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Editorials

Novel in silico technology in combination with microarrays: a state-of-the-art technology for allergy diagnosis and management?

, &
Pages 1559-1561 | Published online: 05 Nov 2014

Abstract

‘Allergen microarrays, in poly-sensitized allergic patients, represent a real value added in the accurate IgE profiling and in the identification of allergen(s) to administer for an effective allergen immunotherapy.’ Allergen microarrays (AMA) were developed in the early 2000s to improve the diagnostic pathway of patients with allergic reactions. Nowadays, AMA are constituted by more than 100 different components (either purified or recombinant), representing genuine and cross-reacting molecules from plants and animals. The cost of the procedure had suggested its use as third-level diagnostics (following in vivo– and in vitro–specific IgE tests) in poly-sensitized patients. The complexity of the interpretation had inspired the development of in silico technologies to help clinicians in their work. Both machine learning techniques and expert systems are now available. In particular, an expert system that has been recently developed not only identifies positive and negative components but also lists dangerous components and classifies patients based on their potential responsiveness to allergen immunotherapy, on the basis of published algorithms. For these characteristics, AMA represents the state-of-the-art technology for allergy diagnosis in poly-sensitized patients.

The large majority of patients with suspected allergic symptoms can be diagnosed by identifying allergen(s) with the standard in vitro and in vivo methods (which include first-level assays, such as the skin prick test, and second-level assays, such as detection of specific anti-allergen IgE). Nonetheless, in a small but consistent number of complex cases (i.e., multiple sensitizations with the standard tests), a third-level approach is needed. Third-level in vitro assays can reliably identify both genuine and cross-reacting components Citation[1]. This information can be obtained by either using single components (both recombinant allergens and purified natural allergen molecules) or a multiplex approach, which is represented by allergen microarrays (AMA). The reasons for a third-level approach are related to improving not only the diagnostic accuracy but also, more importantly, a better definition of the causal allergen(s) involved and, consequently, to decide the allergen(s) to be included in allergen immunotherapy (AIT) prescriptions accurately Citation[2]. This consideration is especially relevant in European countries, where only a few different allergenic extracts are administered, whereas in other countries, such as USA, virtually all the positive allergens are administered and are mixed together Citation[3].

AMAs were developed in early 2000 and immediately represented a very interesting tool for allergy diagnosis. Although the first versions of the assays were considered suitable to support a complete serological IgE sensitization diagnosis, in 2010, the presence of discrepancies between specific IgE on whole extracts (sIgE) and AMA (namely ImmunoCAP ISAC™, Thermo Fisher Scientific ImmunoDiagnostics Division, Milano, Italy) became evident Citation[4]. For example, the Phleum pratense components were well represented in AMAs (and discrepancies are very rare). For other allergens (such as Ragweed, Alternaria, Aspergillus and dog), the agreement between positive sIgE and positive AMA was reported to range between 13 and 30%. The possible explanations for these discrepancies were the sIgE concentration was too low, there was an absence of the relevant component in the AMA panel and the presence of an IgE against the cross-reacting molecules (such as profilins, polcalcins and PR-10) in the whole allergen extract (that resulted in positive standard tests) in the absence of a specific IgE toward genuine allergens (such as Amb a 1, which is present in AMA). These considerations suggested that AMA should be used in already well-studied patients, as a third-level assay Citation[1,5].

The specificity of AMA is extremely high; thus, if an IgE toward an allergenic component is positive, the sIgE to the allergen extract are positive too Citation[4]. Sensitivity is still a matter of debate. Indeed, the correlation analysis of the sIgE and AMA results seems to demonstrate that the sensitivity is not entirely reliable Citation[4]. In this context, other authors have shown that the comparison of single-plexed recombinant molecules with the same components in multiplexed AMA is good Citation[6].

AMA advantages include the availability of a wide spectrum of components (mainly inhalants and food, which are genuine and cross-reactive), the possibility to obtain specific added values Citation[4,7], an accurate description of the IgE profile by the identification of sensitization to cross-reacting versus genuine components Citation[7–10], the direct identification of an IgE that is specific for potentially harmful components Citation[5] and, finally, the need of only a few microliters of serum (in particular for pediatric patients) to obtain a large screening Citation[11–13]. The disadvantages include the cost of a single test, certain difficulties in the interpretation Citation[14,15], the possibility that unexpected results will need to be interpreted and explained to the patients Citation[5] and the presence of discrepancies between a skin prick test sIgE results and AMA results.

Therefore, after more than 10 years of experience using microarrays in allergy diagnoses, in the authors’ opinion, AMA represents a state-of-the-art technology for allergy diagnoses, provided that certain aspects are carefully considered. The authors’ opinion is that the large majority of patients with inhalant allergen sensitization can be well studied using in vivo skin prick test, sIgE and a small panel of components, both recombinant and highly purified from natural allergen extracts Citation[5]. Only in highly poly-sensitized patients, in which not only the risk of a sensitization to cross-reacting components is present but also a pollen-food syndrome is suspected, AMA can be used Citation[5]. The choice between single or multiplexed diagnostics will be based on the number of tests required for the evaluation of the proper number of components. Specifically, if it is more than 10, an AMA should be indicated. Of course, in very complex cases (e.g., a food allergy, anaphylaxis and young pediatric patients), microarrays should be routinely used.

The introduction of a large panel of molecules, which are characterized by specific features, in allergy diagnostics, can be considered a real revolution. For this reason, an article was published to improve the knowledge of the ‘molecular allergy terminology’ Citation[16]. The large number of results produced by a microarray analysis allows for additional data to be obtained by the use of a computer learning machine approach Citation[15]. The interpretation of AMA was particularly investigated in an article Citation[14] that described the development of an expert system, which was dedicated to ImmunoCAP ISAC. A number of added values, which provided results that were different from those that were described at the beginning of our experience, were identified. For example, a cluster analysis Citation[13] of the ratio between genuine and cross-reacting components allowed for the identification of different phenotypes that seem to correspond to an increased risk of AIT failure Citation[8]. Another additional value of AMA, following the interpretation proposed by Allergenius, is the explanation of the discrepancies between sIgE test and AMA results. Indeed, any discrepancies should be carefully managed. For example, it should be useful to check whether other allergen sources that are present in AMA have positive results in their cross-reacting components. Thus, this would be a demonstration of the positivity of the whole extract allergen test.

Microarray technology is improving with time, and not only are the number of spotted components increasing but also the strategy for the component choice is also getting better. Future trends include, for example, MeDALL Citation[17], which is a novel approach with 170 different allergens. Of course, the costs and the interpretation difficulties will be proportionally increased; however, such a tool will have a tremendous impact on allergy diagnoses if used by allergists with specific experience in molecular diagnostics. In addition, the molecule-based approach is expected to be of special relevance in the diagnosis of life-threatening diseases, such as hymenoptera venom allergy Citation[18]. Other approaches also seem to be interesting, including flow cytometry Citation[19], which should allow for an accurate serological analysis using very common laboratory instruments (at least, more common than a microarray reader). To improve the study of the fine relationships between components and IgE, plasmon resonance Citation[20] seems to be a very powerful tool. If MeDALL and flow cytometry are available in a short time, the clinical relevance of IgE avidity for allergens is at present largely unknown. For this reason, it is reasonable to consider that surface plasmon resonance will be a research tool in the near future.

On the basis of these considerations, we need to determine which indications could be suggested for the use of microarray allergy tests in 2014.

These tests should mainly be used for an accurate description of the IgE profile and the allergen identification for AIT in poly-sensitized patients. Although there is a large consensus for the former Citation[5], the latter is still a matter of debate. Clear evidence that the use of components, including AMA, may significantly improve the quality of the administered AIT has been already given Citation[7,9]. Indeed, it was also demonstrated that a better result is obtained using AIT in patients who are sensitized against genuine components Citation[8]. In addition, the biochemical nature of both genuine and cross-reacting components should be considered at the light of AIT identification, for example. Moreover, the role of cross-reactive carbohydrate determinants that are present on components purified from allergen extracts was considered to be relevant for the documented risk of false-positive results Citation[21] and the consequent possible errors in the identification of allergen sources for AIT Citation[10].

AMA technology represents a state-of-the-art method in allergy diagnosis if the patient is characterized by a poly-sensitization, in particular, by a pollen-food syndrome; a molecular strategy for the identification of allergen sources for AIT is used and the user has the scientific culture or specific informatics tools for the interpretation of the AMA results.

Acknowledgements

The article was revised by American Journal Experts only for English grammar, editing and typing.

Financial & competing interests disclosure

The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the article. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.

No writing assistance was utilized in the production of this manuscript.

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