The frequency of single specific immunoglobulin E and allergen mixes with a MAST (multiple-antigen simultaneous test) technique

Introduction

As environmental elements have changed in modern life, the number of patients diagnosed with an allergy disorder has increased steadily. Meanwhile, the physician should objectively assess the origin of complaints and determine whether they are caused by a predicted allergy or an unexpected allergic reaction before treating patients [1].

The term “allergy” describes a hypersensitive immune response to any allergen that is mostly immunoglobulin E (IgE)-mediated. However, in a tiny minority of cases, cellular mechanisms may also be at play. The industrialized world has seen an increase in allergy cases worldwide over the past 50 years [2]. Despite these increases, even in the developed world, the treatment of people with allergic disorders is disorganized and far from ideal.

Because of the high incidence of allergies, allergy should be considered a serious public health issue and classified as a noncommunicable disease. Finally, the declaration of the World Allergy Organisation recommends that further research should be carried out to establish the true burden of Allergic Diseases and Asthma as well as initiating more preventive measures for allergens and air quality [3].

The World Allergy Organization (WAO) is deeply concerned about the growing global burden of allergic diseases and is committed to increased global collaboration and communication, engaging governments and policymakers to channel resources and efforts toward recognizing allergic disease as a public health issue. Studies on the prevalence of IgE-mediated allergies are limited in Turkey, as in the rest of the world. The diagnosis of allergic illnesses depends on determining which allergens are causing the problem in a certain patient. Showing that serum contains allergen-specific immunoglobulin E (sIgE) is a substitute method of identifying and detecting the offending allergen [4]. A widely used technique for the identification of allergies worldwide is the in vivo skin prick test (SPT) [5]. However, when used in clinical settings, this test has certain drawbacks. Patients with allergies who are unable to stop taking their medicine cannot be tested with SPT since the use of antihistamines, for example, can alter the results. In vitro tests intended for clinical use have been developed as an alternative to SPT [6]. An increasing number of clinical settings are using commercially accessible immunochemical diagnostics [7]. Multiple allergens cannot be detected at the same time as singleplex assays and costs per allergen are higher than with multiplex tests, which is one of the major challenges for organizing these types of studies. Several multiplex allergy screening technologies for clinical use have recently been developed. These techniques, however, have significant disadvantages, including a lengthy test time and low sensitivity and specificity [8], [9], [10]. Another drawback of multiplex testing is their limited/non-sensitive detection rate for certain SpIgEs examined in panels. Therefore, increasing the number of allergens does not ensure a better identification of the causing allergens. PROTIA Allergy-Q96^® is a recently developed Multiple-Antigen Simultaneous Test (MAST) technique to identify allergy causes.

The population-based studies conducted in various countries with different methodologies measured a specific allergen or an allergen group, but multiplex frequency studies having various types of allergen groups were not organized over our population, yet. The current study on the child and adult population is part of a larger and first study on the frequency of multiplex allergens measured in a single analysis in Turkey. We aimed to assess the frequency of allergens causing severe allergic diseases in Turkey over a single period to obtain comparable data across the country using MAST technique. The system has a high range of allergen menu/groups (80 single and nine mixed allergens) simultaneous detection which allowed us to measure efficiently the frequency of each type in a routine selected population.

Materials and methods

Results

A total of 100 patients seeking allergy care clinics at different hospitals in Turkey were recruited. The serum samples from those patients (median age, 22 years, range 1–71 years) 40 males and 60 females were tested for the full panel in this study. Characteristics of all participants and positive SpIgE found participants were summarized in Supplementary Material 2. The participants were analyzed in two different groups to evaluate the age distribution of the allergen-SpIgE positivity and negativity. The median age of the pediatric group (1–18 years) was 6 years, whereas the adult group (>18 years) was 32 years, ending in age 71. According to the two age groups, allergen positivity rates were: 62.5 % (n=30) (1–18 years) (p.0.03) and 15.4 % (n=8) (over 18 years), showing a statistically significant p-value of 0.05.

Among participants with allergen-positive, 4 (11.3 %) of them stated that they were allergic to only one SpIgE, and 34 (5.8 %) of them were allergic to more than one. In one patient, the highest allergen type with a number of 59 SpIgE was seen. He was in Class 5 and contained allergens: Bermuda grass and Timothy grass.

As we grouped the patients according to the classification of their allergy level, we did not find any allergen in 62 patients, which means they were in Class 0 (<0.35 kU/mL). The rest (n=38) of the patients were positive for at least one or more sIgE allergens (a relative frequency of 38 %). The grouping of 100 patients according to classification of allergy level was given in Supplementary Material 3. We decided to separate the groups into three which was not the same as the manufacturer’s classification given in Table 1. The first reason was that we did have not enough participants in each group to calculate the differences. Secondly, most clinicians preferred the average assumption of classifications to be more clear in therapy decision-making and mostly relied on allergy skin tests [13]. Whenever we graded allergen amount into three groups to summarize allergen classification, we observed a percentage of low grade (44.7 %) levels compared to medium and high (total 55.3 %) grades with a statistically significant p-value of <0.04 (Table 1).

Overall, a number of 478 SpIgE-positive allergen types were obtained from one hundred patients. Twenty-nine SpIgE were not measured in the patient’s serum samples and the list of them was given according to manufacturer group distribution in Supplementary Material 4. The distribution of each allergen group having measured positive allergens was given in Table 2 with their positivity ratio (%). The highest value was observed at Common Allergen Group, with a ratio of 36.8 % and the lowest value was at animals materials (0.60 %).

The gender distribution of each allergen group was given in Figure 1. Significant differences were observed for indoor acars, and meats, and animal products, with a higher number for males (p<0.001).

Figure 1:

Distribution of the frequencies of the allergen groups according to gender in the total number of positive allergens. Differences were significant in indoor acars, and meats, and animal products (p<0.001).

As we measured each allergen in total participants we found that D.farinea was the most (28 %) allergen followed by grass mix (21 %), house dust (21 %), cow’s milk (20 %), and Acarus siro (20 %) followed the most (Table 3). Among participants who were allergic to at least one or more sIgE allergens, 100 % of them stated that they were the most allergic to grass mix, Timothy grass, beef, and fishes (shell) mix followed by Bermuda grass (89.4 %), potato (68.4 %), and goldenrod (60.5 %). Of them, they were the least allergic to rabbits (5.2 %). We analyzed 11 different allergen groups and wanted to find out the most frequent ones. For that reason, as we summated the positive frequency rates in the allergen-positive group, pollens were the highest (665.4 %), followed by common allergens (354 %). The lowest frequency was seen in animals materials group (13 %).

The assay measures total IgE at the same time in one process. Among 62 patients who were negative for allergens, 61 patients (98.4 %) have a total IgE level in reference ranges, whereas only one patient (35 years old, man) has a value of 127 U/mL which was high (reference range:87 U/mL). Also, for allergen-positive 38 patients, 16 patients (42 %) have a total IgE level in reference ranges, whereas 22 patients (58 %) have high levels. The degree of agreement of total IgE levels in allergen-positive participants divided into two age groups was given in Table 4. A total of significant agreement (kappa value:0.609) was obtained (p<0.001) with a higher degree in adults (kappa value:0.766) vs. pediatric groups (0.462).

Discussion

The multiple allergen simultaneous test is useful in identifying causes of allergy when it is difficult to predict allergens or when the subject is likely to be sensitized to multiple allergens. To solve this problem and to measure the frequency of several types of allergens in Turkey, we used the PROTIA Allergy-Q96^®, a multiplex sIgE measurement assay for total crude allergens.

We received two types of data from serum sIgE assays: qualitative information (0–6 classes) and quantitative measures in SI units (kU/L), under the WHO International Reference Preparation 75/502 for Human IgE [14]. The former is more crucial in the case of medication or occupational allergies [15]. Quantitative sIgE assessment can assist clinicians in identifying culprit allergens that should be included or excluded from allergen-specific immunotherapy and avoidance strategies [16]. Furthermore, this allows for the tracking of variations in sIgE levels in children with food allergies throughout their clinical course [17]. In these cases, sensitivity is important. In other situations, precise sIgE concentration measurements are required. PROTIA Allergy-Q96^® exhibited a good agreement ratio (>85 % for all tested allergens) and high kappa values (>0.7 for all tested allergens), when compared to ImmunoCAP^® FEIA, suggesting that PROTIA Allergy-Q96^® may be used for the quantitative measurement for sIgE [12].

The benefits of multiplex allergen sIgE detection kits include a shorter run time, a smaller amount of blood samples, and more clinical information regarding the culprit allergens. False positivity and false negatives must be minimized for clinical significance [18].

Cross-reactive carbohydrate determinants (CCDs) are simple carbohydrates linked to amino acid chains found in pollens, vegetable foods, insects, and Hymenoptera venoms and are broadly cross-reactive with CCD-specific IgE antibodies [19]. On the PROTIA Allergy-Q96^® Panel, while reactions to antigens were observed, there were no CCD allergens detected. However, some studies showed false positive responses to MAST [20]. So, we did not need to use CCD-blocker treatment in samples with anti-CCD antibodies, and the number of allergens showing positive reactions was a true positive response.

In Turkey, the multiplex sIgE measurement system is popular instead of singleplex, likely due to the reimbursement policy of the Turkey Health Insurance Program, which only permits six singleplex sIgE measurements. Only in exceptional cases, for example, children under 6 years old, 12 measurements have been permitted. Therefore, the multiplex sIgE system that can detect dozens of traditional sIgEs has been prescribed approximately 5–10 times more than the other systems. Considering this real situation in Turkey, PROTIA Allergy-Q96^® could be a useful MAST assay for Turkish allergic patients.

No standardized specific cut-off level for positive results has been defined worldwide until today [20]. Furthermore, distinct cutoff levels were used in earlier research comparing different MAST assays. For instance, several studies used class 1 as the cutoff level for positive results [21], [22], [23], whereas class 2 was adopted as the cut-off level for positive results in other studies [24]. Considering the natural characteristics of semi-quantitative results in MAST assays, comparing various cut-off levels in the paired results may offer clinical hints for a more accurate diagnosis. Therefore, we applied cutoff levels of class 1 to class 6 for positive results which were 38 patients, in total. If we used Class 2 as a cut-off level for positives, then the total number of positive patients would be 32 (32 %), as six patients were Class 1. We preferred to get the sum of Class 1 and 2 (low-grade allergy) and obtained a statistically significant value to medium and high-grade allergy which created a reasonable comparison. The establishment of a standard cut-off class for positive results should be resolved for future studies. Using the adjusted class 1 as the positive cut-off level might increase the positive rate of allergy disease patients dramatically [23]. Using class 1 as a cut-off level requires correction for false positives.

Few studies have investigated the prevalence of allergy in Turkey [25], [26], [27], [28]. They were mostly focused on food allergies, so we did not get enough information about general allergy distribution.

We divided the allergen positivity rates into two age groups, mostly in the pediatric group showing a distribution of 62.5 %, whereas in adults it was 15.4 %. In other studies on the frequency of food allergies; Roehr et al. reported that 31.4 % of 2,354 children aged 0–17 years in Germany [29]. In our study, females have a higher degree of presenting allergy. However, no relationship was reported between gender and the risk of food allergy [30], 31]. We observed significant differences for indoor acars, and meats and animal products, with a higher number for males (p=0.000).

The manufacturers designed the allergens into 11 main groups. The Pollens Group was at the top in allergen-positive patients (665.40 %) followed by the common allergen group. Although the common group had 27 different allergens, there were only 17 allergens were detected (63 %), and the rest (10) were not. The multiplex allergen screening panels need to be modified according to geographical regions and ethical differences in culprit allergens. To address this issue, manufacturers have to design certain panels to identify culprit allergens related to specific allergic diseases.

In this study, the most frequent allergens that gave positive IgE results were dermatophagoides, house dust, and grass mix consistent with an earlier report [30]. Cow’s milk allergy is one of the most common food allergies in children [32]. In this study, the order of food allergy frequency for cow’s milk, rice, cultivated rye, and potato allergies were 20 , 13, 12, and 10 %, respectively nearly similar to other studies [33]. In allergen-positive patients, the order of food allergens differed and the Fish (shell) Group was 100 %, followed by potatoes (68.4 %) and nuts mix (almond, pine nut, and sunflower) (60.5 %). It is known that our country is an intensive hazelnut production region, but it is an interesting finding that hazelnut allergy is rare both in our study (4 % in total) and in the study conducted by Orhan et al. [34]. In the study conducted by Gelincik et al., vegetables and fruits, especially eggs, tomatoes, and strawberries, were found to be the most common allergenic foods. In the same study, cocoa was identified as another frequent nutrient [35]. In our study, although cacao has a low frequency of 3 % in total, it was higher in the allergen-positive group (21 %).

Another problem for future MAST analyzer development is the inclusion of new allergens in the panel list. Candidate allergens should be evaluated based on evidence of ongoing and significant changes in modern people’s environments and socio-behavioral lifestyles [36]. Simultaneously, a cost-effective approach based on epidemiologic findings of geographically different allergy investigations is required for therapeutically efficient allergen identification [37], [38], [39], [40]. Our data support the high positive rate (20 %) for Acarus siro [41], a new allergen detected solely in the Indoors Group. More research into Acarus siro as an indoor allergen in the general population may shed light on the utility of this technique.

Pet allergies have been increasing worldwide [42]. Cat epithelium, dog dander, and egg white showed slightly lower concordance rates, these different results could be sufficiently overcome by careful interpretation of MAST results in association with clinical manifestations. Among the allergenic mold species, Alternaria alternata, Cladosporium herbarum, Aspergillus fumigatus, and Penicillium notatum were not detected in this study. In Europe, it is estimated to be between as low as 4.4 % [43], and 9 % [44] in the general adult population. The diversity, methodological differences, demographic, and cultural conditions related to dietary habits should be taken into consideration in the worldwide prevalence of allergies. Given the numerous positive result frequencies associated with cross-reactivity across comparable allergens as a viable mechanism for explanation [45], [46], [47], the analyzer’s positive proclivity should be interpreted with caution. Furthermore, differences in allergen extraction methods utilized by different manufacturers may have contributed to the phenomena of diverse positive tendencies in each analyzer.

The 100 % beef allergy detection in the allergen-positive group has great value because PROTIA Allergy-Q96^® detects IgE for α-gal using bovine thyroglobulin which is highly important in clinical sight. The α-gal syndrome, allergy to red meat, is generally characterized by urticaria, angioedema, anaphylaxis, and gastrointestinal symptoms occurring 3–6 h after ingesting red meat including beef, pork, or lamb [48]. Skin Prick Test (SPT) and sIgE for crude meat extracts such as beef, pork, or lamb are known to be not valuable for the differential diagnosis of α-gal syndrome [49]. Contrarily, sIgE for α-gal is known to have diagnostic value in α-gal syndrome [50].

The agreement of total IgE levels in allergen-positive participants divided into two age groups was 58 %, total and 75 % in adults and 53.3 % in the pediatric group (median age 5 years). In young children, total IgE is of limited value when evaluating allergy-related disorders. The lack of agreement among the positive tests of the sIgE and total IgE levels for some allergens implies that these tests should not be used interchangeably, and It is likely best to perform both tests in tandem when determining a child’s atopic sensitization diagnosis. The presence of SIgE-positive results and high total IgE concentration prognosticate a more severe course of the disease.

The current study has some limitations. First, the diagnosis of enrolled patients was not known or confirmed by provocation test. Therefore, sensitivity, specificity, PPV, and NPV could not be calculated. Positive results for any allergens cannot be validated due to the lack of a reference method and reference material for the allergy test. To reduce variations between laboratories, quality control materials, and an external quality control survey will be introduced soon [51]. All clinical laboratory tests must be accurate and reproducible, and in vitro, multiple allergen screening procedures with serum must also be validated. We did not have an opportunity to compare the assay results with skin tests in this study. Using well-standardized allergenic extracts, it had been known that the concordance rate of SPT and serum sIgE measurement ranged from 85 to 95 % [19]. The other limitation is that the number of patients was not high enough to make strong conclusions, because the method is a new one that is not known properly and randomly ordered. The strength of our study is that it is the first study that focuses on the general and several SpIgE detection in our population in a single test.

In conclusion, PROTIA Allergy-Q96^®, a multiplex assay for detecting sIgE might be a reliable and cost-effective alternative to a component-resolved diagnosis of allergic patients. The diversity, methodological differences, demographics, and cultural conditions related to dietary habits should be considered in the worldwide prevalence of allergies. Moreover, this study was a retrospective analysis of the characteristics of IgE-mediated allergies for the entire country of Turkey. Careful consideration is required when applying our data to other ethnic or regional populations.