients among 108 children with a suspected wheat allergy.
      • Makela M.J.
      • Eriksson C.
      • Kotaniemi-Syrjanen A.
      • et al.
      Wheat allergy in children - new tools for diagnostics.
      Higher levels of sIgE to wheat or ω-5 gliadins also predicted anaphylaxis to wheat in Japanese infants
      • Koike Y.
      • Yanagida N.
      • Sato S.
      • et al.
      Predictors of persistent wheat allergy in children: a retrospective cohort study.
      and likely predict reactions to food challenges.
      • Yanagida N.
      • Sato S.
      • Takahashi K.
      • et al.
      Increasing specific immunoglobulin E levels correlate with the risk of anaphylaxis during an oral food challenge.
      This is significantly associated with WDEIA.
      • Kennard L.
      • Thomas I.
      • Rutkowski K.
      • et al.
      A multicenter evaluation of diagnosis and management of omega-5 gliadin allergy (also known as wheat-dependent exercise-induced anaphylaxis) in 132 adults.
      Tri tu 14 (from durum wheat) shares about 50% amino acid identity with Tri a 14 (from common wheat), leading to a potential danger for cross-reactivity in WDEIA. Further in vitro cross-binding was found to Pru p 3 from peach.
      • Safi H.
      • Wangorsch A.
      • Lidholm J.
      • et al.
      Identification and molecular characterization of allergenic non-specific lipid-transfer protein from durum wheat (Triticum turgidum).
      Wheat allergy represents not only a food allergy but also a relevant occupational allergy in bakers' asthma. When 101 bakers (40 German, 37 Dutch, and 24 Spanish) with wheat flour allergy were tested by CAP-FEIA combined with inhibition by wheat or rye flour and grass pollen extract, Tri a 27 and Tri a 28 were found to be the most important IgE binders.
      • Sander I.
      • Rihs H.P.
      • Doekes G.
      • et al.
      Component-resolved diagnosis of baker's allergy based on specific IgE to recombinant wheat flour proteins.
      The highest diagnostic accuracy, however, was achieved with a combination of 5 allergens, which allowed discrimination of occupational sensitization from exposure to homologous pollen.

      Buckwheat

      Buckwheat (Fagopyrum esculentum and F. tataricum) is not in the same family (Poaceae) as wheat and is used as an alternative to wheat. However, it also poses a risk for food-induced anaphylaxis. Buckwheat allergy is predicted by concomitant sensitization to legumin, Fag e 2, and Fag e 5.
      • Geiselhart S.
      • Nagl C.
      • Dubiela P.
      • et al.
      Concomitant sensitization to legumin, Fag e 2 and Fag e 5 predicts buckwheat allergy.
      IgE to Fag e 3 correlates with a risk for anaphylaxis.
      • Yanagida N.
      • Sato S.
      • Maruyama N.
      • et al.
      Specific IgE for Fag e 3 Predicts Oral Buckwheat Food Challenge Test Results and Anaphylaxis: a Pilot Study.
      Interestingly, horses may also develop IgE to buckwheat from their diet.
      • Einhorn L.
      • Hofstetter G.
      • Brandt S.
      • et al.
      Molecular allergen profiling in horses by microarray reveals Fag e 2 from buckwheat as a frequent sensitizer.

      Rosaceae fruits

      Peach allergy in northern and southern Italy differs in terms of the molecular allergens relevant for IgE binding. In the north, pollen-derived PR-10 allergens are the primary sensitizers, and anti–Pru p 1 sensitization produces weaker symptoms. In the south, the nsLTP Pru p 3 was long regarded as the major sensitizer as well as an elicitor of allergic reactions. In a study on 133 Pru p 3–positive patients, a south-north gradient in sensitization patterns to peach molecules was reported.
      • Uasuf C.G.
      • Villalta D.
      • Conte M.E.
      • et al.
      Different co-sensitizations could determine different risk assessment in peach allergy? Evaluation of an anaphylactic biomarker in Pru p 3 positive patients.
      Additionally, the authors found a significant correlation between the levels of IgE and symptom severity and were able to differentiate symptomatic from non-symptomatic patients. A suspected cross-sensitizing capacity of cypress to peach could not be confirmed.
      • Uasuf C.G.
      • Villalta D.
      • Conte M.E.
      • et al.
      Different co-sensitizations could determine different risk assessment in peach allergy? Evaluation of an anaphylactic biomarker in Pru p 3 positive patients.
      ,
      • Caimmi D.
      • Barber D.
      • Hoffmann-Sommergruber K.
      • et al.
      Understanding the molecular sensitization for Cypress pollen and peach in the Languedoc-Roussillon area.
      A recent study changed this paradigm by reporting that Pru p 3 sensitization is a marker for severe reactions in central Europe as well as southern Europe.
      • Mothes-Luksch N.
      • Raith M.
      • Stingl G.
      • et al.
      Pru p 3, a marker allergen for lipid transfer protein sensitization also in Central Europe.
      Recently, a new cross-reactive pan-allergen has been characterized that belongs to the gibberellin-regulated protein (GRP) family. BP14 from cypress pollen, Pru p 7 from peach, and Pun g 7 from pomegranate are some representative GRP molecules.
      • Inomata N.
      • Miyakawa M.
      • Aihara M.
      High prevalence of sensitization to gibberellin-regulated protein (peamaclein) in fruit allergies with negative immunoglobulin E reactivity to Bet v 1 homologs and profilin: clinical pattern, causative fruits and cofactor effect of gibberellin-regulated protein allergy.
      • Klingebiel C.
      • Chantran Y.
      • Arif-Lusson R.
      • et al.
      Pru p 7 sensitization is a predominant cause of severe, cypress pollen-associated peach allergy.
      • Poncet P.
      • Aizawa T.
      • Senechal H.
      The subtype of Cupressaceae pollinosis associated with Pru p 7 sensitization is characterized by a sensitization to a cross-reactive gibberellin-regulated protein in cypress pollen: BP14.
      • Senechal H.
      • Santrucek J.
      • Melcova M.
      • et al.
      A new allergen family involved in pollen food-associated syndrome: snakin/gibberellin-regulated proteins.

      Kiwi

      Among 59 people in Sweden with peanut allergy, 39% also reported symptoms to kiwi, likely elicited by cross-reactivity between the 11S and 7S globulins present in both kiwi and peanut, and co-sensitization to the kiwi 2S albumin Act d 13.
      • van Odijk J.
      • Sjolander S.
      • Brostedt P.
      • Borres M.P.
      • Englund H.
      High frequency of IgE sensitization towards kiwi seed storage proteins among peanut allergic individuals also reporting allergy to kiwi.
      Additional reactivities to nuts and legumes other than peanuts were also reported.
      • van Odijk J.
      • Sjolander S.
      • Brostedt P.
      • Borres M.P.
      • Englund H.
      High frequency of IgE sensitization towards kiwi seed storage proteins among peanut allergic individuals also reporting allergy to kiwi.

      Fish

      Parvalbumin is the most frequent cause of fish allergy and has been implicated in cross-reactivity among fish species. Parvalbumin is heat stable, so cooking is not expected to affect reactions to it in sensitized patients.
      • Matricardi P.M.
      • Kleine-Tebbe J.
      • Hoffmann H.J.
      • et al.
      EAACI molecular allergology user's guide.
      Kuehn et al. identified 2 additional allergens: beta-enolase and fructose-bisphosphate aldolase.
      • Kuehn A.
      • Hilger C.
      • Lehners-Weber C.
      • et al.
      Identification of enolases and aldolases as important fish allergens in cod, salmon and tuna: component resolved diagnosis using parvalbumin and the new allergens.
      Among 62 patients, 60% showed IgE to these new allergens. Their specific implications for risk assessment and cross-reactivities still need to be evaluated, but recent papers support the high predictive value of [email protected] for risk assessment in patients with fish and shellfish allergies.
      • Tong W.S.
      • Yuen A.W.
      • Wai C.Y.
      • Leung N.Y.
      • Chu K.H.
      • Leung P.S.
      Diagnosis of fish and shellfish allergies.

      Shellfish

      To identify the clinical relevance of sensitization to shrimp allergens, IgE to recombinant shellfish antigens and IgE and IgG4 to peptides were determined in 86 patients with a positive SPT to shrimp; a group co-sensitized to HDM or cockroach were used as controls.
      • Pascal M.
      • Grishina G.
      • Yang A.C.
      • et al.
      Molecular diagnosis of shrimp allergy: efficiency of several allergens to predict clinical reactivity.