Pilot study: assessing the clinical diagnosis of allergy in atopic children using a microarray assay in addition to skin prick testing and serum specific IgE

Atopic dermatitis (AD) is a common inflammatory skin disease affecting 15–20 % of children in developed countries [1]. It usually starts in the first few months of life and is frequently outgrown by 15 years of age although there is a large amount of variability between children [2].

It is well known that there are close links between food allergy and AD as it is a significant risk factor for the development of food sensitization and food allergy [2–4]. Patients with higher levels of total Immunoglobulin-E (IgE) in their blood, which is often the case in children with AD, are more likely to be sensitised to foods [1, 5]. Kumar et al. found that children with AD had an 8.4-fold higher risk of developing a food allergy [6]. de Benedictis et al. demonstrated that of infants with AD in various countries around the world, 55.5 % were sensitised to at least one allergen and 52.9 % had a raised serum total IgE [7].

Due to the increased likelihood of children with AD being sensitised to various food allergens, it can be difficult to establish whether these children have true food allergy. The European Academy of Allergy and Clinical Immunology (EAACI) guidelines recommend the use of clinical history, skin prick tests (SPT) and serum specific IgE (sp-IgE) tests in the investigation of food allergy but the gold standard for diagnosis remains the oral food challenge [8]. AD is often associated with aeroallergen sensitization and in the context of a high level of total IgE, there are often low levels of sp-IgE to numerous aeroallergens [1]. These are of unclear clinical significance.

Immunocap^® ISAC-112 (Thermo Fisher/Phadia AB, Uppsala, Sweden) is an in vitro assay for the measurement of allergen sp-IgE antibodies in human plasma [9]. It is intended to aid in the diagnosis of IgE-mediated allergic disorders. There is currently no advice on the use of ISAC testing worldwide [8, 10]. The National Institute of Allergy and Infectious Diseases (NIAID) guidelines do not recommend the combined use of SPTs and sp-IgE tests for routine diagnosis of food allergy and instead recommend individual SPT and sp-IgE tests to help identify foods that might be provoking a reaction alongside clinical history [10]. Thus, the utilization of a broad screening test is controversial. Whilst it may facilitate a more accurate clinical allergy diagnosis for specific atopic conditions [11, 12], it has been suggested that identifying patterns of allergen responses for patients with AD is less reliable [12]. Spergel et al. [13] found that the predictive values of food-antigen sp-IgE were not clinically significant in predicting the development of food allergy in children with mild to moderate AD. However, this study performed sp-IgE only for common food allergens, potentially missing less common allergens [13].

The ISAC-112 has the potential as a single test modality, to identify food and aeroallergen sensitizations in at risk atopic children. Hatzler et al. [14] looked at the IgE response to grass pollen antigens and demonstrated that sensitization can occur years before the onset of clinical disease through a process called “molecular spreading”. Therefore, the use of microarrays could be convenient in the setting where common food allergens have not yet been introduced thereby limiting the value of a detailed clinical history.

The aim of this pilot study was to assess the use of current diagnostic modalities (clinical consultation, SPT, sp-IgE) in a specialist allergy clinic in children with AD. We also assessed whether Immunocap^® ISAC sIgE-112 testing could improve the diagnosis of food and aeroallergen allergy in these children.

A total of 30 participants were recruited for this study. The mean age of the children was 3.91 years (SD 3.3) and 60 % (n = 18) were male. Of the group, 53.3 % (n = 16) and 46.7 % (n = 14) had SCORAD scores suggestive of moderate and severe AD, respectively.

Comparison of SPT, sp-IgE and ISAC tests

We compared the percentage of children who had a positive test result on SPT, sp-IgE and ISAC for common food and aeroallergens (Fig. 1). Overall, apart from egg allergen, the sp-IgE tests had a higher percentage of positive results for each allergen compared to SPT and ISAC tests. When comparing SPT and ISAC tests a relatively similar percentage of positive results for allergens were noted. For example, for birch pollen, 36 % had a positive SPT and 36.7 % had a positive ISAC test.

We also looked at the agreement between the three different tests (Table 1). There was good agreement between the SPT and ISAC tests for sensitization to selected food and aeroallergens. There was relatively good agreement between sp-IgE and ISAC tests for house dust mite and Alternaria mould. There was also good agreement between SPT and sp-IgE for house dust mite. Other test and allergen comparisons showed low levels of agreement.

Allergen	SPT and IgE	SPT and ISAC	IgE and ISAC
Peanut	0.42 (0.02)	0.54 (0.003)	0.36 (0.023)
Egg	0.29 (0.072)	0.43 (0.01)	0.19 (0.148)
Cow’s milk	0.39 (0.010)	1.00 (<0.001)	0.38 (0.006)
Cat	0.53 (0.007)	0.86 (<0.001)	0.56 (0.003)
Dog	0.30 (0.029)	0.18 (0.192)	0.38 (0.008)
Birch pollen	0.20 (0.175)	0.65 (0.001)	0.55 (0.003)
Timothy grass pollen	0.28 (0.063)	0.73 (<0.001)	0.44 (0.005)
House dust mite	0.73 (<0.001)	0.58 (0.002)	0.92 (<0.001)
Alternaria mould	−0.25 (0.855)	0.06 (0.390)	0.70 (<0.001)

Figures are Kappa statistics (p values) where represent the level of agreement between allergen sensitization tests having taken into account any agreement that is expected by chance. Results of 0.61–0.99 signify excellent agreement, 0.41–0.60 signifies moderate agreement and 0.01–0.4 signifies only slight to fair agreement [18]

There is little published data on the use of ISAC testing in AD children. Prosperi et al. [12] found that ISAC testing had reasonable discrimination in detecting allergens associated with asthma, rhino-conjunctivitis, wheeze and airway hypersensitivity but it was not useful for detecting allergens associated with eczema in a paediatric cohort. Our study demonstrates that there is a high prevalence of food and aeroallergen sensitization in children with moderate to severe AD. Although the use of the ISAC-112 testing did not significantly change the number of allergic sensitizations or diagnoses compared to traditional test modalities of SPT and sp-IgE tests, the majority of participants had a change in specific allergy diagnoses when all of the ISAC-112 results were utilized.

Our results demonstrate that for the food allergens tested, sp-IgE testing yielded more positive results as compared to SPT and ISAC, despite the ISAC test covering a greater array of pre-set food allergen components. It has been suggested that the binding characteristics of the ISAC-112 are more specific as they bypass the high rates of sensitization that can occur in AD children who have a raised total IgE. However, the number of food and aeroallergen sensitizations was not lower using ISAC tests, which suggests that the ISAC test may not circumvent this issue.

In recent years, several studies have been performed which demonstrate strong correlation between the ISAC-112 test and other single-plex allergy tests (SPT, sp-IgE tests) [19, 20]. More specifically, studies have shown a good correlation between the testing methods for aeroallergens such as grass pollens [21] and house dust mite in allergic rhinitis patients [11]. In our cohort, the results available from the ISAC-112 test resulted in a change in diagnosis of allergic rhino-conjunctivitis to non-allergic rhino-conjunctivitis for six children. This supports the view that the ISAC test may be helpful at differentiating between allergic and non-allergic rhino-conjunctivitis compared to clinical history and single-plex tests. In fact, the WAO-ARIA-GA²LEN consensus guidelines clarify the role that molecular allergy diagnostics such as ISAC-112 can have on resolving genuine versus cross-reactive sensitization in poly-sensitized patients, such as those with AD; hence facilitating a more accurate assessment of the severity of risks to allergens [22].

Another consideration that allergy diagnostic centres may have is the cost of running these diagnostic tests. In our study, SPT cost £15.95 per vial of allergen solution (Alyostal^® Stallergenes), which is used for approximately 60 SPT (£0.27 per allergen per test). Skin prick testing requires a trained health care practitioner, safe medical facilities and time to perform and interpret the tests. Sp-IgE tests cost £14.30 per allergen compared to the ISAC test, which costs £250 for 112 allergen components. Both of these tests require phlebotomy services, laboratory staff and the necessary laboratory equipment. For the patients with a clinical history that identifies likely target allergens, confirmation by SPT and sp-IgE testing may prove sufficient and more cost-effective. However, in the poly-sensitised allergic patient with no identifiable causative allergens, based on costing in our tertiary centre, the ISAC test would prove to be more cost effective if the clinician wanted to test for more than 18 allergens or allergen components. Although this seems like a considerable number of allergens to test for, it is not an uncommon practice in investigating AD children. It is in the children with equivocal histories or poly-allergenicity that the ISAC test may provide clinicians with a more comprehensive overview of sensitization to common food and aeroallergens.

The limitations of this study include small sample size recruited in a tertiary allergy clinic, which makes it difficult to extrapolate results to the general population. In smaller hospitals, ISAC testing may not be available which make these results less applicable to general clinical practice. However, our study focused on children with AD, which is an extremely common childhood atopic phenomena, of which allergic diagnoses can be challenging to diagnose. Although we tested for only a small number of allergens, these represent the most common food and aeroallergens in the UK. We assessed the additional diagnostic value of the ISAC-112 as opposed to the diagnostic performance of each of the modalities in isolation against a gold standard diagnostic provocation test. We also acknowledge that it would be beneficial to compare the different test modality outcomes for allergic diagnoses between AD children and a control population (i.e. children without AD) in future work. Also, the fact that the clinical examiners in this study were experienced specialist paediatric allergists can be seen as both an advantage and limitation of the study; this provides greater validity to the clinical diagnoses made, but different allergy tests may be interpreted differently in less experienced hands.

This pilot study demonstrates that there is a defined role for the use of ISAC testing in diagnosing sensitization and allergy in children with AD, which compares favourably with existing modalities of testing (i.e. SPT, sp-IgE tests). Depending on the clinical setting and the patient population (i.e. those with AD), the ISAC test may be useful in leading to a change in diagnosis and be more cost effective. Further investigation in a larger population, testing each test in isolation as well as against the gold standard (i.e. OFC), is needed.