Ansotegui et al. World Allergy Organization Journal (2020) 13:100080 http://doi.org/10.1016/j.waojou.2019.100080 Open Access IgE allergy diagnostics and other relevant tests in allergy, a World Allergy Organization position paper Ignacio J. Ansotegui, MD, PhDa,1*, Giovanni Melioli, MDb,1, Giorgio Walter Canonica, MDc,1, Luis Caraballo, MD, PhDd,1, Elisa Villa, MD, PhDe,1, Motohiro Ebisawa, MD, PhDf,1, Giovanni Passalacqua, MDg,1, Eleonora Savi, MDh,1, Didier Ebo, MD, PhDi,1, R. Maximiliano Gómez, MD, PhDj,1, Olga Luengo Sánchez, MD, PhDk,1, John J. Oppenheimer, MDl,1, Erika Jensen-Jarolim, MDm,1, David A. Fischer, MDn,1, Tari Haahtela, MDo, Martti Antila, MDp, Jean J. Bousquet, MD, PhDq,r,s,t,u, Victoria Cardona, MD, PhDv, Wen Chin Chiang, MBBSw, Pascal M. Demoly, MD, PhDx,y, Lawrence M. DuBuske, MDz, Marta Ferrer Puga, MDaa, Roy Gerth van Wijk, MD, PhDab, Sandra Nora González Díaz, MD, PhDac, Alexei Gonzalez-Estrada, MDad, Edgardo Jares, MDae, Ayse Füsun Kalpakliog  lu, MDaf, Luciana Kase Tanno, MD, PhD , Marek L. Kowalski, MD, PhD , Dennis K. Ledford, MDai, ag ah Olga Patricia Monge Ortega, MDaj, Mário Morais Almeida, MDak, Oliver Pfaar, MD, PhDal, Lars K. Poulsen, PhDam, Ruby Pawankar, MD, PhDan, Harald E. Renz, MD, PhDao, Antonino G. Romano, MD, PhDap, Nelson A. Rosário Filho, MD, PhDaq, Lanny Rosenwasser, MDar, Mario A. Sánchez Borges, MDas, Enrico Scala, MDat, Gian-Enrico Senna, MDau, Juan Carlos Sisul, MDav, Mimi L. K. Tang, MBBS, PHDaw, Bernard Yu-Hor Thong, MDax, Rudolf Valenta, MDay,az,ba, Robert A. Wood, MDbb and Torsten Zuberbier, MD, PhDbc ABSTRACT Currently, testing for immunoglobulin E (IgE) sensitization is the cornerstone of diagnostic evalu- ation in suspected allergic conditions. This review provides a thorough and updated critical appraisal of the most frequently used diagnostic tests, both in vivo and in vitro. It discusses skin tests, challenges, and serological and cellular in vitro tests, and provides an overview of in- dications, advantages and disadvantages of each in conditions such as respiratory, food, venom, drug, and occupational allergy. Skin prick testing remains the first line approach in most instances; the added value of serum specific IgE to whole allergen extracts or components, as well as the role of basophil activation tests, is evaluated. Unproven, non-validated, diagnostic tests are also dis- cussed. Throughout the review, the reader must bear in mind the relevance of differentiating between sensitization and allergy; the latter entails not only allergic sensitization, but also clinically relevant symptoms triggered by the culprit allergen. Keywords: IgE, Allergy, In vitro tests, Skin tests, Diagnostic strategies a Hospital Quironsalud Bizkaia, Bilbao-Erandio, Spain Received 15 August 2019; Accepted 8 October 2019 *Corresponding author. E-mail: ignacioansotegui@gmail.com 1939-4551/© 2019 The Authors. Published by Elsevier Inc. on behalf of 1 These authors equally contributed to this manuscript World Allergy Organization. This is an open access article under the CC BY- Full list of author information is available at the end of the article NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). http://doi.org/10.1016/j.waojou.2019.100080 2 Ansotegui et al. World Allergy Organization Journal (2020) 13:100080 http://doi.org/10.1016/j.waojou.2019.100080 KEY STATEMENTS immunotherapy (AIT) are required to achieve optimal, long-term outcomes. Allergy diagnosis,  Clinical suspicion of allergic sensitization is hence, may be categorized as precision medicine. confirmed by demonstrating the presence of allergen-specific IgE antibodies in vivo (skin Several types of skin tests are used in allergy tests) or in vitro. diagnostics:  Confirmation of allergen sensitization and the 1) Skin Prick Test (SPT): This represents the first identification of causal allergens are essential for level of approach for the diagnosis of type I, optimizing the management of allergic immediate, IgE-mediated allergy. It is safe, has conditions. high sensitivity and good specificity when per-  Skin prick testing (SPT) is the most frequently formed and interpreted correctly; a specific used method for the detection of IgE antibodies, variant of type I skin tests is prick-to-prick testing due to its rapidity, simplicity and low cost. Skin (PPT) with native allergens. prick tests and other skin test results must be 2) Intradermal Test (IDT): This can be used to interpreted by a clinician with adequate knowl- evaluate both immediate IgE-mediated allergy edge of medical history, clinical findings, and and delayed-type hypersensitivity, according to relevant type I allergens (including environ- the time of read-out. It has increased sensitivity mental, food, animal, insect, fungal, and drug and decreased specificity compared to SPT. allergens). Skin tests should include the relevant allergens in the given geographical area and 3) Patch test: This is used for delayed type, cell- ideally carried out only using standardized mediated, hypersensitivity reactions. It has no allergenic extracts. relevance for IgE-mediated allergy and thus will not be further examined in the present  In vitro tests, including molecular based allergy document. diagnostics, using either in single-plex and in multi-plexed strategies and other more func- The in vitro diagnosis of IgE-mediated allergic tional tests, such as Basophil Activation Tests diseases is useful in the identification of the caus- allow to better define the IgE profile of the pa- ative allergen(s) and usually involves different lab- tient. This approach is in line with the Precision oratory procedures. In particular: Medicine statements. 1) The total IgE assay which is nonspecific and provides only gross information. INTRODUCTION 2) Serum specific IgE assays against allergen Allergic diseases are amongst the most preva- sources/molecules are the most commonly lent diseases worldwide and the burden of these used in vitro diagnostic approach. They can be diseases continues to increase. An accurate diag- performed by a singleplexed or multiplexed nosis coupled with optimal therapy requires the strategy. use of appropriate tests to confirm the allergen 3) The Basophil Activation Test (BAT) which is sensitization and detailed information about quite specific, but complex to perform, and exposure to the putative allergen. Skin tests, therefore limited to selected situations. especially SPT, represent the most reliable and cost-effective tool for the diagnosis and manage- ment of IgE-mediated diseases,. They demonstrate The first part of the present manuscript focuses a good correlation with outcomes of nasal, on skin testing in the diagnosis of IgE-mediated conjunctival, dermal, oral and bronchial allergy and is intended for all practitioners. There challenges. have been criticisms that the procedure is often left to technicians and nurses with limited expertise Once the diagnosis has been established, and and poor attention towards quality control and the relevant allergens have been identified, spe- methodological standardization.1 Surveys have cific treatments, including medications, environ- highlighted the variability of the technical mental control measures and/or allergen methodology2–4 and also the interpretation and Volume 13, No. 2, February 2020 3 communication of results5,6 by different clinically when reactions occur within 30–120 mi- practitioners. In the present document, nutes of exposure. recommendations for its clinical use, technical aspects, reporting, and interpretation of the In general, clinical conditions where SPT is results have been revised and updated. The indicated are the following: second part deals with the in vitro techniques  Asthma; (serum IgE assays and cell-based assays), discus- sing characteristics, performance and indications  Rhinitis/rhinosinusitis/rhino-conjunctivitis/ for the various approaches. The third part includes conjunctivitis; allergen provocation testing. A final part is dedi-  Eczema/atopic dermatitis (in the setting of cated to special situations, where the confirmation selectively high clinical suspicion for underlying or exclusion of an IgE-mediated disease mecha- presence of IgE hypersensitivity to specific nism is required (allergies to foods, drugs, insect allergens); venoms or occupational agents).  Suspected food allergy (oral allergy syndrome, IN VIVO DIAGNOSIS: SKIN TESTS anaphylaxis/acute onset or exacerbation of urti- caria or eczema that is temporally correlated Skin prick test with food ingestion); General information  Suspected drug allergy; The credit for the first skin testing devices goes  Hymenoptera venom allergy (systemic reactions to Charles H. Blackley, who in 1865 abraded a immediately following insect sting); quarter-inch area of skin with a lancet, producing a dermographic reaction. In 1924, Lewis and Grant  Suspected occupational disease or exposure to first described the skin prick test (SPT) method. selected potential allergens; SPT is the simplest in vivo method to assess the  Chronic urticaria in rare selected cases which presence of IgE sensitization in humans. When a strongly suggest an allergen as potential trigger/ specific allergen is introduced through a lancet aggravating factor; into the skin of allergic individuals, dermal mast cells begin to degranulate mainly due to the cross-  Less common disorders, such as eosinophilic linking of allergen-specific IgE bound to their esophagitis, eosinophilic gastroenteritis or membrane receptors. Degranulation leads to the allergic bronchopulmonary aspergillosis, where immediate release of histamine and other media- IgE sensitization is one of the characteristics of its tors, inducing a cutaneous response, clinically pathogenesis. However, there is controversy characterized by a wheal (sometimes with pseu- regarding the utility of SPT for these illnesses. dopods) and surrounding erythema (flare) that can be measured in order to assess the degree of On the contrary, SPT is not routinely indicated in cutaneous sensitivity. Thus SPT represents a sur- the following instances in the absence of other rogate indicator of systemic allergic sensitization existing features of allergic disease: (i.e., nose, lungs, eyes, gut) through the presence of cutaneous reactivity to specific allergens.  Suspected food intolerance (e.g., irritable bowel syndrome, etc.); When should skin prick tests be performed?  Chronic urticaria in the absence of allergic fea- The diagnosis of allergy requires an appropriate tures in the history; medical history and physical examination. If the  Desire to lose weight (according to non- clinical information suggests type I (immediate- conventional approaches, obesity may be due type) allergy, SPTs are indicated to detect the to food intolerance, but no supporting scientific presence of specific IgE to relevant causative al- data have been reported in the literature); lergens: inhalant, food, hymenoptera venoms, drugs and, in some cases, occupational allergens.  Non-specific food-associated symptoms to food Type I hypersensitivity (immediate) is suspected additives/preservatives/colorants; 4 Ansotegui et al. World Allergy Organization Journal (2020) 13:100080 http://doi.org/10.1016/j.waojou.2019.100080  Evaluation of the effectiveness of allergen this antibody. These tests may be viewed as immunotherapy (but may be supportive in Hy- complementary. menoptera venom immunotherapy); The clinician who performs/interprets the SPT and  Non-specific respiratory symptoms to irritants the setting (i.e., smoke, perfumes, detergents, chemicals and other strong odors); A clinician with adequate knowledge of the important, relevant suspected allergens, based  Screening for allergic sensitization patterns in upon the patient's history and the geographic the absence of clinical symptoms (i.e., family area, should decide which specific allergens are history of allergy); tested and interpret the clinical significance of the  Non-specific cutaneous rashes in the absence of test results. SPT must be performed under medical atopic features or other allergic symptoms; supervision, with emergency equipment available migraine, except for the indication of specific for the treatment of anaphylaxis. The risk of sys- hypersensitivity to hormones. However, strong temic reactions in clinically stable patients is scientific data are still missing. extremely low when using standardized respiratory allergen extracts.  Chronic fatigue syndrome. Skin tests reporting form SPTs with great fidelity provide an objective and As for any medical procedure, it is essential that reliable confirmation of allergic sensitization. proper documentation be recorded. An ideal skin However, the clinical relevance of IgE-mediated testing form should list the following information: sensitizations should always be carefully consid-  name and date of birth of the patient; ered since, sometimes, positive SPTs do not directly imply allergic manifestations. A correct  date of the skin test; diagnosis of type I allergic conditions is quite  name, address, and telephone number of the important in order to choose proper avoidance responsible physician; measures and to prescribe allergen immuno- therapy, when needed. When indicated, SPTs are  region tested (e.g., back, forearm); convenient, simple, biologically relevant, repro-  name of technician/nurse/doctor/health profes- ducible, time- and cost-effective, and highly sen- sional trainee who performed the test; sitive. They can be performed in parallel to serum specific IgE (sIgE) detection, and in specific cases,  type of device used; accompanied by other allergen challenge tests to  negative and positive controls (type and con- evaluate the clinical relevance of the allergic sen- centrations), with the respective results; sitizations. SPTs assess the presence of allergen specific IgE bound to mast cells in the dermis.  name of each allergen tested as reported on the These mast cells can bind individual allergen commercial bottle (with genus and species specific IgE molecules for over one year. Allergen identifier), followed by local/common names, specific IgE blood tests measure of the presence of concentration, and manufacturer;  Stop H1-antihistamines the required number of days according to the characteristics of the drugs listed below (most antihistamines 4–5 days, ideally 7 days) before performing skin tests (Table 3).  Stop H2-antihistamines for 24 hours before performing skin tests (Table 3).  Stop anti-depressants with H1-antihistamine activity 1 week before test (ask prescriber if it is possible).  It is not necessary to stop leukotriene antagonists.  If possible, avoid use of high potency topical steroids, optimally three weeks before SPT, in places where skin tests are to be applied. Table 1. Recommendations on medications that possibly interfere with SPT Volume 13, No. 2, February 2020 5  if the allergen extract is diluted, both diluent and molecular weight haptenic chemicals, when trans- dilution should be recorded; formed to complete antigens, can also induce allergic sensitization.  size of the resulting wheal and flare for each allergen after pricking; Allergen extracts are usually obtained from  time point of reading the result, usually 15– natural sources by aqueous extraction. Their 20 minutes; composition and biologic properties may be influenced by the quality and purity of the source  optional: check box for pre-medications poten- material, the methods of extraction and process- tially interfering with the result before starting. ing, as well as the storage conditions. Extracts should not contain more than one allergenic Records of extract source, lot number as well as source (mixes can be used if containing sources expiration date may be kept separately. from a homogeneous taxonomic family) and should not contain interfering preservatives such Drugs possibly interfering with skin prick tests as thiomersal. Before testing, the clinician should verify that the patient has not been taking medications that might Diagnostic extracts made of recombinant or interfere with testing. According to the guideline highly purified allergenic proteins are available in recommendations of the Joint Task Force on some countries where they are approved for al- Practice Parameters of the American Academy of lergy diagnostics. Recombinant and natural Allergy Asthma and Immunology (AAAAI), the allergen preparations have been evaluated and American College of Allergy Asthma and Immu- compared. In general, skin testing with synthesized nology (ACAAI) and the European Academy of allergens is highly specific and avoids the creation Allergy and Immunology (EAACI), certain medica- of false positives by the elimination of cross- tions should be discontinued to avoid suppressive reactive allergens. However, the precise role of effects on the immediate wheal and flare skin test recombinant allergens as an in vivo diagnostic tool response (Tables 1 and 2). remains to be fully determined.  This effect is attributed to a combination of a Allergenic extracts used for diagnostics should decrease in mast cell recruitment and an in- ideally be standardized, both in vivo and in vitro crease of mast cell apoptosis. (meaning that manufacturers should quantify the presence of major allergens in their allergenic ex- Allergen extracts for SPT tracts). Standardization facilitates the comparison The number of skin tests and the selection of of extracts from different manufacturers, lot to lot allergens for skin testing should be determined variability, and the reliability and reproducibility of based on specific clinical history, allergen expo- test results.8 sure pattern (seasonal versus perennial, or spo- radic), distribution of allergenic sources in the local Current standardization is crucial particularly in environment as well as living conditions, occupa- European countries, where strict regulatory rules tion, hobbies, or recreational activities.7 have led sometimes to a problematic and costly registration of some products.9 Allergenic extracts Patient populations tend to be highly mobile, should also be cost effective. hence exposure to different allergens at different places may occur. Stability and potency of the allergen test ex- tracts are also important issues. Since allergen Allergenic extracts consist of mixtures of aller- extract potency deteriorates with time, accelerated genic (proteins, glycoproteins, polysaccharides) by dilution and higher temperatures, allergen skin and non-allergenic components (lipids, salts, pig- test extracts are usually preserved with 50% glyc- ments, metabolites) derived from the allergenic erin. All extracts should be stored in a refrigeration source. Crude extracts thus usually contain both unit at 2–8  C to improve stability. genuine sensitizers and cross-reactive proteins. Allergens in general are mainly proteins or glyco- For diagnostic use, both standardized and non- proteins, but carbohydrates or other low- standardized products are commercially available 6 Ansotegui et al. World Allergy Organization Journal (2020) 13:100080 http://doi.org/10.1016/j.waojou.2019.100080 Day(s) suppressed Drugs Generic drug Despite the intervals indicated, the higher limit of the interval is recommended H1-antihistamines First generation Chlorpheniramine 2–6 Clemastine 5–10 Cyproheptadine 9–11 Dexchlorpheniramine 4 Diphenhydramine 2–5 Hydroxyzine 5–8 Promethazine 3–5 Tripelennamine 3–7 Second generation Azelastine nasal 3–10 Ebastine 3–10 Cetirizine 3–10 Fexofenadine 2 Loratadine 7–10 Desloratadine 3–10 Levocetirizine 3–10 Bilastine 4–5 Levocabastine nasal Do not suppress skin tests Levocabastine ophthalmic Do not suppress skin tests Rupatadine 3–7 Tricyclic antidepressants Desipramine 2 and tranquilizers Imipramine >10 Doxepin 6–11 Doxepin topical 11 H2-antagonists Ranitidine 1 Anti-IgE monoclonal Omalizumab Prick tests can be performed after 6 weeks but antibody false negative results can occur up to one year226 Cysteinyl leukotriene Montelukast Does not suppress skin tests receptor antagonists Zafirlukast Does not suppress skin tests Short term oral 30 mg of prednisone daily Do not suppress skin tests corticosteroids for 1 week Volume 13, No. 2, February 2020 7 Day(s) suppressed Drugs Generic drug Despite the intervals indicated, the higher limit of the interval is recommended Long term and relatively >20 mg/d Possible suppression of immediate skin test high dose corticosteroids reactions Potent topical >3 weeks Suppress immediate skin test over areas where corticosteroids** they have been applied Local anesthetic EMLA (Eutectic Mixture of 1 h before test suppression (only suppresses Local Anesthetic) cream erythema) Table 2. (Continued) Suppressant effects of drugs on immediate skin testing * NB: Where this article reports in fractions of days, the total has been rounded up. Maximum days would apply to most patients, but there may be exceptions where suppression could last longer. Adapted from Bernstein IL, Li JT, Bernstein DI et al. Allergy diagnostic testing: an updated practice parameter. Ann Allergy Asthma Immunol 2008; 100 (Suppl 3):S1–S148.227 with labeling in a variety of potency units (some by IgE cross-reactivity to homologous (cross-reac- examples are provided in Table 3). tive) allergens. Cross-reactivity of pollens is frequent among taxonomically related plants or in Number of skin tests the case of highly conserved proteins across A pan-European study of skin tests, supported different species (Table 4). by the Global Allergy and Asthma European Network (GA2LEN), showed that, for respiratory/ Generally, fewer tests to suspected allergens are conjunctival allergies, it is not necessary to include required in infants and very young children (<2 a large number of allergen extracts for skin tests, at years of age) because children are not likely to be least in Europe. The total number of allergens sensitized to as many allergens as older children tested depends, as mentioned above, on the local and adults. In toddlers, allergic sensitization re- exposure framework. The suggested panel for flects intense and/or prolonged exposure to aller- respiratory allergy includes 18 allergens, as fol- gens encountered early in life, such as foods, lows: alder (Alnus incana), birch (Betula alba/ver- house dust mites, indoor molds, and animal rucosa), cypress (Cupressus sempervirens/ dander rather than pollens.12 arizonica), hazel (Corylus avellana), plane (Platanus Relatively few foods account for most IgE- vulgaris), grass mix (including Poa pratensis, Dac- mediated allergic reactions in both children and tilis glomerata, Lolium perenne, Phleum pratense, adults. The more common food allergens in infants Festuca pratensis, Helictotrichon pratense), olive and young children are: cow's milk, hen's egg, (Olea europea), mugwort (Artemisia vulgaris), peanuts, tree nuts, soybeans, and wheat, whereas ragweed (Ambrosia artemisiifolia), Alternaria the adult counterparts are peanuts, tree nuts, fish, alternata (tenuis), Cladosporium herbarum, Asper- crustaceans, mollusks, fruits, and vegetables. gillus fumigatus, Parietaria, cat (Felis domesticus), However, this generalization does not preclude the dog (Canis familiaris), dust mite (Dermatopha- possibility that larger numbers of tests may be goides pteronyssinus/farinae), and cockroach required, if multiple or hidden food allergies are (Blatella germanica).10 In tropical countries, testing suspected; this must be weighed against a high with Blomia tropicalis is recommended.11 The false positive response rate. PPT (Prick-Prick number of tests performed should be much testing) whereby a fresh food sample is pricked lower when testing infants. followed by immediate pricking of the skin in Information regarding cross-reactivity among suspected vegetable and fruit IgE-mediated re- allergens is important when interpreting results. actions can provide greater sensitivity. Occupa- Cross-reactivity describes the phenomenon tionally related allergy (e.g., latex, rodents, flour, whereby an immediate type skin reaction by a food inhalants, etc.) is a special clinical condition particular allergen (genuine sensitizer) can also be for which a limited number of reliable skin test elicited by other similar allergens and is explained reagents are available. 8 Ansotegui et al. World Allergy Organization Journal (2020) 13:100080 http://doi.org/10.1016/j.waojou.2019.100080 Allergen product Skin prick test concentration Intradermal test concentration Standardized short ragweed 1:20 w/v 1:1000 w/v Standardized cat hair 10,000 BAU/mL 200 BAU/mL Standardized grass pollens 10,000–100,000 BAU/mL 200 BAU/mL Standardized Hymenoptera venoms 100 mg protein/mL 0.1–1 mg protein/mL Standardized mites 10,000 AU/mL 200 AU/mL Non standardized allergens 1:40–1:20 w/v 1:1000 w/v Table 3. Examples of skin test concentrations of standardized and non-standardized allergens. Adapted from Dolen WK, MD. Immunology and Allergy Clinics of North America. Volume 21, number 2 May 2001. Saunders. Selection of allergen products for skin testing by Robert E. Esch, PhD.228 The general recommendations for SPT are reactive. The ulnar side of the arm is more reactive summarized in Table 5. than the radial area. In infants, the back is the preferred site for SPT. It is recommended that tests Available devices for skin prick testing should not be placed within 5 cm of the wrist and There are a variety of devices for performing 3 cm of the antecubital fossa. Skin tests should not skin tests throughout the world, developed in be performed on skin sites with active dermatitis, an attempt to improve reproducibility of the severe dermographism and tattoos. Ageing of the skin prick test method. Devices used generally skin, and sun damage will affect the skin's reac- are designed with a sharp point (0.9 or 1 mm) tivity. The location of each allergenic drop can be and a shoulder to prevent excessive penetration marked with a pen or a test grid. The allergenic into the dermis. The most popular instruments solution placed on the skin should be immediately are the Morrow Brown standardized needle, the pricked. “Greer PickÒ” (DKL), the StallerpointÒ, and the The selected test site is cleansed and dis- PhazetÒ. Puncture tests can also be performed infected with alcohol and allowed to dry. After with a bifurcated smallpox vaccination needle, a marking the skin sites to place the extract drops 23G intravenous needle or with other devices. with numbers, codes, or using a template, the Several plastic devices with multiple heads allergen extracts are applied to the skin, depos- (“multi-headed” skin test devices) have also been iting a drop of allergen extract on the skin of the developed to apply several skin tests at the same forearm of the patient. The puncture device is time, which may limit technician time, and in- passed through the drop at a 45 –60 angle to the crease efficiency. skin, achieving penetration of small amounts of The inter-device wheal size variability is highly allergen extract just below the epidermis. This is significant (Table 6). Potential causes of this called the skin prick test (alternatively, the skin variability include depth of penetration into the device may be passed through the drop at a 90 skin, amount of antigen entering the skin, angle angle to the skin with gentle pressure for 1 second, of penetration, and skill of the investigator. It is this is called a puncture test). The drops must be imperative that the allergist/immunologist placed 2 cm or more apart each other to avoid understands the characteristics of the device mixing or overlapping and therefore false-positive chosen (Table 7). reactions. If mast cells are sensitized with specific IgE in the patient's tissue, the penetration of the Skin prick test technique allergen causes the release of histamine, resulting in a wheal and flare response. SPT must be performed on normal skin. SPT are usually applied, for practical reasons on the volar Because of inter-patient variability in cutaneous surface of the forearm; but other sites are equally reactivity, it is necessary to include negative and effective. The antecubital fossa is the most reactive positive controls at the same time as allergen tests portion of the arm, whereas the wrist is the less in every skin test evaluation. Positive control Volume 13, No. 2, February 2020 9 Cross-reacting Representative generaa groups Grass Pooideae Poa (bluegrass), Bromus (brome), Dactylis (orchard), Festuca (fescue), Pollens Lolium (perennial rye), Agrostis (redtop), Anthoxanthum (sweet vernal), Avena (cultivated oat), Holcus (velvet), Phalaris (reed canary), Phleum (timothy), Agropyron (quack), Elymus (wild rye), Secal e (cultivated rye), Triticum (cultivated wheat) Chloridoideae Cynodon (Bermuda), Bouteloua (blue grama, mosquito Grass), Distichlis (salt) Panicoideae Paspalum (Bahia), Sorghum (Johnson), Panicum (Para grass), Zea (corn) Tree Aceraceae Acer (maples and box elder) Pollens Betulaceae Alnus (alder), Betula (birches), Corylus (hazelnut) Cupressaceae Cupressus (cypress), Juniperus (junipers and cedars), Taxodium (bald- cypress), Cryptomeria (Japanese cedar) Fabaceae Acacia (mimosa), Robinia (locust), Prosopis (mesquite tree) Fagaceae Quercus (oaks), Fagus (beech) Juglandaceae Carya (hickory and pecan), Juglans (walnut) Moraceae Morus (mulberry), Broussonetia (paper mulberry) Oleaceae Olea (olive), Fraxinus (ash), Ligustrum (privet) Pinaceae Pinus (pines) Platanaceae Platanus (sycamore) Salicaceae Populus (cottonwood and poplars), Salix (willows) Ulmaceae Ulmus (elms) Weed Chenopodiaceae Atriplex (scales and saltbush), Chenopodium (lamb's quarter), Salsola Pollens (Russian thistle), Kochia (firebush), Allenrolfea (iodine bush) Asteraceae: Artemisia (mugworts, wormwood, sages) Artemisia Asteraceae: Ambrosia (ragweeds), Xanthium (cocklebur), Iva (poverty weed hemp) Ambrosia Amaranthaceae Amaranthus (careless weed, pigweeds), Acnida (Western water hemp) Plantaginaceae Plantago (plantain) Polygonaceae Rumex (dock and sorrel) Urticaceae Parietaria Table 4. Cross-reacting pollen allergen groups. a. Representative genera are members of the same botanical family or subfamily. Manufacturers currently offer allergen products derived from one or more species of each listed genus solutions (histamine phosphate, used at a con- reactive to histamine, and determine variations in centration of 5.43 mmol/L or 2.7 mg/mL, equiva- technician performance and/or the potency of the lent to 1 mg/mL of histamine base) are used to testing reagent. Histamine dihydrochloride is detect suppression by medications or disease, commercially available and has been approved for detect the exceptional patients who are poorly the use for in vivo testing in Japan. 10 Ansotegui et al. World Allergy Organization Journal (2020) 13:100080 http://doi.org/10.1016/j.waojou.2019.100080  Include skin test allergens relevant to the specific geographical region.  Standardized allergen products should be used for skin testing whenever possible.  Clinical history should guide skin testing, as there is a potential for false positive responses.  More allergens may need to be assessed in areas with greater biodiversity and warmer climates.  Limit the number of allergen used in skin testing, taking into account cross-reactions and eliminating allergens irrelevant in the exposure area.  Include a positive (histamine) and a negative (buffer) controls. Table 5. Recommendations for SPT The negative control (saline or 50% glycerinated than simultaneously performed diluent control is human serum albumin–saline) will also detect required as proof of the presence of cutaneous traumatic reactivity induced by the skin test device hypersensitivity, indicative of the presence of spe- (with a wheal which may approach a diameter of cific IgE. 3 mm with some devices) and/or the technique of the tester or the presence of dermographism. The presence of allergic sensitization (a positive SPT with no correlative allergic disease) is a com- Some of the most common errors in skin prick mon finding, occurring in 8–30% of the population testing are listed in Table 8. when using a local standard panel of aero- allergens. Interestingly, a prospective study13 Measurement and interpretation of skin prick test reported that 60% of skin prick test positive Skin tests should be read at the peak of their (wheal > 4 mm) asymptomatic subjects reaction and in a standardized manner. Whatever developed clinical allergy, thus, the presence of the method, the immediate skin test induces a positive SPT in asymptomatic subjects may response that reaches a peak in 8–10 minutes for predict the subsequent development of allergic histamine and 15–20 minutes for allergens. The symptoms. reading and evaluation of skin tests using an False-positive tests may be provoked by impu- arbitrary scale of 0–4 þ is not recommended un- rities, contaminants, and non-specific mast cell less the specific criteria for the scale are defined on secretagogues in the extract, as well as dermog- the skin test form. The least variable method, which raphism. Devices used and techniques applied is both objective and reproducible, occurs when should also be considered when comparing mea- the wheal's size is measured in millimeters (mm) surements to the negative control. with a ruler. The size of the reaction may be recorded as a mean wheal diameter, D þ d/2 (with The most common cause of false-negative tests D indicating the largest diameter of the wheal and is the ingestion of a drug that inhibits the effect of d indicating the largest diameter orthogonal to D). histamine. In addition, skin reactivity is decreased Other Authors suggests (D þ d)/2. Pseudopods in infants and the elderly and in skin that has suf- are not included in the measurement, but can be fered chronic solar injury. Technical factors that marked separately. A prick/puncture test with a result in false negative results include improper response of at least 3-mm diameter in wheal more technique, too short or too long interval from  Avoid devices or techniques that produce a negative control >3 mm wheal and >10 mm flare, due to the possibility of producing a false positive reaction.  Ideally use the same device to improve reliability, comparability and interpretation.  Strictly avoid deep punctures associated with bleeding. Table 6. Recommendations on SPT devices Volume 13, No. 2, February 2020 11 Devices for which a 3-mm wheal would be significant Devices for which a wheal >3 mm should be used as significant 99th Percentile of reactions 99th Percentile of reactions Device Device at the negative control sites at the negative control sites Quintest (HS) puncture 0 mm DuoTip (Lincoln) 3.5 mm twist Smallpox needle (HS) 0 mm Bifurcated 4.0 mm prick needle(ALO) prick DuoTip (Lincoln) prick 1.5 mm MultiTest 4.0 mm (Lincoln) Puncture - Lancet (HS) puncture 2.0 mm Bifurcated 4.5 mm needle (ALO) puncture Lancet (ALK) Puncture 3.0 mm Quick Test 4.0 mm (Pantrex)- DermaPICK II 0 mm Greer 3.5 mm (Biomedixs) Prick or Track(Greer) puncture a Table 7. Wheal size indicating a positive response to skin tests using various devices . Abbreviations: HS, Hollister Steir; Greer, Greer Laboratories; ALO, Allergy Labs of Ohio; Lincoln, Lincoln Diagnostics; ALK, ALK America. Positive response is defined as a wheal greater than 99% of wheals generated by the administration of saline to the subject's back by the same operator. a. Adapted from Bernstein IL, Li JT, Bernstein DI et al. Allergy diagnostic testing: an updated practice parameter. Ann Allergy Asthma Immunol 2008; 100(Suppl 3):S1–S148.12 application to measurement and extracts of Since the interpretation of SPT results is crucial reduced potency due either to aging of the extract for a proper treatment approach, in order to find or poor original quality of the extract. out “reading keys” for interpretation of test results, a GA2LEN (Global Allergy and Asthma European Network) survey investigated the correlation be- Since the interpretation of skin tests can have tween SPT wheal size and self-reported clinical significant impact on daily life, in terms of avoid- relevance (i.e., symptoms related to asthma, allergic ance measures and therapies, the specialist must rhinitis, atopic dermatitis, and food allergy) for 18 pay attention to different clinical aspects: allergens tested in 3068 patients in 17 European  positive tests may occur in absence of clinical centers. With the exception of Aspergillus fumiga- relevant symptoms (sensitization); tus, with larger wheal sizes the prevalence of allergic  negative skin prick test results can miss the pres- symptoms increased significantly. This correlation ence of IgE-mediated sensitization (e.g., due to was variable among the different allergens, ranging lack of major allergens in commercial extracts); from 40% (Blatella) to 87–89% (grass, Dermatopha- goides pteronyssinus) of the positive SPT wheals  negative SPT results in children do not exclude associated with patient-reported clinical symptoms. the possibility of development of allergic dis- In general, children with positive SPT were less eases in the future. symptomatic than adults in relation to hazel tree (P < 0.001) and dog (P < 0.001); no difference was found for house dust mites. The frequency of When the SPT result is not clear or does not symptoms was slightly higher among skin test pos- correlate with clinical history, a serum specific IgE itive women than skin test positive men for hazel assay or, more rarely a challenge with the culprit tree (P ¼ 0.012), dog (P ¼ 0.031), and Dermato- allergen, may be needed (Table 9). phagoides pteronyssinus (P ¼ 0.064). Furthermore, 12 Ansotegui et al. World Allergy Organization Journal (2020) 13:100080 http://doi.org/10.1016/j.waojou.2019.100080  Tests are placed too close together (<2 cm), and overlapping reactions cannot be separated visually.  Induction of bleeding, possibly leading to false positive results and increased risk of systemic reaction.  Insufficient penetration of skin by puncture instrument, leading to false negative results. This occurs more frequently with plastic devices.  Spreading of allergen solutions during the test of when the solution is wiped away. Table 8. Common errors in skin prick testing. Adapted from Mansmann HC, Jr, Bierman CW, Pearlmann DS (eds). Allergic diseases in infancy, childhood and adolesence.1980: p.289, with permission. Copyright, Elsevier; Bousquet J et al. Practical guide to skin prick test in allergy to aeroallergens. Allergy 2011. when geographical influence was assessed, a lower tests, the intradermal test (IDT) can be considered. frequency of symptoms in the Mediterranean vs. IDTs are frequently used for inhalant allergen Nordic and Central European regions was noted in sensitization in the United States. Challenge relation to positive SPTs to hazel tree and dog; on studies have not confirmed the predictability of the the contrary, no differences were found for grass or test, however, it could be applied in specific situ- house dust mites. Regarding the correlation be- ations (i.e., IDTs to foods are sometimes utilized for tween positive SPTs and physician-reported diag- delayed food anaphylaxis from alpha-gal al- nosis, positive SPT for grass (OR 2.96, 95% CI 2.4– lergy).15 IDTs are applied when assessing 3.7), cat (OR 2.0, CI 1.6–2.6), Dermatophagoides hypersensitivity to drugs or hymenoptera venoms pteronyssinus (OR 1.7, CI 1.4–2.1) and hazel tree (see below). (OR 1.7, CI 1.1–2.5) statistically increased the risk of It is important to consider the relative advan- allergic rhinitis. Positive SPT to Dermatophagoides tages of prick and intradermal testing (see pteronyssinus (OR 2.2, 95% CI 1.8–2.6), cat (OR 1.4, Table 10). CI 1.1–1.8), and grass (OR 1.2, CI 1.0–1.5) signifi- cantly increased the risk of developing asthma, Intradermal testing (IDT) is important to particularly in children (OR 4.2, CI 3.4–5.2). Positive reveal both immediate IgE-mediated allergy SPT reaction to cat (OR 1.3, 95% CI 1.0–1.7) and and delayed-type hypersensitivity. When used grass (OR 1.3, CI 1.0–1.6) were slightly correlated for type 1 allergy diagnosis, it is characterized with a major risk for atopic dermatitis, especially in by an increased risk for adverse reactions, thus children (OR 1.5, CI 1.2–1.9) and in females (OR 1.5, requiring high levels of technical and interpreta- CI 1.2–1.9). Cutaneous positivity to birch (OR 1.7, CI tive expertise: for this reason, it is generally 1.1–2.6) augmented the risk of food allergy, partic- restricted to a clinical setting where emergency ularly in females (OR 1.4, CI 1.1–1.8). For each equipment and treatment are readily avail- allergen, the wheal size in mm (ranging from 3 to able.16,17 Delayed IDT readings are performed 10 mm) with an 80% positive predictive value (PPV) for delayed reactions, but will not be discussed for clinical relevance was calculated and reported in in this manuscript. a GA2LEN “reading key” form that can be a useful tool for interpreting SPT results.14 Indications and contraindications Intradermal test IDT is usually not required for the diagnosis of respiratory allergy. It is mainly indicated in case of General aspects suspected respiratory allergies with negative SPT, In a patient with a strong clinical suspicion of an venom allergy and drug allergy. It has an estab- IgE-mediated disease with negative skin prick lished place in testing b-lactam (in particular,  Consider a skin prick test result to be positive if the wheal diameter is 3 mm greater than the negative control.  Consider a skin prick test result negative if the wheal diameter is less than 3 mm with a positive simultaneous histamine control. Table 9. Recommendations in SPT interpretation Volume 13, No. 2, February 2020 13 Prick test Intradermal test Simplicity þþþþ þþ Speed þþþþ þþ Interpretation of positive and negative reactions þþþþ þþ Discomfort þ þþþ False-positive reactions Possible Likely False-negative reactions Possible Rare Reproducibility þþþ þþþþ Sensitivity þþþ þþþþ Specificity þþþþ þþþ Indicative of IgE antibodies Yes Yes Safety þþþþ þþ Testing of infants Yes Difficult Table 10. Relative advantages/disadvantages of prick and intradermal allergy skin testing. Adapted from Adkinson: Middleton's Allergy: Principles and Practice, 7th ed. 2008. Chapter: 71 – In Vivo Methods for the Study of Allergy. Pascal Demoly, Jean Bousquet, and Antonino Romano.229 penicillin and cephalosporin) allergy, but may also Technique be used for testing a number of other drugs such In IDT, allergens (usually, 0.02 mL) are injec- as insulin, opiates, anesthetics, neuromuscular re- ted intradermally with small needles to produce laxants, proton-pump inhibitors, enzymes, and a small bleb, and the outcome measure is an chemotherapeutic agents.18–20 increase in the size of the wheal with flare re- action at 20 minutes. Allergenic extract must be Contraindications for IDT include: diluted (10–1000 fold or more) from the con- centrations used for SPT. IDT should always be  Diffuse dermatological conditions, such as preceded by SPT including negative and posi- eczema, urticaria, and dermographism; tive controls.  Poor subject cooperation; The skin end point titration can be defined as  Patients being unable to stop antihistamines/ the intradermal injection of allergens at increasing other interfering drug treatments; concentrations to measure their allergic response. This is typically done for venom and drug allergy  Food allergy*, for lack of specificity21,22 assessments but not for inhalant allergens. To  *May be utilized in specific situations (i.e., IDT for avoid severe allergic reactions, testing starts with foods are sometimes utilized for delayed food highly diluted extracts. After 15–20 minutes, the anaphylaxis from alpha-gal)15 injection site is measured in terms of the size of the wheal and flare reaction. The end point, typically, is the concentration of antigen that causes an in- Relative contraindications/precautions: crease in the size of the wheal. For other Au- thors,23 the endpoint is the first dose of antigen  Persistent/unstable asthma; provoking minimal erythema.  Pregnancy (due to risk of anaphylaxis with hy- This method allows one to grossly quantify the potension and uterine contractions); individual's skin response and, subsequently, their  Infants or younger children. degree of allergic sensitivity. Titration methods for 14 Ansotegui et al. World Allergy Organization Journal (2020) 13:100080 http://doi.org/10.1016/j.waojou.2019.100080 1. Skin tests should never be performed unless a physician or other health-care provider professional capable of treating anaphylaxis is available immediately to treat systemic reactions. 2. Emergency equipment and therapies to treat anaphylaxis, including epinephrine, should be available in facilities in which allergy skin testing is performed. 3. The clinical status of the patient should be evaluated before testing. 4. Caution should be exercised and allergy skin testing possibly postponed if subjects are having allergic symptoms. 5. The concentrations and storage of the testing reagents should be appropriate. 6. Negative and positive controls must be performed with each allergy skin test session. 7. Allergy skin tests should be performed on normal skin, preferably the volar surface of the forearm or, if needed, on the upper back. 8. Patients should be evaluated for dermographism to avoid false positive results. 9. The medications taken by the test subject should be recorded and the time of discontinuing medications that would suppress a histamine skin response should be documented. 10. Allergy skin test results should be measured at the appropriate time. Table 11. Type I skin testing summary precautions. Adapted from Adkinson: Middleton’s Allergy: Principles and Practice, 7th ed. 2008. Chapter: 71 – In Vivo Methods for the Study of Allergy. Pascal Demoly, Jean Bousquet, and Antonino Romano.229 IDTs have not been validated.24 allergic disease is the main purpose of the allergy diagnostic evaluation. The identification of biolog- Precautions and contraindications of all type I skin ical sources having allergenic properties dates back testing more than 100 years, and it is still in progress. Raw extracts obtained from allergenic sources have Adverse reactions been used for decades to help demonstrate clini- The prick/puncture test is safe, with systemic cally relevant sensitization by means of skin testing, reactions occasionally observed with commercial as well as provocation testing, such as oral or extracts. Foods were identified as the most rele- mucosal (nasal, conjunctival or bronchial) chal- vant trigger (75%), with nuts having the highest lenges. Since the purification of IgE in 1967, sero- risk. In general, history of severe allergic symptoms logical testing has become a commonly used test in and large skin test reactions were recognized as the evaluation of allergic diseases.25 predictors of possible severe adverse reactions to allergy skin testing (Table 11). Using the first in vitro specific-IgE tests, some drawbacks of in vitro allergy testing were reported,26 Frequency of skin testing but the progressive introduction of high- Skin tests may be repeated for a variety of rea- performance laboratory-based IgE methods, partic- sons including: changes in clinical manifestations ularly after the development of second-generation or exposures; lack of clinical correlation with in vitro systems,27 has greatly increased their sensitization patterns; or the resolution of venom diagnostic accuracy. Currently, automated immunotherapy. However, routine repeated skin workstations reduce the required labor, costs and testing is not recommended. errors, thus improving the consistency of in vitro tests. To date, the results achieved with in vitro Age for performing skin testing specific IgE measurement and skin testing are Prick/puncture tests may be performed at any nearly comparable with some well-known advan- age if indicated, recognizing that positive reactions tages and disadvantages for each diagnostic tend to be smaller in infants and younger children approach.28,29 The main problem with in vitro tests (<2 years), and in the elderly. concerns the intrinsic nature of the crude extracts used, which are often are an unpredictable mixture IN VITRO DIAGNOSIS of allergenic and non-allergenic substances. In addition, different producers have different extracts General concepts and the same producer may have different extract Identification of the causative allergen (usually an from material collected in different years.30 Despite allergenic protein) responsible for the causation of these intrinsic differences which were an obstacle Volume 13, No. 2, February 2020 Classification of Cross reactivity of Risk of Frequency of Evolution of Susceptibility Classification of molecular molecular molecular molecular molecular of molecular allergen extract components components components components components components Inhalants Inhalants (Phl p 1) Genuine (Phl p 1) Potentially Frequent Age related Heat dangerous (Ara (major (Bos d 1) Sensitive: h 1) component) Mal d 1 Phl p 1 Resistant: (Pru p 3) Food Food (Mal d 1) Pan allergen Virtually Rare (minor Related to Low pH Phl p 12 innocuous components) allergy march Sensitive: (Ara h 8) Phl p 2 (Gal d 1) Mal d 1 Resistant: (Pru p 3) Contact (latex) Contact (latex) Cross-reactive Related to Gut (Hev b 6) Bet v 1 allergy march peptidase (Phl p 12) Sensitive: Mal d 1 Resistant: (Pru p 3) Hymenoptera Hymenoptera (Ves v 5) Table 12. Different classification of allergens and molecular components are based on specific functional, clinical or biochemical characteristics (exemplary allergens in parentheses) 15 16 Ansotegui et al. World Allergy Organization Journal (2020) 13:100080 http://doi.org/10.1016/j.waojou.2019.100080 Allergen extract positive but its molecules negative (a) Serum IgE binds only to extract's molecules that are not (yet) available in molecular assays. (b) molecular assay less analytically sensitive than the extract-based assay Allergen extract negative but its molecules positive (a) Serum IgE binds to molecules tested as components which are missing or in low abundance in the extract. (b) Extract assay less analytically sensitive than the molecular assay Allergen extract positive but its genuine components are negative (a) Serum IgE binds only to highly cross-reactive, minor allergenic molecules or CCD determinants Allergen extract less positive than the added molecular assay level (a) Serum IgE binds to molecules tested as components being of low abundance in the extract. Table 13. Interpretation of non-concordance between allergen extract and allergen molecular IgE assay results (modified from Matricardi, Kleine-Tebbe 2016 144) to full standardization,31,32 these “raw” extracts were SPT in vivo and sIgE in vitro started virtually used as allergen source for in vivo and in vitro tests for immediately after the development of laboratory several decades. However, during the first years of methods for the detection of specific IgE. Two the 1990s, molecular allergists began the groups of spirited partisans arose. SPT followers production of recombinant allergens (termed maintained that SPT is in general simple and components) that were thus used as reagents for painless; but sIgE supporters considered that SPT, in vitro diagnostics. It was immediately evident that in small children, can be really difficult, requiring a molecular components did not completely certain amount of co-operation from the patient. correlate with the results of SPT and of sIgE tests for SPT followers maintain that in vivo assessment of- a number of reasons that will be discussed. But, it fers quick results (tens of minutes) but sIgE fol- was also evident that the specific characteristics of lowers showed that modern sIgE requires just a each molecular component, when used in couple of hours. SPT followers indicated that SPT diagnostics, provided a real added value. Indeed, can be performed with virtually any allergen (the we are now able to classify components according prick-to-prick procedure) but sIgE countered by to different strategies, represented by well-defined maintaining that the number of allergens to be molecular characteristics (Table 12). tested in vitro is just limited by the costs. Moreover, sIgE followers noted that sIgE assays can be per- The full assessment of molecular components is formed by any laboratory technician, are not quite complicated: at the moment (September affected by anti-histamine drugs, the results – 2018), in fact, >4700 distinct molecules have been despite certain differences between reagents and described, including >3200 isoforms (such as Ara h producers – are not dependent on the operator's 2.0101, Ara h 2.0102, Ara h 2.0201 and Ara h 2.0202 experience, and potentially dangerous allergens or Ara h 2) (http://www.allergome.org/script/ are not administered to the patient. Nowadays, the statistic.php). This very large number of potential two parties (SPT followers and sIgE followers) are reagents offers unexpected possibilities in allergy still debating.33–35 As a general concept, SPTs are diagnostics. So, in this chapter, the characteristics of more sensitive than in vitro tests, whereas serum in vitro IgE tests will be discussed in light of the most specific IgE detection is more quantitative than recent advances in basic and clinical research. SPT. This discussion could have certain significance only if in vitro assays should replace SPT in the future (for example, if a reagent for DIAGNOSTIC STRATEGY SPT cannot be produced like for a drug). But at Before describing the different methods avail- present, SPT are still available and the top-down able and their extended technical possibilities, it is strategy of allergy diagnosis36 remains the most necessary to analyze the medical and diagnostic frequent approach followed in clinical allergy contexts in which these in vitro methods are used. diagnostics. However, in this context, it should be The comparison of the results obtained by using noted that SPT is considered a functional test by Volume 13, No. 2, February 2020 17 which the skin reactivity to an allergen (no matter experimental conditions or increasing the whether mediated by IgE or not) is measured by sensitivity of the assay itself. Antibodies are the diameter of the wheal. On the contrary, characterized by a spectrum of specificities for different sIgE tests are assays used to detect the the antigens: they are extremely specific under presence of IgE antibodies able to bind an certain experimental conditions but, in other extract allergen or a molecular component. So, conditions, antibodies can efficiently recognize specific IgEs are suitable to identify the presence similar epitopes. So, the answer to this question of serum IgE to one or more allergens provided lies squarely in the hands of the allergist who that IgE are present and detectable (Table 13). must decide whether what is observed is just a Sensitivity and specificity can be evaluated, for sensitization or a real allergy. Keeping in mind all every new laboratory assay, by comparing the these relevant points, it is evident that the results of the novel test to that of a gold comparison between methods may be standard. For specific IgE, a gold standard does sometimes an oversimplification of the problem, not seem to exist. Indeed, it is a common notion causing more confusion rather than shedding that every subject recognizes antigens or light on the argument. Along this line, from a allergens on the basis of her/his genetic practical point of view, an allergist should build substrate: so, certain epitopes are recognized by her/his experience on a well-defined in vitro test, certain haplotypes, while others are not. In in order to have a clear strategy for the interpre- addition, in vitro assays are in general based on tation of the in vitro results. All these consider- an excess of allergen bound to the solid phase: ations are even more relevant when the tools of for this reason, a large low-affinity immune molecular allergy diagnostics are used. Indeed, at response cannot be easily distinguished from a present, if an accurate diagnosis of allergic sensi- high affinity one. tization is required, specific IgE to allergen extracts are not always as precise as wished for, and a more To this it should be added that, like any other specific description of the IgE profile may be serological method, every specific IgE test has mandated. This is particularly true when allergen certain characteristics, related to the structure of immunotherapy (AIT) is provided to the patient. the solid phase (if any), the amount of allergen in Even though large discussions on this topic are still the tube, the time and conditions for the incuba- ongoing, suggestive data seems to indicate that tion, the characteristics of the anti-IgE, the AIT is probably more effective in patients sensi- behavior of the labeled antigen the detection tized to genuine allergens,38–43,42,43 while its method used (like Enzyme Immune Assay [EIA] or activity is less impressive in patients with a Chemiluminescence [CL]), and the dynamic range sensitization to cross-reacting components or of the reading etc. For example, in vitro assays pan-allergens. In this context, the in vitro evidence based on chemiluminescence are more positive (in of specific IgE involvement in the patient's symp- particular in the presence of low levels of specific toms associated with the detection of IgE specific IgE) than assays based on ELISA or similar tech- for genuine components seems to be the entry niques. So, in the presence of a positive result with level diagnostic for patients that could have a real CL and a negative result with ELISA, the question modification of their allergy by AIT. that must be asked is: is this a false positive result or is this result the demonstration of the presence These considerations should be always be taken of a small (but specific) IgE response to the into account. One cannot directly compare the two allergen? In other words, is ELISA underestimating methods (SPT and sIgE tests) as they are pro- the presence of sIgE? Along this line, even the foundly different in many aspects. Specific IgE (to solid phase structure may have an unexpected the allergen extract or to molecular components) effect on the serological result. For example, it has provide a real added value in those cases where been shown that certain solid phases express the allergist requires a precise diagnosis before CCD, thus causing false positive results in patients starting AIT and to identify accurate therapeutic or with sIgE to CCD.37 What is known from basic prophylactic strategies in food and hymenoptera immunology is that a positive result can be allergy. Along this line, it should be clearly noted observed in any situation just by changing the that specific IgE tests are poor or not significant at 18 Ansotegui et al. World Allergy Organization Journal (2020) 13:100080 http://doi.org/10.1016/j.waojou.2019.100080 all for the very large majority of drug allergies. serum IgE levels can be seen also in a proportion Although a few drug reagents are available (in of smokers. particular, antigens derived from penicillin and In the past, total IgE levels were calculated using some peptide hormones) they do not encompass a number of immunoassays that utilize specific the requirements of allergists in this field. antibodies for human IgE as both capture and/or detection reagents. These antibodies, in the vast Total serum IgE majority of cases, are conjugated on a solid phase In 1967, the first solid-phase sandwich immuno- (capture antibody) and/or directly labeled with assay for the measurement of total and specific IgE radio-nuclide, enzyme, or fluorophore. Automated was described.44 The amount of total IgE was platforms significantly improved accuracy and considered in the early studies as the simplest way reproducibility, increasing both specificity and to identify allergic subjects,45,46 but it became sensitivity as well. Commercially available assays evident soon that total IgE levels could not be have been cross-standardized to a common pri- considered a reliable marker of allergy status.47,48 mary human IgE standard (WHO 11/234).51 Total IgE levels significantly higher than the normal IgE values are currently reported in International threshold, are usually associated with atopic Units of IgE per volume (IU/mL); a conversion disorders, but also with other conditions (see factor (1 IU ¼ 2.42 ng) is sometimes applied. below). On the contrary, low or normal values do Nowadays it is very common to be reported in its not exclude the presence of IgE-mediated dis- equivalence of kU/L. eases. As a consequence, total levels of IgE should Different scenarios can be considered. Table 14 be carefully interpreted and not considered as an shows these different conditions. indication for the presence of allergic diseases. Allergen-specific IgE assays Serum IgE concentration is largely age- dependent. Very low levels of IgE are found in General concepts cord serum (<4.8 ng/mL) with a progressive in- crease observed up to the age of 15 years, similar The laboratory methods to serum IgA. Total serum IgE then declines from The measurement of specific IgE recognizing the 2nd through the 8th decades of life. allergenic epitopes can be achieved both through the usage of single reagents (singleplex) or with a Very high IgE levels are observed in parasitic pre-defined panel of a number of molecules to be infestations, rarely in multiple myeloma patients tested simultaneously (multiplex).52 producing IgE, and in some primary immunodefi- ciencies (e.g., Immune dysregulation poly- In general, there are some positive and negative endocrinopathy enteropathy X-linked syndrome aspects for both in vitro allergy testing methods (IPEX), Omenn syndrome, Wiskott-Aldrich syn- that distinguish between the two different drome, Comel-Netherton syndrome, hyper-IgE techniques. syndrome and atypical complete Di George syn- drome).49 Allergic broncho-pulmonary aspergil- The structure of the assay losis (ABPA) is the only clinical condition described Since the time of the original assays,25 the to date, where the presence of high levels of IgE is assay for the detection of specific IgE has been strictly related to disease severity.50 Increased based on the classic sandwich technique with Normal Total IgE level High total IgE level Absent sIgE Non-allergic patient See non-allergic high IgE conditionsa Present sIgE Patients with sensitization(s) Patients with sensitization(s) Table 14. Relationships between Total IgE and specific IgE results. a. This condition may also occur when specific IgE to the relevant allergen are not tested in the in vitro assay, or the specific allergen is missing in the allergen panel used. Volume 13, No. 2, February 2020 Patient's Anti-IgE Enzyme Stop Reading Producer Solid phase Allergens Anti-IgE serum Labelling substrate solution system 125 RAST Sephadex or Extract 0.05 mL/ Polyclonal I none NN Gamma-counter paper sample Phadia Polymer of Extract or 0.04 mL Mouse ß- 4-metilumbelliferil- Na Photometer hydrophilic, recombinant monoclonal Galattosidase ß-D-galattoside Carbonate highly bound anti-human branched covalently to IgE cellulose the solid derivative phase enclosed in a capsule. Siemens Streptavidin- Extract or anti-IgE Alkaline 4-methoxy-4-(3- N.S. Light emission covered recombinant antibody phosphatase phosphatephenyl)- detector polystyrene allergens (mAb ? spiro-(1,2- (chemiluminescence) ball covalently to pAb?) dioxetane-3,20 - conjugated soluble adamantane) with biotinylated streptavidin- polylysine polymers. Hycor Magnetic, Extract or 0.04 mL A mixture Horseradish acridin based N.S. Light emission streptavidin- recombinant of two Peroxidase chemiluminescent detector coated mouse substrate (chemiluminescence) microparticles monoclonal incubated Anti-IgE with a biotinylated allergen Euroimmun Paper Extract or 1000 mL Alkaline Water Scanner recombinant phospatase Table 15. The most commonly used systems for specific IgE detection include the following distinct components 19 20 Ansotegui et al. World Allergy Organization Journal (2020) 13:100080 http://doi.org/10.1016/j.waojou.2019.100080 Number of Total number of Number of Producer molecular Laboratory method available allergens extract allergens component Thermofisher 566 460 106 (of which 28 N Enzyme and 78 R) immunoassay Siemens 439 413 26 (of which 20 N Chemiluminescence and 6R) Hycor 79 69 10 Chemiluminescence Euroimmun (in 316 285 31 of which 20 R and Dot blot 92 strips) 11 N) Table 16. Allergen extracts and molecular components available from different commercial sources in single-plex allergy diagnostics minor variation. The main elements are developed.55,56 In other systems, a liquid-phase represented by the solid phase, the allergen is used with coupled anti-IgE that captures free conjugated with the solid phase, the patient's serum IgE. These systems seemed not to serum, the anti-IgE antibody, the labelling of the recognize low affinity IgE, claimed to have less anti-IgE, and the substrate (if any) for the pro- relevance from a clinical point of view, and have duction of the signal. Washing and stop reagents been rarely used. They are described as research are also present. However, the specific charac- tools.27 Usually, each allergen represents a teristics of the former are fundamental for the distinct reagent, but a multi-allergen assay can results while the latter (washings and stop solu- be achieved by mixing a group of allergens into tion) are less relevant. one reagent. In addition, solid-phase reagents are sometimes supplemented with recombinant The main reagents used in the assay molecules in order to improve the extract's  The reaction site (carrying the allergen) can be: a performance (e.g., latex extract supplemented polyethylene cap with an internal sponge matrix, with rHev b 5).57 a plastic (polyethylene) or glass tube, a plastic  The nature of allergens used in the in vitro test microtiter plate well, a plastic stick, or a carbo- for specific IgE. hydrate filament-coated silicone chip. To ameliorate the antibody-binding capacity, a va- As previously mentioned, allergens can be both riety of carbohydrate-based allergo-sorbents raw extract allergens or single molecules. These (other than Sephadex and paper), such as molecules can be obtained by recombinant DNA agarose and microcrystalline cellulose, can be technology or by biochemical purification from used. The most important advance was the natural extracts (Table 16). Of note, raw extracts development of an encapsulated hydrophilic and highly purified extracts have certain post- carrier polymer to which the allergen is cova- translational modifications (such as glycosylation) lently coupled.53 that are absent in molecules produced in E.coli.  The allergen-containing reagent can be repre- There are two distinct types of molecules used in sented by a solid-phase allergo-sorbent or assays for specific IgE. The first one is represented liquid-phase conjugated allergen. This is the by the so-called “genuine” markers of exposure, most complex and highly variable component in such as Phl p 1 from timothy grass pollen or Par j 2 terms of preparation from raw material, quality from pellitory. These allergenic molecules belong control and validation,54 conferring specificity to a specific biological source and are able to not on the IgE antibody assay. For example, RAST only identify IgE sensitization, but also point to- represented a non-competitive, immuno-radio- wards the presence of the related allergenic metric assay that used allergen coupled on sources in the environment.58,59 On that basis, allergo-sorbent paper discs. Subsequently epidemiological studies became possible. The several other variants of the same assay were first large scale surveys based on the routine use Volume 13, No. 2, February 2020 21 of ISAC biochips,60,61 allowed for the monoclonal antibodies. Combinations of poly- understanding of allergic sensitization in different clonal and monoclonal (mAb) anti-human IgE geographical regions, and provided prevalence and labeled human a-FcεR170 have also been data for the development of molecule-based used to detect human (but also horse, dog, immunotherapy.62 The second group of and cat) IgE. molecules is represented by the so-called “cross-  Antibody labelling and detection methods. Anti- reactive molecules” or “pan-allergens”.63 They are human anti-sera or mAb were originally labeled families of strictly related proteins that are widely with 125I (the original Radio Allergo-Sorbent Test distributed among different species because they – RAST). Nowadays, other labelling techniques are involved in crucial cellular processes. Several using enzymes such as b-Galactosidase (b-Gal), panels of pan-allergens families are now identi- Alkaline phosphatase (AP), and Horseradish fied (e.g. Bet v 1–like molecules, lipid transfer Peroxidase (HPO) are used. A strictly related proteins, profilins, tropomyosins, parvalbumins, reagent is represented by the enzyme sub- lipocalins, serum albumins, 2S albumins, vicilins, strates: from pNPP for AP to “perox” for HPO. and 11S albumins). These panels of homologous The sensitivity of the assay is improved when molecules facilitate the diagnosis of sensitization in specific substrates (named chemiluminescent) individual patients and also enhance the accuracy are used. In this second case, a different reading of epidemiologic research. Furthermore, despite method is necessary: indeed, for substrates the high sequence identity among components of emitting in the optical range, a photometer is every single group of pan-allergens, IgE co- needed, while for chemiluminescence, a fluo- recognition of homologous molecules64 does not rometer is required. always reflect what is predicted on the basis of amino acid sequence, but rather on the  The calibration system (e.g., reference serum molecular 3D-structure.65 The use of several containing a known amount of IgE) defines the representative homologous molecules within level of IgE antibody measured by the assay every single pan-allergen group would provide creating a calibration curve. The reference curve, more information on IgE epitope recognition, as stated above, can be obtained by means of a allergen structures, and, possibly, the identification “heterologous” or “homologous” interpolation of clinical phenotypes.66 The clinical picture, in approach. In the first approach, quantitative fact, depends on which exposures determine the allergen-specific IgE antibodies are expressed in type of sensitization pattern (as previously IUA/mL, where the “A” means “allergen-specific”, mentioned) and may range from total absence of differentiating this measurement from the IU/mL symptoms, (despite the presence of IgE reactivity) utilized for the total IgE assay. In the second, the to severe, life threatening generalized measurement is indicated by arbitrary units us- reactions.67–69 ing a homologous calibration curve.27  The human sample. Both human sera and  The reaction buffer medium (salts, proteins) plasma have been used in diagnostics. In the normalizes pH and gives a protein matrix for the original assays, undiluted serum samples were analyses of interest to warrant the nonspecific used. Following the introduction of novel and binding. automated assays, the sample volume was  The control samples, containing positive serum reduced. It is evident that the concentration of controls and not containing (negative serum the human sample in the test tube determines controls) allergen-specific IgE antibody. the results. Every laboratory method has its specific serum volume and concentration cali-  The data-processing software, for managing of brated on the other reagents, such as the results and data processing. allergen amount and the anti-IgE “detection” antiserum (or monoclonal antibody) Singleplex assay  The anti-human IgE Fc detection reagents (ε The presence of allergen-specific IgE antibody heavy-chain specific) usually are polyclonal rab- in serum identifies sensitized individuals, and a bit, goat, sheep, horse, or murine anti-IgE large fraction of them can be considered allergic if 22 Ansotegui et al. World Allergy Organization Journal (2020) 13:100080 http://doi.org/10.1016/j.waojou.2019.100080 the symptoms they observe are in agreement with From a practical point of view, allergists use the the allergen(s) detected. singleplexed diagnostics in two different manners. The first is related to the results of SPT performed A number of articles have been published to try in the patient, in order to verify whether a positive to define the sensitivity and the specificity of or a negative result is confirmed by the presence in vitro IgE tests. This may prove useful for more of IgE to that allergen. Using this approach, the epidemiological reasons, if, in the future, in vivo allergist focuses her/his attention to a very select SPT are abandoned. From a scientific point of (and small) number of allergens. In recent years, view, it is a common notion that not all allergic the availability of molecular components (identi- reactions are mediated by IgE. Thus, defining the fying both genuine and cross-reacting molecules) sensitivity of an in vitro assay that is well known to has substantially improved the singleplexed strat- be able to detect only allergen specific IgE, by egy by allowing mixing whole allergen extracts comparing it with clinical diagnosis of allergy, and selected components, in order to have a clear could lead to partially wrong conclusions. The description of the IgE profile of the patient. diagnosis of allergy is a clinical diagnosis. The Nevertheless, it is evident that the allergist, by a presence of specific IgE that could better correlate singleplexed and censored choice of the allergen with the clinical picture, could be useful to better to be tested, can only detect IgE for the allergens define the allergic profile of the patient before required. Along this line, in some complex situa- starting AIT. This is particularly true for inhalant tions, for example a polysensitized patient with a allergy, while for food allergy, the picture is much large number of positive results by SPT, the list of more complex. For example, the dynamic range of allergens and components to be tested could be specific IgE to inhalant allergens is wide, while the long. same range for food IgE is significantly smaller. Another approach (more focused on primary Thus, the use of a single interval of reference care) is related to the use of allergen panels. It is values in these two different situations should not evident that panels for adults are different from be accepted in a laboratory medicine setting. panels for children, panels for a respiratory allergy Indeed, not all hormones have the same range are different from panels for a food allergy, and interval of reference values either. Although dis- panels for northern countries are different from crepancies remain, the original reference intervals panels of southern countries, etc. So, the best used for allergens never changed. In the future, a approach, should be that every allergist develops more accurate approach will be required to define her/his list of different panels suitable for her/his reference intervals, particularly in the presence of patients. This could be more difficult in primary low concentrations of IgE. In this context, if should care: for this reason, panels were developed in the be noted that a certain variability between late 1970s but at present their use is limited. A different methods (and laboratories) is expected, producer developed many different panels for as was recently observed.71,72 different ages, climates, and symptoms. However, the choice of each allergist still remains funda- There are a large number of singleplexed di- mental. Panels with a mixture of whole extract al- agnostics systems in the world, and four major lergens and molecular components could be a ones can be identified (Table 15). Each has specific very interesting method, provided that the signifi- characteristics and performances. Probably, this is cance of a positive component is known in primary not the right place to discuss differences between care. different laboratory solutions. Each has pros and cons. But in general, an IgE profile derived from The sensitivity of specific serum IgE antibody one of these four producers is acceptable. As measurements could be considered as compara- stated before, the specific characteristics of each ble to that obtained with skin prick testing for method should be well known to the allergist. So, respiratory and food allergy, but only comple- at least in follow up (in particular in the pediatric mentary to the intra-dermal skin test for drug and age), the use of different methods could venom allergy diagnosis. The accuracy and repro- sometimes generate results that could be difficult ducibility of the modern fully automated systems, to interpret and/or manage. providing reliable quantitative measures, has Volume 13, No. 2, February 2020 23 prompted investigators to assess the possible For example, the IgE profile of dust mites, such as relationship between allergen-specific IgE anti- Dermatophagoides pteronyssinus, can be well body levels in serum and the clinical risk of an detected by Der p 1, Der p 2 and Der p 10. But adverse reaction (probability-based risk assess- only with Der p 23, a fraction of originally extract- ment), especially in the field of food allergy.73–79 positive/component-negative sera becoming Higher risk of adverse reactions to food, as available, could the discrepancies between ex- assessed by the reference standard for the tracts and molecules be reduced. diagnosis of food allergy, the double-blind placebo-controlled food challenge, was As stated before, the singleplexed strategy of associated with defined diagnostic cut-off levels allergy diagnostics is strictly related to the classic of food-specific IgE antibody.74 Unfortunately “top-down” approach: the patient is seen by the conflicting results were obtained in similar allergist, SPT are performed, then if necessary, studies performed by other investigators specific IgE for a selected panel of allergens are attempting to replicate these findings,75 tested and, in a minority of situations, the study is probably because predictive values should be deepened by using allergen components, in order carefully defined for each specific population to have a very accurate picture of the IgE profile. separately.76 More recently, a totally different approach, the In the last several years, with the continued so called “bottom-up” approach, was introduced development of molecular diagnostics, it has in allergy diagnostics. Patients are originally become possible to perform singleplex assays with screened by the use of a very large panel of re- the use of recombinant or purified allergens (i.e., agents and, based on those results, the allergist not only with allergenic extracts), thus increasing then decides how many and which other compo- sensitivity, specificity, and diagnostic accuracy of nents to test to improve the diagnosis and suggest the tests.8 The choice of using diagnostic a treatment. This approach was considered icon- recombinants in singleplex instead of in oclastic by classic allergists, but it may have certain multiplex, is made on a case-by-case basis advantages, at least in a precision medicine (considering previous history and clinical profile) setting, where the best description of the patient's and in an allergen-dependent manner (i.e., situation is required for any further decision. allergen source and availability of single In certain situations the use of recombinants is recombinants). more useful and convenient, as compared with The use of whole allergens and molecular whole allergen extracts, such as in cases of multi- components has pros and cons as it does in SPT. It ple IgE sensitivities, particularly when assessing the is evident that the sensitivity of molecular compo- following: nents, when compared with that of whole aller- - potential clinical risk and severity of allergic gens, is lower. This is due to many reasons. First, manifestations; only in rare situations, a single component is so frequently positive in sensitized patients that the - presence of cross-reactivities; molecular component is representative of the - primary IgE sensitization; sensitization. For example, Par j 2 is a major allergen of Parietaria judaica, and in clinical prac- - polysensitization that is difficult to interpret. tice, the correlation between these two reagents is complete. But, for example, Phl p 1, one of the A representative example is insuring that a major components of Phleum pratense is positive complete screening includes specific IgE for Bet v in 70–80% of patients characterized by a positive 1, Bet v 2, and Bet v 4 recombinants to assess the reaction to the extract. To reach 90%, Phl p 1 type of sensitivity to birch (primary sensitization, should be associated with the other major possibility of cross-reactivities, and risk assessment component, Phl p 5. Second, not every molecular for potential allergic reactions to cross-reactive allergen is available in diagnostics. For this reason, foods). Another is the detection of sIgE for Der p at present, it is virtually impossible to describe a 10 which is associated with IgE sensitivity to the positive IgE reaction only by molecular allergen. tropomyosin protein family, increasingly the 24 Ansotegui et al. World Allergy Organization Journal (2020) 13:100080 http://doi.org/10.1016/j.waojou.2019.100080 likelihood of reactions to ingestion of crustaceans and the amount of IgE present is calculated from and mollusks due to dust mite sensitivity, even if the amount of fluorescence. With over 4000 sci- tropomyosin sensitization is primarily driven by entific articles showing its clinical value, Immuno- seafood and not mite.80 CAP at the moment is perceived as “reference standard” for in vitro IgE testing.81 However, the Qualitative, semi-quantitative, and/or quantita- presence of this antibody only proves tive IgE antibody immuno-assays are currently sensitization, not allergy, the latter being available. sensitization in the context of clinical symptoms A qualitative assay generates only negative or upon exposure to the allergen. positive results. It does not provide any measure- The “CAP inhibition” technique is a variation of ment of IgE concentration, but it is defined by the the direct CAP: allergic serum (containing sIgE) is presence of the analyte above a given positive first mixed with the soluble unknown allergen; then threshold level of the assay. If levels are close to a standard amount of the solid-phase (immobi- the cut-off point of the system, they can be lized) allergen is added. The CAP inhibition is considered as “borderline”. important to evaluate the total allergenic activity of A semi-quantitative assay produces a series of a diagnostic or therapeutic extract. Furthermore, it increasing classes (e.g., from I to VI), thus defining is still performed to distinguish multiple sensitiza- the amount of the response. These assays are not tions from cross-reactive sensitivities, in particular characterized by linearity, dilution recovery, or as a diagnostic tool in Hymenoptera venom al- parallelism of quantitative assays. lergy; however, the development of molecular- based diagnostics in allergy has significantly A quantitative assay provides the measurement reduced the need of CAP inhibition performance of IgE antibody concentration, on the basis of the for certain Authors,82 while, for others, the CAP interpolation from a multipoint calibration inhibition still remains fundamental.83–85 curve,77,78 obtained using both heterologous and/ ELISA assays can represent a valid alternative to or homologous methods. A reliable quantitative FEIAs, being relatively simple and inexpensive for assay should report results in units traceable to the assessment of serum total and specific IgE for an internationally recognized standard (e.g., various common allergens. Their usefulness is WHO 11/234).51 Unfortunately, it is not possible more greatly appreciated where ImmunoCAP is to use individual homologous calibrated IgE not available. Academic medical centers may be antibodies, for each of the hundreds of biologic able to provide in vitro allergy assays in areas sources tested. Therefore, the calibration method without ImmunoCAP.86 Various systems currently generally adopted is a heterologous interpolation employed in assessing allergen specific IgE often of specific IgE antibody from a single total serum have substantial allergen by allergen discordance IgE reference curve. Quantitative systems results especially in the lower end of the allergen are reported in gravimetric (mg/L [total serum IgE specific IgE assay range. This is likely due to assays]) or international units (kU/L [total serum variance in the allergens employed in these IgE assays] or kUA/L [allergen-specific IgE anti- systems. body assays]). Most specific IgE blood tests are immunoassays Multiplex assay that include enzyme-linked immunosorbent assays Genomic microarrays were developed and (ELISAs), fluorescent enzyme immunoassays introduced about fifteen years ago in biomedicine (FEIAs), chemiluminescent assays, or radioallergo- to monitor the expression of many genes in par- sorbent assays (RASTs). Since 2010, the National allel and to investigate differences in the level of Institute of Allergy and Infectious Diseases (NIAID) / mRNA expression from thousands of genes at the National Institutes of Health (NIH) have recom- same time, in order to gain information about mended discontinuation of the RAST as a diag- transcriptional changes involved in specific path- nostic tool for allergy in favor of more sensitive ways.87 Genomic microarrays have provided fluorescence enzyme-labeled assays, in which a insight into transcriptional gene expression of fluorescent antibody binds to the patient's sIgE, specific biologic pathways in the normal state Volume 13, No. 2, February 2020 25 and in disease.88 The proof of concept of the highly glycosylated protein cross-react with IgE possible application of a proteomic microarray specific for CCD, but also nCry j 1, nCup a 1, nCyn approach in the diagnosis of allergic sensitization d 1, nPla a 1, and nJug r 2 can be non-specifically appeared in the literature in 2002,89 followed by positive in patients sensitized to CCD. For this several other reports90–92 verifying the same reason, the real clinical significance of a positive array, widely known as the ISAC (Immuno Solid- nJug r 2 must be carefully evaluated in the context phase Allergen Chip) system. A number of other of other component results and the patient's clin- studies have been published on the development ical picture. of microarray prototypes,.93–106 Allergen microarrays were also used to evaluate Currently, IgE detection by means of microarray the presence of specific IgE in fluids different from systems is classified as an in vitro diagnostic (IVD) serum or plasma. Valenta and co-workers analyzed tool.27 A few years ago the first version of the ISAC the presence of IgE in samples of breast milk.113 microarray became commercially available. It had Leonardi et al.114 recently showed that in vernal 74 different allergenic proteins spotted on the keratoconjunctivitis (VKC), ISAC is able to detect microarray (ISAC74). The number of allergens the presence of specific IgE to grass, trees, mites, immobilized on this microarray is growing and a and animals but also food allergen-specific IgE in version with 112 allergens (ISAC112) is currently tears. What was particularly interesting was that in available. The small amount of material necessary some patients, specific IgE were absent in serum for the process of identification (from 0.1 to but detectable in tears. The presence of specific 1 ng)107 allows the use of both recombinant and IgE only in tears of VKC patients reinforces the highly purified natural molecules. concept of possible local sensitization. These two examples of the use of microarray technology for A study compared ImmunoCAP sIgE singleplex specimens different from plasma or serum opens tests and the ISAC 112 IgE multiplex assay in 101 new fields not only for research but also in clinics. patients sensitized to grass pollens. The ISAC Another interesting and innovative use of multiplex test correlated well with ImmunoCAP allergen microarray is the monitoring of Allergen singleplex results, with a positive percent agree- Immunotherapy. Indeed, it has been recently ment (PPA) and negative percent agreement (NPA) observed115 that allergen microarrays are useful to of corresponding allergens varying between 60 monitor the development of allergen-specific IgG and 100% for PPA and 78–97% for NPA.108 responses during SIT, both against the allergen Interestingly, a deeper analysis of the ISAC 112's present in the SIT vaccine as well as against cross- characteristics also demonstrated that it is a highly reactive allergens. This application of the tech- reproducible and accurate method which may be nique may finally offer a general-purpose tool for considered as a single analyte assay in the view of monitoring the immunological effects of AIT, the EN ISO 15,189 accreditation procedure.109 resulting in better control of the treatment and, This was further demonstrated in another even more, a better understanding of therapeuti- work.110 Other authors108 observed that results cally positive and negative results. This data was obtained by allergen microarray correlated well further supported by the article of Schmid JM with ImmunoCAP singleplexed results. In et al.116 that demonstrated that pre-treatment addition, technical improvement seems to allergen component-specific IgE appears to ameliorate some of the less reproducible determine the induction of IgG4 during the properties of the allergen microarray. Monroe updosing phase. Induced IgG4 seems to suppress et al.111 showed that their use of a calibrated IgE levels on ISAC, resulting in a marked decrease fluorescence enhancement (CaFE) technique in ISAC-measured specific IgE levels after updos- seems to more accurately measure probe density ing of SCIT. They conclude that the decrease in and bound target for a variety of antibody- ISAC IgE levels can be used to monitor the antigen pairs. blocking effect of allergen-specific immuno- therapy-induced non-IgE antibodies. At the same time, some at least partially unex- pected cross-reactions between components of A number of studies have been performed in ISAC were identified.112 Not only can nPhl p4, a order to define the conditions where allergen 26 Ansotegui et al. World Allergy Organization Journal (2020) 13:100080 http://doi.org/10.1016/j.waojou.2019.100080 microarray can or cannot be useful in different aureus toxins, and Maltose binding protein clinical pictures. For example, D'Amelio et al.117 (MBP). The clinical features of the MeDALL argued whether the performance of ImmunoCAP microarray were further evaluated during the so- ISAC 112 is sufficient to diagnose peach and called “allergen march”, from childhood to apple allergies. They concluded that although the adolescence.124,125 The prevalence of allergic sensitivity of the peach components in ISAC is sensitization increased in all three diagnostic improvable, it can be sufficient in their region.115 tests from age 10–16 years. It was similar by SPT The same authors118 concluded that the and ImmunoCAP but significantly higher with the diagnostic performance of ISAC was adequate MeDALL-chip at 10 years. All three tests were for hazelnut and walnut allergy but not for comparable for identification of allergic sensitiza- peanut allergy. Finally, in another different tion among children with current rhinitis or situation,119 even if the standard ImmunoCAP asthma.125 has, for apple and peach, a wider number of A different approach has been developed by an available components (in particular Mad d 3, a English/Swedish company that designed and lipid transfer protein [LTP]), the evaluation of Pru further implemented a microarray where not only p 3 (largely homologous to Mal d 3) may support single allergen components but also extractive the identification of an apple sensitivity even if whole components were spotted.126 This “the presence of sIgE against Pru p 3 in LTP combination of extractive allergens and sensitized patients can be due to cross-reactivity recombinant components was tested with other and should therefore not be used to predict clin- three allergy test methods (SPT, ImmunoCAP, ical symptoms”. and ISAC 112) and a total of 3485 pairwise test On the contrary, in idiopathic anaphylaxis120,121 results were analyzed and compared. The four the ISAC array contributed to the diagnosis in 20% methods showed comparable results with a of patients and may offer additional information positive/negative agreement of 81–88% for any where a careful allergy history and follow-on pair of test methods compared, which is in line testing have not revealed the cause of the with data in the literature. The most prevalent anaphylaxis. allergens (cat, dog, mite, timothy, birch and peanut) and their individual allergen components Microarray technology is progressively showed agreement between methods with improving, as shown by the MeDALL-chip which correlation coefficients between 0.73 and 0.95. was developed within the MeDALL European All four methods revealed deviating individual project (Mechanisms of the Development of AL- patient results for a minority of patients. These Lergy) and is based on 170 relevant allergens. The results indicate that microarray platforms are MeDALL chip provided new information for some efficient and useful tools to characterize the allergens and seemed to be more sensitive in specific IgE profile of allergic patients using a detecting allergic sensitization than ImmunoCAP small volume of serum sample. The results sIgE or SPT.119,122 produced by the Microtest system were in agreement with diagnostic tests in current use. In the period when ISAC 103 and ISAC 112 were used in clinics, other different attempts to modify More recently, a different tool, termed ALEX (as the strategy of multiplexed molecular diagnostics in ALlergen EXplorer) was developed by Macro- were developed. The main attempt was related to ArrayDX in Vienna (Austria). The ALEX test is per- the works of the MeDALL group that, in strict formed using an array of allergens spotted on a cooperation with industry, developed a novel solid phase by way of nano-particles. ALEX con- microarray by adding more than 70 new compo- tains 282 reagents (157 allergen extracts and 125 nents to the standard panel of ISAC 112.123 These recombinant or highly purified molecules). So, in new components were comprised of: Peanut this chip, second level diagnostics (represented by allergens, Almond, cashew and pistachio extract allergens) and third level diagnostics (rep- allergens, Cow's milk allergens, Wheat allergens, resented by single molecules) are all available. Olive pollen allergens, Mite allergens, Dog ALEX has been evaluated and the quality of ALEX allergens, Insect venom allergens, Staphylococcus results has been described.127 In consideration of Volume 13, No. 2, February 2020 27 the very large number of allergens and to thirty different micro-beads can be carried out components and the significant complexity of the just using the Fluorescence-Activated Cell Sorting interpretation of the results (at least for non- (FACS) facilities. The opportunity of multiple fluo- professional molecular allergists), ALEX has been rescence parameters evaluation could also permit linked to a new version of the expert system, the measurement of distinct antibody isotypes Allergenius, originally developed for the interpre- involved in antigen recognition in the same tation of ISAC results.128 In its present form, ALEX sample.136 seems to be a good reagent for the “bottom-up” Some criticisms have arisen against the multi- strategy of allergy diagnostics. plex approach, including poor flexibility in the list Another group of tests are represented by the of allergens and the possible lack of relevant al- multiple allergen simultaneous tests (MAST)- lergens. The allergist is forced to test the entire set immunoblot, such as EUROLINE, which is a of molecules in the commercial product, and it is commercially available assay for component not possible to test a “patient-tailored” set of resolved allergy diagnostics based on the immu- molecules. Despite this, the allergen array based noblot technique. One “line blot” consists of approach is in line with the modern concept of several membrane chips containing allergen “precision medicine”. However, this approach may components coated in single lines. The membrane sometimes generate more questions than answers chips principle allows for optimized coating con- and complicate the management of, and the rec- ditions of the respective components. Due to the ommendations for, a specific patient.137 A recent simultaneous determination of sIgE to different paper compared the classic top-down approach allergen components, a sensitization profile can be (represented by the visit of the patient, then SPT, generated in a single test run using low amounts of then — if considered useful — by specific IgE to serum. This technique allows the detection of whole extract allergens and/or single components specific IgE quickly and efficiently with the use of and, finally, in a very select number of cases, the immunoblot strips containing optimized combi- use of multiplexed IgE detection assays) and the nations of relevant allergen components. Each so-called “bottom-up” strategy, represented by the profile is tailored to a specific indication: several immediate analysis of the patient's IgE profile (the indication-specific profiles for molecular allergy patient's phenotype) followed by in vitro and diagnostics are already available for inhalant al- in vivo tests useful to better describe the patient's lergens, insect venoms, and food allergens. When endotype and how they can relate to therapeutic compared to ImmunoCAP, substantial agreement management.138 between MAST and ImmunoCAP was found for inhalant, food, and venom allergens thus repre- Informatics senting a valid diagnostic alternative.129 The introduction of Expert System technologies A flow cytometric bead array (CBA) has been to support Molecule Based Allergy Diagnostics developed to detect soluble factors.130,131 Naked (MBAD) has also led to the introduction of new fluorescent micro-beads (Flex Set), can also be concepts to the diagnostic approach.128 Indeed, customized to detect specific antibodies,132,133 ISAC seems to be redundant to some extent: for but not many reports on this specific application example, the number of profilins and LTPs are found in the literature.134 CBA Flex Set, measured seem to be higher than needed. unlike other micro-bead-based systems,135 is Nevertheless, it has been recently observed that designed for flow cytometry, an instrument a hierarchy of cross-reacting components can be widely used in almost all routine or research identified using large-scale MBAD assays.139 It was laboratories. The feasibility of the CBA Flex Set shown that IgE reactivity of PR-10 proteins is for specific IgE detection in human sera using characterized by a hierarchical interrelationship: allergenic molecules coupled on the fluorescent Bet v 1 > Mal d 1 > Cor a 1.04 > Ara h 8 > Pru p micro-beads, thus obtaining a “flexible” micro- 1 > Aln g 1 > Api g 1 > Act d 8 > Gly m 4. For this array for testing IgE, has been demonstrated.136 reason, it is evident that having many cross- The system was named Allergen Bead Array reacting components indicates a lot more than (ABA), and the simultaneous measurement of up having only a few. Along this line, the rule is that if 28 Ansotegui et al. World Allergy Organization Journal (2020) 13:100080 http://doi.org/10.1016/j.waojou.2019.100080 the number of positive components is >40% of the strategy used in ALEX, where both extracts and total number of components of a given family, the single components are assayed in the same patient can be considered sensitized to the whole allergen array. family of cross-reacting molecules.128 In addition, A different approach that also works was used the first sensitizer in the member of the family of by Prosperi et al. By using the techniques of ma- cross-reacting components can be identified as chine learning, they studied how microarray results the one with the highest IgE score. Other added can be applied to allergic diseases. A reasonable values can be derived from these rules: for discrimination ability for asthma, rhino- example, if a discrepancy is identified between the conjunctivitis, wheeze and airway hyper-reactivity, results of SPT (or sIgE) of a certain extract of but not for eczema, starting from ISAC phenotyp- allergen and the ISAC results (namely, a positive ing, was observed142 SPT result with negative specific components derived from that allergen), an expert system can evaluate whether other cross-reacting components Interpretation of multiplexed in vitro allergy assays. (belonging to other allergen sources but cross- The interpretation of the results of a 112- reacting with components well known to be allergen assay, and even more a 282 allergen detectable in the whole allergen) are also positive. assay, may be challenging, even for the experi- So, for example, if Ambrosia is positive in SPT but enced and trained allergen-array user. Some Amb a 1 is negative, other cross reacting compo- points should be carefully considered. nents need evaluation. Profilins, PR-10, and CBP  Detection of sIgE is indicative of a sensitization, are all well represented in Ambrosia but are not and not of an allergy. Thus, a positive sIgE present in ISAC. If at least one of these cross- response in the absence of a history of allergic reacting components is positive, then a discrep- symptoms or a negative provocation should be ancy is not considered. On the contrary, if all the referred to as clinically irrelevant.143 possible cross-reacting components are negative, a clear discrepancy is evident. By using this  Clear differences have been described144 approach, the number of apparent discrepancies between allergen extracts and molecular is reduced significanly. Another added value is components (revised in Table 13). Sometimes related to the capacity of an expert system to these discrepancies are quantitative, and sIgE evaluate the sum of the score of genuine inhalants levels to the allergen extract are lower than for and the sum of the score of inhalant components the individual allergens when components are belonging to cross-reacting families. It has been in low abundance in the extract. In cases when demostrated that patients sensitized to genuine sIgE to allergen extract are positive but its components are able to better respond to AIT than genuine components are negative, sensitization people sensitized to cross-reacting compo- to minor allergenic molecules or CCD nents.43,140 In addition, by the use of applying determinants responsible for cross-reactivity cluster analysis techniques to data from should be ruled out. Of note, the recent sug- thousands of ISAC patients,141 it has been clearly gestion to introduce an inhibitor of CCD in the shown that there are five different clusters of sample diluent37 in order to reduce the signal patients: Groups I and II (characterized by a related to reactivity to CCD is routinely used in sensitization to a large fraction of genuine the novel allergen array ALEX.127 This seems to components) are the only optimal targets for AIT, be particularly relevant when highly purified while Groups III and IV have a worse expectation components derived from natural extracts — of success. Group V relates to food allergies. and, for this reason, characterized by the Starting from this evidence, a new era presence of a post translational glycosylation — characterized by the integration between artificial are used. For example, in ImmunoCAP ISAC, intelligence tools and MBAD seems ready to Walnut nJug r 2, Bermuda grass nCyn d 1, begin. Of note, many of these problems (in Timothy grass nPhl p 4, Japanese cedar nCry j particular discrepancies between the result of the 1, Arizona cypress nCup a 1, and Plane nPla a allergen extract and those of relevant 2 may be positive for their content of glycidic components) can be easily overcome by the chains This point was also noted in a AIT Volume 13, No. 2, February 2020 29 algorithm40 that suggested a specific strategy to symptoms.148 According to this, the assessment follow before administering AIT for pollens in of IgE sensitization to the three key allergens — Mediterranean regions. Bet v 1 homologues, LTPs and profilins — is of paramount importance for the interpretation of  This introduces another relevant point, repre- molecular diagnosis to fruits and vegetables, sented by geographical differences in sensitiza- especially in the Mediterranean area.149 tion. For example, Ole e 1 would be a marker of genuine sensitization to olive pollen in the South  An advantage of multiplex analysis is also one of Spain but a marker of genuine sensitization to its main pitfalls: the generation of an extensive ash tree in Northern France. An example of a IgE sensitization profile, detecting IgE to unex- geographic distribution of sensitization has been pected allergens, which may sometimes induce recently published145 confusion in the clinician if there is no suggestive pre-test clinical history. That is often the case in  Differences in high or low risk markers and insect venom allergy. Due to the high prevalence component combinations create differences in of insect venom sensitization in approximately the interpretation of risk phenotypes. Generally, 25% of the population, nonspecific screening allergens resistant to heat and digestion, like would generate an abundance of clinically irrel- seed-storage proteins or lipid transfer proteins, evant results and mainly serve to unsettle pa- often trigger more severe allergic reactions and tients and their physicians.150 No have been proposed as markers for severe re- recommendations are currently available on actions. Again, the specific relevance of each of how to effectively manage these cases,137 but these markers of severity will vary according to it seems reasonable to act in the same way as local molecular profiles (e.g. in USA and Northern with other clinically irrelevant sensitizations to Europe Ara h 2 is the best predictive marker for food or respiratory allergens that don't need severe reactions to peanut, while in the Mediter- any intervention other than to follow the ranean area it is Ara h 9). On the other hand, Bet v- patient to detect possible future reactions. On 1 homologues and profilins are labile allergens, the other hand, the detection of silent which typically induce local symptoms such as sensitivities may give the allergist the chance to oral allergy syndrome (OAS) and have been pro- investigate other hypersensitivities and to alert posed as markers of mild reactions. the patient towards possible risks.151 In the  However, the clinician needs to be aware that case of sensitization to allergens responsible there may be exceptions to this rule in situations for food-pollen cross-reactive syndromes, the when large quantities of allergens are clinician should re-interrogate the patient for consumed, cofactors are associated, or in re- symptoms upon consumption of foods contain- gions with large quantities of pollen exposure. ing those allergens. However, sensitization itself Examples of this are severe anaphylactic re- should not drive avoidance measures.152 It is actions reported in patients mono-sensitized to important to state that according to current Bet v1 homologues when drinking apple juice guidelines the indication of an elimination diet after performing exercise,146 or severe reactions should be recommended only if food allergy in patients mono-sensitized to profilin in areas due to cross-reactions is based on a clear his- with over-exposure to grass pollen.147 In tory or on a clinical observation after oral prov- addition to considering the individual markers ocation tests.153 of severe allergic reactions, component  A further approach can be based on some sim- combinations can define phenotypes with ple rules from proteomics. It has been defined different clinical expression. It has been that a cross-reaction may occur if the homology recently reported that, in an Italian cohort, between two or more molecular allergens is sensitization to more than 5 nsLTPs out of the 8 >70%. This is a very simple rule that can be easily present in ImmunoCAP ISAC Ò is related to a verified by comparing the sequences of two higher incidence of food-induced systemic re- components which are suspected to be related. actions, while co-sensitization to PR-10 or profilin It is evident that components belonging to the pan-allergens is associated with milder same family (such as PR-10) are highly 30 Ansotegui et al. World Allergy Organization Journal (2020) 13:100080 http://doi.org/10.1016/j.waojou.2019.100080 homogeneous, such as Act c 8 (Green Kiwi), Ara studies of sIgE/FcεRI-independent effector cell h 8 (Peanut), Aspa o 17kD (Asparagus officinalis), activation, such as by off-target occupancy of the Cas s 1 (Chestnut), Fag s 1 (Fagus sylvatica), Fic c MRGPRX2 receptors,156 as basophils, unlike 1 (Ficus carica), Jug r 5 (Walnut), Mor a 1 (White cutaneous mast cells, barely express this Mulberry), Ost c 1 (Ostrya carpinifolia), Rub i 1 receptor.157 (Rubus idaeus), Sol a l 4 (Tomato), Tri fg 4 (Trigonella foenum-graecum), Vig r 1 (Vigna Over the last two decades several important radiata), etc. In these cases, the presence of advances have been achieved, announcing the cross reactions always should be considered. BAT as an increasingly attractive in vitro diagnostic Other situations can be different. For example, in tool, to be applied in selected contexts. However, the presence of a weak positivity to Ambrosia a., some methodological aspects need to be taken the presence of a strong IgE reaction to Arte- into consideration in order to interpret the data misia v. should be verified, because of the correctly: presence of a Amb a 1 like protein in Artemsia. o The BAT is a flow-cytometric based assay: Finally, a well known exception is represented by therefore, the investigator must be trained in LTP from Par j 2, that when compared with Pru p flow-cytometric techniques, and proper equip- 3 (a prototypic nsLTP) shares a 26% identity and ment is required. The principles of the BAT and a 52% positivity, while Pru p 3 compared with HistaFlow technique, that studies intracellular Mal d 3 (another nsLTP) has an identity ¼ 77% histamine content, are summarized and positivity ¼ 83%, clearly justifying the pres- elsewhere.155,158,159 ence of a cross reaction. o There are several surface-marker combinations, Cellular assays: basophil activation test which allow the correct identification of baso- phils. They include the combinations CCR3þ/ Cellular assays assess selected and defined CD3-, or CD123þ/HLA-DR-, or IgEþ/CD203cþ. functions of effector cells within the allergic The only lineage-specific basophil marker is cascade, and therefore play an increasing role CD203c. The precise identification of the popu- within in vitro allergy diagnostic tests. This is lation of basophils is a prerequisite for a valid particularly true in case of equivocal and/or interpretation of test results. negative results obtained with other in vitro and in vivo tests, and in case of discrepant results. In o In a second step, the appearance and/or up- this context, the basophil activation test (BAT) has regulation of the desired activation/degranula- gained increasing interest within the scientific tion marker is investigated. CD63 or (lysosomal- community and supplanted traditional histamine associated membrane protein [LAMP-3]), is a release assays.154 degranulation marker that appears during compounded degranulation of the cell. CD203c, General aspects or ectonucleotide pyrophosphatase/phospho- diesterase 3 (ENPP-3), is not only a lineage Basophils, like mast cells, are recognized as specific marker of basophils, but it also serves as important effector cells in immediate hypersensi- an activation marker. In the resting basophil, the tivity responses. Basophils express the high-affinity expression of CD203c is low, and activation re- IgE receptor (FcεRI), and thus they carry specific sults in a rapid and marked increase in CD203c. IgE (sIgE) antibodies on their surface and degra- Other activation/degranulation markers can also nulate when the allergen cross-links these sIgE/ be used, amongst them analysis of the intracel- FcεRI complexes. This degranulation of basophils lular histamine content using an enzyme affinity can be detected and quantified by flow cytometry method (HistaFlow).159 Fig. 1 illustrates the techniques.155 Since mast cells, a tissue-resident principle of the BAT/HistaFlow. cell also expressing FcεRI, are not accessible for in vitro diagnostic tests, the basophil represents a o Although commercial basophil activation assays unique alternative to study sIgE/FcεRI-dependent are available, these are rarely thoroughly vali- degranulation. Moreover, comparative analyses dated and require additional investigation between mast cells and basophils might benefit before they can enter mainstream application. Volume 13, No. 2, February 2020 31 Fig. 1 The principle of the BAT/HistaFlow o Standardization and harmonization of the BAT experiments, spanning different stimulation techniques and interpretation of outcome values concentrations and receiver operating charac- and results are still lacking. This is particularly teristic (ROC) analysis are required to calculate important in the definition of decision cut-off allergen-specific cut-offs. Obviously, viability values. Until international agreement and stan- and cytotoxicity studies are mandatory to dardization has been achieved, each laboratory exclude false-negative results. should establish its own cut-off values. The best o Pre-analytical considerations are of utmost current available definition of a positive test importance, in order to obtain valid test results. result is based on the frequency of activated In this regard, the BAT is best performed not basophils following stimulation. For proteina- later than 6–12 months after clinical reaction. ceous allergens, a test result of at least 15% However, BAT can stay positive over years, even activated basophils is generally considered as a for drugs. reliable cut-off value, eventually together with a stimulation index (compared to the negative o It has been demonstrated that antihistamines do control) of at least 2. For drugs and small NOT influence the outcome in BAT.160 chemical substances, which are known to be less Therefore, unlike for skin and provocation tests, potent stimulators, lower decision thresholds these drugs should not be discontinued. frequently apply. Anyhow, it is recommended to o A key problem still remains, the so-called abandon arbitrarily chosen decision thresholds. “anergic”, that is, non-responders in the BAT. As a matter of fact, broad dose-finding 32 Ansotegui et al. World Allergy Organization Journal (2020) 13:100080 http://doi.org/10.1016/j.waojou.2019.100080 About 10% of donors present “anergic” baso- higher diagnostic accuracy for the BAT (sensitivity phils. A paper published in 2017 showed how 86%, specificity 88%). inactivation of basophils (“basophil anergy”) These data have been confirmed in a study, seems to be associated with a down regulation where basophil activation was statistically of basophil Syk and an apparent reduction in the correlated with double-blind placebo-controlled incidence of allergic rhinitis.161 For this, anti-IgE food challenge (DBPCFC) outcomes and can be used as a positive control in BAT to severity scores, and compared to SPT and identify non-responders. serum sIgE.166 o Of great advantage, particularly in comparison Furthermore, the BAT assay has been investi- to in vitro IgE antibody diagnostic tests, is the gated and correlated with the degree of cow's milk fact that the BAT assesses IgE-dependent as well tolerance in 132 subjects divided into 3 groups: as IgE-independent mechanisms. Activation of baked-milk-reactive, baked-milk-tolerant or the basophils is not only obtained through IgE- “outgrown milk allergy”, based on the oral food dependent signal transduction pathways, but it challenge outcomes. BAT showed a significantly can also be the consequence of a non-IgE higher diagnostic accuracy compared to specific- mediated reaction. In addition, as already IgE measurements (sensitivity 86%, specificity exemplified, comparisons between mast cell 88%). The basophil reactivity was significantly and basophil activation can benefit identification higher in baked-milk-reactive patients rather than and investigation of alternative activation path- in baked-milk-tolerant subjects (P < 0.01), and ways, such as occupancy of the MRGPRX- statistically higher in baked-milk-tolerant in- receptor. dividuals than in those who have overcome their allergy (P < 0.05).167 BAT in food allergy Similar results were obtained with other foods, The application of BAT in food allergy has to be such as egg and tree nut allergens. A study group seen in the overall context of molecular di- compared the basophil allergen threshold sensi- agnostics.162 A major issue that is not addressed tivity (CD-sens) to peanut allergen-specific IgE here is the allergen source. BAT might antibody levels in relation to the DBPCFC significantly differ according to the variety outcome. BATs were performed with both peanut employed (e.g. it has been proven for extract and purified rAra h 2: they showed positive peanut).163 Nevertheless, BAT can benefit results in 92% of the samples, in agreement with a diagnosis in difficult cases despite not very positive DBPC food challenge. Negative outcome reliable in plant-derived food allergy. in CD-sens was associated with negative results in DBPCFC.168–170 Cow's milk allergy is one of the most common food allergies in early childhood. In a study by Although these data must be confirmed by Rubio et al.164 several diagnostic methods were additional studies, this initial set of results indicates compared in order to assess the diagnostic value that at least for certain food allergens and in for each. The comparison of skin test, anti-cow's certain populations, the BAT plays a significant role milk IgE antibody measurement, and BAT in establishing the diagnosis. revealed that BAT yielded the highest specificity (90%) and sensitivity (91%), the highest positive BAT in drug allergy predictive value with 81%, and the highest Needless to say, one of the major applications negative predictive value with 96%. of BAT is immediate drug hypersensitivity, as (in contrast to food allergy) there is no need for diffi- In another study165 food allergy was assessed in cult extract preparation, and other in vitro tests are a cohort of 120 patients with suspected irritable frequently lacking and skin tests still associated bowel disease. The presence of food allergy was with considerable uncertainty. Moreover, it has established by double-blind, placebo-controlled been shown that drug challenges based on skin food challenges. The comparison of specific IgE testing are not absolutely predictive to give a measurements and BAT revealed a significantly green light for safe subsequent re-exposure.171 Volume 13, No. 2, February 2020 33 To date, as reviewed in Mangodt et al and Ebo systemic reaction, especially with inhalation et al,172,173 BAT has mainly been studied in challenges. immediate hypersensitivity drug reactions due to neuromuscular blocking agents (NMBAs), As such, these challenges should be standard- antibiotics (b-lactams and fluoroquinolones), and ized to some extent, in order to control environ- iodinated contrast media (ICM). BAT sensitivity mental conditions (e.g., temperature, humidity), generally ranges between 50% and 60%, and and can be performed repeatedly in selected in- specificity is 80%, except for fluoroquinolones dividual patients. However, an international stan- where lower sensitivity of the BAT probably dardization of protocols for these provocation tests reflects an alternative mechanistic endotype.157 has been identified as an important unmet Presently, BAT appears to be the only reliable need,180 which has been followed by the technique to diagnose IgE-mediated opiate European Academy of Allergy and Clinical hypersensitivity.174,175 Immunology (EAACI) in a Position Paper on “standardization of nasal allergen challenges“181 and “conjunctival allergen provocation test: guidelines for daily practice“.182 In the “Guideline BAT in venom allergy on the clinical development of products for The potential and limitations of BAT in the specific immunotherapy for the treatment of diagnosis of venom allergy should be viewed in allergic diseases” the European Medicines the context of recent developments in molecular Agency (EMA) has stated that in phase II “dose diagnostic tests that have greatly improved diag- finding trials on allergen immunotherapy (AIT), nosis in difficult cases,176,177 mainly patients provocation tests may be used as primary demonstrating double positive sIgE results and at endpoints”. Besides that, they can be used in risk for inappropriate venom immunotherapy. In pharmacodynamic trials or in Phase III (pivotal) addition, BAT can benefit diagnosis in patients trials to support proof of efficacy.183 with negative results. BAT can be performed using native extracts but also with recombinant Allergen Exposure Chambers (AECs) have the venom proteins that are increasingly advantage of mimicking real-life allergen expo- characterized and cloned. Another application of sures by challenging not only single target organs BAT in venom allergy is monitoring of treatment. but the entire patient. In particular the conjunctival From these follow-up studies it emerges that it is mucosa as well as upper and lower airways are important to distinguish between basophil sensi- exposed simultaneously. The number of technically tivity and reactivity, as the effect of venom immu- validated AEC facilities has increased during the notherapy on basophils is mainly seen when the past two decades, and they are frequently used in cells are stimulated with suboptimal clinical trials.183,184 Through the 2016 revision of concentrations.178 the “Allergic Rhinitis: Developing Drug Products for Treatment Guidance for Industry“ document, the Center for Drug Evaluation and Research (CDER) of the U.S. Department of Health and ALLERGEN PROVOCATION TEST Human Services Food and Drug Administration Allergen extracts can be applied to the (FDA) underlines the role of AECs as useful tools conjunctival, bronchial or nasal mucosa in order to in clinically assessing allergic rhinitis.185 In line provoke symptoms and to clinically demonstrate with this, the EMA acknowledges AEC facilities as relevance of IgE-mediated sensitization (as identi- a promising tool for the evaluation of efficacy in fied by skin-testing or by in vitro parameters) in AIT but calls for further clinical validation.183 patients with allergic rhinoconjunctivitis and/or Therefore, a published EAACI Position paper allergic asthma.179,180 Allergen provocation tests overviewed current concepts and unmet needs in are useful in confirming the diagnosis of AECs aimed to enhance progress towards a underlying allergic disease, if by clinical history, broader use of these facilities in the future.186 skin tests and specific IgE determinations are not Taken together, allergen challenge tests can be conclusive. There is, however, the potential for a ideally used to demonstrate the clinical relevance 34 Ansotegui et al. World Allergy Organization Journal (2020) 13:100080 http://doi.org/10.1016/j.waojou.2019.100080 of inhalant allergens and play a central role in the (likelihood of allergic reaction) and to the time diagnostic methods for allergenic diseases. How- elapsed between the last reaction and testing, ever, a thorough standardization of these proced- since the sensitivity of the latter decreases pro- ures as well as further validation is needed. Even gressively with time.189–191 though the number of AECs world-wide is still limited, these facilities may play an important role Skin testing for drug allergy as diagnostic and therapy-monitoring tools in the A variety of factors need consideration for future.185 performing and interpreting drug allergy skin testing. Positive and negative control tests should SPECIAL CASES IN ALLERGY DIAGNOSIS be performed simultaneously, usually with hista- mine and normal saline respectively. Skin testing IgE testing in drug allergy with drugs should be performed in a setting with IgE-mediated drug allergy accounts for only a sufficient resources to facilitate management of limited portion of immune-mediated adverse drug systemic reactions. Patients may sign an informed reactions. Even when the clinical picture is consis- consent document, which might be mandatory in tent with an immediate drug reaction suggesting some countries. Tests should be started by SPTs, the involvement of IgE, the sensitivity of skin prick/ and if these are negative, followed by IDT starting intradermal testing and serum specific IgE assays with the lowest concentrations, until a positive remains low for many drug groups. The reasons reaction is obtained or until the maximum con- include the following:187 centration is reached. Whenever the test is per- formed with a drug for which the appropriate  Drugs seldom are complete antigens, but concentrations have not been validated, it may be frequently act as haptens which require protein necessary to test control subjects, such as the binding to become a complete antigen, in this physician and clinical staff, to rule out non-specific case an “allergen” with IgE-binding capability. responses.  Some drugs trigger allergic reactions through In b-lactam skin testing, it is recommended to their metabolites. use the major and minor determinants of benzyl-  Some immediate reactions may not be IgE- penicillin, as well as the culprit drug and possibly mediated. other b-lactams, to ascertain any cross-reac- tivity.189 Benzylpenicilloyl-poly-L-lysine (PPL, Pre- All these factors contribute to challenges in penÔ, AllerQuest LLC, Plainville, CT, USA) and consistently reproducing the pathogenic mecha- benzylpenicilloyl-octa-L-lysine (BP-OL, DAPÔ, Dia- nism(s) involved in allergic drug reactions.188 ter, Leganes, Spain) are the major determinants in use today, while the minor determinants are ben- Taking into account these limitations, skin zylpenicillin and sodium benzylpenilloate (DAPÔ). testing with suspected drugs is still a widely used However, they are not commercially available in all first-line procedure to assess the possibility of a countries. The sensitivity of skin testing with b-lac- drug allergy. One of the major problems is that tams differs among studies; it can reach 80% in concentration ranges allowing for an acceptable immediate reactions (i.e., occurring within 1 hour sensitivity but lacking an irritant effect have only of the last drug administration).187 For most been reported for a limited number of drugs, such antibiotics other than b-lactams, the value of skin as b-lactams, neuromuscular blocking agents tests appears to be less clear or not yet (NMBAs), platinum salts, iodinated contrast media, validated. SPT and IDT with undiluted and heparins (see Table 8). Others have been intravenous quinolones are irritative. Higher published only as isolated case reports or small dilutions have to be used in order to avoid these case series. irritating results. Recommended highest non- The positive and negative predictive value of irritative dilutions in the literature vary greatly. In skin testing with drugs or specific-IgE determina- any case, the sensitivity of skin testing with quino- tion varies, according to each specific drug, to the lones appears to be low and recommendations on selection criteria of the patients to be tested concentrations are currently not possible. In Volume 13, No. 2, February 2020 35 immediate hypersensitivity reactions to macro- that only 7.8–36% of suspected hypersensitivity lides, positive skin tests appear to be rare. In some (both immediate and delayed) to NBLAs could be studies, undiluted concentrations of i.v. macrolides confirmed by skin and/or provocation tests, hence have been reported to be irritating in the SPT. the need of a common guideline on a standard- Table 17 summarizes the concentrations ized diagnostic approach in the pediatric recommended for the drugs most frequently population.192 involved in IgE-mediated reactions. For neuromuscular blocking agents (NMBAs), In pediatric patients, the definitive diagnosis of skin tests are generally considered to have a high b-lactam and non b-lactam antibiotics (NBLAs) hy- sensitivity. Cross-reactivity is reported with NMBAs persensitivity frequently depends on drug provo- in up to 60–70% of cases,193 even if it is possible cation tests. Studies including children showed that different results are observed. Drug SPT IDT Reference b-lactam antibiotics 189,190 Amoxicillin 20 mg/ml 20 mg/ml 190 Ampicillin 20 mg/ml 20 mg/ml Penicillin G 10,000 UI/ml 10,000 UI/ml Cephalosporins 2–20 mg/ml 20 mg/ml230 Aztreonam 2 mg/ml 2 mg/ml 190 Imipenem/cilastin 0.5 mg/ml each 0.5 mg/ml each Meropenem 1 mg/ml 1 mg/ml 190 Neuromuscular blocking agents (NMBAs) Atracurium 1 mg/ml 0.01 mg/ml Cisacurium 2 mg/ml 0.02 mg/ml Mivacurium 0.2 mg/ml 0.002 mg/ml Rocuronium 10 mg/ml 0.05 mg/ml Vecuronium 4 mg/ml 0.4 mg/ml Pancuronium 2 mg/ml 0.2 mg/ml Suxamethonium 10 mg/ml 0.1 mg/ml 190 Platinum salts Carboplatin 10 mg/ml 1 mg/ml Oxaliplatin 1 mg/ml 0.1 mg/ml Cisplatin 1 mg/ml 0.1 mg/ml 190 Iodinated contrast media Undiluted 1/10 in normal saline Heparins Undiluted 1/10 in normal saline Pyrazolones (Metamizol) and other NSAIDs Undiluted 0.1 mg/ml Table 17. Highest drug concentrations recommended for skin testing. *Adapted from K. Brockow et al.: Skin test concentrations for systemically administered drugs - an ENDA/EAACI Drug Allergy Interest Group position paper.231 36 Ansotegui et al. World Allergy Organization Journal (2020) 13:100080 http://doi.org/10.1016/j.waojou.2019.100080 If a skin test with the NMBA to which the patient a cohort of 37 patients with suspected hypersen- was exposed is positive, the remaining available sitivity reactions to ICM. Only one subject experi- NMBAs should be tested, in order to rule out enced a mild adverse reaction. Further studies cross-sensitizations and identify safe alternatives. however are needed to confirm the safety and NMBAs can induce a non-specific direct histamine predictive value of this procedure.198 release in the skin, increasing the risk of false positive tests, especially in intradermal testing.194 Heparin preparations should be used in a 1:10 There remains the possibility of low specificity dilution for intradermal tests. Immediate-type in- based upon exposure and sensitization to tradermal-test positivity may be observed in up to common products (cosmetics and cough 10% using this dilution. Lower concentrations medicine) which lead to false positive results.195 decrease the sensitivity of these tests.199 Hagau and colleagues demonstrated that sub- For non-steroidal anti-inflammatory drugs jects with a history of antibiotic hypersensitivity (NSAIDs), the sensitivity of skin testing is low except reactions seem to have an increased incidence of for pyrazolones.200 In fact, the vast majority of positive skin tests for NMBAs, in particular atracu- immediate hypersensitivity reactions (e.g., rium (P ¼ 0.02). These data suggest that there is a urticaria, bronchospasm, and anaphylaxis) to subset of patients that might be at higher risk for NSAIDs, excluding pyrazolones, are not due to an developing intraoperative anaphylaxis after un- IgE-mediated mechanism, but are related to an dergoing general anesthesia, compared to the aberrant arachidonic acid metabolism. In immedi- general population.196 With regard to platinum ate hypersensitivity reactions to pyrazolones, posi- salts (carboplatin, oxaliplatin, and cisplatin), there tive skin tests may occur in up to 40% of patients. is sufficient information to recommend the use of Nevertheless, some cases have been published for undiluted drugs for SPT and a tenfold dilution for NSAIDs other than pyrazolones, in which skin tests IDT.197 have rendered positive results (Table 17). Although immediate corticosteroid allergy is infrequent, skin Skin testing with iodinated contrast media (ICM) tests may be useful for diagnosis. However, skin is suggested in patients who have previously testing or sIgE is not generally recognized of value experienced contrast-related reactions. On the for ICM or NSAIDs allergies in the USA. contrary, there are no indications in non-ICM- exposed and/or non-previous-reactors for testing as the predictability of these skin tests has not In vitro testing for drug-specific IgE been confirmed. In those with positive reactions, The possibility of proving the existence of serum testing a panel of different compounds is recom- specific IgE to the suspected drug in an allergic mended to detect any cross-reactivity. SPT should reaction would make it possible to avoid per- be performed using undiluted solutions. For IDT, forming in vivo tests, reducing the possibility of preparations should be used in a 1:10 dilution, as unwanted adverse reactions. Nevertheless, only a undiluted contrast media may be irritating.198 few IgE assays are commercially available for drug Sese et al. published their experience with a low- allergy diagnosis, with variable predictive values. dose intravenous provocation test with ICM that, In some cases, individual laboratory assays, such as added to skin tests, seem to have a NPV of 80% in ELISA or FEIA, are used. Table 18 summarizes the Drug Techniques Sensitivity Specificity NMBA RIA/RAST/CAP FEIA 47–97% 91–100% b-lactams RAST/CAP FEIA/Sepharose-RIA 0–87% 67–97% Quinolones Sepharose-RIA 32–55% 100%, ND Propyphenazone ELISA 58% ND Table 18. Summary of IgE testing for drug allergy in different drug groups ELISA, enzyme-linked immunosorbent assay; FEIA, fluorometric enzyme immunoassay; RAST radioallergosorbent test; RIA, radioimmunoassay; ND, not done. Adapted from.232 Volume 13, No. 2, February 2020 37 value of IgE testing for drug allergy for the most the allergen source is the only available diagnostic relevant studied drug groups. However, there is tool. An interesting example in this respect is latex, an honest difference of opinion regarding the which has been reported as an important allergen interpretation of these results between US and for health care workers,202,203 in which an European allergists. outbreak of an allergy "epidemic" was observed in the years following the increased utilization of BAT for drug allergy rubber gloves in the 1980s. Latex, the raw This topic is discussed in the BAT chapter of this product of natural rubber, contains more than 200 polypeptides, and as of today 14 proteins, article. Hev b 1 to Hev b 14 have been given an IUIS nomenclature designation, with many isoforms IgE assays for occupational allergens and variants within these. Additional latex Occupational allergen sources may be materials proteins have been isolated but have not that a large part of the general population is received a designated allergen name. Health exposed to, but only occupational settings provide care workers seem to be particularly frequently sufficiently high, prolonged and relevant exposure sensitized to Hev b 5 and b 6.02, but many other to cause sensitization and, eventually, allergic dis- sensitizing allergens have been reported.203 The eases. Two examples are flour dust and industrial ideal diagnostic work-up, if the reagents are detergent enzymes where the majority of reported available, is for a two-step process, where the cases come from industries and bakeries where sensitization to the whole extract is first demon- large amounts of these source materials are strated, after which the sensitization to individual handled. allergens can be analyzed. While the sensitization to the whole extract is of relevance for the occu- IgE-testing plays a particularly important role in pational exposure (since the patient is of course occupational allergy because of the relatively rare exposed to the whole product), the elucidation of occurrence of the individual allergenic sources, the sensitization to individual allergenic molecules and thus a correspondingly low number of stan- may be of relevance in advising the patient about dardized extracts for skin testing.201 Even locally cross-reactivities that may be experienced outside or ad hoc produced skin test extracts may suffer of work. Latex sensitization to Hev b 6.02 may from problems of toxicity that may limit the use confer cross-reactivity to endochitinases contained of skin test as a diagnostic procedure. The lack of in avocado, chestnut, banana, and sweet pepper, pre-produced extracts, however, may also be a whereas sensitization to Hev b 5 may cause cross- problem for IgE-testing in settings where it is not reactivity to kiwi fruit. Another aspect of cross- possible to develop new assays based on an reactivity that should be considered is the possi- extract of the occupational material that is sus- bility that the patient may be sensitized to pollens pected of causing allergy. For this reason, the which may cause recognition of cross-reacting basophil histamine release assay or BAT may be a carbohydrate determinants (CCD)204 or profilins helpful technique, since it is possible to make an (Hev b 8 in latex, Phl p 12 in Timothy grass).205 extract of the suspected material and use it for challenging the basophils. It is pertinent though, A very important area of occupational allergy that the proper controls (no activation of basophils occurs with exposure to proteins in the handling of from healthy persons, no interference with the food whether it is in the industrial, workshop- assay in general) are carried out. based (bakeries, butchers) or catering sector. The The range of occupations in which IgE-mediated most important exposure route is likely to be via diseases have been reported is vast and varied, inhalation of dust or aerosols created during the and it is important to realize that Occupational manufacturing process, but also absorption via the Allergy is a field with not only protein allergens but skin should be considered. A list of the foods that also small molecules, examples of which are given have been reported to act as occupational aller- below. In many cases the specific allergen(s) from gens in causing asthma include food from both the an occupational exposure have been identified, plant kingdom (cereals, seeds, whole plants, veg- but there are also cases where a whole extract of etables, fruits, spices, teas) and the animal 38 Ansotegui et al. World Allergy Organization Journal (2020) 13:100080 http://doi.org/10.1016/j.waojou.2019.100080 kingdom (seafood and fish, meats from mammals in enzyme production plants,215 the and birds, milk and eggs).206,207 This list was pharmaceutical industry, 216 and bakeries.217 updated in 2014.207 Other enzymes such as those used in the Probably the best-described occupational al- detergent industry have also been known to cause lergy to food is baker's asthma where the patient problems. The initial proteases used in detergents may be sensitized to the flour but also to additives were produced by fermentation of Bacillus subtilis, in the form of enzymes (fungal alpha-amylase and thus called subtilisins. Soon after the intro- added to improve the leavening of the dough) or duction of a subtilisin in detergent products, se- insect pests in the form of cockroaches that may be vere IgE-mediated asthma reactions appeared contaminants. IgE-reactivity to wheat flour may among workers in detergent factories.218 Strict serve as an interesting lesson regarding occupa- exposure control programs reduced the tional versus non-occupational reactivity. Wheat exposures to low levels (15 ng/m3 range), which IgE-mediated allergy manifests itself as food al- reduced sensitization and prevented the onset of lergy and as occupational inhalant allergy (baker's allergic symptoms.219 Large scale studies of asthma),208 and identical allergens seem to be sensitization and allergic symptoms suggest that responsible in both allergies, although their sensitization comes first, and only in some cases relative importance differs.209,210 Nevertheless, leads to development of symptoms. It has also healthy patients may also display positive skin been suggested that not only enzymes, but also test or specific IgE towards wheat, due to the whole microorganisms used in the food and feed cross-reactivity between grass pollen and cereals industries may cause an allergy risk. Cleaning which may have an impact on the specificity of the workers having asthma and/or rhinitis should also diagnostic tests.74,211 This was confirmed in a be evaluated, even though only a minority could study, showing up to 35% positivity against wheat actually be sensitized. for both skin prick tests and sIgE tests, when Other exposures to high molecular weight al- testing grass pollen allergic subjects that lergens may be seen in occupations with live ani- tolerated eating wheat and other cereals and mals such as farms or experimental animal facilities without a history of baker's asthma.212 Thus also (where rodent urinary proteins seem to be a strong for occupational allergens, specificity remains an sensitizing allergen) and with plants from the important issue. farming and the gardening industry.220 In addition to the raw products involved, food Finally, occupational allergies may develop to additives and contaminants including insects, small organic molecules, that are either complete fungi and parasites should also be considered. To allergens with a symmetric structure such as chlor- compensate for a low content of natural amylases, hexidine221 or incomplete allergens that act as bioindustrially produced a-amylases are added to haptens by binding to carrier molecules, which are wheat flour to improve the leavening of the dough. generally human proteins.222 Examples are One such a-amylase is derived from Aspergillus ethylene oxide, formaldehyde, isocyanates, and oryzae and formulated as the product FungamylÒ, phthallic anhydride that are used for the which has been used as an additive to flour for production of polyurethane.223 The isocyanate more than 40 years. This preparation of a-amylase group compromise different chemical species, and complies with the FAO/WHO JECFA recom- there seems to be a certain cross-reactivity.224 mended specifications for food grade enzymes,213 Notably, in these cases, skin test or specific IgE in and it is generally considered as safe for human serum are not useful tools for diagnosis.225 ingestion. Occupational exposure to enzyme dust, however, may cause type I allergic In conclusion, IgE-based diagnostic methods for sensitization and allergic symptoms like asthma, occupational allergies are not, in principle, rhinitis, and urticaria may be elicited on different from other types of allergies. The large subsequent exposure.214 Preparations of a- number of different allergenic sources for which amylase derived from Aspergillus oryzae, occupational sensitizations have been reported, including FungamylÒ, in several cases, have however, has made the research area more been reported to cause sensitization of workers extensive, and for many occupations the specific Volume 13, No. 2, February 2020 Test Description Scientific evidence References 233–235 Specific IgG Food specific IgG or IgG4 panels are available as The titer of specific IgG-antibodies does not antibodies diagnostic tool for food allergy (NOTE: healthy correlate with oral food challenges. In children with subjects can produce specific IgG and IgG4 to proven milk allergy (positive oral challenge) no commonly eaten foods without being allergic). increase in specific IgG was detected. There is no evidence that IgG subclasses are a reliable diagnostic tool. Seventy-three patients were challenged in a double-blind manner to the IgG4 positive food, with no adverse reactions reported. 236,237 Cytotoxic test This is an in vitro test in which food allergens (up to In controlled conditions the reproducibility and 180) are put into contact with whole blood. Any diagnostic efficiency were largely insufficient. change in leukocytes' shape indicates a positive reaction. Hair analysis Nutritional deficiencies, detectable in hair (e.g., zinc, Nine allergic patients to fish (positive challenge) and 238,239 magnesium) are due to food allergy/intolerance. 9 healthy controls underwent the test. The test did not recognize allergic patients, and additional allergies were found without clinical significance. Hair samples of two teenagers were tested in 13 different laboratories, which reported different results. 240 Iridology Anatomical/morphological changes in the iris may A systematic review on iridology concluded that this suggest systemic diseases. test as a diagnostic tool is not supported by scientific evidence. 241,242 Kinesiology The patient holds a vial with a specific food in one According to two controlled studies the scientific hand, while the examiner tests the muscular strength evidence suggests that this diagnostic modality is of the opposite arm by applying a light pressure. not validated, and kinesiology as a diagnostic tool is Food allergy/intolerance is indicated by a decreased no better than random guessing. muscular contraction when the offending substance is held. 243,244 Electrodermal The patient is placed in a circuit where a According to two double-blind placebo-controlled testing galvanometer measures skin conductance. Vials with studies this method failed to distinguish between food extract are sequentially inserted into the circuit. allergic and non-allergic patients, and between A positive response consists of a drop in positive and negative tests. Poor reproducibility was conductance. always reported. Table 19. Unproven diagnostic approaches 39 40 Ansotegui et al. World Allergy Organization Journal (2020) 13:100080 http://doi.org/10.1016/j.waojou.2019.100080 allergens are still not complete. In the past Furthermore, the development of cellular assays different allergy "epidemics" such as latex, indus- such as the BAT may improve the diagnostic ac- trial enzymes, and proteins from experimental an- curacy of testing, particularly for food, venoms, imals have gradually been contained even though and drugs. However, BAT, standardized extracts, new sensitizations still occur. On the other hand component testing, multiplex testing, and micro- new processes, and particularly those that involve arrays are only available for specific allergens, handling of, and exposure to, proteins, may also thus, having limited clinical availability. create new and hitherto unknown exposures that Great effort is now focused on the standardiza- may cause new IgE-mediated allergies. tion process, both for testing and production of immunotherapy extracts. Standardization will Unproven diagnostic approaches result in optimal, reproducible, accurate testing that will improve patient outcomes and general Food allergy is a frequent allergic disorder, as 6– health care. Additional population studies are 8% of children and 2–3% of adults are affected. necessary to verify if more specific testing, such as However, the public perception of food allergy/ component assays, are relevant before these as- intolerance is higher, as one out of three people says can be recommended. The ultimate value of believes they are allergic or intolerant to one or allergy testing depends upon the pretest proba- more foods. This perception is at least in part bility derived from history and physical examina- based on the results of unproven diagnostic ap- tion that precedes the testing, as the clinical proaches (Table 19). Therefore these unreliable judgment of the healthcare provider influences diagnostic approaches may be costly for patients, greatly the predictive value of allergy testing. No delaying appropriate diagnosis and therapy. test can substitute for the importance of an adequate assessment, and remote testing should CONCLUSION be discouraged to limit misdiagnosis. For type I IgE-mediated allergic disease, skin tests are still considered the first-line approach for POSITION PAPER REVIEW AND indicating the presence of allergen-specific IgE ENDORSEMENT antibodies on the surface of mast cells in the skin of a sensitized patient. Skin testing is a simple and This position paper was reviewed and endorsed generally safe method, reliable in skilled hands; by the following national member societies of the the results are reproducible when standardized World Allergy Organization. extracts are employed. As complementary or American College of Allergy, Asthma and alternative diagnostics tools, in vitro serum IgE Immunology detection with the use of highly purified allergen or recombinants, in singleplex or multiplex Australasian Society of Clinical Immunology and manner, is an alternative diagnostic procedure. Allergy Serum IgE testing entails no risk to the patient Belarus Association of Allergology & Clinical other than a blood draw and is preferable if the Immunology patient has an unstable or uncontrolled medical condition, is at high risk of anaphylaxis, is taking Belgian Society of Allergology and Immunology essential medication that interferes with testing, is Canadian Society of Allergy and Clinical very young such that the procedure would be Immunology unduly stressful, or has a skin condition that limits available skin for testing. The development of Croatian Society of Allergology and Clinical screening tests with multiple allergens or multiplex Immunology tests that identify multiple specific-IgEs with a small Cuban Society of Allergy, Asthma and Clinical blood volume makes this testing more appealing Immunology to very young children. However, food allergy panels need to be adapted and improved in order Czech Society of Allergology and Clinical to be cost-effective and more specific. Immunology Volume 13, No. 2, February 2020 41 Egyptian Society of Pediatric Allergy and H2: Histamine 2 receptor; HPO: Horseradish Peroxidase; Immunology IDT: Intradermal Test; IgE: immunoglobulin E; ISAC: Immuno-Solid phase Allergen Chip; IUIS: International Hungarian Society of Allergology and Clinical Union of Immunological Societies; IVD: in vitro diagnostic Immunology tool; kUA/L: kilo Units of Allergen/Liter for allergen-specific IgE antibody assays; LAMP-3: Lysosomal-Associated Indian College of Allergy, Asthma and Applied Membrane Protein; mAb: Monoclonal Antibody; Immunology MRGPRX2: Mas-related G protein receptor 2; MBAD: Molecule Based Allergy Diagnostics; NIH: National In- Japanese Society of Allergology stitutes of Health (USA); NMBAs: NeuroMuscular Blocking Agents; NPA: Negative Percent Agreement; NSAIDs: Non- Korean Academy of Allergy, Asthma and Clin- Steroidal Anti-Inflammatory Drugs; pNPP: p-Nitro- ical Immunology phenylphosphate; PPA: Positive Percent Agreement; PPT: Prick-Prick Test; sIgE: specific IgE; RAST: Radio Allergo Kuwait Society of Allergy and Clinical Sorbent Test; SCAR: severe cutaneous adverse drug re- Immunology actions; SPT: Skin prick test; w/v: weight /volume Paraguayan Society of Immunology and Allergy Polish Society of Allergology DECLARATIONS Ethics approval and consent to participate Portuguese Society of Allergology and Clinical Not applicable. Immunology Consent for publication Romanian Society of Allergology and Clinical Not applicable. Immunology Availability of data and materials Not applicable. Spanish Society of Allergology and Clinical Funding Immunology Not applicable. Swiss Society of Allergology and Immunology Competing interest Uruguayan Society of Allergology E Jensen-Jarolim discloses she is a shareholder, and in- ventor on EP 2894478, with Biomedical International RþD The Jordanian Society for Allergy and Clinical GmbH Vienna; has received a grant and lecture honoraria Immunology reviewed and approved of publica- from Bencard GmbH Germany; has received lecture hon- oraria from Sanofi GmbH, Roxall, Meda, and Novartis; and tion of the position paper. The Austrian Society of is a joint Editor-in-Chief of this journal. Allergology and Immunology also reviewed the R Valenta has received research grants from Viravaxx, paper. Vienna, Austria, and serves as a consultant for this company. Abbreviations All other authors have declared they have no competing AAAAI: American Academy of Allergy Asthma and Immu- interests related to this work. nology; ABA: Allergen Bead Array; ACAAI: American Col- Authors’ contributions lege of Allergy Asthma and Immunology; AIT: allergen IJA, GM, GWC, LC, EV, ME, GP, ES, DE, RMG, OLS, JO, EJJ, immunotherapy; AEC: Allergen Exposure Chambers; Anti- and DF equally contributed to this manuscript, IgE: Antibody against IgE; AP: Alkaline Phosphatase; AU/ coordinating the different sections and reviewing all the mL: Allergenic Units milliLiter; BAT: Basophil Activation content. All other authors critically reviewed the Test; BAU/mL: Biologic Allergenic Units milliLiter; CaFE: manuscript. Calibrated Fluorescence Enhancement; CBA: Cytometric Bead Array; CCD: Cross-reactive Carbohydrate De- Acknowledgements terminants; CDER: Center for Drug Evaluation and The authors wish to thank the allergy societies from the Research (USA); CL: Chemiluminescence; DBPCFC: Dou- membership of the World Allergy Organization who ble-Blind Placebo-Controlled Food Challenge; EAACI: Eu- provided critical review of the draft and the WAO Board of ropean Academy of Allergy and Immunology; EIA: Enzyme Directors for supporting this work. Immune Assay; ELISA: Enzyme Linked Immuno Sorbent Analysis; EMEA: European MEdicine Agencies; ENPP-3: Author details a EctoNucleotide Pyrophosphatase/Phosphodiesterase 3; Hospital Quironsalud Bizkaia, Bilbao-Erandio, Spain. b FACS: Fluorescence-Activated Cell Sorting; FDA: Food and Department of Biomedical Sciences, Humanitas Drug Administration (U.S. Department of Health and Hu- University, Pieve Emanuele, Milan, Italy. cDepartment of man Services); FEIA: Fluorescent Enzyme Immunoassays; Biomedical Sciences, Humanitas University, Pieve FcεRI: High affinity IgE receptor; H1: Histamine 1 receptor; Emanuele, Personalized Medicine, Asthma and Allergy, 42 Ansotegui et al. World Allergy Organization Journal (2020) 13:100080 http://doi.org/10.1016/j.waojou.2019.100080 Humanitas Research Hospital, Rozzano, Milan, Italy. Missouri at Kansas City, School of Medicine, Kansas City, d Institute for Immunological Research, University of MO, USA. asCentro Médico Docente La Trinidad, Caracas, Cartagena, Cartagena, Colombia. eAzienda Sanitaria Venezuela. atExperimental Allergy Unit, Istituto Locale di Vercelli, S.C. Pneumologia, Vercelli, Italia. fClinical Dermopatico dell'Immacolata, Rome, Italy. auOspedale Research Center for Allergy and Rheumatology, Civile Maggiore, Verona, Italy. avAllergy & Asthma Institute, Sagamihara National Hospital, Sagamihara, Kanagawa, Asunción, Paraguay. awRoyal Children's Hospital, Japan. gAllergy and Respiratory Diseases, IRCCS Policlinico Department of Allergy & Immunology, Parkville, Victoria, San Martino, University of Genoa, Genoa, Italy. hOspedale Australia. axTan Tock Seng Hospital, Deptartment of Civile, Gossolengo, PC, Italy. iDepartment of Immunology - Rheumatology, Allergy & Immunology, Singapore, Allergology - Rheumatology, Antwerp University Hospital, Singapore. ayDivision of Immunopathology, Department of Antwerp University, Department Immunology and Pathophysiology and Allergy Research, Center for Allergology, AZ Jan Palfijn Gent, Ghent, Belgium. Pathophysiology, Infectiology and Immunology, Medical j Universidad Católica de Salta, Fundación Ayre, Buenos University of Vienna, Vienna, Austria. azNRC Institute of Aires, Argentina. kAllergy Section, Department of Internal Immunology FMBA of Russia, Moscow, Russia. ba Medicine, Vall d’Hebron University Hospital, Barcelona, Laboratory of Immunopathology, Department of Clinical Spain. lPulmonary & Allergy Associates, Denville, NJ, USA. Immunology and Allergy, Sechenov First Moscow State m Institute for Pathophysiology and Allergy Research, Medical University, Moscow, Russia. bbJohns Hopkins Center of Pathophysiology, Infectiology and Immunology, University School of Medicine, Johns Hopkins Hospital, The Interuniversity Messerli Research Institute, University of Baltimore, MD, USA. bcCampus Charite Mitte, Klinik fur Veterinary Medicine Vienna, Medical University Vienna, Dermatologie & Allergologie, Berlin, Germany. Vienna, Austria. nFischer Medicine Professional Corporation, Barrie, Ontario, Canada. oSkin and Allergy Hospital, University of Helsinki, Helsinki, Finland. pClínica REFERENCES de Alergía, Sorocaba, São Paulo, Brazil. qMACVIA-France, Montpellier, France. rINSERM, Villejuif, France. sUniversité 1. Dreborg S. Diagnosis of food allergy: tests in vivo and in vitro. Versailles St-Quentin-en-Yvelines, Montigny le Bretonneux, Pediatr Allergy Immunol. 2001;12(Suppl 14):24–30. France. tEuforea, Brussels, Belgium. uCHU Montpellier, 2. Portnoy J. Diagnostic testing for allergies. Ann Allergy Asthma France. vUniversitat Autónoma de Barcelona, Hospital Immunol. 2006;96(1):3–4. Universitario Vall d'Hebron, Servicio de Medicina Interna, 3. Oppenheimer J, Nelson HS. Skin testing: a survey of allergists. Sección de Alergología, Barcelona, Spain. wMount Ann Allergy Asthma Immunol. 2006;96(1):19–23. Elizabeth Medical Centre, Chiang Children's Allergy & Asthma Clinic, Singapore, Singapore. xUniversity Hospital 4. Malling HJ, Allesen-Holm P, Karved LS, Poulsen LK. Proficiency testing of skin prick testers as part of a quality Montpellier, Montpellier, France. ySorbonne Université, assurance system. Clin Transl Allergy. 2016;6:36. https://doi. Paris, France. zImmunology Research Institute of New org/10.1186/s13601-016-0126-7. eCollection 2016. England, Gardner, MA, USA. aaThe Unidad de Educación Médica, Department of Medical Education, School of 5. Portnoy JM. What do allergy skin tests really mean? Ann Medicine, Clinica Universitad de Navarra, Navarra, Spain. Allergy Asthma Immunol. 2002;89(4):335–336. ab Erasmus MC, Sector Allergologie, Rotterdam, the 6. McCann WA, Ownby DR. The reproducibility of the allergy Netherlands. acUniversidad Autónoma de Nuevo León, skin test scoring and interpretation by board-certified/board- Hospital Universitario, Monterrey, Nuevo León, Mexico. eligible allergists. Ann Allergy Asthma Immunol. 2002;89(4): ad Department of Allergy, Mayo Clinic, Jacksonville, FL, 368–371. USA. aeFemeca Femedica, Buenos Aires, Argentina. 7. Pawankar R, Holgate ST, Canonica GW, Lockey RF, Blaiss M, af Kirikkale Universitesi, Tip Fakultesi, Kirikkale, Turkey. eds. WAO White Book on Allergy 2013 Update, World Allergy ag Hospital Sírio Libanês, University of São Paulo, São Paulo, Organization; 2013. https://www.worldallergy.org/wao-white- Brazil. ahFaculty of Medicine, Department of Clinical book-on-allergy. Immunology & Allergy, Medical University of Łódz, Łódz, 8. Canonica GW, Ansotegui IJ, Pawankar R, et al. A WAO - ARIA - Poland. aiUniversity of South Florida College of Medicine, GA(2)LEN consensus document on molecular-based allergy, Tampa, FL, USA. ajAllergology Unit, San Juan de Dios diagnostics. World Allergy Organ J. 2013;6(1):17. https://doi. Hospital, University of Costa Rica, San José, Costa Rica. org/10.1186/1939-4551-6-17. ak Hospital CUF-Descobertas, Lisbon, Portugal. 9. Klimek L, Hoffmann HJ, Renz H, et al. Diagnostic test allergens al Department of Otorhinolaryngology, Head and Neck used for in vivo diagnosis of allergic diseases are at risk: a Surgery, Section of Rhinology and Allergy, University European Perspective. Allergy. 2015;70(10):1329–1331. Hospital Marburg, Philipps-Universität Marburg, Marburg, https://doi.org/10.1111/all.12676. Epub 2015 Aug 19. Germany. amGentofte University Hospital, Lab for 10. Heinzerling L, Mari A, Bergmann KC, et al. The skin prick test - Allergology, Allergy Clinic, Hellerup, Denmark. anNippon European standards. Clin Transl Allergy. 2013;3(1):3. https:// Medical School, Dept. of Otolaryngology, Tokyo, Japan. doi.org/10.1186/2045-7022-3-3. ao University Hospital GI & MR GmbH, Institute of 11. Matricardi PM, Kleine-Tebbe J, Hoffmann HJ, et al. EAACI Laboratory Medicine & Pathology, Standort Marburg, molecular allergology user's guide. Pediatr Allergy Immunol. Marburg, Germany. apStallergenes, Rome, Italy. 2016;27(Suppl 23):1–250. https://doi.org/10.1111/pai.12563. aq University of Parana, Curitiba, PR, Brazil. arUniversity of Volume 13, No. 2, February 2020 43 12. Oppenheimer J, Nelson HS. Skin testing. Ann Allergy Asthma human IgE antibodies of defined allergen specificities. Immunol. 2006;96(2 Suppl 1). S6-12. (CLSI-ILA20-A3). J Allergy Clin Immunol February. 2015;135(2 - Suppl). AB8. 13. Bodtger U, Poulsen LK, Malling HJ. Asymptomatic skin sensitization to birch predicts later development of birch 28. Nolte H, DuBuske LM. Performance characteristics of a new pollen allergy in adults: a 3-year follow-up study. J Allergy automated enzyme immunoassay for the measurement of Clin Immunol. 2003;111(1):149–154. allergen-specific IgE. Summary of the probability outcomes comparing results of allergen skin testing to results obtained 14. Haahtela T, Burbach GJ, Bachert C, et al. Clinical relevance is with the HYTEC system and CAP system. Ann Allergy Asthma associated with allergen-specific wheal size in skin prick Immunol. 1997;79(1):27–34. testing. Clin Exp Allergy. 2014;44(3):407–416. https://doi.org/ 10.1111/cea.12240. 29. Kam KL, Hsieh KH. Comparison of three in vitro assays for serum IgE with skin testing in asthmatic children. Ann Allergy. 15. Commins SP, Satinover SM, Hosen J, et al. Delayed 1994;73(4):329–336. anaphylaxis, angioedema, or urticaria after consumption of red meat in patients with IgE antibodies specific for 30. Asero R, Scala E, Villalta D, et al. Shrimp allergy: analysis of galactose-alpha-1,3-galactose. J Allergy Clin Immunol. commercially available extracts for in vivo diagnosis. 2009;123(2):426–433. https://doi.org/10.1016/j.jaci.2008.10. J Investig Allergol Clin Immunol. 2017;27(3):175–182. 052. Epub Dec 13. 31. Ciardiello MA, Giangrieco I, Tuppo L, et al. Influence of the 16. Bernstein DI, Wanner M, Borish L, Liss GM. Twelve-year survey natural ripening stage, cold storage, and ethylene treatment of fatal reactions to allergen injections and skin testing: 1990- on the protein and IgE-binding profiles of green and gold 2001. J Allergy Clin Immunol. 2004;113(6):1129–1136. kiwi fruit extracts. J Agric Food Chem. 2009;57(4):1565–1571. https://doi.org/10.1021/jf802966n. 17. Amin HS, Liss GM, Bernstein DI. Evaluation of near-fatal reactions to allergen immunotherapy injections. J Allergy Clin 32. Hildebrandt S, Steinhart H, Paschke A. Comparison of Immunol. 2006;117(1):169–175. different extraction solutions for the analysis of allergens in hen's egg. Food Chem. 2008;108(3):1088–1093. https://doi. 18. Rueff F, Przybilla B, Bilo MB, et al. Clinical effectiveness of org/10.1016/j.foodchem.2007.11.051. Epub Nov 29. hymenoptera venom immunotherapy: a prospective observational multicenter study of the European academy of 33. Jensen-Jarolim E, Jensen AN, Canonica GW. Debates in allergology and clinical immunology interest group on insect allergy medicine: molecular allergy diagnosis with ISAC will venom hypersensitivity. e63233 PLoS One. 2013;8(5). https:// replace screenings by skin prick test in the future. World doi.org/10.1371/journal.pone.0063233. Print 2013. Allergy Organ J. 2017;10(1):33. 19. Schwindt CD, Hutcheson PS, Leu SY, Dykewicz MS. Role of 34. Larenas-Linnemann D, Luna-Pech JA, Mosges R. Debates in intradermal skin tests in the evaluation of clinically relevant Allergy Medicine: allergy skin testing cannot be replaced by respiratory allergy assessed using patient history and nasal molecular diagnosis in the near future. World Allergy Organ J. challenges. Ann Allergy Asthma Immunol. 2005;94(6):627–633. 2017;10(1):32. 20. Romano A, Warrington R. Antibiotic allergy. Immunol Allergy 35. Mothes-Luksch N, Jordakieva G, Hinterholzl L, et al. Allergy Clin N AM. 2014;34(3):489–506, 2014 Aug. diagnosis from symptoms to molecules, or from molecules to symptoms: a comparative clinical study. World Allergy Organ 21. James JMBA. Food allergy: current diagnostic methods and J. 2018;11(1):22. interpretation of results. In: Kemp SFLR, ed. Diagnostic Testing of Allergic Disease. New York: Marcel Dekker Inc.; 36. Kowalski ML, Ansotegui I, Aberer W, et al. Risk and safety 2000:199–215. requirements for diagnostic and therapeutic procedures in allergology: world Allergy Organization Statement. World 22. Sampson HA. Food allergy. Part 2: diagnosis and Allergy Organ J. 2016;9(1):33. management. J Allergy Clin Immunol. 1999;103(6):981–989. 37. Hemmer W, Altmann F, Holzweber F, Gruber C, Wantke F, 23. Turkeltaub PC. Precutaneous and intracutaneous diagnostic Wohrl S. ImmunoCAP cellulose displays cross-reactive tests of IgE-mediated diseases (immediate hypersensitivity. carbohydrate determinant (CCD) epitopes and can cause false- In: Kemp SFLR, ed. Diagnostic Testing of Allergic Disease. positive test results in patients with high anti-CCD IgE antibody New York: Marcel Dekker Inc; 2000:66. levels. J Allergy Clin Immunol. 2018;141(1):372–381 e3. 24. Gungor A, Houser SM, Aquino BF, et al. A comparison of skin 38. Valenta R, Niederberger V. Recombinant allergens for endpoint titration and skin-prick testing in the diagnosis of immunotherapy. J Allergy Clin Immunol. 2007;119(4):826– allergic rhinitis. Ear Nose Throat J. 2004;83(1):54–60. 830. 25. Wide L, Bennich H, Johansson SG. Diagnosis of allergy by an 39. Passalacqua G, Melioli G, Bonifazi F, et al. The additional in-vitro test for allergen antibodies. Lancet. 1967;2(7526): values of microarray allergen assay in the management of 1105–1107. polysensitized patients with respiratory allergy. Allergy. 26. Cox L, Williams B, Sicherer S, et al. Pearls and pitfalls of 2013;68(8):1029–1033. allergy diagnostic testing: report from the American College 40. Douladiris N, Savvatianos S, Roumpedaki I, Skevaki C, of allergy, asthma and immunology/American academy of Mitsias D, Papadopoulos NG. A molecular diagnostic allergy, asthma and immunology specific IgE test Task Force. algorithm to guide pollen immunotherapy in southern Ann Allergy Asthma Immunol. 2008;101(6):580–592. Europe: towards component-resolved management of 27. Hamilton RGMP, Hovanec-Burns D, Mark Van Cleve M, allergic diseases. Int Arch Allergy Immunol. 2013;162(2):163– et al. Analytical performance characteristics, quality 172. assurance and clinical utility of immunological assays for 44 Ansotegui et al. World Allergy Organization Journal (2020) 13:100080 http://doi.org/10.1016/j.waojou.2019.100080 41. Camacho GD, Arjona AMM, Padial JF, Jesus RC. How 58. Fernandez-Rivas M, Bolhaar S, Gonzalez-Mancebo E, et al. molecular diagnosis may modify immunotherapy Apple allergy across Europe: how allergen sensitization prescription in multi-sensitized pollen-allergic children. J profiles determine the clinical expression of allergies to plant Allergol Immunopathol (Madr). 2018 Nov-Dec;46(6):552– foods. J Allergy Clin Immunol. 2006;118(2):481–488. Epub 556. https://doi.org/10.1016/j.aller.2018.03.002. Epub 2018 2006 Jun 27. Jul 13. 59. Barber D, de la Torre F, Feo F, et al. Understanding patient 42. Sastre J, Landivar ME, Ruiz-Garcia M, Andregnette- sensitization profiles in complex pollen areas: a molecular Rosigno MV, Mahillo I. How molecular diagnosis can change epidemiological study. Allergy. 2008;63(11):1550–1558. allergen-specific immunotherapy prescription in a complex https://doi.org/10.1111/j.1398-9995.2008.01807.x. pollen area. Allergy. 2012;67(5):709–711. 60. Scala E, Alessandri C, Bernardi ML, et al. Cross-sectional 43. Schmid-Grendelmeier P. [Recombinant allergens. For routine survey on immunoglobulin E reactivity in 23,077 subjects use or still only science?]. Hautarzt. 2010;61(11):946–953. using an allergenic molecule-based microarray detection system. Clin Exp Allergy. 2010;40(6):911–921. https://doi.org/ 44. Wide L, Bennich H, Johansson SG. Diagnosis of allergy by an 10.1111/j.365-2222.010.03470.x. Epub 2010 Mar 1. in-vitro test for allergen antibodies. Lancet. 1967;2(7526): 1105–1107. 61. Melioli G, Marcomini L, Agazzi A, et al. The IgE repertoire in children and adolescents resolved at component level: a 45. Kjellman NM, Johansson SG, Roth A. Serum IgE levels in cross-sectional study. Pediatr Allergy Immunol. 2012;23(5): healthy children quantified by a sandwich technique (PRIST). 433–440. Clin Allergy. 1976;6(1):51–59. 62. Valenta R, Niederberger V. Recombinant allergens for 46. Wittig HJ, Belloit J, De Fillippi I, Royal G. Age-related serum immunotherapy. J Allergy Clin Immunol. 2007;119(4):826– immunoglobulin E levels in healthy subjects and in patients 830. Epub 2007 Mar 1. with allergic disease. J Allergy Clin Immunol. 1980;66(4):305– 313. 63. Sastre J. Molecular diagnosis in allergy. Clin Exp Allergy. 2010;40(10):1442–1460. 47. Meyers DA, Postma DS, Panhuysen CI, et al. Evidence for a locus regulating total serum IgE levels mapping to 64. Ferreira F, Hawranek T, Gruber P, Wopfner N, Mari A. Allergic chromosome 5. Genomics. 1994;23(2):464–470. cross-reactivity: from gene to the clinic. Allergy. 2004;59(3): 243–267. 48. Cookson WO, Young RP, Sandford AJ, et al. Maternal inheritance of atopic IgE responsiveness on chromosome 65. Scala E, Alessandri C, Palazzo P, et al. IgE recognition patterns 11q. Lancet. 1992;340(8816):381–384. of profilin, PR-10, and tropomyosin panallergens tested in 3, 113 allergic patients by allergen microarray-based 49. Ozcan E, Notarangelo LD, Geha RS. Primary immune technology. e24912 PLoS One. 2011;6(9). https://doi.org/10. deficiencies with aberrant IgE production. J Allergy Clin 1371/journal.pone.0024912. Epub 2011 Sep. 15. Immunol. 2008;122(6):1054–1062. https://doi.org/10.1016/j. jaci.2008.10.023. quiz 63-4. 66. Asero R, Monsalve R, Barber D. Profilin sensitization detected in the office by skin prick test: a study of 50. Greenberger PA. Allergic bronchopulmonary aspergillosis. prevalence and clinical relevance of profilin as a plant J Allergy Clin Immunol. 2002;110(5):685–692. food allergen. Clin Exp Allergy. 2008;38(6):1033–1037. 51. Thorpe SJ, Heath A, Fox B, Patel D, Egner W. The 3rd https://doi.org/10.1111/j.1365-2222.2008.02980.x. Epub International Standard for serum IgE: international 2008 Apr 13. collaborative study to evaluate a candidate preparation. Clin 67. Kleine-Tebbe J, Vogel L, Crowell DN, Haustein UF, Vieths S. Chem Lab Med. 2014;52(9):1283–1289. https://doi.org/10. Severe oral allergy syndrome and anaphylactic reactions 1515/cclm-2014-0243. caused by a Bet v 1- related PR-10 protein in soybean, 52. Lee S, Lim HS, Park J, Kim HS. A new automated multiple SAM22. J Allergy Clin Immunol. 2002;110(5):797–804. allergen simultaneous test-chemiluminescent assay 68. Ballmer-Weber BK, Holzhauser T, Scibilia J, et al. Clinical (MAST-CLA) using an AP720S analyzer. Clin Chim Acta. characteristics of soybean allergy in Europe: a double-blind, 2009;402(1-2):182–188. https://doi.org/10.1016/j.cca.2009. placebo-controlled food challenge study. J Allergy Clin 01.014. Immunol. 2007;119(6):1489–1496. Epub 2007 Mar 26. 53. Ewan PW, Coote D. Evaluation of a capsulated hydrophilic 69. Zuidmeer L, van Ree R. Lipid transfer protein allergy: primary carrier polymer (the ImmunoCAP) for measurement of food allergy or pollen/food syndrome in some cases. Curr specific IgE antibodies. Allergy. 1990;45(1):22–29. Opin Allergy Clin Immunol. 2007;7(3):269–273. 54. PNJ M, RG H, RE E, et al. Analytical Performance 70. Stedman K, Lee K, Hunter S, Rivoire B, McCall C, Wassom D. Characteristics and Clinical Utility of Immunological Assays for Measurement of canine IgE using the alpha chain of the Human Immunoglobulin E (IgE) Antibodies and Defined human high affinity IgE receptor. Vet Immunol Immunopathol. Allergen Specificities. 2009. 2001;78(3-4):349–355. 55. Dolen WK. Skin testing and immunoassays for allergen- 71. Wojtalewicz N, Kabrodt K, Goseberg S, Schellenberg I. specific IgE. Clin Rev Allergy Immunol. 2001;21(2-3):229–239. Evaluation of the manufacturer-dependent differences in sIgE 56. Hamilton RG, Franklin Adkinson Jr N. In vitro assays for the results for indoor allergens. Ann Allergy Asthma Immunol. diagnosis of IgE-mediated disorders. J Allergy Clin Immunol. 2018 Oct;121(4):490–495. https://doi.org/10.1016/j.anai. 2004;114(2):213–225. quiz 26. 2018.07.016. Epub 2018 Jul 17. 57. Lundberg M, Chen Z, Rihs HP, Wrangsjo K. Recombinant 72. Goikoetxea MJ, Sanz ML, Garcia BE, et al. Recommendations spiked allergen extract. Allergy. 2001;56(8):794–795. for the use of in vitro methods to detect specific Volume 13, No. 2, February 2020 45 immunoglobulin E: are they comparable? J Investig Allergol 88. MacBeath G, Schreiber SL. Printing proteins as microarrays Clin Immunol. 2013;23(7):448–454. quiz 2 pp. preceding 55. for high-throughput function determination. Science. 2000;289(5485):1760–1763. 73. Jahn-Schmid B, Harwanegg C, Hiller R, et al. Allergen microarray: comparison of microarray using recombinant 89. Hiller R, Laffer S, Harwanegg C, et al. Microarrayed allergen allergens with conventional diagnostic methods to detect molecules: diagnostic gatekeepers for allergy treatment. allergen-specific serum immunoglobulin E. Clin Exp Allergy. FASEB J. 2002;16(3):414–416. Epub 2002 Jan 14. 2003;33(10):1443–1449. 90. Untersmayr E, Lukschal A, Hemmer W, et al. Exercise with 74. Sampson HA. Utility of food-specific IgE concentrations in latex sport bands represents a risk for latex allergic patients. predicting symptomatic food allergy. J Allergy Clin Immunol. Immunol Lett. 2008;115(2):98–104. Epub 2007 Nov 6. 2001;107(5):891–896. 91. Wohrl S, Vigl K, Zehetmayer S, et al. The performance of a 75. Celik-Bilgili S, Mehl A, Verstege A, et al. The predictive component-based allergen-microarray in clinical practice. value of specific immunoglobulin E levels in serum for the Allergy. 2006;61(5):633–639. outcome of oral food challenges. Clin Exp Allergy. 92. Ott H, Schroder CM, Stanzel S, Merk HF, Baron JM. 2005;35(3):268–273. Microarray-based IgE detection in capillary blood samples of 76. Eigenmann PA. Are specific immunoglobulin E titres reliable patients with atopy. Allergy. 2006;61(9):1146–1147. for prediction of food allergy? Clin Exp Allergy. 2005;35(3): 93. Alcocer MJ, Murtagh GJ, Wilson PB, Progias P, Lin J, 247–249. Archer DB. The major human structural IgE epitope of the 77. Yunginger JW, Ahlstedt S, Eggleston PA, et al. Quantitative Brazil nut allergen Ber e 1: a chimaeric and protein microarray IgE antibody assays in allergic diseases. J Allergy Clin approach. J Mol Biol. 2004;343(3):759–769. Immunol. 2000;105(6 Pt 1):1077–1084. 94. Cerecedo I, Zamora J, Shreffler WG, et al. Mapping of the IgE 78. Wickman M, Ahlstedt S, Lilja G, van Hage Hamsten M. and IgG4 sequential epitopes of milk allergens with a peptide Quantification of IgE antibodies simplifies the classification of microarray-based immunoassay. J Allergy Clin Immunol. allergic diseases in 4-year-old children. A report from the 2008;122(3):589–594. https://doi.org/10.1016/j.jaci.2008.06. prospective birth cohort study–BAMSE. Pediatr Allergy 040. Immunol. 2003;14(6):441–447. 95. Constantin C, Quirce S, Poorafshar M, et al. Micro-arrayed wheat 79. Park KH, Lee J, Sim DW, Lee SC. Comparison of singleplex seed and grass pollen allergens for component-resolved specific IgE detection immunoassays: ImmunoCAP phadia diagnosis. Allergy. 2009;64(7):1030–1037. https://doi.org/10. 250 and immulite 2000 3gAllergy. Ann Lab Med. 2018;38(1):23– 1111/j.1398-9995.2009.01955.x. Epub 2009 Feb 11. 31. 96. Cretich M, di Carlo G, Longhi R, et al. High sensitivity protein 80. Barber D, Arias J, Boquete M, et al. Analysis of mite allergic assays on microarray silicon slides. Anal Chem. 2009;81(13): patients in a diverse territory by improved diagnostic tools. 5197–5203. https://doi.org/10.1021/ac900658c. Clin Exp Allergy. 2012;42(7):1129–1138. 97. Fall BI, Eberlein-Konig B, Behrendt H, Niessner R, Ring J, 81. Boyce JA, Assa'ad A, Burks AW, et al. Guidelines for the Weller MG. Microarrays for the screening of allergen-specific diagnosis and management of food allergy in the United IgE in human serum. Anal Chem. 2003;75(3):556–562. States: report of the NIAID-sponsored expert panel. J Allergy 98. Gadermaier G, Wopfner N, Wallner M, et al. Array-based Clin Immunol. 2010;126(6 Suppl):S1–S58. https://doi.org/10. profiling of ragweed and mugwort pollen allergens. Allergy. 1016/j.jaci.2010.10.007. 2008;63(11):1543–1549. https://doi.org/10.1111/j.1398- 82. Hamilton RG, Wisenauer JA, Golden DB, Valentine MD, 9995.2008.01780.x. Adkinson Jr NF. Selection of Hymenoptera venoms for 99. Gaudin JC, Rabesona H, Choiset Y, et al. Assessment of the immunotherapy on the basis of patient's IgE antibody cross- immunoglobulin E-mediated immune response to milk- reactivity. J Allergy Clin Immunol. 1993;92(5):651–659. specific proteins in allergic patients using microarrays. Clin 83. Savi E, Peveri S, Makri E, Pravettoni V, Incorvaia C. Comparing the Exp Allergy. 2008;38(4):686–693. https://doi.org/10.1111/j. ability of molecular diagnosis and CAP-inhibition in identifying 365-2222.008.02952.x. Epub 2008 Feb 26. the really causative venom in patients with positive tests to 100. Heyries KA, Loughran MG, Hoffmann D, Homsy A, Blum LJ, Vespula and Polistes species. Clin Mol Allergy. 2016;14:3. Marquette CA. Microfluidic biochip for chemiluminescent 84. Savi E, Incorvaia C, Boni E, et al. Which immunotherapy product detection of allergen-specific antibodies. Biosens is better for patients allergic to Polistes venom? A laboratory Bioelectron. 2008;23(12):1812–1818. https://doi.org/10. and clinical study. PLoS One. 2017;12(7). e0180270. 1016/j.bios.2008.02.025. Epub Mar 7. 85. Quercia O, Cova V, Martini M, et al. CAP-inhibition, molecular 101. Lebrun SJ, Petchpud WN, Hui A, McLaughlin CS. diagnostics, and total IgE in the evaluation of Polistes and Development of a sensitive, colorometric microarray assay for vespula double sensitization. Int Arch Allergy Immunol. allergen-responsive human IgE. J Immunol Methods. 2018;177(4):365–369. 2005;300(1-2):24–31. Epub 2005 Apr 13. 86. Negrini AC, Troise C, Voltolini S. ELISA in diagnosis of 102. Ott H, Baron JM, Heise R, et al. Clinical usefulness of respiratory allergy. A comparison with RAST and skin tests. microarray-based IgE detection in children with suspected Allergy. 1985;40(4):238–241. food allergy. Allergy. 2008;63(11):1521–1528. https://doi. org/10.1111/j.1398-9995.2008.01748.x. 87. Schulze A, Downward J. Analysis of gene expression by microarrays: cell biologist's gold mine or minefield? J Cell Sci. 103. Ott H, Folster-Holst R, Merk HF, Baron JM. Allergen 2000;113(Pt 23):4151–4156. microarrays: a novel tool for high-resolution IgE profiling in 46 Ansotegui et al. World Allergy Organization Journal (2020) 13:100080 http://doi.org/10.1016/j.waojou.2019.100080 adults with atopic dermatitis. Eur J Dermatol. 2010;20(1):54– 118. Goikoetxea MJ, D'Amelio CM, Martinez-Aranguren R, et al. Is 61. https://doi.org/10.1684/ejd.2010.0810. Epub 2009 Oct 2. microarray analysis really useful and sufficient to diagnose nut allergy in the mediterranean area? J Investig Allergol Clin 104. Ott H, Schroder C, Raulf-Heimsoth M, et al. Microarrays of Immunol. 2016;26(1):31–39. recombinant Hevea brasiliensis proteins: a novel tool for the component-resolved diagnosis of natural rubber latex 119. Javaloyes G, Goikoetxea MJ, Garcia Nunez I, et al. Pru p 3 allergy. J Investig Allergol Clin Immunol. 2010;20(2):129–138. acts as a strong sensitizer for peanut allergy in Spain. J Allergy Clin Immunol. 2012;130(6):1432–4 e3. 105. Shreffler WG, Lencer DA, Bardina L, Sampson HA. IgE and IgG4 epitope mapping by microarray immunoassay reveals 120. Heaps A, Carter S, Selwood C, et al. The utility of the ISAC the diversity of immune response to the peanut allergen, Ara allergen array in the investigation of idiopathic anaphylaxis. h 2. J Allergy Clin Immunol. 2005;116(4):893–899. Clin Exp Immunol. 2014;177(2):483–490. 106. Melioli G, Bonifazi F, Bonini S, et al. The ImmunoCAP ISAC 121. Cardona V, Ansotegui IJ. Component-resolved diagnosis in molecular allergology approach in adult multi-sensitized anaphylaxis. Curr Opin Allergy Clin Immunol. 2016;16(3): Italian patients with respiratory symptoms. Clin Biochem. 244–249. 2011;44(12):1005–1011. 122. Skrindo I, Lupinek C, Valenta R, et al. The use of the MeDALL- 107. Deinhofer K, Sevcik H, Balic N, et al. Microarrayed allergens chip to assess IgE sensitization: a new diagnostic tool for for IgE profiling. Methods. 2004;32(3):249–254. allergic disease? Pediatr Allergy Immunol. 2015;26(3):239– 246. https://doi.org/10.1111/pai.12366. 108. Huss-Marp J, Gutermuth J, Schaffner I, et al. Comparison of molecular and extract-based allergy diagnostics with multiplex 123. Lupinek C, Wollmann E, Baar A, et al. Advances in allergen- and singleplex analysis. Allergo J Int. 2015;24:46–53. microarray technology for diagnosis and monitoring of allergy: the MeDALL allergen-chip. Methods. 2014;66(1):106– 109. Lambert C, Sarrat A, Bienvenu F, et al. The importance of EN 119. ISO 15189 accreditation of allergen-specific IgE determination for reliable in vitro allergy diagnosis. Allergy. 124. Skrindo I, Lupinek C, Valenta R, et al. The use of the MeDALL- 2015;70(2):180–186. chip to assess IgE sensitization: a new diagnostic tool for allergic disease? Pediatr Allergy Immunol. 2015;26(3):239– 110. Martinez-Aranguren R, Lizaso MT, Goikoetxea MJ, et al. Is 246. the determination of specific IgE against components using ISAC 112 a reproducible technique? PLoS One. 2014;9(2). 125. Wickman M, Lupinek C, Andersson N, et al. Detection of IgE e88394. reactivity to a handful of allergen molecules in early childhood predicts respiratory allergy in adolescence. 111. Monroe MR, Reddington AP, Collins AD, et al. Multiplexed EBioMedicine. 2017;26:91–99. method to calibrate and quantitate fluorescence signal for allergen-specific IgE. Anal Chem. 2011;83(24):9485–9491. 126. Williams P, Onell A, Baldracchini F, Hui V, Jolles S, El- Shanawany T. Evaluation of a novel automated allergy 112. Villalta D, Conte M, Asero R, Da Re M, Stella S, Martelli P. microarray platform compared with three other allergy test Isolated IgE reactivity to native walnut vicilin-like protein methods. Clin Exp Immunol. 2016 Apr;184(1):1–10. https:// (nJug r 2) on ISAC microarray is due to cross-reactive doi.org/10.1111/cei.12721. Epub 2016 Jan 13. carbohydrate epitopes. Clin Chem Lab Med. 2013;51(10): 1991–1995. 127. Heffler E, Puggioni F, Peveri S, Montagni M, Canonica GW, Melioli G. Extended IgE profile based on an allergen 113. Hochwallner H, Alm J, Lupinek C, et al. Transmission of macroarray: a novel tool for precision medicine in allergy allergen-specific IgG and IgE from maternal blood into breast diagnosis. World Allergy Organ J. 2018;11(1):7. milk visualized with microarray technology. J Allergy Clin Immunol. 2014;134(5):1213–1215. 128. Melioli G, Spenser C, Reggiardo G, et al. Allergenius, an expert system for the interpretation of allergen microarray 114. Leonardi A, Borghesan F, Faggian D, Plebani M. Microarray- results. World Allergy Organ J. 2014;7(1):15. based IgE detection in tears of patients with vernal keratoconjunctivitis. Pediatr Allergy Immunol. 2015;26(7): 129. Konopka E, Ceregra A, Maciorkowska E, et al. Specific IgE 641–645. antibodies in young children with atopic dermatitis– correlation of multiple allergen simultaneous immunoblot 115. Wollmann E, Lupinek C, Kundi M, Selb R, Niederberger V, test and ImmunoCap system. Clin Lab. 2016;62(5):815–821. Valenta R. Reduction in allergen-specific IgE binding as measured by microarray: a possible surrogate marker for 130. Morgan E, Varro R, Sepulveda H, et al. Cytometric bead array: effects of specific immunotherapy. J Allergy Clin Immunol. a multiplexed assay platform with applications in various 2015;136(3):806–809 e7. areas of biology. Clin Immunol. 2004;110(3):252–266. 116. Schmid JM, Wurtzen PA, Dahl R, Hoffmann HJ. Pretreatment 131. Varro R, Chen R, Sepulveda H, Apgar J. Bead-based IgE sensitization patterns determine the molecular profile of multianalyte flow immunoassays: the cytometric bead array the IgG4 response during updosing of subcutaneous system. Methods Mol Biol. 2007;378:125–152. https://doi. immunotherapy with timothy grass pollen extract. J Allergy org/10.1007/978-1-59745-323-3_9. Clin Immunol. 2016;137(2):562–570. 132. Ferbas J, Thomas J, Hodgson J, Gaur A, Casadevall N, 117. D'Amelio CM, Goikoetxea MJ, Martinez-Aranguren R, et al. Is Swanson SJ. Feasibility of a multiplex flow cytometric bead the performance of ImmunoCAP ISAC 112 sufficient to immunoassay for detection of anti-epoetin alfa antibodies. diagnose peach and apple allergies? Ann Allergy Asthma Clin Vaccine Immunol. 2007;14(9):1165–1172. Epub 2007 Jul Immunol. 2016;116(2):162–163. 18. Volume 13, No. 2, February 2020 47 133. Fulton RJ, McDade RL, Smith PL, Kienker LJ, Kettman Jr JR. 150. Jakob T, Forstenlechner P, Matricardi P, Kleine-Tebbe J. Advanced multiplexed analysis with the FlowMetrix system. Molecular allergy diagnostics using multiplex assays: Clin Chem. 1997;43(9):1749–1756. methodological and practical considerations for use in research and clinical routine: Part 21 of the Series Molecular 134. Kettman JR, Davies T, Chandler D, Oliver KG, Fulton RJ. Allergology. Allergo J Int. 2015;24:320–332. Classification and properties of 64 multiplexed microsphere sets. Cytometry. 1998;33(2):234–243. 151. Macchia D, Melioli G, Pravettoni V, et al. Guidelines for the use and interpretation of diagnostic methods in adult food 135. King EM, Vailes LD, Tsay A, Satinover SM, Chapman MD. allergy. Clin Mol Allergy. 2015;13:27. Simultaneous detection of total and allergen-specific IgE by using purified allergens in a fluorescent multiplex array. 152. Rosario NLAL. Does sensitization to food allergens in patients J Allergy Clin Immunol. 2007;120(5):1126–1131. Epub 2007 with rhinitis mean food allergy? J Allergy Ther. 2014;2(3):167. Sep. 7. 153. Werfel T, Asero R, Ballmer-Weber BK, et al. Position paper of 136. Pomponi D, Bernardi ML, Liso M, et al. Allergen micro-bead the EAACI: food allergy due to immunological cross- array for IgE detection: a feasibility study using allergenic reactions with common inhalant allergens. Allergy. molecules tested on a flexible multiplex flow cytometric 2015;70(9):1079–1090. immunoassay. e35697 PLoS One. 2012;7(4). https://doi.org/ 154. Demoly P, Lebel B, Arnoux B. Allergen-induced mediator 10.1371/journal.pone.0035697. Epub 2012 Apr 17. release tests. Allergy. 2003;58(7):553–558. 137. Incorvaia C, Mauro M, Ridolo E, Makri E, Montagni M, 155. Bridts CH, Sabato V, Mertens C, Hagendorens MM, De Ciprandi G. A pitfall to avoid when using an allergen Clerck LS, Ebo DG. Flow cytometric allergy diagnosis: microarray: the incidental detection of IgE to unexpected basophil activation techniques. Methods Mol Biol. allergens. J Allergy Clin Immunol Pract. 2015;3(6):879–882. 2014;1192:147–159. 138. Canonica GW, Ferrando M, Baiardini I, et al. Asthma: 156. McNeil BD, Pundir P, Meeker S, et al. Identification of a mast- personalized and precision medicine. Curr Opin Allergy Clin cell-specific receptor crucial for pseudo-allergic drug Immunol. 2018;18(1):51–58. reactions. Nature. 2015;519(7542):237–241. 139. Westman M, Lupinek C, Bousquet J, et al. Early childhood IgE 157. Van Gasse AL, Sabato V, Uyttebroek AP, et al. Immediate reactivity to pathogenesis-related class 10 proteins predicts moxifloxacin hypersensitivity: is there more than currently allergic rhinitis in adolescence. J Allergy Clin Immunol. meets the eye? Allergy. 2017;72(12):2039–2043. 2015;135(5), 1199-11206 e1-11. 158. Ebo DG, Sainte-Laudy J, Bridts CH, et al. Flow-assisted allergy 140. Schmid-Grendelmeier P. [Pollen allergy and diagnosis: current applications and future perspectives. immunotherapy]. Ther Umsch. 2012;69(4):239–248. Allergy. 2006;61(9):1028–1039. 141. Melioli G, Passalacqua G, Canonica GW, Baena-Cagnani CE, 159. Ebo DG, Bridts CH, Mertens CH, Hagendorens MM, Matricardi P. Component-resolved diagnosis in pediatric Stevens WJ, De Clerck LS. Analyzing histamine release by allergic rhinoconjunctivitis and asthma. Curr Opin Allergy flow cytometry (HistaFlow): a novel instrument to study the Clin Immunol. 2013;13(4):446–451. degranulation patterns of basophils. J Immunol Methods. 142. Prosperi MC, Belgrave D, Buchan I, Simpson A, Custovic A. 2012;375(1-2):30–38. Challenges in interpreting allergen microarrays in relation to 160. Sturm GJ, Kranzelbinder B, Sturm EM, Heinemann A, Groselj- clinical symptoms: a machine learning approach. Pediatr Strele A, Aberer W. The basophil activation test in the Allergy Immunol. 2014;25(1):71–79. diagnosis of allergy: technical issues and critical factors. 143. Hamilton RG, Kleine-Tebbe J. Molecular allergy diagnostics: Allergy. 2009;64(9):1319–1326. analytical features that support clinical decisions. Curr Allergy 161. Puan KJ, Andiappan AK, Lee B, et al. Systematic Asthma Rep. 2015;15(9):57. characterization of basophil anergy. Allergy. 2017;72(3):373– 144. Matricardi PM, Kleine-Tebbe J. Molecular allergology 384. https://doi.org/10.1111/all.12952. Epub 2016 Aug 8. between precision medicine and the choosing wisely 162. Faber M, Sabato V, De Witte L, et al. State of the art and initiative. Clin Exp Allergy. 2016;46(5):664–667. perspectives in food allergy (part I): diagnosis. Curr 145. Scala E, Villalta D, Uasuf CG, et al. An atlas of IgE sensitization Pharmaceut Des. 2014;20(6):954–963. patterns in different Italian areas. A multicenter, cross- 163. Sabato V, van Hengel AJ, De Knop KJ, et al. Basophil sectional study. Eur Ann Allergy Clin Immunol. 2018 activation reveals divergent patient-specific responses to Sep;50(5):217–225. https://doi.org/10.23822/EurAnnACI. thermally processed peanuts. J Investig Allergol Clin 1764-1489.67. Epub 2018 Jul 24. Immunol. 2011;21(7):527–531. 146. Roseler S, Balakirski G, Plange J, et al. [Anaphylaxis to PR-10 164. Rubio A, Vivinus-Nebot M, Bourrier T, Saggio B, Albertini M, proteins (Bet v1 homologues)]. Hautarzt. 2013;64(12):890–892. Bernard A. Benefit of the basophil activation test in deciding 147. Alvarado MI, Jimeno L, De La Torre F, et al. Profilin as a when to reintroduce cow's milk in allergic children. Allergy. severe food allergen in allergic patients overexposed to grass 2011;66(1):92–100. https://doi.org/10.1111/j.398-9995.2010. pollen. Allergy. 2014;69(12):1610–1616. 02432.x. 148. Scala E, Till SJ, Asero R, et al. Lipid transfer protein 165. Carroccio A, Brusca I, Mansueto P, et al. A cytologic assay for sensitization: reactivity profiles and clinical risk assessment in diagnosis of food hypersensitivity in patients with irritable an Italian cohort. Allergy. 2015;70(8):933–943. bowel syndrome. Clin Gastroenterol Hepatol. 2010;8(3):254– 260. https://doi.org/10.1016/j.cgh.2009.11.010. Epub Nov 149. Fernandez-Rivas M. Fruit and vegetable allergy. Chem 22. Immunol Allergy. 2015;101:162–170. 48 Ansotegui et al. World Allergy Organization Journal (2020) 13:100080 http://doi.org/10.1016/j.waojou.2019.100080 166. Song Y, Wang J, Leung N, et al. Correlations between 180. Pfaar O, Demoly P, Gerth van Wijk R, et al. Recommendations basophil activation, allergen-specific IgE with outcome and for the standardization of clinical outcomes used in allergen severity of oral food challenges. Ann Allergy Asthma immunotherapy trials for allergic rhinoconjunctivitis: an Immunol. 2015;114(4):319–326. https://doi.org/10.1016/j. EAACI Position Paper. Allergy. 2014;69(7):854–867. https:// anai.2015.01.006. doi.org/10.1111/all.12383. Epub 2014 Apr 25. 167. Ford LS, Bloom KA, Nowak-Wegrzyn AH, Shreffler WG, 181. Auge J, Vent J, Agache I, et al. EAACI Position paper on the Masilamani M, Sampson HA. Basophil reactivity, wheal size, standardization of nasal allergen challenges. Allergy. and immunoglobulin levels distinguish degrees of cow's milk 2018;73(8):1597–1608. tolerance. e1-3 J Allergy Clin Immunol. 2013;131(1):180–186. 182. Fauquert JL, Jedrzejczak-Czechowicz M, Rondon C, et al. https://doi.org/10.1016/j.jaci.2012.06.003. Epub Jul 20. Conjunctival allergen provocation test : guidelines for daily 168. Sato S, Tachimoto H, Shukuya A, et al. Utility of the peripheral practice. Allergy. 2017;72(1):43–54. blood basophil histamine release test in the diagnosis of 183. European Medicines Agency (EMA), Committee for hen's egg, cow's milk, and wheat allergy in children. Int Arch Medicinal Products for Human Use (CHMP). Guideline on the Allergy Immunol. 2011;155(Suppl 1):96–103. https://doi.org/ Clinical Development of Products for Specific Immunotherapy 10.1159/000327490. Epub 2011 Jun 1. for the Treatment of Allergic Diseases; 2009:3–13. Ref. CHMP/ 169. Glaumann S, Nopp A, Johansson SG, Rudengren M, EWP/18504/20 https://www.ema.europa.eu/en/documents/ Borres MP, Nilsson C. Basophil allergen threshold sensitivity, scientific-guideline/guideline-clinical-development-products- CD-sens, IgE-sensitization and DBPCFC in peanut-sensitized specific-immunotherapy-treatment-allergic-diseases_en.pdf. children. Allergy. 2012;67(2):242–247. https://doi.org/10. 184. Rosner-Friese K, Kaul S, Vieths S, Pfaar O. Environmental 1111/j.398-9995.2011.02754.x. Epub 2011 Nov 30. exposure chambers in allergen immunotherapy trials: current 170. Hoffmann-Sommergruber K, Pfeifer S, Bublin M. Applications status and clinical validation needs. J Allergy Clin Immunol. of molecular diagnostic testing in food allergy. Curr Allergy 2015;135(3):636–643. Asthma Rep. 2015;15(9):56. https://doi.org/10.1007/s11882- 185. U.S. Department of Health and Human Services Food and Drug 015-0557-6. Administration Center for Drug Evaluation and Research 171. Sabato V, Ebo DG. Hypersensitivity to neuromuscular (CDER). Allergic Rhinitis: Developing Drug Products for blocking agents: can skin tests give the green light for Re- Treatment Guidance for Industry; 2016. http://www.fda.gov/ exposure? J Allergy Clin Immunol Pract. 2018;6(5):1690– downloads/drugs/guidancecomplianceregulatoryinformation/ 1691. guidances/ucm071293.pdf. 172. Mangodt EA, Van Gasse AL, Decuyper I, et al. In vitro 186. Pfaar O, Calderon MA, Andrews CP, et al. Allergen exposure diagnosis of immediate drug hypersensitivity: should we go chambers: harmonizing current concepts and projecting the with the flow. Int Arch Allergy Immunol. 2015;168(1):3–12. needs for the future - an EAACI Position Paper. Allergy. 2017;25(10):13133. 173. Ebo DG, Faber M, Elst J, et al. In vitro diagnosis of immediate drug hypersensitivity during anesthesia: a review of the 187. Drug allergy: an updated practice parameter. Ann Allergy literature. J Allergy Clin Immunol Pract. 2018;6(4):1176–1184. Asthma Immunol. 2010;105(4):259–273. https://doi.org/10. 1016/j.anai.2010.08.002. 174. Leysen J, De Witte L, Sabato V, et al. IgE-mediated allergy to pholcodine and cross-reactivity to neuromuscular blocking 188. Ebo DG, Clarke RC, Mertes PM, Platt PR, Sabato V, agents: lessons from flow cytometry. Cytometry B Clin Cytom. Sadleir PHM. Molecular mechanisms and pathophysiology of 2013;84(2):65–70. perioperative hypersensitivity and anaphylaxis: a narrative review. Br J Anaesth. 2019 Jul;123(1):e38–e49. https://doi. 175. Van Gasse AL, Hagendorens MM, Sabato V, Bridts CH, De org/10.1016/j.bja.2019.01.031. Epub 2019 Mar 8. Clerck LS, Ebo DG. IgE to poppy seed and morphine are not useful tools to diagnose opiate allergy. J Allergy Clin 189. Blanca M, Romano A, Torres MJ, et al. Update on the Immunol Pract. 2015;3(3):396–399. evaluation of hypersensitivity reactions to betalactams. Allergy. 2009;64(2):183–193. https://doi.org/10.1111/j.398- 176. Ebo DG, Van Vaerenbergh M, de Graaf DC, Bridts CH, De 9995.2008.01916.x. Clerck LS, Sabato V. In vitro diagnosis of Hymenoptera venom allergy and further development of component 190. Brockow K, Garvey LH, Aberer W, et al. Skin test resolved diagnostics. Expert Rev Clin Immunol. 2014;10(3): concentrations for systemically administered drugs – an 375–384. ENDA/EAACI Drug Allergy Interest Group position paper. Allergy. 2013;68(6):702–712. https://doi.org/10.1111/all. 177. Blank S, Bilo MB, Ollert M. Component-resolved diagnostics 12142. Epub 2013 Apr 25. to direct in venom immunotherapy: important steps towards precision medicine. Clin Exp Allergy. 2018;48(4):354–364. 191. Torres MJ, Blanca M, Fernandez J, et al. Diagnosis of immediate allergic reactions to beta-lactam antibiotics. 178. Nullens S, Sabato V, Faber M, et al. Basophilic histamine Allergy. 2003;58(10):961–972. content and release during venom immunotherapy: insights by flow cytometry. Cytometry B Clin Cytom. 2013;84(3):173– 192. Kuyucu S, Mori F, Atanaskovic-Markovic M, et al. 178. Hypersensitivity reactions to non-betalactam antibiotics in children: an extensive review. Pediatr Allergy Immunol. 179. Agache I, Bilo M, Braunstahl GJ, et al. In vivo diagnosis of 2014;25(6):534–543. https://doi.org/10.1111/pai.12273. allergic diseases–allergen provocation tests. Allergy. 2015;70(4):355–365. https://doi.org/10.1111/all.12586. Epub 193. Laxenaire MC, Gastin I, Moneret-Vautrin DA, Widmer S, Gueant JL. 2015 Feb 12. Cross-reactivity of rocuronium with other neuromuscular blocking agents. Eur J Anaesthesiol Suppl. 1995;11:55–64. Volume 13, No. 2, February 2020 49 194. Moneret-Vautrin DA, Mertes PM. Anaphylaxis to general 210. Tatham AS, Shewry PR. Allergens to wheat and related anesthetics. Chem Immunol Allergy. 2010;95:180–189. cereals. Clin Exp Allergy. 2008;38(11):1712–1726. https://doi. https://doi.org/10.1159/000315951. Epub 2010 Jun 1. org/10.1111/j.1365-2222.2008.03101.x. Epub 2008 Sep 24. 195. Johansson SG, Florvaag E, Oman H, et al. National 211. Scibilia J, Pastorello EA, Zisa G, et al. Wheat allergy: a pholcodine consumption and prevalence of IgE-sensitization: double-blind, placebo-controlled study in adults. J Allergy a multicentre study. Allergy. 2010;65(4):498–502. https://doi. Clin Immunol. 2006;117(2):433–439. org/10.1111/j.398-9995.2009.02193.x. Epub 2009 Oct 1. 212. Martens M, Schnoor HJ, Malling HJ, Poulsen LK. Sensitization 196. Hagau N, Gherman N, Cocis M, Petrisor C. Antibiotic-induced to cereals and peanut evidenced by skin prick test and immediate type hypersensitivity is a risk factor for positive specific IgE in food-tolerant, grass pollen allergic patients. allergy skin tests for neuromuscular blocking agents. Allergol Clin Transl Allergy. 2011;1(1):15. https://doi.org/10.1186/ Int. 2016;65(1):52–55. https://doi.org/10.1016/j.alit.2015.07. 2045-7022-1-15. 007. Epub Aug 29. 213. Kanda N, Hau CS, Tada Y, Sato S, Watanabe S. Decreased 197. Castells MC, Tennant NM, Sloane DE, et al. Hypersensitivity serum LL-37 and vitamin D3 levels in atopic dermatitis: reactions to chemotherapy: outcomes and safety of rapid relationship between IL-31 and oncostatin M. Allergy. desensitization in 413 cases. J Allergy Clin Immunol. 2012;67(6):804–812. https://doi.org/10.1111/j.398-9995. 2008;122(3):574–580. https://doi.org/10.1016/j.jaci.2008.02. 2012.02824.x. Epub 2012 Apr 10. 044. Epub May 27. 214. Johnsen CR, Sorensen TB, Ingemann Larsen A, et al. Allergy 198. Sese L, Gaouar H, Autegarden JE, et al. Immediate risk in an enzyme producing plant: a retrospective follow up hypersensitivity to iodinated contrast media: diagnostic study. Occup Environ Med. 1997;54(9):671–675. accuracy of skin tests and intravenous provocation test with 215. Quirce S, Cuevas M, Diez-Gomez M, et al. Respiratory allergy low dose. Clin Exp Allergy. 2016;46(3):472–478. https://doi. to Aspergillus-derived enzymes in bakers' asthma. J Allergy org/10.1111/cea.12703. Clin Immunol. 1992;90(6 Pt 1):970–978. 199. Bircher AJ, Harr T, Hohenstein L, Tsakiris DA. Hypersensitivity 216. Baur X, Czuppon AB. Allergic reaction after eating alpha- reactions to anticoagulant drugs: diagnosis and management amylase (Asp o 2)-containing bread. A case report. Allergy. options. Allergy. 2006;61(12):1432–1440. 1995;50(1):85–87. 200. Gomez E, Blanca-Lopez N, Torres MJ, et al. Immunoglobulin 217. Pepys J, Longbottom JL, Hargreave FE, Faux J. Allergic E-mediated immediate allergic reactions to dipyrone: value reactions of the lungs to enzymes of Bacillus subtilis. Lancet. of basophil activation test in the identification of patients. Clin 1969;1(7607):1181–1184. Exp Allergy. 2009;39(8):1217–1224. https://doi.org/10.1111/ j.1365-2222.2009.03237.x. Epub 2009 Apr 7. 218. Schweigert MK, Mackenzie DP, Sarlo K. Occupational asthma and allergy associated with the use of enzymes in the 201. van Kampen V, de Blay F, Folletti I, et al. EAACI position detergent industry–a review of the epidemiology, toxicology paper: skin prick testing in the diagnosis of occupational type and methods of prevention. Clin Exp Allergy. 2000;30(11): I allergies. Allergy. 2013;68(5):580–584. https://doi.org/10. 1511–1518. 1111/all.12120. Epub 2013 Feb 15. 219. Larsen AI, Johnsen CR, Frickmann J, Mikkelsen S. Incidence 202. Turjanmaa K, Alenius H, Reunala T, Palosuo T. Recent of respiratory sensitisation and allergy to enzymes among developments in latex allergy. Curr Opin Allergy Clin employees in an enzyme producing plant and the relation to Immunol. 2002;2(5):407–412. exposure and host factors. Occup Environ Med. 2007;64(11): 203. Cullinan P, Brown R, Field A, et al. Latex allergy. A position 763–768. paper of the British society of allergy and clinical 220. Paulsen E, Skov PS, Bindslev-Jensen C, Voitenko V, immunology. Clin Exp Allergy. 2003;33(11):1484–1499. Poulsen LK. Occupational type I allergy to christmas cactus 204. Aalberse RC. Food allergens. Environ Toxicol Pharmacol. (schlumbergera). Allergy. 1997;52(6):656–660. 1997;4(1-2):55–60. 221. Nagendran V, Wicking J, Ekbote A, Onyekwe T, Garvey LH. 205. Santos A, Van Ree R. Profilins: mimickers of allergy or IgE-mediated chlorhexidine allergy: a new occupational relevant allergens? Int Arch Allergy Immunol. 2011;155(3):191– hazard? Occup Med (Lond). 2009;59(4):270–272. https://doi. 204. https://doi.org/10.1159/000321178. Epub 2011 Feb 2. org/10.1093/occmed/kqp042. Epub 2009 Mar 26. 206. Cartier A. The role of inhalant food allergens in occupational 222. Wisnewski AV, Liu J, Redlich CA. Antigenic changes in human asthma. Curr Allergy Asthma Rep. 2010;10(5):349–356. albumin caused by reactivity with the occupational allergen https://doi.org/10.1007/s11882-010-0130-2. diphenylmethane diisocyanate. Anal Biochem. 2010;400(2): 251–258. https://doi.org/10.1016/j.ab.2010.01.037. Epub Feb 1. 207. Raulf M, Buters J, Chapman M, et al. Monitoring of occupational and environmental aeroallergens– EAACI 223. Wisnewski AV, Jones M. Pro/Con debate: is occupational asthma position paper. Concerted action of the EAACI IG induced by isocyanates an immunoglobulin E-mediated occupational allergy and aerobiology & air pollution. Allergy. disease? Clin Exp Allergy. 2010;40(8):1155–1162. https://doi. 2014;69(10):1280–1299. org/10.1111/j.1365-2222.2010.03550.x. Epub 2010 Jun 7. 208. Baur X. Baker's asthma: causes and prevention. Int Arch 224. Wisnewski AV. Developments in laboratory diagnostics for Occup Environ Health. 1999;72(5):292–296. isocyanate asthma. Curr Opin Allergy Clin Immunol. 2007;7(2):138–145. 209. Palosuo K. Update on wheat hypersensitivity. Curr Opin Allergy Clin Immunol. 2003;3(3):205–209. 50 Ansotegui et al. World Allergy Organization Journal (2020) 13:100080 http://doi.org/10.1016/j.waojou.2019.100080 225. Jares EJ, Baena-Cagnani CE, Gomez RM. Diagnosis of 243. Lewith GT, Kenyon JN, Broomfield J, Prescott P, Goddard J, occupational asthma: an update. Curr Allergy Asthma Rep. Holgate ST. Is electrodermal testing as effective as skin prick 2012;12(3):221–231. tests for diagnosing allergies? A double blind, randomised block design study. BMJ. 2001;322(7279):131–134. 226. Corren J, Shapiro G, Reimann J, et al. Allergen skin tests and free IgE levels during reduction and cessation of omalizumab 244. Semizzi M, Senna G, Crivellaro M, et al. A double-blind, therapy. J Allergy Clin Immunol. 2008;121(2):506–511. https:// placebo-controlled study on the diagnostic accuracy of an doi.org/10.1016/j.jaci.2007.11.026. electrodermal test in allergic subjects. Clin Exp Allergy. 2002;32(6):928–932. 227. Bernstein IL, Li JT, Bernstein DI, et al. Allergy diagnostic testing: an updated practice parameter. Ann Allergy Asthma Immunol. 2008;100(3 Suppl 3):S1–S148. 228. WK D. Immunology and Allergy Clinics of North America. Philadelphia, Pensylvania: Saunders Company; 2001. 229. Demoly PPV, Bousquet J. In Vivo Methods for Study of Allergy: Skin Tests, Techniques and Interpretation. sixth ed. New York: Mosby; 2003. 230. Torres MJ, Romano A, Celik G, et al. Approach to the diagnosis of drug hypersensitivity reactions: similarities and differences between Europe and North America. Clin Transl Allergy. 2017;7:7. 231. Brockow K, Romano A. Skin tests in the diagnosis of drug hypersensitivity reactions. Curr Pharmaceut Des. 2008;14(27): 2778–2791. 232. Ebo DG, Leysen J, Mayorga C, Rozieres A, Knol EF, Terreehorst I. The in vitro diagnosis of drug allergy: status and perspectives. Allergy. 2011;66(10):1275–1286. https://doi.org/ 10.1111/j.1398-9995.2011.02661.x. Epub 2011 Jun 7. 233. Burks AW, Williams LW, Casteel HB, Fiedorek SC, Connaughton CA. Antibody response to milk proteins in patients with milk-protein intolerance documented by challenge. J Allergy Clin Immunol. 1990;85(5):921–927. 234. Firer MA, Hoskings CS, Hill DJ. Humoral immune response to cow's milk in children with cow's milk allergy. Relationship to the time of clinical response to cow's milk challenge. Int Arch Allergy Appl Immunol. 1987;84(2):173–177. 235. Antico A, Pagani M, Vescovi PP, Bonadonna P, Senna G. Food-specific IgG4 lack diagnostic value in adult patients with chronic urticaria and other suspected allergy skin symptoms. Int Arch Allergy Immunol. 2011;155(1):52–56. 236. Chambers VV, Glaser J. The incidence of subsequent ragweed pollinosis in symptom-free persons having positive reactions to ragweed pollen extract. J Allergy. 1958;29(3):249–257. 237. Lehman CW. The leukocytic food allergy test: a study of its reliability and reproducibility. Effect of diet and sublingual food drops on this test. Ann Allergy. 1980;45(3):150–158. 238. Sethi TJ, Lessof MH, Kemeny DM, Lambourn E, Tobin S, Bradley A. How reliable are commercial allergy tests? Lancet. 1987;1(8524):92–94. 239. Barrett S. Commercial hair analysis. Science or scam? J Am Med Assoc. 1985;254(8):1041–1045. 240. Ernst E. Iridology: a systematic review. Forschende Komplementärmed. 1999;6(1):7–9. 241. Garrow JS. Kinesiology and food allergy. Br Med J. 1988;296(6636):1573–1574. 242. Ludtke R, Kunz B, Seeber N, Ring J. Test-retest-reliability and validity of the Kinesiology muscle test. Complement Ther Med. 2001;9(3):141–145.