1. INTRODUCTION
Respiratory allergies (RA) are the most common allergies worldwide, still increasing in frequency and severity. The World Health Organization estimated patients suffering from allergic asthma (AA) at 235 million in 2013 and the numbers for allergic rhinitis (AR) at about 400 million in 2006.1,2 Although the number of patients with RA drastically increased on a global scale, the increase is greatest in Western countries. In 2011, the European Federation of Allergy (EFA) reported that RA affected around 20-30% of the European population. More specifically, around 68 million patients suffered from AA and 113 million from AR.3 To emphasize the increasing prevalence, The European Academy of Allergy and Clinical Immunology (EAACI) reported in 2015 that half the European population will suffer from chronic allergic diseases by 2025.4Alarmingly, a vast majority of individuals affected by allergies are children and young adults.5 Although not being associated with high morbidity and mortality, RA has generated a burden on society due to both the impact on the quality of life and the cost of treatment. A quick and precise diagnosis to identify the causative allergens is of fundamental importance to establish the most adequate treatment.6
In addition to the history and the clinical examination, looking for IgE sensitization is an essential step in the diagnosis of complaints compatible with allergy. Data suggests that over 50% of allergic patients are multi-sensitized, making diagnosis even more challenging.7 Traditionally, two methods are applied:in vivo by executing a skin prick test (SPT) or in vitroby the detection of sIgE in blood by using the ImmunoCAP methodology.8 SPT is quick and results are immediately known, but requires trained clinical personnel to perform it. Although SPT is sensitive, minimally invasive and modest in costs, in vitro methods like ImmunoCAP and ELISA have some advantages including direct quantitation, improved safety, and the possibility of long-term storage of specimens. Notwithstanding, the in vitro procedures present limitations that are an impediment for large studies and for prospective studies to be performed over several years. For instance, measurements for each allergen and sample has to be performed separately, which is time-consuming and is therefore associated with higher costs and increased possibility for technical errors. Comparing studies of the two above mentioned test methods, the SPT seems to be more sensitive (less false-negative results), while sIgE immunoassays seem to be more specific (less false-positive results).9,10
During the last decade, there was a gradual evolution from performing singleplex towards multiplex immunoassays. These multiplex immunoassays provide several advantages over singleplex immunoassays including increased efficiency at reduced expense, lower sample volume needed making it interesting from a pediatric standpoint, greater output (number of markers assessed) per sample volume and higher throughput predicting more detailed diagnostics, thereby facilitating personalized medicine.11 Currently, various singleplex as well as multiplex immunoassays are available to investigate RA-associated immunological protein markers (e.g. by Luminex, Fireplex, Meso Scale Discovery (MSD)), but none provided an assay to assess sIgE levels in multiplex. In this study, we utilized the MSD multiplex immunoassays considering that this method ensures all the above-mentioned advantages, with only 25 µL of serum volume required for analysis. This particular advantage cannot be emphasized enough, since taking blood samples from children remains a demanding technique and ethically sensitive issue. By using the MSD multiplex immunoassay, a finger prick could be sufficient to collect enough sample volume to conduct the test. Likewise, biomonitoring studies could also benefit from this advantage. Additionally, the MSD multiplex immunoassay is a highly flexible method as you can interchange the allergen panel according to the individual needs of the patient. Both individual allergens and allergen mixtures could be spotted on either a complete or partial 96-well plate, making the test even more compelling for either individual diagnosis as well as for biomonitoring studies. Novel techniques like the ImmunoCAP Rapid Point-of-care, which can be performed on children and adults, still require 110µL of sample volume.12,13 Moreover, this technique is not flexible as it operates via a fixed allergen panel.
To date, validation of multiplex immunoassays for in vitrotesting in clinical settings is limited. In this regard, this study aimed to investigate the possibility of diagnosing respiratory allergies with the MSD multiplex immunoassay while simultaneously comparing this method to both an SPT and an ImmunoCAP assay. Statistical analysis demonstrated high comparability of the MSD multiplex immunoassay and the ImmunoCAP assay. Both tests appear reliable in terms of sensitivity and specificity. Moreover, the reproducibility of the MSD multiplex immunoassay was determined, including intra- and inter-assay variation and reproducibility over time between different sampling moments. The reproducibility parameters established significantly high Pearson correlations. Finally, a shorter version of the protocol, including 2h instead of overnight incubation with the samples, provides results within 8h after blood collection.